从兔股骨头中提取总RNA的方法特点

目的:建立一种高效、快速的骨组织总RNA提取方法。方法:实验于2005-01/2006-01在昆明医学院实验动物中心和中国科学院昆明动物研究所中科院细胞与分子进化重点实验室完成。取健康新西兰大白兔1只,截取其股骨头,迅速置于液氮罐中保存,于研钵中研磨,使骨组织始终保存于液氮中,继续研磨,如此重复3次,然后利用Trizol使骨细胞结构迅速破坏,将粉末转入离心管,室温静置5min。随后加入氯仿等有机溶剂处理、离心,使RNA与细胞DNA、蛋白质及其他成分分离从而得到总RNA。最后鉴定RNA的质量、纯度及产率,取2μL提取出的RNA在体积分数为0.008的甲醛变性琼脂糖凝胶上进行电泳,主要观察RNA的28S、18S及5S三个条带是否清晰,有无降解和DNA污染。以99μLDEPC水稀释1μLRNA样品,紫外分光光度计测量其吸光度(A)值,A260/A280之比值表示RNA的纯度,同时根据吸光度值计算其质量浓度。结果:①对提取的兔股骨头RNA进行琼脂糖凝胶电泳,可显示清晰的28S、18S两个条带,5S条带亦可见,表明了RNA是完整的。②用紫外分光光度法测定兔股骨头中提取出的RNAA260/A280,结果表明由本法提取的RNA纯度高,无DNA和蛋白质的污染。③经紫外分光光度计吸收定量,每毫克兔股骨头组织能提取1.0～1.2μg的总RNA。结论:本法提取骨组织总RNA方便、快捷,质量高,可用于骨组织的分子生物学研究。     

缺血预处理程序中心肌环磷酸腺苷含量及环磷酸腺苷依赖蛋白激酶活性的变化

目的:制备大鼠在体缺血再灌注模型,观察缺血预处理程序中心肌环磷酸腺苷含量及环磷酸腺苷依赖蛋白激酶活性的变化。方法:实验于2005-03/2006-10在解放军沈阳军区总医院医学实验动物中心和全军心血管研究所实验室完成。实验分组:选用健康雌性SD大鼠36只,根据预适应程序分为第1,2,3次缺血,第1,2,3次再灌注,每一时间点6只大鼠。实验过程:用手术套管法造成左冠状动脉主干缺血及再灌注。所有实验动物在实验程序结束后,取出心脏迅速置液氮保存备用。实验评估:用放射免疫法测环磷酸腺苷水平,生化法测环磷酸腺苷依赖蛋白激酶活性变化。结果:36只大鼠均进入结果分析。①环磷酸腺苷含量:第1次再灌注组低于第1次缺血组[(0.325±0.015),(0.395±0.024)pmol/g,t=6.06,P<0.001],第2次再灌注组低于第2次缺血组[(0.523±0.017),(0.708±0.067)pmol/g,t=6.56,P<0.001],第3次再灌注组低于第3次缺血组[(0.567±0.031),(0.712±0.038)pmol/g,t=7.24,P<0.001]。②环磷酸腺苷依赖蛋白激酶活性:第1次再灌注组低于第1次缺血组[(10.115±1.000),(16.351±0.849)pkat/g,t=11.12,P<0.001],第2次再灌注组低于第2次缺血组[(11.877±2.213),(14.869±0.619)pkat/g,t=3.31,P<0.01],第3次再灌注组低于第3次缺血组[(11.745±0.987),(14.766±0.329)pkat/g,t=7.09,P<0.001]。③缺血预处理程序中心肌环磷酸腺苷含量及环磷酸腺苷依赖蛋白激酶活性随缺血及再灌注呈周期性波动。在5min缺血预处理时表现为明显增高,而在间隔的再灌注程序中恰呈相反改变,有明显下降的趋势。结论:环磷酸腺苷及环磷酸腺苷依赖蛋白激酶的周期性波动变化可能是激发心肌缺血预处理的机制之一,环磷酸腺苷可能在预处理保护作用中起一些作用。     

Culture confirmed multidrug resistant tuberculosis: diagnostic delay, clinical features, and outcome

Aims: To determine the delay in diagnosis of multidrug resistant (MDR) tuberculosis (TB), the correlation between drug susceptibility patterns of adult-child contact pairs, the effectiveness of treatment, and the outcome in these children. Methods: MDR M tuberculosis culture results of children were prospectively collected during a four year period in the Western Cape Province of South Africa, an area with a TB incidence of 589/100 000 population, and a new MDR TB rate of 0.94%. Folder reviews were done to retrieve clinical information. Children not already on treatment at our MDR TB clinic or TB hospital were recalled and appropriate treatment was started. Follow up was done for as long as possible. Results: Thirty nine children, median age 4.5 years at first TB diagnosis and 6.2 years on MDR culture confirmation, were seen. Delay in starting appropriate MDR treatment after TB diagnosis was a median of 2 days if MDR TB source cases were taken into account, but 246 days if the drug susceptibility pattern of the source case was not considered, and 283 days if there was no known tuberculosis source case. Correlation between the drug susceptibility results of the child''s and adult source case''s isolates was 68%. Seventeen children had smear positive tuberculosis, of whom 13 had cavitatory pulmonary disease. Eight children had central nervous system TB. Thirty six children were treated for MDR tuberculosis, of whom four died. Conclusions: Obtaining a detailed contact history is essential as a delay in starting appropriate MDR antituberculosis treatment has potentially serious consequences.     

