OBJECTIVEThe aim of this study is to establish a transgenic mouse model for cleft palate relevant gene thyroid transcription factor-2 (TTF-2), which can be used to study palatal shelf development when the expression pattern and regular activation of TTF-2 is altered.

METHODSThe C57BL/6J mouse TTF-2 gene was cloned through polymerase chain reaction (PCR) from the mouse genomic DNA. The TTF-2 gene was inserted into the expression vector pBROAD3-mcs to construct the recombinant expression vector pBROAD3-TTF-2. This expression vector was then microinjected into the male pronuclei of the fertilized mouse ovum. Thus, the TTF-2 transgenic mice model was established. The genotype of the transgenic mice was identified by PCR and Southern blot analysis. Immunohistochemistry identified the consistent expression of TTF-2 gene during its palatal shelf development.

RESULTSTTF-2 genes were microinjected into 982 fertilized ova. A total of 580 two-cell-stage embryos cultured and transplanted into the oviducts of 48 pseudopregnant female mice. Overall, 68 embryos were obtained for analysis. The genotype of the mice was determined through PCR and Southern blot analysis using genomic DNA extracted from tail biopsies of the transgenic fetus. A total of 13 TTF-2 transgenic mice were detected. The expression of TTF-2 gene during the palatal shelf development of the transgenic mice was consistently detected by immunohistochemistry.

CONCLUSIONThe recombinant expression vector pBROAD3-TTF-2 was integrated into mouse genome through microinjection. The transgenic mouse in the palatal shelf that consistently expressed TTF-2 was successfully established and displayed a cleft palate phenotype.

Animals ; Cleft Palate ; Disease Models, Animal ; Female ; Forkhead Transcription Factors ; Genotype ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Thyroid Gland

To investigate the epidemic and evolutionary trends of enterovirus (EV) in the external environment of Yunnan Province, China, molecular typing was performed on 4 EV strains that were isolated from environmental sewage in Yunnan. The VP1 region of isolates was amplified by RT-PCR using universal enterovirus primers, and the amplified VP1 region was sequenced for GenBank BLAST search and genotype analysis. The 4 EV strains were identified as ECHO7. Their nucleotide and amino acid homologies with the VP1 sequences of 68 ECHO7 strains retrieved from GenBank were measured by Mega software analysis. Our findings showed that ECHO7 strains from environmental sewage and population samples were in different evolutionary branches. These strains showed typical geographical and temporal differences; In addition, there were different transmission chains at the same time and in the same area. ECHO7 strains isolated from sewage water and patients with acute flaccid paralysis during the same period in Yunnan belonged to different clusters and evolved at different speeds. Special concerns are needed for this problem. Continuous molecular biological surveillance of human EV in the external environment of Yunnan will provide strong support for early warning of EV diseases.
China ; Databases, Genetic ; Enterovirus ; genetics ; isolation & purification ; Evolution, Molecular ; Humans ; Molecular Epidemiology ; Sequence Analysis ; Sewage ; virology

BACKGROUND:Half-ring sulcated external fixator is a bone external fixation device.Its structure is relatively simple,with multi-plane fixed,and small occupying space.So far,there are few experimental reports about construction of tibial defect model by it in large animals.OBJECTIVE:To validate the practicality and repeatability of half-ring sulcated external fixator in establishing tibial defect models in goats.DESIGN,TIME AND SETTING:The randomized controlled observation was performed at the Animal Laboratory of the Third Military Medical University from March 2005 to February 2007.MATERIALS:Nine healthy adult(Animal Center of Third Military Medical University);Half-ring sulcated external fixator,made by Professor Li of the Third Military Medical University;Osseous pin,ψ2.5 mm,Shanghai Medicai Instruments Co.,Ltd.METHODS:After animals were anesthetized,two needles were threaded in the same plane of cancellous bone about 1.5-2.0 cm above the superior articular surface of the tibia,named group 1.The angle between the two needles was 40°.One needle was threaded at the place of 3.0 to 5.0 cm under the group 1 and parallel with optional needle of the group 1,named group 2.Needles of group 4 were threaded at the cancellous bone that was 2.0 cm from the joint surface of inferior extremity of the tibia.One needle was threaded at the place of 3.0 to 5.0 cm from group 4 and parallel with optional needle of the group 4.The included angle between these two needles of group 2 and group 3 was 60°.There were totally 6 needles.Tibia was fixed by half-ring sulcated external fixator.According to the length of the tibia from X-ray,tibia and periosteum were amputated by wire saw between the two needles in the middle and inferior segment of the tibia to make segmental bone and periosteal defects of 20 percent of tibia.MAIN OUTCOME MEASURES:①Postoperative general state of the animals and effect of half-ring sulcated external fixator.②Self-repair of bone defects by X-ray and Lane-Sandhu scoring standards.RESULTS:Of the 9 selected goats,1 died of postoperative infection,and 1 developed pinhole infection and supplemented.All the others survived and were included in final analysis.The goats were awake within 2-6 hours after surgery and able to stand up to eat.The loosening nuts were screwed up.No external fixation failure or loosening was found.No skin was necrotic.Injured limb could touch the ground in 2 days,but could not bear load until 2 weeks.The goats could walk in three weeks with a slight limp,then moved freely without claudication in four weeks after the operation.At the 5th week after operation,the radiographs showed little periosteal reaction.At the 10th week after operation,sclerosis was found in the fracture ends,and medullary cavity started to close.Fifteen weeks after operation,the medullary cavity closed and periosteal reaction did not aggravate.Inaddition.Lane score was 0 at any time point.No bone formation was found in the histological examination at 5,10 and 15 weeks after operation,and Lane score was 0 at any time point.CONCLUSION:The goat model established in goat tibia with 20%defects by the half-ring sulcated external fixator shows no bone healing after 15 weeks by X-ray or histological exanimation,indicating this is a practical and repetitive method to establish animal models of large segmental defect for bone tissue engineering.

