Blinded and unblinded internal pilot study designs for clinical trials with count data

Internal pilot studies are a popular design feature to address uncertainties in the sample size calculations caused by vague information on nuisance parameters. Despite their popularity, only very recently blinded sample size reestimation procedures for trials with count data were proposed and their properties systematically investigated. Although blinded procedures are favored by regulatory authorities, practical application is somewhat limited by fears that blinded procedures are prone to bias if the treatment effect was misspecified in the planning. Here, we compare unblinded and blinded procedures with respect to bias, error rates, and sample size distribution. We find that both procedures maintain the desired power and that the unblinded procedure is slightly liberal whereas the actual significance level of the blinded procedure is close to the nominal level. Furthermore, we show that in situations where uncertainty about the assumed treatment effect exists, the blinded estimator of the control event rate is biased in contrast to the unblinded estimator, which results in differences in mean sample sizes in favor of the unblinded procedure. However, these differences are rather small compared to the deviations of the mean sample sizes from the sample size required to detect the true, but unknown effect. We demonstrate that the variation of the sample size resulting from the blinded procedure is in many practically relevant situations considerably smaller than the one of the unblinded procedures. The methods are extended to overdispersed counts using a quasi‐likelihood approach and are illustrated by trials in relapsing multiple sclerosis.     

Adjusted regression trend test for a multicenter clinical trial

Studies using a series of increasing doses of a compound, including a zero dose control, are often conducted to study the effect of the compound on the response of interest. For a one-way design, Tukey et al. (1985, Biometrics 41, 295-301) suggested assessing trend by examining the slopes of regression lines under arithmetic, ordinal, and arithmetic-logarithmic dose scalings. They reported the smallest p-value for the three significance tests on the three slopes for safety assessments. Capizzi et al. (1992, Biometrical Journal 34, 275-289) suggested an adjusted trend test, which adjusts the p-value using a trivariate t-distribution, the joint distribution of the three slope estimators. In this paper, we propose an adjusted regression trend test suitable for two-way designs, particularly for multicenter clinical trials. In a step-down fashion, the proposed trend test can be applied to a multicenter clinical trial to compare each dose with the control. This sequential procedure is a closed testing procedure for a trend alternative. Therefore, it adjusts p-values and maintains experimentwise error rate. Simulation results show that the step-down trend test is overall more powerful than a step-down least significant difference test.     

羌活药材活性成分的提取工艺研究

选择提取次数、溶剂倍数、提取时间为考察因素,以提取浸膏量及三种主要活性物质(紫花前胡苷、异欧前胡素、羌活醇)含量为考察指标,探讨羌活药材活性成分的乙醇加热回流提取工艺。在首先考察单因素对提取效果影响的基础上采用正交试验法,优选出羌活药材活性成分的最佳提取工艺参数:加6倍量95%乙醇,加热回流提取3次,每次提取时间为1 h。     

苏云金杆菌“79007”菌株发酵工艺的研究

对苏云金杆菌79007菌进行了生长条件的研究,获得了该菌最佳发酵培养基配方和最优发酵工艺。最佳培养基配方(g/100 mL):豆饼粉2.5,淀粉2.0,酵母粉2.0,玉米浆2.0,添加剂Ⅰ号0.1,葡萄糖1.0,KH2PO40.3,CaCO30.1,CaCl20.05,MgCl20.01,CuCl20.005,ZnSO40.002,CoCO30.005;15 m3发酵罐最优工艺:通气量1∶0.5~1.1,培养温度28~33℃,搅拌转速0~200 r/min。40 m3发酵罐连续4批试验表明,平均发酵效价6 053.5 IU/μL、平均晶体蛋白含量达0.5%,产品质量达到原粉优质品。     

Shape matters in sampling plant diversity: Evidence from the field

The identification of shape and size of sampling units that maximises the number of plant species recorded in multiscale sampling designs has major implications in conservation planning and monitoring actions. In this paper we tested the effect of three sampling shapes (rectangles, squared, and randomly shaped sampling units) on the number of recorded species. We used a large dataset derived from the network of protected areas in the Siena Province, Italy. This dataset is composed of plant species occurrence data recorded from 604 plots (10 m × 10 m), each divided in a grid of 16 contiguous subplot units (2.5 m × 2.5 m). Moreover, we evaluated the effect of plot orientation along the main environmental gradient, to examine how the selection of plot orientation (when elongated plots are used) influences the number of species collected. In total, 1041 plant species were recorded from the study plots. A significantly higher species richness was recorded by the random arrangement of 4 subplots within each plot in comparison to the ‘rectangle’ and ‘square’ shapes. Although the rectangular shape captured a significant larger number of species than squared ones, plot orientation along the main environmental gradient did not show a systematic effect on the number of recorded species. We concluded that the choice of whether or not using elongated (rectangular) versus squared plots should dependent upon the objectives of the specific survey with squared plots being more suitable for assessing species composition of more homogeneous vegetation units and rectangular plots being more suited for recording more species in the pooled sample of a large area.     

