Objective The prokaryotic expression vector of NADP(H)-dependent retinol dehydrogenase/reductase(NRDR)B1 was constructed for the detection of NRDRB1 protein,and to prepare its polyclonal antibodies,in order to lay the foundation to study the function of NRDRB1.Methods The coding region of NRDRB1 was constructed to the Gateway-based expression vector(pDEST 14),which was transformed into the Escherichia coli(BL21-AI)for the native protein expression.Overexpression of the recombinant was induced at mid-log growth phase of BL21-AI(A600=0.6)using 0.2% L-arabinose.After supersonication the inclusion bodies of NRDRB1 were purified.New Zealand rabbits were immunized with NRDRB1 as the immunogen,which was recovered from SDS-PAGE gel and subscapsularly injected.The titer of the antiserum was determined by dot blot assay.The antibody was purified by HiTrap Protein G column,and its activity and specificity were assessed by Western blotting and immunohistochemistry.Results The prokaryotic expression vector pDEST 14 with NRDRB1 was constructed.The constructs were sequenced by dideoxynucleotide method.NRDRB1 was overexpressed in strain BL2l-AI.The concentration of recovered NRDRB1 was 0.42mg/ml with a recovery rate of 52.3%.All the immunized rabbits produced high-titer antisera after the second booster.The titer of the antiserum was 1∶2 000 with a detection limit of 6.4ng NRDRB1.The purified antibody had a high specificity.Conclusion The present study provides an effective method of preparing polyclonal antibody against NRDRB1.The purified NRDRB1 native protein and the specific polyclonal antibody of NRDRB1 would be valuable for the study on the biological function of NRDRB1.

Objective To explore the possible correlations under the interaction of multi-genes action at different stages and analyze the primary histophotomacrographic changes of hind buds tissue in congenital clubfoot and the pathodynamic developmental procedure.MethodsSeventy-seven female Wistar rats were administered with retinoic acid on the 10th day after pregnancy. And the hindlimb, buds and spinal cord were detected through transmission electron microscopic and molecular biological experiments.ResultsThere was clubfoot-like deformity in 61.8% of the experimental animals. Persistence of the embryonic position of the talus and tibia in fetuses was observed. Poor overlapping between talus and calcaneus was seen. Cell apoptosis at the anterior corner of spinal cord and hind buds were seen.ConclusionCongenital clubfoot deformities appear at early stage and exaggerate along with developmental procedure.

Objective To explore the effects of protein kinase C alpha(PKC?) cDNA on the expression of genes of multidrug resistance (MDR) in renal cell carcinoma(RCC) 786-0 cell line.Methods LR/BR recombination reactions were used to generate mammalian expression vectors of PKC? cDNA with C-terminal fused green fluorescent protein(GFP),and vectors were transfected into human RCC cell with Lipofectimine 2000.Western blot method and inverted fluorescent microscope were used to determine the expression of PKC? in RCC cells transfected by PKC? cDNA.RT-PCR was used to determine the expression of MDR-related genes MDR1,MRP1 and LRP in RCC cells transfected by PKC? cDNA.Results After transfection of 786-0 cells with pcDNA-DEST47-PKC?-GFP vectors,the results of Western blot showed that PKC? was highly expressed in human RCC 786-0 cells;and inverted fluorescent microscopy showed that GFP was highly expressed in RCC 786-0 cells.The results of semi-quantitative RT-PCR analysis showed that the expression level of MDR1 was higher in RCC cells transfected by PKC? cDNA than in RCC cells.Conclusions The expression of MDR1 mRNA in 786-0 cell line can be up-regulated by PKC? cDNA,which suggests PKC? cDNA can induce the increase of MDR in renal cell carcinoma.

Objective To summarize and investigate the lethal factor of acute obstructive suppurative cholangitis (AOSC).Methods The clinical data of 56 patients with AOSC were retrospectively analyzed.Results Six cases died,5 cases with acidosis,5 cases with thrombocytopenia and 5 cases with temperature change obviously,4 cases with heart,lung and kidney disease or diabetes,5 cases with operation and operation time ≥ 150 min,5 cases with from onset to treatment time ≥72 h.Eighteen cases of elderly patients ≥70 years old,4 cases died.The patients whose age≥70 years,temperature ≥39 ℃ or ＜ 36 ℃,combined with acidosis,platelet counts ≤6.0 × 1012/L,with heart,lung,kidney diease or diabetes,time of anesthesia and operation ≥ 150 min and from onset to treatment time ≥72 h had higher death rate (P ＜ 0.05).Conclusion Age,obvious temperature abnormalities,significantly platelet decrease,with heart,lung,kidney diease or diabetes,acidosis,long time of anesthesia and operation and from onset to treatment time ≥ 72 h are the lethal factor of AOSC.

