Objective To construct a eukaryofic expression vector for A PP695 gene carrying green fluorescence protein (GFP) gene. Methods RT-PCR was used to amplify the full-length APP695 cDNA. The PCR products and the eukaryotic expression vector pIRES2-EGFP were digested by restriction endonueleases, and the digested APP695 gene was inserted into the digested eukaryotic expression vector. The positive recombinants were identified by PCR analysis, Nhe Ⅰ and Hind Ⅲ digestion and sequence analysis. Results The 2088-bp DNA fragment was amplified by PCR from the plasmid pCB6, and an identical DNA fragment was also amplified from the recombinants. The products of double restriction enzyme digestion were A PP695 gene with a 5.3-kb DNA fragment. Sequence analysis confirmed successful insertion of A PP695 gene into pIRES2-EGFP vector. Conclusion The eukaryotie expression vector pIRES2/APP695-EGFP has been successfully constructed.

Objective To establish and test the reliability and validity of the disease related psychological pressure scale of pregnant women with chronic HBV infection,so as to provide mental stress assessment tool for the pregnant women.Methods Item pool was developed after the interview and comprehensively retrieved literatures,then after expert review and rebuilt,the scale items were fixed.Totals of 368 pregnant women with chronic HBV infection disease were investigated and the reliability and validity of scale were test.Results The scale had 5 factors and 24 items,and the correlation coefficient between each factor score and total score was 0.712 - 0.894.The correlation coefficient among each factor was 0.409 - 0.631 which was significantly lower than that between each factor score and total score ( P ＜ 0.01 ).The cumulative contribution rate of 5 factors was 64.055％,and tes-retest reliability of 5 factors was 0.856,0.887,0.828,0.813.The Cronbach' s α of internal consistency of 5 factors was 0.788 - 0.865,and the Cronbach' s α of scale was 0.932.Conclusions The scale has good reliability and validity,and can be used for measuring the disease related pregnancy psychological pressure for chronic HBV infection pregnant women.

OBJECTIVETo compare results of the growth and development of the upper dental arch and the velopharyngeal closure of the cleft patients treated by two methods.

METHODSThe dental cast of patient and X-ray films were measured and the statistical medical records were analyzed.

RESULTSThe transverse distance of upper dental arch was found to be wider in group A than in group B. The anterior-posterior distance of the dental arch in bilateral cleft group was shorter in group A than in group B. The difference of the two groups were gradually lessened as age increases. Bony bridge in alveolar gap was 63% and 83.3% in unilateral and bilateral cleft group respectively. 15% of cases in group A and 35.2% in group B needed pharyngeal flap.

CONCLUSIONSThe stable upper dental arch in group A can opposes the pressure from the lip muscles, this maintains the width of the arch. But A-P distance of upper dental arch in BCLP in group A should be followed up after the age of 9 years. Pharyngeal flap is needed less in group A than in group B.

Alveolar Process ; growth & development ; Child ; Child, Preschool ; Cleft Lip ; surgery ; Cleft Palate ; surgery ; Humans ; Infant ; Orthodontics, Corrective ; Palate, Soft ; surgery

OBJECTIVETo explore safety, indications and advantages of mapping and ablation of arrhythmia in children guided by Carto and Ensite system.

METHODSGuided by Carto system, radiofrequency catheter ablation (RFCA) was performed on 8 pediatric patients with tachycardia whose mean age was (6.2 + or - 1.7) years, mean weight was (18.0 + or - 2.0) kg. Guided by Ensite system, RFCA was performed on 10 pediatric patients with arrhythmia, 8 of them were ablated guided by Ensite Array system: 6 cases with premature ventricular contractions (PVCs), 2 cases with right atrial tachycardia, their mean age was (11.3 + or - 1.2) years, and mean weight (40.0 + or - 5.0) kg. The other two cases with W-P-W syndrome were ablated guided by Ensite Navx system.

RESULTGuided by Carto system, 8 cases were successfully mapped and ablated: 6 cases had incision atrial tachycardia, 1 case had left atrial tachycardia and 1 case had right atrial tachycardia. In 1 case with incision atrial tachycardia the condition recurred after 3 months, and was ablated again successfully. Guided by Ensite Array system, 6 cases with PVCs (in 2 originating from the right ventricular inflow tract and in 4 originating from the right ventricular outflow tract) and 2 cases with right atrial tachycardia were successfully mapped and ablated, PVCs of the first 6 cases were reduced from (32 333 + or - 4509) 24 h to (0-4)/24 h after ablation. In 1 case with automatic atrial tachycardia, mapping could not be done by Ensite Array system, because P wave could not be identified from T wave. Single bolus of adenosine 20 mg was given within 30 s to let ventricles stop for 2 s (cardio-ventricular pacing standby) until T wave vanished, mapping and ablation were operated again successfully, but another atrial tachycardia occurred 1 day later. Guided by Ensite Navx system, 2 cases with W-P-W syndrome were successfully ablated, operation under X-rays lasted for 8 and 10 min. In none of the 9 patients the disease recurred after follow-up for 6 months.