Combining adaptive designs with control of the false discovery rate--a generalized definition for a global p-value

When combining adaptive designs with control of the False Discovery Rate one has to keep in mind that the most frequently used procedure for controlling the False Discovery Rate--the explorative Simes procedure--is a stepwise multiple testing procedure. At the interim analysis of an adaptive design it is however not yet known what the boundaries for rejection of hypotheses in the final analysis will be as these boundaries depend on the size of the final p-values. Therefore classical adaptive designs with a predefined stopping criterion for early rejection of hypotheses are not well suited. We propose a generalized definition of a global p-value for a two-stage adaptive design permitting a flexible decision for stopping at the interim analysis. By means of a simulation study in the field of genetic epidemiology we illustrate how applying such a two-stage design can reduce costs.     

Adaptive seamless clinical trials using early outcomes for treatment or subgroup selection: Methods,simulation model and their implementation in R

Adaptive seamless designs combine confirmatory testing, a domain of phase III trials, with features such as treatment or subgroup selection, typically associated with phase II trials. They promise to increase the efficiency of development programmes of new drugs, for example, in terms of sample size and/or development time. It is well acknowledged that adaptive designs are more involved from a logistical perspective and require more upfront planning, often in the form of extensive simulation studies, than conventional approaches. Here, we present a framework for adaptive treatment and subgroup selection using the same notation, which links the somewhat disparate literature on treatment selection on one side and on subgroup selection on the other. Furthermore, we introduce a flexible and efficient simulation model that serves both designs. As primary endpoints often take a long time to observe, interim analyses are frequently informed by early outcomes. Therefore, all methods presented accommodate interim analyses informed by either the primary outcome or an early outcome. The R package asd , previously developed to simulate designs with treatment selection, was extended to include subgroup selection (so-called adaptive enrichment designs). Here, we describe the functionality of the R package asd and use it to present some worked-up examples motivated by clinical trials in chronic obstructive pulmonary disease and oncology. The examples both illustrate various features of the R package and provide insights into the operating characteristics of adaptive seamless studies.     

曼地亚红豆杉RAPD反应体系与程序的优化

以曼地亚红豆杉为研究对象,采用L16(45)正交组合实验和单因素梯度实验对MgCl2、dNTP、随机引物、Taq酶、模板DNA浓度和退火温度、循环次数等影响RAPD扩增的重要因素进行优化,以期建立最优的RAPD反应体系与程序。实验结果表明,各因素最适条件为:25μLPCR反应体系中10×Buffer2.5μL,MgCl21.5mmol/L,dNTP0.2mmol/L,随机引物0.6μmol/L,Taq酶1.0U,模板DNA80ng;退火温度为37℃,循环次数为45次。     

Structure-activity relationships of rationally designed AMACR 1A inhibitors

α-Methylacyl-CoA racemase (AMACR; P504S) is a promising novel drug target for prostate and other cancers. Assaying enzyme activity is difficult due to the reversibility of the ‘racemisation’ reaction and the difficulties in the separation of epimeric products; consequently few inhibitors have been described and no structure–activity relationship study has been performed. This paper describes the first structure–activity relationship study, in which a series of 23 known and potential rational AMACR inhibitors were evaluated. AMACR was potently inhibited (IC₅₀ = 400–750 nM) by ibuprofenoyl-CoA and derivatives. Potency was positively correlated with inhibitor lipophilicity. AMACR was also inhibited by straight-chain and branched-chain acyl-CoA esters, with potency positively correlating with inhibitor lipophilicity. 2-Methyldecanoyl-CoAs were ca. 3-fold more potent inhibitors than decanoyl-CoA, demonstrating the importance of the 2-methyl group for effective inhibition. Elimination substrates and compounds with modified acyl-CoA cores were also investigated, and shown to be potent inhibitors. These results are the first to demonstrate structure–activity relationships of rational AMACR inhibitors and that potency can be predicted by acyl-CoA lipophilicity. The study also demonstrates the utility of the colorimetric assay for thorough inhibitor characterisation.     

