Objective To investigate clinically chromosome micro imbalance in children with unexplained mental retardation(MR) or development delay(DD) by using high resolution microarray comparative genomic hybridization(Array-CGH),to identify chromosome micro imbalance which might be associated with MR/DD,and evaluate the effectiveness of Array-CGH in etiological diagnosis of children with unexplained MR/DD.Methods One hundred and twenty-six children with unexplained MR/DD were recruited for this study by Array-CGH to detect chromosome micro imbalance.All chromosome micro imbalances were verified with database of genomic variation(DGV),Database of Chromosomal Imbalance and Phenotype in Humans using Ensembl Resources(DECIPHER) and literature review,to determine if the chromosome micro imbalances found in these children were associated with MR/DD.Results Twenty eight clinically relevant chromosome micro imbalances were detected among 26 children out of 126 children with unexplained MR/DD.The diagnostic yield for the MR/DD children was 20.6％ (26/126 cases).These chromosome micro imbalances were undetectable by chromosome analysis.All MR/DD children with chromosome micro imbalances had face dysmorphism and/or surface,organ dysmorphism.The most common abnormality was Prader-Willi syndrome/Angelman syndrome(3/26 cases,11.5％),which was followed by DiGeroge syndrome(2/26 cases,7.6％),Cri-du chat syndrome(2/26 cases,7.6％) and 16p11.2 deletion syndrome(2/26 cases,7.6％).Conclusions Chromosome micro imbalance is one of the most common causes of unexplained MR/DD.Array-CGH can detect disease associated with chromosome micro imbalance as a useful evaluation to help differential diagnosis of children with unexplained MR/DD.Screening for chromosome micro imbalance should be firstly carried out in those MR/DD children with face dysmorphism and/or surface,organ dysmorphism.

Objective To evaluate the application of fluorescence PCR in detecting ureA gene of Helicobacter pylori(HP)in feces.Methods Fluorescence PCR was used to detect ureA gene of HP in feces from 50 patients,including 23 confirmed by gastric biopsy urease test and histological staining.Bacterium culture and serum antibody detection were also performed, and chi-square test was used to compare the sensitivity,specificity,positive predictive value and negative predictive value among three methods.Results The sensitivity,specificity,positive predictive value and negative predictive value of fluorescence PCR were 1.00,0.96,96.O%and 100.0%,while those for HP culture were 0.78,1.00,100.0%and 84.0%,and thee for serum antibody detection Was 0.96,0.74,76.O%and 95.0%.There were significant differences in sensitivity and negative predictive value between PCR and bacterium culture (X2=5.60 and 4.44,P<0.05),and significant differences in specificity and positive predictive value between PCR and serum antibody detection(X2=5.28 and 4.08,P<0.05).Conclusion ureA gene detection in feces by fluorescence PCR is of value for the diagnosis of HP infection.

Objective To clone the cheA and cheY genes of Helicobacter pylori for construction of their prokaryotic expression systems, and to establish chemotactic model in vitro of H. pylori for determing chemotaxis-inducing substances and to understand the effects of specific antibody and closantel on inhibiting chemotactic behavior of the microbe. Methods The segments of entire cheA and cheY genes were amplified by PGR and then sequenced after T-A cloning. Prokaryotic expression systems of the genes were subsequent-ly constructed. SDS-PAGE plus Bio-Rad Gel Image Analyzer were used to examine the expression of target recombinant proteins rCheA and rCheY, and Ni-NTA affinity chromatography was performed to extract rCheA and rCheY. Rabbits were immunized with rCheA and rCheY to obtain antisera and IgG in each of the anti-sera was extracted by saturated ammonium sulfate precipitation and DEAE-32 ion exchange chromatography. Immunodiffusion assay was performed to measure the titers of antisera and their IgGs. Chemotactic model in vitro of H. pylori based on hard-agar plus method was established to determine the chemotaxis-inducing effects of eleven candidate substances. Simultaneously, the effects of rCheA-lgG and closantel sodium on blocking the bacterial chemotactic behavior were also observed. Results The segments with expected sizes of cheA and cheY genes were obtained by PCR, and their nucleotide and putative amino acid sequences were 100% idenities to the reports. The constructed prokaryotic systems could efficiently express rCheA and rCheY. The two rabbit antisera and IgG aginst rCheA and rCheY had 1 : 4 and 1 : 2 immunodiffusion titers, respectively. Hydrochloric acid, sulfuric acid and acetic acid were able to induce chemotactic movement of H. pylori. Both rCheA-IgG and closantel sodium with certain concentrations could weaken the chemotactic ability of H. pylori(P<0.05). Conclusion The prokaryotic expression systems of H. pylori cheA and cheY genes were successfully generated in this study. Hydrogen ion (H~+) is the inducer for chemotaxis of H. py-lori. rCheA-IgG, as well as closantel sodium can inhibit H~+-induced chemotaxis of H. pylori.

