Estimating the accuracy of polymerase chain reaction-based tests using endpoint dilution

Polymerase chain reaction (PCR)-based tests for various microorganisms or target DNA sequences are generally acknowledged to be highly "sensitive," yet the concept of sensitivity is ill-defined in the literature on these tests. We propose that sensitivity should be expressed as a function of the number of target DNA molecules in the sample (or specificity, when the target number is 0). However, estimating this "sensitivity curve" is problematic, since it is difficult to construct samples with a fixed number of targets. Nonetheless, using serially diluted replicate aliquots of a known concentration of the target DNA sequence, we show that it is possible to disentangle random variations in the number of target DNA molecules from the underlying test sensitivity. We develop parametric, nonparametric, and semiparametric (spline-based) models for the sensitivity curve. The methods are compared on a new test for M. genitalium.     

环境雌激素实验室模拟暴露方法

从实验鱼类、暴露系统、暴露方式、评价指标四个方面对国内外环境雌激素的实验室暴露研究方法进行了介绍,为环境雌激素的暴露机制、毒性机理及雌激素效应的研究提供了理论依据和实验指导。     

Highly selective l-threonine 3-dehydrogenase from Cupriavidus necator and its use in determination of l-threonine

l-Threonine level in blood plasma is a biomarker of some diseases and nitrogen imbalance in the body. The determination of l-threonine is interesting and is required for diagnosis and management of inherited metabolic disorder. This is the first report of the specific enzymatic determination of l-threonine by a newly discovered l-threonine 3-dehydrogenase (ThrDH, EC 1.1.1.103) from Cupriavidus necator NBRC 102504. ThrDH, a key enzyme in l-threonine catabolism in microorganisms and animals, catalyzes the NAD⁺-dependent oxidation of l-threonine to 2-amino-3-oxobutyrate. ThrDH from C. necator was purified to homogeneity and fully characterized. l-Threonine and dl-2-amino-3-hydroxyvalerate are the only substrates for ThrDH among other l-amino acids, alcohols, and amino alcohols. The primary amino acid structure of ThrDH belongs to the extended short-chain alcohol dehydrogenase superfamily and is related to GDP-mannose-3′,5′-epimerase (GME) from Arabidopsis thaliana. Both enzymes have a glycine-rich NAD⁺-binding domain at the N terminal and conserved catalytic triad of YxxxK residues, but substrate-binding residues of GME were not found in the ThrDH sequence. ThrDH significantly differs from known bacterial and archaea ThrDHs that belong to zinc-binding medium chain alcohol dehydrogenase because of low sequence similarity and the lack of a zinc-binding domain in the sequence. A specific, quantitative, and sensitive enzymatic endpoint method for l-threonine determination was developed by using a ThrDH microplate assay. The assay was successfully applied for determination of l-threonine in human serum and plasma. Our specific determination is simple, convenient, inexpensive, accurate, and suitable for mass screening determination of l-threonine in a number of samples.     

经阿托伐他汀治疗的急性冠脉综合征患者CD4+T、CD4+CD28+T水平变化及与预后的关系

目的:探讨经阿托伐他汀治疗的急性冠脉综合征(ACS)的CD4~+T、CD4~+CD28~+T水平变化及与预后的关系。方法:选择128例ACS患者,随机分为对照组(64例)和观察组两组(64例),其中对照组患者给予常规治疗,观察组患者在上述基础上外加阿托伐他汀治疗。比较两组患者的细胞因子、CD4~+T及CD4~+CD28~+T水平变化,随访6个月,观察两组患者预后终点事件发生情况。并将观察组患者根据预后是否并发终点事件,将其分为预后良组(未并发终点事件)和预后不良组(并发终点事件)两组,分析不同预后ACS患者CD4~+T、CD4~+CD28~+T水平变化及与预后发生终点事件的相关性。结果:治疗后两组的高敏C反应蛋白(hs-CRP)、干扰素-γ(IFN-γ)水平均明显降低,白细胞介素-10(IL-10)、转化生长因子β1(TGF-β1)水平均明显升高,且观察组改善更为明显,差异均有统计学意义(P0.05)。两组患者治疗后CD4~+T明显升高,CD8~+T、CD4~+CD28~+T明显降低,且观察组上述指标改善更为明显,差异均有统计学意义(P0.05)。随访6月中,对照组患者预后有23例终点事件发生,观察组患者预后有26例终点事件发生,差异无统计学意义(P0.05)。与预后良好组相比,预后不良组患者的CD4~+T降低,CD4~+CD28~+T升高,差异均有统计学意义(P0.05)。经阿托伐他汀治疗的ACS患者预后发生终点事件与CD4~+T水平呈现负相关(r=-0.682,P=0.000),与CD4~+CD28~+T水平呈现正相关(r=0.733,P=0.000)。结论:经阿托伐他汀治疗的ACS患者预后发生终点事件与CD4~+T水平呈现负相关,与CD4~+CD28~+T水平呈现正相关,可为临床ACS患者预后的预测提供参考。     