Discovery of potent PTP1B inhibitors via structure-based drug design,synthesis and in vitro bioassay of Norathyriol derivatives

Protein tyrosine phosphatase 1B (PTP1B) has recently been identified as a potential target of Norathyriol. Unfortunately, Norathyriol is not a potent PTP1B inhibitor, which somewhat hinders its further application. Based on the fact that no study on the relationship of chemical structure and PTP1B inhibitory activity of Norathyriol has been reported so far, we attempted to perform structural optimization so as to improve the potency for PTP1B. Via structure-based drug design (SBDD), a rational strategy based on the binding mode of Norathyriol to PTP1B, we designed 26 derivatives with substitutions at the four phenolic hydroxyl groups of Norathyriol. By chemical synthesis and in vitro bioassay, we identified seven PTP1B inhibitors that were more potent than Norathyriol, of which XWJ24 showed the highest potency (IC₅₀: 0.6 μM). We also found out that XWJ24 was a competitive inhibitor and showed the 4.5-fold selectivity over its close homolog, TC-PTP. Through molecular docking of XWJ24 against PTP1B, we highlighted the essential role of its hydrogen bond with Asp181 for PTP1B inhibition and identified a potential halogen bond with Asp48 that was not observed for Norathyriol. The current data indicate that our SBDD strategy is effective to discover potent PTP1B-targeted Norathyriol derivatives, and XWJ24 is a promising lead compound for further development.     

Structures composing protein domains

This review summarizes available data concerning intradomain structures (IS) such as functionally important amino acid residues, short linear motifs, conserved or disordered regions, peptide repeats, broadly occurring secondary structures or folds, etc. IS form structural features (units or elements) necessary for interactions with proteins or non-peptidic ligands, enzyme reactions and some structural properties of proteins. These features have often been related to a single structural level (e.g. primary structure) mostly requiring certain structural context of other levels (e.g. secondary structures or supersecondary folds) as follows also from some examples reported or demonstrated here. In addition, we deal with some functionally important dynamic properties of IS (e.g. flexibility and different forms of accessibility), and more special dynamic changes of IS during enzyme reactions and allosteric regulation. Selected notes concern also some experimental methods, still more necessary tools of bioinformatic processing and clinically interesting relationships.     

Alternative Confidence Regions for Bonferroni‐Based Closed‐Testing Procedures that are not Alpha‐Exhaustive

This article complements the results in Guilbaud (Biometrical Journal 2008; 50 :678–692). Simultaneous confidence regions were derived in that article that correspond to any given multiple testing procedure (MTP) in a fairly large class of consonant closed‐testing procedures based on marginal p‐values and weighted Bonferroni tests for intersection hypotheses. This class includes Holm's MTP, the fixed‐sequence MTP, gatekeeping MTPs, fallback MTPs, multi‐stage fallback MTPs, and recently proposed MTPs specified through a graphical representation and associated rejection algorithm. More general confidence regions are proposed in this article. These regions are such that for certain underlying MTPs which are not alpha‐exhaustive, they lead to confidence assertions that may be sharper than rejection assertions for some rejected null hypotheses H when not all Hs are rejected, which is not the case with the previously proposed regions. In fact, various alternative confidence regions may be available for such an underlying MTP. These results are shown through an extension of the previous direct arguments (without invoking the partitioning principle), and under the same general setup; so for instance, estimated quantities and marginal confidence regions are not restricted to be of any particular kinds/dimensions. The relation with corresponding confidence regions of Strassburger and Bretz (Statistics in Medicine 2008; 27 :4914–4927) is described. The results are illustrated with fallback and parallel‐gatekeeping MTPs.     

Probing the chemical mechanism and critical regulatory amino acid residues of Drosophila melanogaster arylalkylamine N-acyltransferase like 2

Arylalkylamine N-acyltransferase like 2 (AANATL2) catalyzes the formation of N-acylarylalkylamides from the corresponding acyl-CoA and arylalkylamine. The N-acylation of biogenic amines in Drosophila melanogaster is a critical step for the inactivation of neurotransmitters, cuticle sclerotization, and melatonin biosynthesis. In addition, D. melanogaster has been used as a model system to evaluate the biosynthesis of fatty acid amides: a family of potent cell signaling lipids. We have previously showed that AANATL2 catalyzes the formation of N-acylarylakylamides, including long-chain N-acylserotonins and N-acyldopamines. Herein, we define the kinetic mechanism for AANATL2 as an ordered sequential mechanism with acetyl-CoA binding first followed by tyramine to generate the ternary complex prior to catalysis. Bell shaped k_{cat,app – acetyl-CoA} and (k_cat/K_m)_{app – acetyl-CoA} pH-rate profiles identified two apparent pK_a,app values of ∼7.4 and ∼8.9 that are critical to catalysis, suggesting the AANATL2-catalyzed formation of N-acetyltyramine occurs through an acid/base chemical mechanism. Site-directed mutagenesis of a conserved glutamate that corresponds to the catalytic base for other D. melanogaster AANATL enzymes did not produce a substantial depression in the k_cat,app value nor did it abolish the pK_a,app value attributed to the general base in catalysis (pK_a ∼7.4). These data suggest that AANATL2 catalyzes the formation of N-acylarylalkylamides using either different catalytic residues or a different chemical mechanism relative to other D. melanogaster AANATL enzymes. In addition, we constructed other site-directed mutants of AANATL2 to help define the role of targeted amino acids in substrate binding and/or enzyme catalysis.     