Objective To observe the effects of training on intrinsic hand muscles after digital flexor tendonrepair. Methods A total of 60 patients with digital flexor tendon repair were randomly assigned into an experimentgroup (n = 30) and a control group (n = 30). Patients in the experiment group received intrinsic hand muscles train-ing, in addition to the passive mobilization of the flexor tendons and active mobilization of the extensor tendons admin-istered to those in the control group. Minnesota manual dexterity test and Purdue pegboard test were adopted to as-sessment the outcome after 3 months of training. Results After 3 months of training, there were significant differ-ences between the two groups in terms of the results of Minnesota manual dexterity test and Purdue pegboard test( P < 0.01 ). Conclusion Intrinsic hand muscles training after digital flexor tendon repair can improve fine move-ment and coordination of hand.

Objective To determine the effect of cheA gene of Helicobacter pylori in the bacterial chemotaxis in vitro and colonization in vivo. Methods The entire cheA and cheY genes were amplified and cloned from genomic DNA of H. pylori NCTC11637 strain. Subsequently, the prokaryotic expression systems of cheA and cheY genes were generated and the target recombinant proteins rCheA and rCheY were extracted by Ni-NTA affinity chromatography. Rabbits were immunized with either rCheA or rCheY for obtaining antisera, and rCheA-IgG and rCheY-IgG in the antisera were prepared using ammonium sulfate precipitation plus DEAE-52 column chromatography. A suicide plasmid of cheA gene was constructed and then a cheA gene knock-out mutant ( cheA - ) was generated based on homologous recombinant exchange using the suicide plasmid. The cheA- mutant was identified using PCR and sequencing. The phosphorylation levels of CheA and CheY molecules of cheA - and wild-type strain were determined by using rCheA-IgG and rCheY-IgG anchoring the target proteins and protein phosphorylation detection kit. The differences of chemotaxis in vitro and colonization in vivo between cheA- mutant and wild-type strain were compared using chemotactic model and BALB/c infection model of H. pylori. Results The cheA gene knock-out in genome of cheA- mutant was confirmed by the results of PCR and sequencing. After treated with 0. 001-0. 1 mol/L HCI for 10 min, the phosphorylation levels of CheA and CheY molecules of wild-type strain were rapidly descended from ( 59.6 ±11.5) μmol and (55.5 ± 10.2) μmol to ( 10.8 ± 2.6) and (5. 5 ± 1.2) μmol (P ＜ 0.05 ), while the phosphorylation of CheY molecule of cheA - mutant was no markedly changed with a persistent lower level ( P ＞0.05). The diameters [(10-20) ± (2-3) mm] of chemotactic aggregative rings of cheA- mutant were significantly less than those [(16-24) ± (2-3)mm] of wild-type strain (P ＜0.05). The positive isolation rate (90%) of H. pylori in gastric biopsy specimens of mice that infected with wild-type strain was remarkably higher than that (40%) of mice that infected with cheA- mutant (P ＜0.05). The result of fluorescence quantitative was also showed that the numbers (6.3 × 103 ±2.1 × 103 copies/mg) of H. pylori in gastric biopsy specimens of wild-type strain infected mice were significantly larger than those (8.3 × 101 ±3. 1 × 101 copies/mg) in gastric biopsy specimens ofcheA- mutant infected mice (P＜0.05). Conclusion The cheA gene of H. pylori has an important role in the bacterial chemotaxis in vitro and colonization in vivo.

ObjectiveTo optimize the condition of multiple fluorescence quantitative PCR and establish a new assay of four human herpes virus (HHV) detected by AllGlo quadruple fluorescence quantitative PCR.MethodsFour HHV including HSV-1,HSV-2,EBV and CMV were identified by sequence analysing the qualitative PCR production.Furthermore,they were quantitatively detected by AllGlo and TaqMan multiple fluorescence quantitative PCR respectively.ResultsBoth the positive rate and specificity of AllGlo and TaqMan in detecting single HHV achieved 100％.And AllGlo single fluorescence quantitative PCR prevailed over TaqMan's by Ct of 1-3.Four HHV can be simultaneously detected by AllGlo quadruple fluorescence quantitative PCR,comparing to the only two by TaqMan.ConclusionAllGlo fluorescence quantitative PCR assay allows a higher throughput,sensitivity and specificity than TaqMan in detection and thus provides a board prospect.

