Improved two‐stage group sequential procedures for testing a secondary endpoint after the primary endpoint achieves significance

In two‐stage group sequential trials with a primary and a secondary endpoint, the overall type I error rate for the primary endpoint is often controlled by an α‐level boundary, such as an O'Brien‐Fleming or Pocock boundary. Following a hierarchical testing sequence, the secondary endpoint is tested only if the primary endpoint achieves statistical significance either at an interim analysis or at the final analysis. To control the type I error rate for the secondary endpoint, this is tested using a Bonferroni procedure or any α‐level group sequential method. In comparison with marginal testing, there is an overall power loss for the test of the secondary endpoint since a claim of a positive result depends on the significance of the primary endpoint in the hierarchical testing sequence. We propose two group sequential testing procedures with improved secondary power: the improved Bonferroni procedure and the improved Pocock procedure. The proposed procedures use the correlation between the interim and final statistics for the secondary endpoint while applying graphical approaches to transfer the significance level from the primary endpoint to the secondary endpoint. The procedures control the familywise error rate (FWER) strongly by construction and this is confirmed via simulation. We also compare the proposed procedures with other commonly used group sequential procedures in terms of control of the FWER and the power of rejecting the secondary hypothesis. An example is provided to illustrate the procedures.     

Integrating cell‐free biosyntheses of heme prosthetic group and apoenzyme for the synthesis of functional P450 monooxygenase

Harnessing the isolated protein synthesis machinery, cell‐free protein synthesis reproduces the cellular process of decoding genetic information in artificially controlled environments. More often than not, however, generation of functional proteins requires more than simple translation of genetic sequences. For instance, many of the industrially important enzymes require non‐protein prosthetic groups for biological activity. Herein, we report the complete cell‐free biogenesis of a heme prosthetic group and its integration with concurrent apoenzyme synthesis for the production of functional P450 monooxygenase. Step reactions required for the syntheses of apoenzyme and the prosthetic group have been designed so that these two separate pathways take place in the same reaction mixture, being insulated from each other. Combined pathways for the synthesis of functional P450 monooxygenase were then further integrated with in situ assay reactions to enable real‐time measurement of enzymatic activity during its synthesis. Biotechnol. Bioeng. 2013; 110: 1193–1200. © 2012 Wiley Periodicals, Inc.     

Estimation of population allele frequencies from next‐generation sequencing data: pool‐versus individual‐based genotyping

Molecular markers produced by next‐generation sequencing (NGS) technologies are revolutionizing genetic research. However, the costs of analysing large numbers of individual genomes remain prohibitive for most population genetics studies. Here, we present results based on mathematical derivations showing that, under many realistic experimental designs, NGS of DNA pools from diploid individuals allows to estimate the allele frequencies at single nucleotide polymorphisms (SNPs) with at least the same accuracy as individual‐based analyses, for considerably lower library construction and sequencing efforts. These findings remain true when taking into account the possibility of substantially unequal contributions of each individual to the final pool of sequence reads. We propose the intuitive notion of effective pool size to account for unequal pooling and derive a Bayesian hierarchical model to estimate this parameter directly from the data. We provide a user‐friendly application assessing the accuracy of allele frequency estimation from both pool‐ and individual‐based NGS population data under various sampling, sequencing depth and experimental error designs. We illustrate our findings with theoretical examples and real data sets corresponding to SNP loci obtained using restriction site–associated DNA (RAD) sequencing in pool‐ and individual‐based experiments carried out on the same population of the pine processionary moth (Thaumetopoea pityocampa). NGS of DNA pools might not be optimal for all types of studies but provides a cost‐effective approach for estimating allele frequencies for very large numbers of SNPs. It thus allows comparison of genome‐wide patterns of genetic variation for large numbers of individuals in multiple populations.     

Benzofurazan‐based fluorophore for the selective determination of flupentixol dihydrochloride: Application to content uniformity testing

One of the most commonly used drugs in treatment of schizophrenia is flupentixol dihydrochloride, therefore it is important to develop a simple, low cost and sensitive spectrofluorimetric method for the estimation of flupentixol dihydrochloride. The yellow fluorescent product that is generated from the nucleophilic substitution reaction of the free lone pair of the alcoholic hydroxyl group of the drug and 4‐chloro‐7‐nitrobenzofurazan (NBD‐Cl) in Mcllvaine buffer pH 7.0 was estimated at 510 nm (λ_ex 460 nm). The variables that affect the development of the reaction product were explored and optimized. The linear range of this method was 0.5–2.5 μg ml^?1 with a limit of quantitation equal to 0.29 μg ml^?1. Our method was successfully applied for the assurance of flupentixol in tablet form with average percentage recovery of 99.08 ± 1.01% without obstruction from the basic excipients exhibits. Furthermore, our strategy was extended to study the content uniformity testing of flupentixol in Fluaxnol^® tablets.     

