Design considerations for two-stage enrichment clinical trials

When there is a predictive biomarker, enrichment can focus the clinical trial on a benefiting subpopulation. We describe a two-stage enrichment design, in which the first stage is designed to efficiently estimate a threshold and the second stage is a “phase III-like” trial on the enriched population. The goal of this paper is to explore design issues: sample size in Stages 1 and 2, and re-estimation of the Stage 2 sample size following Stage 1. By treating these as separate trials, we can gain insight into how the predictive nature of the biomarker specifically impacts the sample size. We also show that failure to adequately estimate the threshold can have disastrous consequences in the second stage. While any bivariate model could be used, we assume a continuous outcome and continuous biomarker, described by a bivariate normal model. The correlation coefficient between the outcome and biomarker is the key to understanding the behavior of the design, both for predictive and prognostic biomarkers. Through a series of simulations we illustrate the impact of model misspecification, consequences of poor threshold estimation, and requisite sample sizes that depend on the predictive nature of the biomarker. Such insight should be helpful in understanding and designing enrichment trials.     

An enzymatic method for microdetermination of aphidicolin: a promising anticancer drug

We have developed a method, based on the in vitro inhibition of purified human DNA polymerase alpha, the major enzyme of DNA replication, which allows the rapid and accurate determination of pmol amounts of aphidicolin, a promising anticancer drug. The efficacy of this simple method was verified by the determination of aphidicolin in the liver, spleen, blood and urine of mice treated parenterically with the drug. Given its sensitivity and the avoidance of radioactive tracers, this enzymatic method is suitable for the determination of the drug in body fluids and tissue biopsies from living humans. It allows the detection and quantitation of aphidicolin in the presence of inactive metabolite(s) with very similar chemical structure(s) such as those generated by liver microsomal oxidases. The technique will also be useful to monitor the purification of the drug from cultures of Cephalosporium aphidicola.     

Circular dichroism detection in HPLC

A circular dichroism-based detection system presents several advantages in the HPLC analysis of chiral compounds because of the selective monitoring of optically active molecules. Its use allows reliable determination of enantiomeric excesses and elution order. To this end, the application of empirical, semiempirical, and nonempirical methods to get stereochemical information from the CD signal is reported. Furthermore, recording the CD spectra on line and evaluation of the dissymetry factor make the CD detection very powerful in characterizing the stereochemistry of chiral eluates.     

Attraction of the grasshopper, Melanoplus sanguinipes, to host plant odors and volatile components

The attraction of the polyphagous grasshopper, Melanoplus sanguinipes (F.), to odors from plant foliage or to chemical components of these odors was tested in a glass Y-tube olfactometer. Humidified air was passed at equal flow rates through a sample chamber containing plant material and an empty control chamber and then through the Y-tube to the holding chamber containing the test insect. Solutions of volatile chemicals in water were metered at a constant rate into the sample air stream with a syringe pump. Insects moving upwind were recorded after entering either the sample or control arm of the Y-tube. Both nymphs and adults were strongly attracted to the odor of chopped and intact seedling foliage of perennial ryegrass and wheat. Chopped leaves but not intact leaves of sorghum and alfalfa also were significantly attractive. The major components of ryegrass odor from both chopped and stem-cut leaves were Z-hex-3-en-1-yl acetate, Z-hex-3-en-1-ol, and pent-1-en-3-ol, in that order, with lesser amounts of E-hex-2-enal. Chopped leaves released many more minor components. The major components when tested individually or in binary and ternary mixtures were significantly attractive to grasshoppers compared to humidified air but not to the degree of whole plant odor. However, a quartenary mixture simulating the odor blend had levels of attractancy equal to that of chopped grass odor. Blends of these 5- and 6-carbon unsaturated alcohols, esters, and aldehydes volatilizing from green plants probably play an important role as olfactory cues for orientation of grasshoppers to food plants.

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Résumé L'attraction de M. sanguinipes (F.) par l'odeur du feuillage et des composés chimiques de ces odeurs a été examinée dans un olfactomètre en tube de verre en Y. Des flux identiques d'air humidifié étaient introduits dans une enceinte contenant le matériel végétal et dans une enceinte témoin, et, via le tube en Y, dans l'enceinte contenant l'insecte à étudier. Des solutions aqueuses de substances volatiles étaient introduites à un taux constant dans le courant d'air grâce à une seringue. Les insectes se déplaçant contre le vent étaient enregistrés après pénétration dans une branche déterminée du tube en Y. Larves et adultes étaient fortement attirés par l'odeur de feuillage intact ou coupé de semis de Lolium perenne et de blé (Triticum aestivum). Les feuilles coupées, mais non les feuilles intactes de sorgho (Sorghum bicolor) et de luzerne (Medicago sativa) étaient, elles aussi, significativement attractives. Les principales substances constituant de l'odeur de feuilles de Lolium perenne, tant coupées qu'intactes, étaient dans l'ordre de Z-hex-3-en-1-yl acétate, le Z-hex-3-en-1-ol et le pent-1-en-3-ol, et dans une moindre mesure, E-hex-2-enal. Les feuilles coupées libéraient beaucoup plus de composés secondaires. Les principaux constituants testés, seuls ou en mélanges binaires ou ternaires, étaient significativement attractifs, mais moins que l'odeur totale de la plante. Toutefois, un mélange quaternaire, simulant le mélange odorant, avait le même pouvoir attractif que l'odeur de la plante entière. L'union de ces alcools, esters et aldéhydes à 5- et 6-carbones non saturés, volatilisés à partir des plantes vertes, jouent probablement un grand rôle comme signaux orientateurs lors de l'attraction des criquets par leur plantes alimentaires.