Objective To assess the efficiency and reliability of clinical genetic diagnosis of methylmalonic acidemia(MMA) using new generation sequencing platform (HiSeq2000).Methods 1.Nine patients diagnosed with clinical signs of MMA were recruited.DNA library from the patients were mixed with designed gene capture probe.The whole exons region of 48 genes related to organic acid metabolism were screened using the gene capture combined with high-throughput sequencing.2.The joints were removed and the low quality data were filtered,the data were analyzed by means of SNP and InDel.To avoid the false positive,the abnormal sites were verified using the Sanger sequencing method.3.The detection of the organic acid in the urine was performed through gas chromatography-mass spectrometry and other auxiliary examinations.Results 1.Gene mutation:7 gene mutations of MMACHC were identified in 7 patients.Seven mutations:c.482G ＞ A,c.567_568insT,c.609G ＞ A,c.440_441del,c.80A ＞ G,c.315C ＞ G,c.90G ＞ Awere screened.The mutation c.440_441del had not been reported before,and others were all related to the disease.Two gene mutations of mutase apoenzyme(MUT) were identified in 1 case,all of which were introns:.c.754-1G ＞ C,c.1677-1G ＞ A.The novel mutation was c.754-1G ＞ C.No gene mutation was identified in 1 patient.2.Clinical manifestation:all of the patients were development delay,but the degrees were different;3 patients with convulsion; 1 patient with headache and central facial paralysis;1 patient with repeated intractable metabolic acidosis;1 patient with repeated hemolysis.Electroencephalogram of the all patients were abnormal;the result of cranial MRI of the 8 patients were abnormal;In all patients,urine level of methylmalonic acid significantly increased (273.4-146 022.8 times).Blood homo cysteine of 8 patients were significantly increased(27.13-396.84 μmol/L,normal ＜ 20 μmol/L).3.Sanger sequencing:there were no false positive exists.Conclusions 1.There were not a correlation between the clinical manifestation and gene mutation of the patients with MMA.The c.609G ＞ A was the hotspot mutation of MMACHC gene in Chinese patients with MMA and homocysteinemia.2.The mutations c.440_441del and c.754-1G ＞ C were presumed to be novel mutations.3.Gene capture technology combined with next-generation sequencing technology could be used to interrogate the wealth of data available in the human genome and lay the foundations for counseling of gene.This platform can be readily and timely adopted by clinical molecular diagnosis of MMA and represents a high throughput,high sensitivity,high efficiency and other characteristics approach for screening common genetic diseases.

BACKGROUND:Compared with conventional medications, drug micro-capsule system can control the release of drugs and have wel target properties and biocompatibility. The drugs can be concentrated at the focus and play an important role in clinic. OBJECTIVE:To prepare dacarbazine magnetic micro-capsules with different capsule materials and gelatin complex by coacervation, and to optimize capsule materials and preparation process. METHODS:Fe 3 O 4 RESULTS AND CONCLUSION:The solution complex coacervation method was better than the emulsion coacervation method. As for the solution complex coacervation method, the optimal capsule material was gelatin-sodium alginate, with drug embedding rate 37.90%, the yield rate 72.31%, and the average magnetization intensity 8.53 emu/g. The second material was gelatin-chitosan. As a capsule material, the gelatin was better than chitosan with single coagulation method. Drug embedding rate was 51.58%, the yield rate was 64.50%, and the average magnetization was 6.93 emu/g. Single coagulation method was better than coacervation method. complex coacervation, we prepared the gelatin-Arabic gum magnetic micro-capsule, gelatin-sodium alginate magnetic micro-capsules, gelatin-sodium carboxymethyl cel ulose magnetic micro-capsules, and gelatin-chitosan magnetic micro-capsules. With the emulsion complex coacervation method, we further prepared the gelatin-Arabic gum magnetic micro-capsule, gelatin-sodium alginate magnetic micro-capsules, gelatin-sodium carboxymethyl cel ulose magnetic micro-capsules, and gelatin-chitosan magnetic micro-capsules. The magnetic gelatin micro-capsules and magnetic chitosan micro-capsules were prepared with single coagulation method. The micro-capsules were determined for the embedding rate, the magnetic susceptibility, the micro-capsule size and the release performance, to define the optimal preparation technology of dacarbazine magnetic micro-capsules.