Promising antibacterial agents against multidrug resistant Staphylococcus aureus

Rapid emergence of multidrug resistant Staphylococcus aureus infections has created a critical health menace universally. Resistance to all the available chemotherapeutics has been on rise which led to WHO to stratify Staphylococcus aureus as high tier priorty II pathogen. Hence, discovery and development of new antibacterial agents with new mode of action is crucial to address the multidrug resistant Staphylococcus aureus infections. The egressing understanding of new antibacterials on their biological target provides opportunities for new therapeutic agents. This review underlines on various aspects of drug design, structure activity relationships (SARs) and mechanism of action of various new antibacterial agents and also covers the recent reports on new antibacterial agents with potent activity against multidrug resistant Staphylococcus aureus. This review provides attention on in vitro and in vivo pharmacological activities of new antibacterial agents in the point of view of drug discovery and development.     

Potential chemical transformation of phosphinic acid derivatives and their applications in the synthesis of drugs

The chemical transformation of phosphinic acid is a well-considered mature area of research on account of the historical significant reactions such as Kabachnik–Fields, Mannich, Arbuzov, Michaelis–Becker, etc. Considerable advances have been made over last years especially in metal-catalyzed, free-radical processes and asymmetric synthesis using catalytic enantioselective. As a result, the aim of this synopsis is to make the reader familiar with advances in the approaches of phosphinic acids toward the synthesis of highly functionalized and valuable buildings blocks. Another purpose of this survey is to provide the current status of the applications of phosphinic acids in the synthesis of drugs.     

Decision theoretic designs for phase II clinical trials with multiple outcomes

In many phase II clinical trials, it is essential to assess both efficacy and safety. Although several phase II designs that accommodate multiple outcomes have been proposed recently, none are derived using decision theory. This paper describes a Bayesian decision theoretic strategy for constructing phase II designs based on both efficacy and adverse events. The gain function includes utilities assigned to patient outcomes, a reward for declaring the new treatment promising, and costs associated with the conduct of the phase II trial and future phase III testing. A method for eliciting gain function parameters from medical collaborators and for evaluating the design's frequentist operating characteristics is described. The strategy is illustrated by application to a clinical trial of peripheral blood stem cell transplantation for multiple myeloma.     

Defects in mitochondrial DNA replication and oxidative damage in muscle of mtDNA mutator mice

A causal role for mitochondrial dysfunction in mammalian aging is supported by recent studies of the mtDNA mutator mouse (“PolG” mouse), which harbors a defect in the proofreading-exonuclease activity of mitochondrial DNA polymerase gamma. These mice exhibit accelerated aging phenotypes characteristic of human aging, including systemic mitochondrial dysfunction, exercise intolerance, alopecia and graying of hair, curvature of the spine, and premature mortality. While mitochondrial dysfunction has been shown to cause increased oxidative stress in many systems, several groups have suggested that PolG mutator mice show no markers of oxidative damage. These mice have been presented as proof that mitochondrial dysfunction is sufficient to accelerate aging without oxidative stress. In this study, by normalizing to mitochondrial content in enriched fractions we detected increased oxidative modification of protein and DNA in PolG skeletal muscle mitochondria. We separately developed novel methods that allow simultaneous direct measurement of mtDNA replication defects and oxidative damage. Using this approach, we find evidence that suggests PolG muscle mtDNA is indeed oxidatively damaged. We also observed a significant decrease in antioxidants and expression of mitochondrial biogenesis pathway components and DNA repair enzymes in these mice, indicating an association of maladaptive gene expression with the phenotypes observed in PolG mice. Together, these findings demonstrate the presence of oxidative damage associated with the premature aging-like phenotypes induced by mitochondrial dysfunction.     