0.05).Seroquel showed high incidence rate of drowsiness,dizziness and weight gain(26.1%,23.9%,17.3%),chlorpromazine showed high incidence rate of extrapyramidal side effect,pyknocardia and erect collapse(60.9%,39.1%,32.6%).CONCLUSIONS:There was no significant difference of curative effect for the treat-ment of schizophrenia between seroquel-treated group and chlorpromazine-treated group,but seroque showed low side effect and high safety.

Objective To explore the effect of home-based cardiac rehabilitation training on ejection fraction, blood lipid and exercise endurance in patients with coronary heart disease. Methods Randomized controlled trials (RCTs) on the effect of home-based cardiac rehabilitation training on ejection fraction, blood lipid and exercise endurance in patients with coronary heart disease were searched via computer in PubMed, Web of Science, Embase, Cochrane Library database, Wanfang database, CNKI database, VIP database and CBMdisc. After literature screening, data extraction and quality evaluation, Revman5.3 software was used for meta-analysis. Results A total of 11 articles were included. Meta analysis showed that, compared with the conventional treatment group, home-based cardiac rehabilitation training could effectively improve patients' ejection fraction[MD=1.81,95%CI(0.68,2.95),P=0.002],exercise endurance[MD=21.97,95% CI(1.80, 42.14), P=0.03], and reduce cholesterol level[MD=-0.15,95%CI(-0.23,-0.07), P＜ 0.01]. However, there was no significant improvement on high-density lipoprotein, low-density lipoprotein, triglyceride (P＞0.05). Sensitivity analysis showed that the overall results of the study were relatively stable, and individual study had no significant influence on the overall results. Conclusions Home-based cardiac rehabilitation training can significantly improve patients' ejection fraction and exercise endurance and reduce cholesterol level, but in terms of improving patients' high density lipoprotein, low density lipoprotein and triglyceride, more large sample, multi-center long-term studies are needed to verify.

OBJECTIVEThis report presented an overview on the epidemiology of enterovirus in Yunnan province, the People's Republic of China.

METHODSA total of 210 strains of non-polioviruses isolated under acute flaccid paralysis surveillance during a 5-year study period from 1997 to 2000 and 2004 were examined. Of the 210 non-polioviruses strains, a total of 12 strains of adenoviruses were serologically identified. The remaining 198 isolates were used for molecular typing, and the viral genomes of 195 nonpolio enteroviruses (NPEVs) were translated to corresponding amino acid sequences and compared with those of the prototype strains.

RESULTSBased on molecular typing, 5 isolates were classified into 5 serotypes of human enterovirus A species while 158 isolates into 34 serotypes of B and 32 isolates into 6 serotypes of C species. However, we did not isolate any viruses which belonged to human enterovirus D species. Thus, under acute flaccid paralysis surveillance, human enterovirus B species accounted for 75.2% of the 210 isolates and was considered as the predominant one, followed by human enterovirus C (12.2%), adenovirus (5.7%), and human enterovirus A (2.4%).

CONCLUSIONAlthough the epidemiological characteristics of NPEVs from Yunnan province remained "unknown", the molecular typing method had provided us a breakthrough to understand the epidemiology of these viruses.

China ; epidemiology ; Enterovirus ; classification ; genetics ; isolation & purification ; Enterovirus Infections ; epidemiology ; Genes, Viral ; Humans ; Molecular Epidemiology ; Serotyping

Objective To investigate the association of HLA-A and -B alleles with syphilis in Shandong Han population. Methods The allele frequencies of HLA-A and -B were detected in 205 patients with syphilis and 5844 normal human controls by PCR-sequence specific oligonucleotide probe (PCR-SSOP)method. Results The patients with syphilis showed a higher frequency of HLA-A*02, B*15, B*40 alleles(all P＜0.01, Pc＜0.05) and a lower frequency of HLA-A*26 allele (P= 0.003, Pc = 0.039) than the normal human controls did. There was an increased frequency of HLA-B*15 and B*40 alleles in patients with symptomatic syphilis (both P＜0.01, Pc＜0.05), as well as an elevated frequency of HLA-A*02, 11, 29, B*15 and 40 alleles (all P＜0.01, Pc＜0.05) and a decreased frequency of HLA-A*30 and 33 in patients with asymptomatic syphilis(P=0.002, 0.026, Pc=0.001, 0.013 respectively), compared with the normal human controls. The frequency of HLA-A*30 allele was significantly higher in patients with symptomatic syphilis than in those with asymptomatic syphilis (P = 0.001, Pc = 0.013). Conclusions There seems to be an association between HLA-A*02, B* 15 and B*40 alleles and syphilis, between HLA-A*30 allele and symptomic syphilis, and between HLA-A*02, 11 and 29 alleles and asymptom1atic syphilis, in Shandong Han population.