CONCLUSIONCarto system is suitable for mapping and ablation in pediatric patients with continuous tachycardia, especially with incision atrial tachycardia; Ensite Array system fits children older than 10 years with right heart discontinuous arrhythmia; and Ensite NavX system can set up model and display endocardial anatomic structure quickly. Compared with two-dimensional mapping system, the three-dimensional mapping system (Carto and Ensite) can display the origin of arrhythmia and activation sequence clearly, decrease difficulty of operation efficiently and diminish operation time under X-ray.

Arrhythmias, Cardiac ; physiopathology ; surgery ; Catheter Ablation ; methods ; Child ; Child, Preschool ; Electrophysiologic Techniques, Cardiac ; methods ; Humans ; Imaging, Three-Dimensional ; Treatment Outcome

Objective To investigate the molecular genetic causes and their characteristics of deafness in Ningxia province,we established screening of three common hereditary deafness genes in 336 deaf and hard-of-hearing patients in this district.Methods Peripheral blood samples were obtained from a total of 336 patients with non-syndromic sensorineural hearing loss in parts of special education schools in Ningxia province to extract genomic DNA.The mitochondrial DNA 12S rRNA m.1555A ＞ G mutation was screened by PCR Alw26I digestion and sequence analysis PCR and direct sequencing were used to analyze the coding region of GJB2 and exons 8 and 19 of SLC26A4.Statistical analysis was performed by using SPSS 11.0 software.Frequencies of different GJB2 or SLC26A4 mutations were compared between Han and Hui people.Results Among these 336 patients,seven cases (2.08％,7/336) were found to carry mtDNA 12S rRNA m.1555A ＞ G homozygous mutation,45 cases ( 13.39％ ) were caused by GJB2 mutations and 28 cases (8.33％ ) had two mutated alleles (homozygote and compound heterozygote) of SLC26A4.In detail,16.67％ (56/336) patients carried GJB2 mutations including 11 single mutant carriers.The allele frequency of c.235delC and c.299_300delAT were 9.52％ (64/672) and 2.68％ ( 18/672),respectively,making up 81.19％ (82/101) of all pathogenic mutated alleles for GJB2.The single mutant allele carriers of SLC26A4 is 32,and two types (c.919-2A ＞ G and c.2168A ＞ G) accounted for 95.29％ (24/27)mutations,totally.We also found that statistically significant differences in c.919-2A ＞ G and c.2168A ＞ G frequencies between Han and Hui people ( c.919-2A ＞ G,x2 =8.229,P =0.004 ; c.2168 A ＞ G,x2 =5.277,P =0.022).However,there was no statistically significant difference in GJB2 mutation between Han and Hui people.Conclusions GJB2 mutation was a primary causc for non-syndromic sensorineural hearing loss in Ningxia province,and c.235delC was the most common mutant forms of GJB2.c.919-2A ＞ G and c.2168A ＞ G were common mutant forms of SLC26A4,their frequencies were also statistically significant differences between Han and Hui people.

Background and Objective:Recently,the theory of cancer stem cells(CSCs)has presented new targets and orientations for tumor therapy.The major difficulties in researching CSCs include their isolation and purification.The aim of this study is to identify and characterize the side population(SP)cells in small cell lung cancer(SCLC)cell line H446,which Iays the foundation for the isolation and purification of CSCs.Methods:Fluorescence-activated cell sorting(FACS)was used to sort SP and non-SP (NSP)cells from H446,Both subgroups were cultivated to survey the capacity to form into suspended tumor cell spheres.Reverse transcriptionpolymerase chain reaction(RT-PCR)and real-time PCR were used to evaluate the expression levels of the mRNA of CD133,ABCG2,and nucleostemin in both subgroups.The capacity of proliferation and the differences in drug resistance of both subgroups and unso rted cells were tested by the MTT method.The differentiation ability of both subgroups was determined by FACS.Proliferation was determined by subcutaneous tumor formation in nude mice.Results:The percent of Hoechst 33342 negative cells was about(5.1±0.2)%in H446 by fluorescence microscopy.The percent of SP cells was(6.3±0.1)%by flow cytometry.SP cells had a stronger capability of fOrming into tumOr spheres than NSP cells.The mRNA expression Ievels of ABCG2,CD133,and nucleostemin in SP cells were 21.60±0.26,7.10±0.14,and 1.02±0.08 folds higher than that in NSP cells(P＜0.01,P＜0.01,and P＞0.05,respectively).In vivo,SP cells showed better proliferative ability and tougher viability when treated with drugs.SP cells can differentiate into NSP cells,but NSP cells cannot differentiate into SP cells.SP cells had a greater ability to form tumors.Conclusions:The H446 cell line contained some SP cells with stem cell properties.CD133 and ABCG2 may be cancer stem celI markers of SCLC.