Approval policies for modifications to machine learning‐based software as a medical device: A study of bio‐creep

Successful deployment of machine learning algorithms in healthcare requires careful assessments of their performance and safety. To date, the FDA approves locked algorithms prior to marketing and requires future updates to undergo separate premarket reviews. However, this negates a key feature of machine learning—the ability to learn from a growing dataset and improve over time. This paper frames the design of an approval policy, which we refer to as an automatic algorithmic change protocol (aACP), as an online hypothesis testing problem. As this process has obvious analogy with noninferiority testing of new drugs, we investigate how repeated testing and adoption of modifications might lead to gradual deterioration in prediction accuracy, also known as “biocreep” in the drug development literature. We consider simple policies that one might consider but do not necessarily offer any error‐rate guarantees, as well as policies that do provide error‐rate control. For the latter, we define two online error‐rates appropriate for this context: bad approval count (BAC) and bad approval and benchmark ratios (BABR). We control these rates in the simple setting of a constant population and data source using policies aACP‐BAC and aACP‐BABR, which combine alpha‐investing, group‐sequential, and gate‐keeping methods. In simulation studies, bio‐creep regularly occurred when using policies with no error‐rate guarantees, whereas aACP‐BAC and aACP‐BABR controlled the rate of bio‐creep without substantially impacting our ability to approve beneficial modifications.     

On confidence bounds for the ratio of net differences in the "gold standard" design with reference, experimental, and placebo treatment

The three-arm clinical study including a placebo has been recommended to be the preferred study design for the comparison of an experimental treatment relative to a reference treatment. In a confirmatory three-arm study multiplicity issues arise that are not present in two-arm studies. In the past a successful demonstration of superiority of the reference over placebo has been regarded a prerequisite validation step for the demonstration of superiority of the experimental treatment over placebo. However, for an investigator this last comparison is the most critical one. In a clinical study the demonstration of superiority of the experimental treatment over placebo is a result of its own value and this should therefore not be made dependent on tests that are of higher priority in a hierarchical test procedure. This can be achieved through a symmetrical formulation of Fieller's method for constructing confidence intervals for ratio of the expected values from normally distributed variables. In the symmetrical formulation the different meanings of nominator and denominator disappear, and simultaneous statements for comparisons between the experimental treatment and placebo, reference treatment and placebo, and reference and experimental treatment can be made. This is accomplished by moving the discussion on confidence sets from the line of real numbers to the unit circle which allows representing confidence sectors always as connected sets, gives always simple geometrical interpretations, and is easy to be transformed back to the real numbers if the traditional calculations behave well. The proposed procedures provides additional insight in existing methods but does obviously not answer the clinical question whether or not the demonstration of superiority of the reference treatment over placebo is a necessary validation step for further comparisons.     

Microdevice‐based solid‐phase polymerase chain reaction for rapid detection of pathogenic microorganisms

We demonstrate the integration of DNA amplification and detection functionalities developed on a lab‐on‐a‐chip microdevice utilizing solid‐phase polymerase chain reaction (SP‐PCR) for point‐of‐need (PON) DNA analyses. First, the polycarbonate microdevice was fabricated by thermal bonding to contain microchambers as reservoirs for performing SP‐PCR. Next, the microchambers were subsequently modified with polyethyleneimine and glutaraldehyde for immobilizing amine‐modified forward primers. During SP‐PCR, the immobilized forward primers and freely diffusing fluorescence‐labeled reverse primers cooperated to generate target amplicons, which remained covalently attached to the microchambers for the fluorescence detection. The SP‐PCR microdevice was used for the direct identifications of two widely detected foodborne pathogens, namely Salmonella spp. and Staphylococcus aureus, and an alga causing harmful algal blooms annually in South Korea, Cochlodinium polykrikoides. The SP‐PCR microdevice would be versatilely applied in PON testing as a universal platform for the fast identification of foodborne pathogens and environmentally threatening biogenic targets.     

