Development of GFP-based biosensors possessing the binding properties of antibodies

Proteins that can bind specifically to targets that also have an intrinsic property allowing for easy detection could facilitate a multitude of applications. While the widely used green fluorescent protein (GFP) allows for easy detection, attempts to insert multiple binding loops into GFP to impart affinity for a specific target have been met with limited success because of the structural sensitivity of the GFP chromophore. In this study, directed evolution using a surrogate loop approach and yeast surface display yielded a family of GFP scaffolds capable of accommodating 2 proximal, randomized binding loops. The library of potential GFP-based binders or ″GFAbs″ was subsequently mined for GFAbs capable of binding to protein targets. Identified GFAbs bound with nanomolar affinity and required binding contributions from both loops indicating the advantage of a dual loop GFAb platform. Finally, GFAbs were solubly produced and used as fluorescence detection reagents to demonstrate their utility.     

Heredity mode of genetic polymorphism in aldehyde oxidase activity in Donryu strain rats

Donryu strain rats show genetic polymorphisms in the aldehyde oxidase gene, resulting in the phenotypic expression of ultrarapid metabolizers with homozygous nucleotide sequences (337G, 2604C), extensive metabolizers with heterozygous nucleotide sequences (377G/A, 2604C/T), and poor metabolizers with homozygous nucleotide sequences (377A, 2604T). In the mating experiments the ratio of the number of ultrarapid metabolizers, extensive metabolizers, and poor metabolizers rats in the F1 generation from the heterozygous F0 extensive metabolizers male and female rats was roughly 0.6 : 1.5 : 1, and the ratio converged to approximately 1 : 2 : 1 in the F2 generation from the heterozygous F1 extensive metabolizers male and female rats. On the contrary, all the F2 generation from homozygous F1 ultrarapid metabolizers male and female rats or from homozygous F1 poor metabolizers male and female rats had the ultrarapid metabolizers or the poor metabolizers genotypes and phenotypes. The genotypes completely agreed with the phenotypes in all individuals of F0, F1, and F2 generations. The results indicate that the genetic polymorphism of aldehyde oxidase in Donryu strain rats obeys Mendelian heredity. The reason for a low ratio of the ultrarapid metabolizers rats in the commercially available Donryu strain rats?—?not more than several per cent?—?compared with the ratio expected from the Mendelian rule is unknown.     

Estimating efficacy in clinical trials with clustered binary responses

When non-compliance occurs in a clinical trial, it may be of interest to supplement the intent-to-treat analysis with an analysis of the efficacy (or biological effect) of therapy. Sommer and Zeger (1991) developed a method for estimating efficacy applicable to the case of a binary response variable and all-or-none compliance that assumes independent subject responses. We extend this approach to accommodate within-cluster correlations as may be expected in a cluster-randomized design. The method is illustrated using data from a controlled village-randomized clinical trial conducted in Indonesia to investigate the effect of vitamin A supplementation on mortality in children. We find that within-cluster correlations for these data are very small and that taking into account the clustering does not substantially affect inferences in this case. Additional calculations show that small within-cluster correlations (though larger than those found in the vitamin A data) may have a large impact on efficacy inferences. We also present the results of a simulation study that demonstrates the validness of the proposed approach for finite sample sizes.     

Up-front randomization and common standard arm: a proposal for comparing AML treatment strategies between different studies

Most multicenter randomized AML studies use randomizations of patients early or later in complete remission which necessarily occur with exclusions and positive selection of patients included. Since the exclusion criterias are regimen related and do not follow common standards, incomparabilities between treatment results across different studies are produced by these late randomizations. In order to overcome this problem, we here propose a cooperation of studies on the basis of a general up-front randomization with attribution of 10% patients from each study to a common standard arm. A validation of complete treatment strategies according to intent-to-treat against the standard arm and thus also across the studies is provided by this inter-group model which may contribute to accelerate the therapeutic progress in AML.     

Pseudo cluster randomization: a treatment allocation method to minimize contamination and selection bias

In some clinical trials, treatment allocation on a patient level is not feasible, and whole groups or clusters of patients are allocated to the same treatment. If, for example, a clinical trial is investigating the efficacy of various patient coaching methods and randomization is done on a patient level, then patients who are receiving different methods may come into contact with each other and influence each other. This would create contamination of the treatment effects. Such bias might be prevented by randomization on the coaches level. The patients of a coach constitute a cluster and all the subjects in that cluster receive the same treatment. Disadvantages of this approach may be reduced statistical efficiency and recruitment bias, as the treatment that a subject will receive is known in advance. Pseudo cluster randomization avoids this, because in pseudo cluster randomization, not everybody in a certain cluster receives the same treatment, just the majority. There are two groups of clusters: in one group the majority of subjects receive treatment A, while a limited number receive treatment B. In the other group of clusters the proportions are reversed. The statistical properties of this method are described. When contamination is present, the method appears to be more efficient than randomization on a patient level or on a cluster level.     