Subsequent pregnancy outcome after preterm breech delivery,a population based cohort study

Objective: The objective of this study is to investigate the effect of the mode of delivery in women with preterm breech presentation on neonatal and maternal outcome in the subsequent pregnancy.

Methods: Nationwide population-based cohort study in the Netherlands of women with a preterm breech delivery and a subsequent delivery in the years 1999–2007. We compared planned caesarean section versus planned vaginal delivery for perinatal outcomes in both pregnancies.

Results: We identified 1543 women in the study period, of whom 259 (17%) women had a planned caesarean section and 1284 (83%) women had a planned vaginal delivery in the first pregnancy. In the subsequent pregnancy, perinatal mortality was 1.1% (3/259) for women with a planned caesarean section in the first pregnancy and 0.5% (6/1284) for women with a planned vaginal delivery in the first pregnancy (aOR 1.8; 95% CI 0.31–10.1). Composite adverse neonatal outcome was 2.3% (6/259) versus 1.5% (19/1284), (aOR 1.5; 95% CI 0.55–4.2). The average risk of perinatal mortality over two pregnancies was 1.9% (10/518) for planned caesarean section and 2.0% (51/2568) for planned vaginal delivery, (OR 0.98; 95% CI 0.49–1.9).

Conclusion: In women with a preterm breech delivery, planned caesarean section does not reduce perinatal mortality, perinatal morbidity, or maternal morbidity rate over the course of two pregnancies.     

Phenotypic expansion of Bosch–Boonstra–Schaaf optic atrophy syndrome and further evidence for genotype–phenotype correlations

Bosch–Boonstra–Schaaf Optic Atrophy Syndrome (BBSOAS) is an autosomal dominant neurodevelopmental disorder caused by loss‐of‐function variants in NR2F1 and characterized by visual impairment, developmental delay, and intellectual disability. Here we report 18 new cases, provide additional clinical information for 9 previously reported individuals, and review an additional 27 published cases to present a total of 54 patients. Among these are 22 individuals with point mutations or in‐frame deletions in the DNA‐binding domain (DBD), and 32 individuals with other types of variants including whole‐gene deletions, nonsense and frameshift variants, and point mutations outside the DBD. We corroborate previously described clinical characteristics including developmental delay, intellectual disability, autism spectrum disorder diagnoses/features thereof, cognitive/behavioral anomalies, hypotonia, feeding difficulties, abnormal brain MRI findings, and seizures. We also confirm a vision phenotype that includes optic nerve hypoplasia, optic atrophy, and cortical visual impairment. Additionally, we expand the vision phenotype to include alacrima and manifest latent nystagmus (fusional maldevelopment), and we broaden the behavioral phenotypic spectrum to include a love of music, an unusually good long‐term memory, sleep difficulties, a high pain tolerance, and touch sensitivity. Furthermore, we provide additional evidence for genotype–phenotype correlations, specifically supporting a more severe phenotype associated with DBD variants.     

Defining near misses: Towards a sharpened definition based on empirical data about error handling processes

Medical errors in health care still occur frequently. Unfortunately, errors cannot be completely prevented and 100% safety can never be achieved. Therefore, in addition to error reduction strategies, health care organisations could also implement strategies that promote timely error detection and correction. Reporting and analysis of so-called near misses – usually defined as incidents without adverse consequences for patients – are necessary to gather information about successful error recovery mechanisms. This study establishes the need for a clearer and more consistent definition of near misses to enable large-scale reporting and analysis in order to obtain such information. Qualitative incident reports and interviews were collected on four units of two Dutch general hospitals. Analysis of the 143 accompanying error handling processes demonstrated that different incident types each provide unique information about error handling. Specifically, error handling processes underlying incidents that did not reach the patient differed significantly from those of incidents that reached the patient, irrespective of harm, because of successful countermeasures that had been taken after error detection. We put forward two possible definitions of near misses and argue that, from a practical point of view, the optimal definition may be contingent on organisational context. Both proposed definitions could yield large-scale reporting of near misses. Subsequent analysis could enable health care organisations to improve the safety and quality of care proactively by (1) eliminating failure factors before real accidents occur, (2) enhancing their ability to intercept errors in time, and (3) improving their safety culture.     

Day care in relation to respiratory-tract and gastrointestinal infections in a German birth cohort study

AIM: To describe day care attendance in Germany today (in former East and former West Germany). To investigate longitudinally whether children attending day care centres have an increased risk of acquiring common cold, bronchitis, pneumonia, otitis media or diarrhea. METHODS: Questionnaire information was collected when the children in the cohort were 6, 12, 18, 24 months, and 4 and 6 years old. Day care within the first and first 2 years of life was investigated longitudinally with GEE (generalised estimating equations) methods in relation to common cold, bronchitis, pneumonia, otitis media and diarrhea within the first 6 years of life. RESULTS: Day care centre attendance is more common in former East than in former West Germany; this difference is evident even 10-12 years after German reunification. Children attending a day care centre were more likely to have common cold, bronchitis, pneumonia, otitis media and diarrhea within the first 2-3 years of life. With the exception of common cold, from year 4 onwards these associations were not statistically significant anymore and even reversed for some of the infections. CONCLUSIONS: Children attending day care centres were at an increased risk of respiratory and gastrointestinal infections within the first years of life. However, around school age these differences disappeared or even partly reversed.