Re‐Formulating Non‐inferiority Trials as Superiority Trials: The Case of Binary Outcomes

Non‐inferiority trials are conducted for a variety of reasons including to show that a new treatment has a negligible reduction in efficacy or safety when compared to the current standard treatment, or a more complex setting of showing that a new treatment has a negligible reduction in efficacy when compared to the current standard yet is superior in terms of other treatment characteristics. The latter reason for conducting a non‐inferiority trial presents the challenge of deciding on a balance between a suitable reduction in efficacy, known as the non‐inferiority margin, in return for a gain in other important treatment characteristics/findings. It would be ideal to alleviate the dilemma on the choice of margin in this setting by reverting to a traditional superiority trial design where a single p ‐value for superiority of both the most important endpoint (efficacy) and the most important finding (treatment characteristic) is provided. We discuss how this can be done using the information‐preserving composite endpoint (IPCE) approach and consider binary outcome cases in which the combination of efficacy and treatment characteristics, but not one itself, paints a clear picture that the novel treatment is superior to the active control (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Enzymatic analysis of uridine diphosphate N-acetyl-D-glucosamine

The Methanococcus maripaludis MMP0352 protein belongs to an oxidoreductase family that has been proposed to catalyze the NAD⁺-dependent oxidation of the 3′′ position of uridine diphosphate N-acetyl-d-glucosamine (UDP-GlcNAc), forming a 3-hexulose sugar nucleotide. The heterologously expressed MMP0352 protein was purified and shown to efficiently catalyze UDP-GlcNAc oxidation, forming one NADH equivalent. This enzyme was used to develop a fixed endpoint fluorometric method to analyze UDP-GlcNAc. The enzyme is highly specific for this acetamido sugar nucleotide, and the procedure had a detection limit of 0.2 μM UDP-GlcNAc in a 1-ml sample. Using the method of standard addition, UDP-GlcNAc concentrations were measured in deproteinized extracts of Escherichia coli, Saccharomyces cerevisiae, and HeLa carcinoma cells. Equivalent concentrations were determined by both enzymatic and chromatographic analyses, validating this method. This procedure can be adapted for the high-throughput analysis of changes in cellular UDP-GlcNAc concentrations in time series experiments or inhibitor screens.     

Endpoint error in smoothing and differentiating raw kinematic data: An evaluation of four popular methods

‘Endpoint error’ describes the erratic behavior at the beginning and end of the computed acceleration data which is commonly observed after smoothing and differentiating raw displacement data. To evaluate endpoint error produced by four popular smoothing and differentiating techniques, Lanshammar's (1982, J. Biomechanics 15, 99–105) modification of the Pezzack et al. (1977, J. Biomechanics, 10, 377–382) raw angular displacement data set was truncated at three different locations corresponding to the major peaks in the criterion acceleration curve. Also, for each data subset, three padding conditions were applied. Each data subset was smoothed and differentiated using the Butterworth digital filter, cubic spline, quintic spline, and Fourier series to obtain acceleration values. RMS residual errors were calculated between the computed and criterion accelerations in the endpoint regions. Although no method completely eliminated endpoint error, the results demonstrated clear superiority of the quintic spline over the other three methods in producing accurate acceleration values close to the endpoints of the modified Pezzack et al. (1977) data set. In fact, the quintic spline performed best with non-padded data (cumulative error=48.0 rad s⁻²). Conversely, when applied to non-padded data, the Butterworth digital filter produced wildly deviating values beginning more than the 10 points from the terminal data point (cumulative error=226.6 rad s⁻²). Each of the four methods performed better when applied to data subsets padded by linear extrapolation (average cumulative error=68.8 rad s⁻²) than when applied to analogous subsets padded by reflection (average cumulative error=86.1 rad s⁻²).