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 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).

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 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.

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 study the rule of spontaneous behavior and to explore the effect on emotion of mice peripherally infected with influenza A WSN33 virus( H1N1).Methods: Mice were intranasal inoculated with H1N1 WSN33 or saline.Then mice bodyweight change,and total distance movement,average movement speed distance in the central area and feces in the open field test in 5 minutes were recorded in two weeks.Results: Mice following WSN33 infection bodyweight declined sharply until day 7 post-inoculation,and mice bodyweight recovered from influenza infection at day 8 post-inoculation.Total distance movement of mice following H1N1 WSN33 infection decreased in the open field test,and difference of the reduction was significant from day 5 to day 10 post-inocu-lation.The average movement speed had no statistical difference.The range of numbers of fecal grains was large, and they were no significant difference.Conclusion:The total distance movement decreased,but average movement speed did not change following mice infected with H1N1 WSN33.They told us that mice infected with H1N1 WSN33 had anxiety,depressed and nervous emotion which is more evident in acute stage and early recovery stage,whereas the change of the nervous emotion was small and not obvious.

Objective To investigate the clinical efficacy and safety of oral high-dose methylprednisolone in the treatment of infantile spasms (IS).Methods The clinical data of 38 children with infantile spasms were analyzed retrospectively who treated with oral administration of high-dose methylprednisone in Department of Neurology,Wuhan Children's Hospital,Tongji Medical College,Huazhong University of Science and Technology from January 2016 to April 2017.Results (1) Twenty patients (52.6％) of all the 38 patients were seizure-free after 2 weeks of treatment,and 16 cases (42.1％) were seizure-free at the end of treatment.(2) All the 38 cases were typical or atypical hyperarrhythmia.After treatment of 2 weeks,25 cases (65.8％) of hyperarrhythmia disappeared;at the end of the treatment,30 cases (78.9％) of hyperarrhythmia disappeared.(3) Adverse effects mainly were weight gain,Cushing signs,increased appetite,irritability,drowsiness,co-infection,electrolyte disturbance.(4) Follow-up of 3 to 12 months,the recurrence rate was low and the development quotient had improved.Conclusions Oral high dose methylprednisolone in the treatment of IS is effective,safe and has a low recurrence rate.It can be recommended in clinicalal application.

Objective To explore the neuroimmunomedulation mechanism of ICAM-1 and LFA-1 in children with febrile seizures (FS).Methods 40 children with FS were dividedinto simple FS(SFs)groupin20 cases and complex FS(CFS)groupin20 cases,and 30 health children matched with regard to age and sex were enrolled into control group.The real-time fluorescence quantitative PCR wag used to detect the expression of PBMC ICAM-1 mRNA.At the same time,the PBMC LFA-1 mRNA expression wfs studied with Send-QuantitativeRT-PCR analysis.Results The levels of PBMC ICAM-1 mRNA in SFS group were significantly higher than those in control group and CF$group(P<0.05).The levels ofPBMC ICAM-1 mRNA showed downtrend between CFS group and control group.but there was no statistical difference between the two groups(P>0.05).The levels of PBMC LFA-1 mRNA grey-scales in SFS group were significantly higher than those in control group and CFS group(P<0.05).In addition,the levels of PBMC LFA-1 mRNA in CFS group showed downtrend than those in control group,but there wti8 no statistical difference between the two groups(P>0.05).Conclusions The gene expression levels of PBMC ICAM-I/LFA-I in SFS group were different from those in CFS group.Inflammable immunopathology damage induced by ICAM-1/LFA-1 may play an important role in the pathogenesis of SFS.On the contrary,ICAM-1/LFA-1 may have seme neuroprotective effects on the pathogenesis of CFS.