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.     

海州湾近岸海域的微生物群落结构和抗生素抗性基因时空变化

【背景】近岸海域抗生素抗性基因(antibiotic resistance genes, ARGs)的污染和累积将直接影响海产品质量和安全,海州湾作为江苏省的四大渔场之一,是江苏渔业发展的主要载体,有多条大小河流注入,沿岸为重要农业区,对公众健康产生重大影响。【目的】对海州湾夏秋季的水样及沉积物展开微生物及ARGs检测。【方法】基于宏基因组测序技术开展海州湾夏秋两季近岸6个站点中水体和沉积物中ARGs种类和相对丰度以及微生物群落的组成研究。【结果】变形菌门(Proteobacteria)和放线菌门(Actinobacteria)是夏秋季度两种介质中最优势的门类,水样中优势的科级细菌为红细菌科(Rhodobacteraceae),沉积物样品中为脱硫杆菌科(Desulfobacteraceae);夏季水样中的ARGs相对丰度要明显高于秋季,但沉积物中不同季节的ARGs相对丰度未表现出明显的变化趋势;在水样中主要门级微生物群落的抗性机制主要是抗生素靶位替换和抗生素靶位保护,沉积物样品则以抗生素灭活机制为主,而主要科级微生物群落的抗性机制更加多样;冗余分析(redundancyanalysis...     

杂交试验中的最佳样本含量

杂交试验是一项费时费钱的工作,因此在进行试验之前如能进行严密的设计,给出试验所需的样本大小是十分必要的,统计学中常见的估计样本容理的公式不宜应用于杂交试验,本文分两种情况给出了杂交试验中样本容量的估计公式,据此估计出的样本容量安排杂交试验,可在满足试验者要求的条件下,使试验的总成本最低或使试畜的总头数最少。     

DIET OF HARBOR PORPOISES IN THE KATTEGAT AND SKAGERRAK SEAS: ACCOUNTING FOR INDIVIDUAL VARIATION AND SAMPLE SIZE

Stomach contents of 112 bycaught harbor porpoises ( Phocoena phocoena ) collected between 1989 and 1996 in the Kattegat and Skagerrak seas were analyzed to describe diet composition and estimate prey size, to examine sample size requirements, and to compare juvenile and adult diets. Although porpoises preyed on a variety of species, only a few contributed substantially to the diet. Atlantic herring ( Clupea harengus ) was the dominating prey species for both juveniles and adults. Our results, in combination with those from previous studies, suggest that where herring is a dominant food source, porpoises prey primarily on size classes containing mature or maturing individuals. Further, we also show that Atlantic hagfish ( Myxine glutinosa ) may be an important food resource, at least for adult porpoises. Examination of sample size requirement showed that, depending on the taxonomic level used to describe the diet, a minimum of 35–71 stomachs are needed to be confident that all common prey species will be found.     

Strategies for microarray analysis of limiting amounts of RNA.

One of the critical limitations of current microarray technologies for use in expression analyses is the relatively large amount of input RNA required to generate labelled cDNA populations for array analysis. In situations where RNA is limiting, the options for expression profiling are to increase cDNA labelling and hybridisation efficiency, or to use an amplification strategy to generate enough RNA/cDNA for use with a standard labelling method. Sample amplification approaches must preserve the representation of the relative abundances of the different RNAs within the starting population and must also be highly reproducible. This review evaluates current signal and sample amplification technologies, including those that can be used to generate labelled cDNA populations for array analysis from as little as a single cell.     

On-line immunoanalysis of monoclonal antibodies during a continuous culture of hybridoma cells

The monoclonal-antibody production of an immobilized hybridoma cell line cultivated in a fluidized-bed reactor was monitored on-line for nearly 900 h. The monoclonal antibody concentration was determined by an immuno affinity-chromatography method (ABICAP). Antibodies directed against the product, e.g. IgG, were immobilized on a micro-porous gel and packed in small columns. After all IgG present in the sample was bound to the immobilized antibodies, unbound proteins were removed by rinsing the column. Elution of the bound antibodies followed and the antibodies were determined by fluorescence. The analytical procedure was automated with a robotic device to enable on-line measurements. The correlation between the on-line determined data and antibody concentrations measured by HPLC was linear. A sampling system was constructed, which was based on a pneumatically actuated in-line membrane valve integrated into the circulation loop of the reactor. Separation of the cells from the sample stream was achieved by a depth filter made of glass-fibre, situated outside the reactor. Rapid obstruction of the filter by cells or cell debris and contamination of the sample system was avoided by intermittent rinsing of the sample system with a chemical solution. The intermittent rinsing of the filter, which had a surface of 4.8 cm², resulted in an operational capacity of up to 40 samples (1.0 l total sample volume). Both the sampling system and the analytical device functioned without failure during this long-term culture. The culture temperature was varied between 34 and 40 °C. Raising the temperature from 34 up to 37 °C resulted in a simultaneous increase of growth and specific antibody production rate. Specific metabolic rates of glucose, lactate, glutamine and ammonium stayed constant in this temperature range. A further enhancement of temperature up to 40 °C had a negative effect on the growth rate, whereas the specific monoclonal antibody production rate showed a small increase. The other specific metabolic rates also increased in the temperature range between 38 to 40 °C. This revised version was published online in July 2006 with corrections to the Cover Date.     

A thin quartz cell suitable for vacuum ultraviolet absorption and circular dichroism measurements

The design of a thin quartz cell suitable for absorption and circular dichroism measurements in the vacuum ultraviolet is described. Important features of the cell are (1) that it can be disassembled for cleaning and reproducibly reassembled with path lengths up to 0.3 mm, and (2) that strain in the windows from the compressed sample can be relieved by a sample overflow port. The latter feature allows the cell to be used for circular dichroism as well as absorption measurements.     

New preparation techniques for molecular and in‐situ analysis of ancient organic micro‐ and nanostructures

Organic microfossils preserved in three dimensions in transparent mineral matrices such as cherts/quartzites, phosphates, or carbonates are best studied in petrographic thin sections. Moreover, microscale mass spectrometry techniques commonly require flat, polished surfaces to minimize analytical bias. However, contamination by epoxy resin in traditional petrographic sections is problematic for the geochemical study of the kerogen in these microfossils and more generally for the in situ analysis of fossil organic matter. Here, we show that epoxy contamination has a molecular signature that is difficult to distinguish from kerogen with time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS). This contamination appears pervasive in organic microstructures embedded in micro‐ to nano‐crystalline carbonate. To solve this problem, a new semi‐thin section preparation protocol without resin medium was developed for micro‐ to nanoscale in situ investigation of insoluble organic matter. We show that these sections are suited for microscopic observation of Proterozoic microfossils in cherts. ToF‐SIMS reveals that these sections are free of pollution after final removal of a <10 nm layer of contamination using low‐dose ion sputtering. ToF‐SIMS maps of fragments from aliphatic and aromatic molecules and organic sulfur are correlated with the spatial distribution of organic microlaminae in a Jurassic stromatolite. Hydrocarbon‐derived ions also appeared correlated with kerogenous microstructures in Archean cherts. These developments in analytical procedures should help future investigations of organic matter and in particular, microfossils, by allowing the spatial correlation of microscopy, spectroscopy, precise isotopic microanalyses, and novel molecular microanalyses such as ToF‐SIMS.