Detecting Selection in the HIV-1 Genome during Sexual Transmission Events

Little is known about whether and how variation in the HIV-1 genome affects its transmissibility. Assessing which genomic features of HIV-1 are under positive or negative selection during transmission is challenging, because very few virus particles are typically transmitted, and random genetic drift can dilute genetic signals in the recipient virus population. We analyzed 30 transmitter–recipient pairs from the Zurich Primary HIV Infection Study and the Swiss HIV Cohort Study using near full-length HIV-1 genomes. We developed a new statistical test to detect selection during transmission, called Selection Test in Transmission (SeTesT), based on comparing the transmitter and recipient virus population and accounting for the transmission bottleneck. We performed extensive simulations and found that sensitivity of detecting selection during transmission is limited by the strong population bottleneck of few transmitted virions. When pooling individual test results across patients, we found two candidate HIV-1 genomic features for affecting transmission, namely amino acid positions 3 and 18 of Vpu, which were significant before but not after correction for multiple testing. In summary, SeTesT provides a general framework for detecting selection based on genomic sequencing data of transmitted viruses. Our study shows that a higher number of transmitter–recipient pairs is required to improve sensitivity of detecting selection.     

PSNAV2.0-TNFα重组质粒的构建及其在体外的表达

目的:在前期微囊化基因工程细胞制备平台的基础上,构建分泌型人肿瘤坏死因子α的真核表达载体PSNAV2.0-TNFα重组质粒,并鉴定其蛋白的体外瞬时表达,为进一步利用该基因进行微囊化细胞移植治疗和改善疾病奠定基础。方法:实验于2006-06/2007-05在解放军总医院老年医学研究所细胞生物学实验室完成。①以含有人肿瘤坏死因子αcDNA序列的质粒为模板,通过PCR扩增获得人肿瘤坏死因子α基因片段;将其定向插入真核表达载体PSNAV2.0中,获得重组质粒PSNAV2.0-TNFα。采用SalⅠ和EcoRⅠ双酶切法、PCR法及插入片段序列测定法鉴定该质粒。②利用阳离子脂质体介导法,将其转染到人胚胎肾细胞HEK-293细胞中,构建可持续分泌人肿瘤坏死因子α的基因工程细胞,采用RT-PCR法和Western blot法检测转染细胞培养上清液中人肿瘤坏死因子蛋白的体外瞬时表达。结果:①通过SalⅠ和EcoRⅠ双酶切、PCR及测序鉴定证明:在HEK-293中插入片段正确。②采用RT-PCR和Western blot法检测表明HEK-293细胞培养上清中有人肿瘤坏死因子α蛋白,Mr17000。结论:成功构建了重组质粒PSNAV2.0-TNFα真核表达载体,转染HEK-293细胞后可有效分泌人肿瘤坏死因子α蛋白,并能分泌到细胞外。     

人羊膜间充质细胞具有分化成软骨及成骨细胞的潜能

目的:人羊膜间充质细胞具有比骨髓间充质干细胞更强的扩增能力和免疫原性低等优势。建立体外适宜的诱导培养条件,观察人羊膜间充质细胞定向分化为软骨细胞和成骨细胞的能力。方法:实验于2005-09/2006-12在贵州省细胞工程重点实验室完成。①材料来源:经产妇知情同意,无菌采集健康足月分娩新生儿胎盘6份,实验经医院医学伦理委员会批准。②实验方法:采用机械法剥离羊膜组织,二步酶消化法分离收获人羊膜间充质细胞,按2.2×10~8L~(-1)密度接种,传至第1～2代用于诱导分化实验。向软骨细胞诱导分化时,人羊膜间充质细胞按3×10~8L~(-1)密度接种,诱导培养液为含体积分数0.01的胎牛血清、10 mg/L转化生长因β1、100 nmol/L地塞米松、50 mg/L抗坏血酸、1%培养基添加物。向成骨细胞诱导分化时,人羊膜间充质细胞按6×10~7L~(-1)密度接种,诱导培养液为含体积分数0.1的胎牛血清、100 nmol/L地塞米松、50 mg/L抗坏血酸、5 mmol/Lβ-甘油磷酸。③实验评估:原代细胞用流式细胞仪分析表型,免疫细胞化学染色进行波形蛋白表达鉴定。分别于体外诱导第7,14,21,28天采用免疫细胞化学法检测软骨特异性Ⅱ型胶原的表达,细胞化学法检测蛋白聚糖的表达,钙-钴法检测成骨细胞特异性碱性磷酸酶的表达,茜素红S检测钙盐沉积情况。结果:①免疫组化与表型特征:人羊膜间充质细胞高表达间充质干细胞表面标志CD29、CD44和间充质细胞标志波形蛋白。②向软骨细胞诱导分化:诱导14 d后,人羊膜间充质细胞由长梭型逐渐变为多角形,可检测到Ⅱ型胶原蛋白表达及软骨细胞特异性细胞外基质蛋白聚糖。③向成骨细胞诱导分化:诱导21 d后,可观察到人羊膜间充质细胞的胞浆内有碱性磷酸酶表达,且可见钙盐沉积。结论:人羊膜间充质细胞具有分化成软骨细胞和成骨细胞的特性,可作为骨及软骨组织工程种子细胞的新来源。     