Objective To study the genotype distribution of extended-spectrum β-lactamases (ESBLs) in ESBLs-producing Escherichia coli (E.coli) isolates from posthepatitic cirrhosis' patients with bloodstream infection.Methods E.coli were isolated in bloodstream from patients with posthepatitic cirrhosis between January and December in 2011.The strains were identified by VITEK-Ⅱ.The antibiol susceptibility tests were performed with K-B method.β-lactamases genes were detected multi-PCR,PCR,sequence and blast.Results A total of 79 non-duplicate clinical isolates of E coli were consecutively collected from liver cirrhosis' patients with bloodstream infection.There were 20 isolates produced TEM-1 type β-lactamases and 1 isolate produced SHV-1 typeβ-lactamases.40 clinical isolates were detected to produce CTX-M type ESBLs,there were 20 CTX-M-1 group and 26 CTX-M-9 group,including 6 stains habouring both CTX-M-1 and CTX-M-9 group.Eight CTX-M genotypes were confirmed by sequencing of the PCR products,including CTX-M-3,CTX-M-14,CTX-M-15,CTX-M-24,CTX-M-28,CTX-M-31,CTX-M-65 and CTX-M-79.Conclusion CTX-M genotype ESBLs was the most popular extended-spectrum β-lactamases in E.coli isolated from liver cirrhosis' patients with bloodstream infection.The CTX-M-14 is the dominant epidemic type.

Acquired Immunodeficiency Syndrome ; epidemiology ; virology ; Adolescent ; Adult ; Aged ; Aged, 80 and over ; China ; epidemiology ; Female ; Humans ; Male ; Middle Aged ; Young Adult

OBJECTIVEThe paper is a study on the clinical symptoms and pathogeny of ectrodactyly and absence of radius side part palm and split foot malformation of some patients in one family.

METHODSBased on the patient family investigation,a normal control group and a patient group were established. Then, polymerase chain reaction technique was used for DNA sequencing and analysis of the two groups for their exons 5-8 gene group DNA of P63 gene.

RESULTSThe medical examination found that the patients' upper bilateral limbs are short of thumbs, forefingers and middle fingers, and have radius side part palm and double lower limbs foot clefts malformation. The pathogeny research revealed that the PCR expansion pieces of the exons 5-8 of P63 are 284 bp, 259 bp, 245 bp and 259 bp respectively, and the size of the expansion piece of the patients was the same as that of the normal people group. However, a respective comparison between the DNA serial of the expansion piece of the patient and that of the normal people group and that of the P63 gene in the human gene bank showed that mutation occurs at the number 665 base pair of exon 5 of P63, namely a mutation from G to A.

CONCLUSIONThe ectrodactyly, absence of radius side part palm and split foot malformation are caused by the mutation of base pair at number 665 of the exon 5 of P63.

Exons ; genetics ; Female ; Foot Deformities, Congenital ; genetics ; pathology ; Genetic Predisposition to Disease ; Hand Deformities, Congenital ; genetics ; pathology ; Humans ; Male ; Membrane Proteins ; genetics ; Mutation ; Pedigree ; Polymerase Chain Reaction

OBJECTIVETo determine a dengue fever outbreak in Yiwu city, Zhejiang Province in 2009 and to trace the origin of the pathogen.

METHODSThe dengue virus IgM, IgG antibodies and viral nucleic acid were detected and virus was isolated using 40 serum samples from the suspected patients. The viral RNA of the isolated virus strains was extracted and the E gene was amplified by RT-PCR. The amplicons were sequenced and the phylogenetic and homological analyses were also constructed.

RESULTSAmong 40 serum samples from dengue fever suspected patients, 17 were positive from for dengue IgM (42.5%); 4 were IgG positive (10.0%); 34 samples were dengue virus RNA positive (85.0%), 28 dengue virus type 3 (D3) strains were isolated (70.0%). The complete coding region of envelope genes (E) from 13 D3 strains was all 1479 nt without any insertion or deletion, which encoded with 493 amino acids (aa). E gene from the 13 D3 strains from Zhejiang in 2009 (D3/ZJ/2009) was 100.0% identical. The strain from Saudi Arabia shared the highest similarity with the D3 strain, 99.3% and 100.0% of their E genes and deduced amino acids were identical, respectively, whereas they were 93.4% and 97.4% between D3/ZJ/2009 strain and its prototype strain (D3/H87/1956), and 93.6% and 97.4% between D3/ZJ/2009 and a D3 strain isolated in Guangxi Province in 1980. The phylogenetic tree of E genes also indicated that D3/ZJ/2009 had maximum similarity with the D3/Saudi Arabia/2004. They all belonged to the D3/GIII branch, which was originated from Indian Subcontinent.

CONCLUSIONThe outbreak of dengue fever in Zhejiang in 2009 was caused by type 3 dengue virus III genotype. The virus was most likely originated from Saudi Arabia.

Adolescent ; Adult ; Aged, 80 and over ; China ; epidemiology ; Dengue ; epidemiology ; virology ; Dengue Virus ; classification ; genetics ; isolation & purification ; Disease Outbreaks ; Female ; Humans ; Male ; Middle Aged