Objective To screen the differently expressed genes in great saphenous varicose vein using cDNA microarray. Methods Varicose veins from 5 cases and normal veins near the saphenofemoral junction were collected, total RNA was isolated and mRNA was purified by oligotex. mRNA from varicose veins and normal veins were reversely transcribed to cDNA with the incorporation of fluorescent dUTP to prepare the hybridization probes, which were hybridized to cDNA microarray of 4 096 human genes. RT-PCR and Western blot were used to identify differently expressed genes. Results One hundred and sixty-eight genes were shown in differently expressed profile with 96 upregulated and 72 downregulated genes, in all 5 varicose samples there were 28 genes upregulated and 11 downregulated. The differently expressed genes were involved in the functions of apoptosis, signal conduct, protooncogene and anti-oncogene. The expression of caspase 9 and MAP3K were identified by RT-PCR and Western blot. Conclusion Disease-related gene screening by cDNA microarray in great saphenous varicose vein affords an insight into the pathogenesis of varicosis.

Objective To evaluate the application value of PCR-reverse dot blot hybridization in the identification of Candida and the detection of Candida albicans drug-resistant genes.Methods The vaginal secretion samples from 285 patients with candidal vaginitis and 50 healthy women were collected.The identification of Candida species and their drug susceptibility were detected by the bioMérieux Yeast identification cards and MIC method(Zhengzhou Antu kit),respectively.The identification of Candida species and the mutation of Candida albicans,drug-resistant genes were also detected by the Shenzheng Yaneng test kit(PCR-reverse dot blot hybridization).The drug-resistant genes were also identified by PCR and nucleic acid sequencing.Based on the culture identification,MIC method and nucleic acid sequencing as the contrast methods,the sensitivity,specificity and accuracy of PCR-reverse dot blot hybridization in the identification of Candida species and the mutation detection of Candida albicans drug-resistant genes were evaluated.Results Compared with the bioMérieux Yeast identification method,the sensitivity,specificity,positive predictive value,negative predictive value and total coincidence rate of PCR-reverse dot blot hybridization for detecting six kinds of Candida species,including Candida albicans,Candida glabrata,Candida tropicalis,Candida parapsilosis,Candida krusei and Candida guilliermondii,were above 95％,96％,96％,98％ and 97％,respectively.There was no significant difference in detecting six kinds of Candida species between the two methods (x2 =0.44,0,0,0,0 and 0,respectively,P ＞ 0.05),and there was good consistency between them (Kappa ＞ 0.9).Compared with the MIC method,the sensitivity,specificity,positive predictive value,negative predictive value and total coincidence rate of PCR-reverse dot blot hybridization for detecting the drug resistance of Candida albicans were 98％,88％,98％,88％ and 96％,respectively.There was no significant difference in detecting the drug resistance of Candida albicans between the two methods (x2 =0.17,P ＞ 0.05),and there was good consistency between them (Kappa ＞ 0.8).The results of PCR-reverse dot blot hybridization in detecting the mutation sites of six kinds of Candida albicans drug-resistant genes were 100％ of coincidence with that of the nucleic acid sequencing method.Conclusion The PCR-reverse dot blot hybridization has high consistency with the culture method and the nucleic acid sequencing method in the identification of Candida species and the mutation detection of Candida albicans drug-resistant genes,which is more early and rapid than the traditional detection methods,and may be applied to the auxiliary diagnosis of vulvovaginal candidiasis (VVC).

The article introduced the content of medical genetics in USMLE,and compared it with Chinese official teaching material. Medical genetics is an important part of USMLE and the USMLE questions come from real clinical cases and pay more attention to clinical experience. To adapt to the international trend,Chinese teaching material can clarify the genetic-related illness in detail. Clinical case is a good guide for PBL(Problem-Based learning)teaching. The medical genetics were called for in Chinese Practicing Physician Qualifications Test.