Insights into the Enhanced Cycle and Rate Performances of the F‐Substituted P2‐Type Oxide Cathodes for Sodium‐Ion Batteries

A series of F‐substituted Na_2/3Ni_1/3Mn_2/3O_2?xF_x (x = 0, 0.03, 0.05, 0.07) cathode materials have been synthesized and characterized by solid‐state ¹⁹F and ²³Na NMR, X‐ray photoelectron spectroscopy, and neutron diffraction. The underlying charge compensation mechanism is systematically unraveled by X‐ray absorption spectroscopy and electron energy loss spectroscopy (EELS) techniques, revealing partial reduction from Mn⁴⁺ to Mn³⁺ upon F‐substitution. It is revealed that not only Ni but also Mn participates in the redox reaction process, which is confirmed for the first time by EELS techniques, contributing to an increase in discharge specific capacity. The detailed structural transformations are also revealed by operando X‐ray diffraction experiments during the intercalation and deintercalation process of Na⁺, demonstrating that the biphasic reaction is obviously suppressed in the low voltage region via F‐substitution. Hence, the optimized sample with 0.05 mol f.u.^?1 fluorine substitution delivers an ultrahigh specific capacity of 61 mAh g^?1 at 10 C after 2000 cycles at 30 °C, an extraordinary cycling stability with a capacity retention of 75.6% after 2000 cycles at 10 C and 55 °C, an outstanding full battery performance with 89.5% capacity retention after 300 cycles at 1 C. This research provides a crucial understanding of the influence of F‐substitution on the crystal structure of the P2‐type materials and opens a new avenue for sodium‐ion batteries.     

Upper bound estimators of the population size based on ordinal models for capture‐recapture experiments

Capture‐recapture studies have attracted a lot of attention over the past few decades, especially in applied disciplines where a direct estimate for the size of a population of interest is not available. Epidemiology, ecology, public health, and biodiversity are just a few examples. The estimation of the number of unseen units has been a challenge for theoretical statisticians, and considerable progress has been made in providing lower bound estimators for the population size. In fact, it is well known that consistent estimators for this cannot be provided in the very general case. Considering a case where capture‐recapture studies are summarized by a frequency of frequencies distribution, we derive a simple upper bound of the population size based on the cumulative distribution function. We introduce two estimators of this bound, without any specific parametric assumption on the distribution of the observed frequency counts. The behavior of the proposed estimators is investigated using several benchmark datasets and a large‐scale simulation experiment based on the scheme discussed by Pledger.     

A matrix‐based method of moments for fitting the multivariate random effects model for meta‐analysis and meta‐regression

Multivariate meta‐analysis is becoming more commonly used. Methods for fitting the multivariate random effects model include maximum likelihood, restricted maximum likelihood, Bayesian estimation and multivariate generalisations of the standard univariate method of moments. Here, we provide a new multivariate method of moments for estimating the between‐study covariance matrix with the properties that (1) it allows for either complete or incomplete outcomes and (2) it allows for covariates through meta‐regression. Further, for complete data, it is invariant to linear transformations. Our method reduces to the usual univariate method of moments, proposed by DerSimonian and Laird, in a single dimension. We illustrate our method and compare it with some of the alternatives using a simulation study and a real example.     

The P2X7 receptor as a route for non‐exocytotic glutamate release: dependence on the carboxyl tail

P2X7 receptors trigger Ca²⁺‐dependent exocytotic glutamate release, but also function as a route for non‐exocytotic glutamate release from neurons or astrocytes. To gain an insight into the mechanisms involving the P2X7 receptor as a direct pathway for glutamate release, we compared the behavior of a full‐length rat P2X7 receptor, a truncated rat P2X7 receptor in which the carboxyl tail had been deleted, a rat P2X7 receptor with the 18‐amino acid cysteine‐rich motif of the carboxyl tail deleted, and a rat P2X2 receptor, all of which are expressed in HEK293 cells. We found that the P2X7 receptor function as a route for glutamate release was antagonized in a non‐competitive way by extracellular Mg²⁺, did not require the recruitment of pore‐forming molecules, and was dependent on the carboxyl tail. Indeed, the truncated P2X7 receptor and the P2X7 receptor with the deleted cysteine‐rich motif both lost their function as a pathway for glutamate release, while still evoking intracellular Ca²⁺ elevation. No glutamate efflux was observed through the P2X2 receptor. Notably, HEK293 cells (lacking the machinery for Ca²⁺‐dependent exocytosis), when transfected with P2X7 receptors, appear to be a suitable model for investigating the P2X7 receptor as a route for non‐exocytotic glutamate efflux.