Objective To establish a rapid,accurate and specific method to detect the common mycobacteria based on multiplex real-time PCR.Methods The dual priming oligonucleotide ( DPO)primers and TaqMan probes labeled with FAM,ROX,HEX or JOE fluoresceins at 5' end and eclipse at 3' end respectively were designed to detect the 16S rRNA of mycobacteria.Both specificity and sensitivity were estimated on multiplex real-time PCR detecting genome DNA from 4 mycobacterial model species.Sixty eight early morning sputum specimens collected from hospitalized patients in the Red Cross Hospital of Hangzhou were detected by multiplex real-time PCR,bacterial culture and smear microscopy simultaneously.The positive rates were analyzed by chi-square.Results Mycobacteria including Mycobacterium tuberculosis and three common non-tuberculosis mycobacteria spp.were identified by multiplex real-time PCR accurately and specifically,with the limited load at 101 cfu/ml.In 68 sputum specimens,31 were positive (positive rate 45.6％ ) by this method,which was significant higher than that by smear microscopy ( positive rate 14.7％,x2 =15.4,P ＜0.05 ).The positive cases were identified as 28 Mycobacterium tuberculosis,1 Mycobacterium avium and 2 Mycobacterium intracellulare in agreement with the culture results.One case,which is detected by culture,but not by PCR,was identified as Mycobacterium chelonae by sequencing.Conclusion The multiplex real-time PCR characterizing as sensitive,specific and time-saving for Mycobacterium tuberculosis and common non-tuberculosis mycobacteria could be chosen as the rapid laboratory test of mycobacterial infection.

Objective To explore the protective effect and mechanism of the dihydromyricetin (DHM) on cognitive dysfunction in Alzheimer’s disease (AD) rat model. Methods The AD model of rats was established by injecting Aβ1-42 oligomolymer into the hippocampus. According to the random number table,30 successfully constructed AD model rats were divided into AD group,AD+DHM1 group and AD+DHM2 group,with 10 in each group. And the rats in the three groups were intraperitoneally injected with nor-mal saline,100 mg/kg DHM and 200 mg/kg DHM for 21 days,respectively. Another 10 rats with body mass matching were taken as the control group. Morris water maze was used to evaluate the spatial learning and memory ability of rats in each group,the expression of inflammatory cytokines were detected by Elisa,and the expressions of AMPK and SIRT1 proteins were detected by Western blot. Results Compared with the con-trol group,the escape incubation period of rats in AD group was prolonged,and the difference was statistically significant (day 5 :(10. 36±2. 80)s,(22. 40±2. 98)s;t=-18. 63,P<0. 05). Compared with AD group,the escape latency of rats in AD+DHM1 group and AD+DHM2 group were shortened (day 5:AD+DHM1 group (15. 68±3. 06) s,AD+DHM2 group (18. 85±3. 22) s; t=10. 65,4. 13,both P<0. 05). Compared with AD group,rats in AD+DHM1 group and AD+DHM2 group had more crossing times ((1. 87± 0. 76),( 2. 75± 0. 63) and (3. 78±0. 71);t=-6. 86,-9. 83,both P<0. 05),and the target quadrant residence time were ex-tended ((17. 08±1. 99) s,(16. 33±4. 33) s,(22. 59±4. 21) s;t= 28. 5,8. 63,both P<0. 05). Compared with the control group,the levels of IL-1β,IL-6 and TNF-α in the serum and hippocampus of the AD group were significantly increased (serum: t=4. 98, 7. 87, 5. 43, all P<0. 05; hippocampus: t=11. 13, 30. 50, 23. 38,all P<0. 05). Compared with the AD group,the levels of IL-1β,IL-6 and TNF-α in the serum and hippocampus of the AD+DHM1 group and the AD+DHM2 group were significantly decreased,the difference was statistically significant ( serum: AD+DHM1 group t=-4. 13,-10. 70,-9. 22, AD+DHM2 group t=-1. 75,-3. 63,-18. 75,all P<0. 05;hippocampus:AD+DHM1 group t=-69. 13,-15. 13,-6. 50,AD+DHM2 group t=-10. 25,-39. 00,-8. 00,all P<0. 05). Compared with the control group,the expression of p-AMPK/AMPK protein and SIRT1 protein in the AD group were decreased. The expression of the two pro-teins in the AD+DHM1 group and the AD+DHM2 group were increased,comparing with those of AD group, and the difference was statistically significant(all P<0. 05). Conclusion DHM exerts protective role in AD model rats,which may be related to the activation of AMPK/SIRT1 pathway and the inhibition of inflammato-ry response.