Rest Myocardial Perfusion Imaging in a Patient with Atypical Chest Pain and a Nondiagnostic Electrocardiogram

ACC/AHA guidelines assign a class I indication for use of myocardial perfusion imaging (MPI) for the evaluation of chest pain in patients with acute coronary syndromes and a nondiagnostic ECG. However, MPI is not a widely used modality for the evaluation of patients who present to the ER with chest pain and an intermediate pretest probability for coronary artery disease. We report a case in which resting MPI was pivotal in diagnosing acute myocardial infarction and expedited the appropriate reperfusion strategy. Copyright © 2010 Wiley Periodicals, Inc.     

Assessment of a daily online implanted fiducial marker‐guided prostate radiotherapy process

The aims of this study were to investigate whether intrafraction prostate motion can affect the accuracy of online prostate positioning using implanted fiducial markers and to determine the effect of prostate rotations on the accuracy of the software‐predicted set‐up correction shifts. Eleven patients were treated with implanted prostate fiducial markers and online set‐up corrections. Orthogonal electronic portal images were acquired to determine couch shifts before treatment. Verification images were also acquired during treatment to assess whether intrafraction motion had occurred. A limitation of the online image registration software is that it does not allow for in‐plane prostate rotations (evident on lateral portal images) when aligning marker positions. The accuracy of couch shifts was assessed by repeating the registration measurements with separate software that incorporates full in‐plane prostate rotations. Additional treatment time required for online positioning was also measured. For the patient group, the overall postalignment systematic prostate errors were less than 1.5 mm (1 standard deviation) in all directions (range 0.2–3.9 mm). The random prostate errors ranged from 0.8 to 3.3 mm (1 standard deviation). One patient exhibited intrafraction prostate motion, resulting in a postalignment prostate set‐up error of more than 10 mm for one fraction. In 14 of 35 fractions, the postalignment prostate set‐up error was greater than 5 mm in the anterior–posterior direction for this patient. Maximum prostate rotations measured from the lateral images varied from 2° to 20° for the patients. The differences between set‐up shifts determined by the online software without in‐plane rotations to align markers, and with rotations applied, was less than 1 mm (root mean square), with a maximum difference of 4.1 mm. Intrafraction prostate motion was found to reduce the effectiveness of the online set‐up for one of the patients. A larger study is required to determine the magnitude of this problem for the patient population. The inability in the current software to incorporate in‐plane prostate rotations is a limitation that should not introduce large errors, provided that the treatment isocentre is positioned near the centre of the prostate.     

Toxic epidermal necrolysis. A comprehensive approach. Multidisciplinary management in a burn center

Toxic Epidermal Necrolysis (TEN) is a life-threatening disorder with reported mortality rates of 25-60 percent in pediatric patients. The authors report on their experience in managing six children using a standardized treatment protocol in the intensive care unit of a regional burn center. Areas of sloughed skin were covered with porcine xenograft (pig skin) until reepithelialization was complete. There was one death in the series, and one child had ophthalmologic complications. Treatment in a multidisciplinary burn center is recommended for children with TEN.     

Man at risk. Preventive strategies and risk management for patient safety

Anaesthesia-related risk has been significantly reduced within the last decade. Nevertheless the risk and the possibility of dying or suffering permanent damage still exist. To improve patient safety, risk assessment and analysis must lead to the development of preventive strategies. For this purpose anaesthesia can rely on the concepts of other "high reliability" organisations such as aviation or nuclear power plants. Analyses of critical incidents in the different fields confirm that next to technical problems human factors account for most of the preventable mishaps. Human factors are responsible for individual mistakes as well as for organisational errors. Therefore besides traditional concepts of risk reduction (e.g. guidelines) new strategies (e.g. full-scale simulation) must be applied to minimise the negative impact of human factors on patient safety. Risk management has to consider technical, organisational and human factors to implement a higher standard of patient safety.