Mode of action of allatostatins in the regulation of juvenile hormone biosynthesis in the cockroach,Diploptera punctata

The FGLamide allatostatins (FGL/ASTs) are a family of neuropeptides with pleiotropic functions, including the inhibition of juvenile hormone (JH) biosynthesis, vitellogenesis and muscle contraction. In the cockroach, Diploptera punctata, thirteen FGLa/ASTs and one allatostatin receptor (AstR) have been identified. However, the mode of action of ASTs in regulation of JH biosynthesis remains unclear. Here, we determined the tissue distribution of Dippu-AstR. And we expressed Dippu-AstR in vertebrate cell lines, and activated the receptor with the Dippu-ASTs. Our results show that all thirteen ASTs activated Dippu-AstR in a dose dependent manner, albeit with different potencies. Functional analysis of AstR in multiple cell lines demonstrated that activation of the AstR receptor resulted in elevated levels of Ca²⁺ and cAMP, which suggests that Dippu-AstR can act through the Gα_q and Gα_s protein pathways. The study on the target of AST action reveals that FGL/AST affects JH biosynthesis prior to the entry of acetyl-CoA into the JH biosynthetic pathway.     

Shifts in thermoregulatory strategy during ontogeny in harp seals (Pagophilus groenlandicus)

Heat balance can be difficult for young and/or small animals in polar regions because environmental conditions in combination with small body size or physiological immaturity can increase heat loss. We investigated how thermoregulatory patterns change with ontogeny in 5 age classes of harp seal (Pagophilus groenlandicus) from birth to post-molt to further understand the timing of thermoregulatory development in relation to their potential vulnerability to ongoing fluctuations in the extent and stability of Arctic pack ice. We measured changes in the amount, conductivity, and resistance of the seal pups׳ insulative layers (blubber and fur), the potential for endogenous heat-generation by shivering (muscle enzyme activity), and nonshivering thermogenesis (NST; brown adipose tissue (BAT) uncoupling protein 1 (UCP1) expression and mitochondrial density). There was no significant difference in blubber conductivity among age classes, though the amount of blubber insulation significantly increased from birth to weaning. Pelage conductivity was low (0.12±0.01 W m⁻¹ °C⁻¹) except in 9-day old pups (0.40±0.08 W m⁻¹ °C⁻¹); the significantly higher conductivity may signal the beginning of the molt, and this age group may be the most vulnerable to early water entry. Citrate synthase activity significantly increased (49.68±3.26 to 75.08±3.52 μmol min⁻¹ g wet weight⁻¹) in the muscle; however it is unlikely that increasing a single enzyme greatly impacts heat generation. BAT of younger pups contained UCP1, though expression and mitochondrial density quickly declined, and the ability of pups to produce heat via NST was lost by weaning. While total thermal resistance did not differ, neonatal and early nursing animals gained the majority of their thermal resistance from lanugo (82.5±0.03%); however, lanugo is not insulative when wet, and NST may be important to maintain euthermia and dry the coat if early immersion in water occurs. By late nursing, blubber seems sufficient as insulation (75.87±0.01% of resistance after 4 weeks), but high conductivity of fur may be responsible for retention of UCP1 expression. Weaned animals rely on blubber insulation, and no longer need NST, as wetted fur is no longer a threat to euthermia.     

The oxidant-antioxidant equilibrium,activities of selected lysosomal enzymes and activity of acute phase protein in peripheral blood of 18-year-old football players after aerobic cycle ergometer test combined with ice-water immersion or recovery at room temperature

The goal of the study was to evaluate the effect of an aerobic exercise bout followed by ice-water immersion or recovery at room temperature on the redox state, activities of selected lysosomal enzymes and activity of α1-antitrypsin (AAT) in the blood of healthy sportsmen. Eleven amateur football players aged 18 were randomly assigned to two similar 30-min aerobic cycle ergometer tests followed by a recovery at room temperature (20 °C; Experiment 1) or ice-water immersion (3 °C, 5 min; Experiment 2). Peripheral blood was collected three times during both study experiments: before (baseline), as well as 20 and 40 min after the recovery or immersion. The concentrations of thiobarbituric acid reactive substances in blood plasma (plTBARS) and erythrocytes (erTBARS) were measured. The erythrocytic activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) were also determined. In the blood serum, the activities of acid phosphatase (AcP), arylsulphatase (ASA), cathepsin D (CTS D) and AAT were evaluated. The activities of AcP, ASA, CTS D and AAT changed similarly during both experiments. The GPx activity decreased 40 min after the exercise/recovery compared to the baseline activity and was lower than 40 min after the exercise/immersion. The exercise followed by the recovery or immersion had no significant effect on the serum lysosomal and AAT activities in the studied men. The exercise/recovery reduced the hydrogen peroxide concentration in the men's erythrocytes, however the exercise/immersion demonstrated the opposite effect.