     

Electrochemical detection of nucleic base mismatches with ferrocenyl naphthalene diimide

Electrochemical detection of nucleic base mismatches was attempted successfully with ferrocenyl naphthalene diimide (FND) in a model system with 20-meric double-stranded oligonucleotides with or without a mismatch(es). Thus, dA(20) or a 20-meric sequence of the lac Z gene was immobilized on a gold electrode and complementary oligonucleotides with different numbers of mismatches were allowed to hybridize in the presence of FND to give rise to an electrochemical signal. The signal intensity varied depending on the number of unpaired bases on the DNA duplex. From experiments with a quartz crystal microbalance, eight molecules of FND were found to bind to the 20-meric double-stranded oligos and this number decreased as the number of mismatches increased. These findings were further supported by matrix-assisted laser desorption ionization time-of-flight mass spectroscopy. This novel method will be useful for the analysis of single-nucleotide polymorphisms present on human genes.     

基于SD的生态安全指标阈值的确定及应用——以上海崇明岛为例

结合区域复合生态系统的特点,建立由经济、人口、资源、环境和生态5个子系统71个参数构成的上海崇明岛区域生态安全的系统动力学模型,并以变量"净GDP"为参考依据,通过情景仿真进行生态安全趋势分析和方案比较,确定生态安全的指标阈值;通过系统模拟对阈值进行验证,认为阈值在反映系统动态发展趋势上,是一定发展模式下的生态安全的特征参照系。这一尝试克服了目前生态安全指标阈值的研究中以参照法为主的静态性缺憾。将确定的阈值应用于崇明岛生态安全的评价和因子分析中,发现由于崇明岛生态岛的定位和生态保障措施的实施,近几年生态安全综合得分呈现逐步提高的特点,但目前(2007年)得分为0.523,仍处于中等水平;并以阈值为标准的因子检验发现各主要安全因子差异较大,主要的制约因子主要体现为环境治污水平和能源利用等方面。     

NADH荧光法快速检测细菌总数

基于细菌胞内NADH的荧光特性及其在胞内含量稳定的特性, 建立一种快速检测细菌总数的新方法。该荧光法的NADH检测限为1 nmol/L, NADH含量在10 nmol/L~0.2 mmol/L间与荧光强度呈良好线性关系(R2 =0.9905)。经离心获得菌体细胞, 热Tris-HCl法提取胞内NADH, 以 342 nm为激发波长, 461 nm为发射波长测定提取液荧光强度, 1 h内可检测到样品1×104 CFU/mL菌数。结果表明该方法快速、灵敏、简便、重复性好, 可适用于食品卫生与安全、环境检测等领域活细菌数量的定量检测。     

Diversity of an ectomycorrhizal fungal community studied by a root tip and total soil DNA approach

Molecular methods based on soil DNA extracts are increasingly being used to study the fungal diversity of ectomycorrhizal (EM) fungal communities in soil. Contrary to EM root tip identification, the use of molecular methods enables identification of extramatrical mycelia in soil. To compare fungal diversity as determined by root tip identification and mycelial identification, six soil samples were analysed. Root tips were extracted from the six samples and after amplification, the basidiomycete diversity on the root tips was analysed by denaturing gradient gel electrophoresis (DGGE). The soil from the six samples was sieved, total soil DNA was extracted and after amplification, the basidiomycete diversity in the soil fractions was analysed by DGGE. Fourteen different bands were excised from the DGGE gel and sequenced; fungal taxon names could be assigned to eight bands. Out of a total of 14 fungal taxa detected in soil, 11 fungal taxa were found on root tips, of which seven were EM fungal taxa. To examine whether the sieving treatment would affect EM species diversity, two different sieve mesh sizes were used and in addition, the organic soil fraction was analysed separately. DGGE analysis showed no differences in banding pattern for the different soil fractions. The organic fraction gave the highest DGGE band intensities. This work demonstrates that there is a high correspondence between basidiomycete diversity detected by molecular analysis of root tips and soil samples, irrespective of the soil fraction being analysed.