A method has been developed by solid phase extraction-high performance liquid chromatography coupled with photodiode array detector for simultaneous determination of 10 kinds of cyanotoxins in lake water, including microcystin-RR (MC-RR), MC-YR, MC-HtyR, MC-LR, MC-WR, MC-LA, MC-LY, MC-LW, MC-LF and nodularin ( NOD ).The samples were enriched and purified by a HLB solid phase extraction column.The separation was performed on a Waters C18 column (250 mm×4.6 mm, 5 μm) with the gradient elution of acetonitrile and water containing 0.05% trifluoroacetic acid.The flow rate of the mobile phase was 0.8 mL/min.The column temperature was 30℃ and the injection volume was 20 μL.The photodiode array detector was scanned from 190 nm to 300 nm and the detection wavelength was 238 nm.Multiple qualitative analyses were completed according to retention time, full scanning spectrum of photodiode array detector and purity analysis of chromatographic peak.Good linearity was observed in the cyanotoxin concentration range of 0.05 mg/L-2.0 mg/L with correlation coefficients ( R) from 0.9998 to 0.9999.The limits of detection for 10 cyanotoxins were in the range of 0.005 μg/L-0.020 μg/L.The recoveries were in the range of 85.1%-105.3% at the three spiked levels of 0.1 mg/L, 1.0 mg/L and 1.8 mg/L with the relative standard deviations ( RSD ) of 0.8% -9.2%.The method was characteriZed by wide applicability, convenient operation, quick analytical rate, high sensitivity, good accuracy and high recovery.The qualitative accuracy was improved by using multiple qualitative analyses.This method was successfully applied to determination of 10 kinds of cyanotoxins in lake water.

Objective To analyze alterations in electrocardiogram (ECG) of adenosine test in 99Tcm-MIBI myocardial perfusion imaging(MPI)SPECT study. Methods A total of 641 patients were in cluded in the study. The patients each underwent 99Tcm-MIBI MPI with adenosine test. The ECGs were taken before, during, and after adenosine infusion. Results In all, abnormal ECGs were found in 205(32.0%) patients. During adenosine infusion, 20.6%(132/641) of patients suffered from arrhythmia,29.5%(39/132) had atrial premature beats, 34. 1% (45/132) had premature ventricular beats, and 6. 1% (8/132) had sinoatrial block. In addition, 5.3% (7/132) had first-, 24.2% (32/132) had second-, and 0.8%(1/132) had third-degree atrioventricular block (AVB). After adenosine infusion, 4.4%( 28/641) of patients suffered from arrhythmia, 57.1% (16/28) had atrial premature beats, 39.3%(11/28) had premature ventricular beats, and 3.6% (1/28) had sinoatrial block. The perfusion images showed ischemia in 36 patients and infarction in 8 patients. Adenosine infusion was terminated in 39 patients (6. 1%) because of poorly tolerated side effects. However, no death or acute myocardial infarction occurred in the study. Conclusions Adenosine pharmacologic test for 99TcmMIBI MPI may result in relatively high incidence of arrhythmia in ECG monitoring.

OBJECTIVETo observe the alteration of fibroblast growth factor 10 (Fgf10) and fibroblast growth factor receptor 2 (Fgfr2b) signal in mouse embryonic palate after dexamethasone and vitamin B12 exposure.

METHODSDams were divided teratogenetic group, antagomistic group and control group and were respectively injected dexamethasone, dexamethasone and vitamin B12, and normal sodium. Dams were killed and fetus was collected at embryo 12.5 and 13.5 day. The expression of Fgf10 and Fgfr2b and mesenchymal cells proliferation of mouse embryonic by western blotting and BrdU assay were checked.

RESULTSFgf10 and Fgfr2b expression was down-regulated and mesenchymal cells proliferation was inhibited significantly after dexamethasone exposure. After vitamin B12 treatment, Fgf10 and Fgfr2b expression did not restore, but cells proliferation was recovered.

CONCLUSIONDexamethasone and vitamin B12 affected the expression of Fgf10 and Fgfr2b of mouse embryonic palate and mesenchyme cells proliferation, but the change was disaccord.

Animals ; Cell Proliferation ; Dexamethasone ; Fibroblast Growth Factor 10 ; Mice ; Receptor, Fibroblast Growth Factor, Type 2 ; Signal Transduction ; Vitamin B 12