Objective To analyze the clinical features and gene mutation in mthylmalonic acidemia (MMA) accompanied by homocysteinemia (cblC), and review the relevant literatures. Methods The clinical features of 3 cases of MMA diagnosed by gene detection were retrospectively analyzed, and meanwhile the pertinent literatures of pathogenesis of MMA, especially combined with late-onset cblC and its gene detection, were reviewed. Results Patient 1 (26 days old) suffered from intermittent convulsions for 3 days, with isosuccinic acid 175.8 μmol/L, C3/C2 rate 1.363, homocysteine >?65 μmol/L and abnormal EEG. MMACHC gene detection found an exon deficiency (delEXON1), which has not been reported. Patient 2 ( 12 year old) was hospitalized for limb shaking, hyperspasmia and vomiting. His isosuccinic acid level was 334.3 μmol/L, C3/?C2 rate was 0.37, homocysteine >?65 μmol/L, and had abnormal EEG. MMACHC gene detection found the mutations of c.482G?>?A and c.609G?>?A. Patient 3 was hospitalized for intermittent convulsions for 20 days, whose isosuccinic acid, C3/?C2 rate, and homocysteine were increased. MMACHC gene detection found the mutations of c.394C?>?T and c.540del8 and c.540del8 had not been reported. Review of literatures discovered that MMA was combined with epileptic seizure in some patents, which further validate that the mutation in MMACHC gene c.482G?>?A may be related to the late-onset of cblC. Conclusions Gene detection contributes to the diagnosis of MMA; the mutation of MMACHC gene c.482G>A may be related to the late-onset of cblC; delEXON1 and c.540del8 are new mutations which have not been reported.

Objective To compare the efficiency and safety between Aripiprazole and other traditional drugs for Tourette's syndrome treatment.Methods Databases such as China National Knowledge Infrastructure,Wanfang,VIP,China Biology Medicine Disc,PubMed and Web of Science were electronically searched for studies on Aripiprazole for Tourette's syndrome treatment.According to the inclusion and exclusion criteria,studies,extracted data,and assessed quality were screened.Meta-analysis was performed by using Stata 11.0 software.Results Four studies about Aripiprazole for Tourette's syndrome treatment with 396 patients (Aripiprazole group:201 cases;control group:195 cases) were synthetically and quantitatively analyzed.Meta-analysis results showed that Aripiprazole was better than other traditional agents (placebo,Tiapride,Haloperidol) [standardized mean difference (SMD) =0.21,95％ CI:0.10-0.32].The subgroup by time of treatment analysis results indicated that Aripiprazole was superior to other drugs in 2 weeks (SMD =0.28,95％ CI:0.06-0.50).There was no significant difference in the efficacy between Aripiprazole and other drugs for treatment of Tourette's syndrome in 4 weeks and 8 weeks after treatment (SMD =0.16,0.28;95％ CI:-0.05-0.38、-0.20-0.76).The subgroup by matched drugs results suggested that Aripiprazole was better than Tiapride (SMD =0.29,95 ％ CI:0.15-0.43),but was not significantly different from Haloperidol (SMD =-0.03,95％ CI:-0.28-0.22).There was no significant difference in side effects incidence between Aripiprazole and traditional drugs for treatment of Tourette's syndrome (RR =0.83,95 ％ CI:0.36-1.89).Conclusions Compared with the conventional drugs,Aripiprazole has better therapeutic efficacy in the treatment of Tourette's syndrome in children in 2 weeks.Aripiprazole is better than Tiapride,but equal to Haloperidol in the treatment of Tourette's syndrome.The safety of Aripiprazole needs to be further verified.

Objective To investigate the clinical features of paroxysmal kinesigenic dyskinesia (PKD) and the mutation features of its pathogenic gene proline-rich transmenbrane protein 2 (PRRT2). Method The clinical manifestations and genetic tests of one case of PKD were retrospectively analyzed, and the related literatures were reviewed. Results A 10 year and 9 month male patient was recruited. The age of dyskinesias onset was 7 year and 6 month. The descriptions of the attacks were abnormal involuntary movements which were induced by sudden voluntary movements and presented with dystonia. The frequency of the attacks was three to ifve times per day with the duration lasting ten to twenty seconds, and there is no loss of consciousness. Treatment with oxcarbazepine is effective. A heterozygous mutation in PRRT2 gene, c.649_650insC (p. 217fs224X), was found by genetic testing, and the mutation was inherited from the patient’s mother who showed no symptom of PKD. Conclusion The onset age of PKD could be in the childhood and adolescence. The attack is provoked by sudden movements and the duration time is short. Treatment with antiepileptic drug is effective. The test of PRRT2 gene may help diagnosis. Mutation c.649_650insC is the hotspot mutation of the gene.