孟德尔遗传糖尿病研究进展

糖尿病是由多种遗传和环境因素相互作用而引起的一组慢性代谢异常综合征.一些特殊类型的糖尿病,如:青年发病的成年型糖尿病、矮妖精貌综合征、脂肪萎缩性糖尿病和Rabson-Mendenhall综合征为单基因致病并符合孟德尔遗传.本文对孟德尔遗传糖尿病的研究进展加以综述.     

Comparison of randomization techniques for clinical trials with data from the HOMERUS-trial

Background. Several methods of randomization are available to create comparable intervention groups in a study. In the HOMERUS-trial, we compared the minimization procedure with a stratified and a non-stratified method of randomization in order to test which one is most appropriate for use in clinical hypertension trials. A second objective of this article was to describe the baseline characteristics of the HOMERUS-trial. Methods. The HOMERUS population consisted of 459 mild-to-moderate hypertensive subjects (54% males) with a mean age of 55 years. These patients were prospectively randomized with the minimization method to either the office pressure (OP) group, where antihypertensive treatment was based on office blood pressure (BP) values, or to the self-pressure (SP) group, where treatment was based on self-measured BP values. Minimization was compared with two other randomization methods, which were performed post-hoc: (i) non-stratified randomization with four permuted blocks, and (ii) stratified randomization with four permuted blocks and 16 strata. In addition, several factors that could influence outcome were investigated for their effect on BP by 24-h ambulatory blood pressure monitoring (ABPM). Results. Minimization and stratified randomization did not lead to significant differences in 24-h ABPM values between the two treatment groups. Non-stratified randomization resulted in a significant difference in 24-h diastolic ABPM between the groups. Factors that caused significant differences in 24-h ABPM values were: region, centre of patient recruitment, age, gender, microalbuminuria, left ventricular hypertrophy and obesity. Conclusion. Minimization and stratified randomization are appropriate methods for use in clinical trials. Many outcome factors should be taken into account for their potential influence on BP levels. Recommendation. Due to the large number of potential outcome factors that can influence BP levels, minimization should be the preferred method for use in clinical hypertension trials, as it has the potential to randomize more outcome factors than stratified randomization.     

Gold is not always good enough: the shortcomings of randomization when evaluating interventions in small heterogeneous samples

The three criteria for valid inference in therapeutic intervention evaluation are achieving control, avoiding systematic error, and minimizing random error. The randomized, double-blind, controlled trial has appropriately been accepted as the methodological gold standard because it is the only method with the potential to avoid systematic error resulting from unbalanced distributions of recognized and unrecognized determinants of outcome. This potential is not always realized, however, particularly with small, heterogeneous patient samples—which undermines the rationale for randomization in these circumstances. Minimization is one possible strategy to attain validity in such circumstances, but the acceptability of nonrandomized strategies is currently hampered by deference to the concept of randomization. For each intervention evaluation, research design should be considered afresh, focusing on the criteria determining validity rather than particular methodological elements.     

Misrepresenting random sampling? A systematic review of research papers in the Journal of Advanced Nursing

AIM: This paper discusses the theoretical limitations of the use of random sampling and probability theory in the production of a significance level (or P-value) in nursing research. Potential alternatives, in the form of randomization tests, are proposed. BACKGROUND: Research papers in nursing, medicine and psychology frequently misrepresent their statistical findings, as the P-values reported assume random sampling. In this systematic review of studies published between January 1995 and June 2002 in the Journal of Advanced Nursing, 89 (68%) studies broke this assumption because they used convenience samples or entire populations. As a result, some of the findings may be questionable. DISCUSSION: The key ideas of random sampling and probability theory for statistical testing (for generating a P-value) are outlined. The result of a systematic review of research papers published in the Journal of Advanced Nursing is then presented, showing how frequently random sampling appears to have been misrepresented. Useful alternative techniques that might overcome these limitations are then discussed. REVIEW LIMITATIONS: This review is limited in scope because it is applied to one journal, and so the findings cannot be generalized to other nursing journals or to nursing research in general. However, it is possible that other nursing journals are also publishing research articles based on the misrepresentation of random sampling. The review is also limited because in several of the articles the sampling method was not completely clearly stated, and in this circumstance a judgment has been made as to the sampling method employed, based on the indications given by author(s). CONCLUSION: Quantitative researchers in nursing should be very careful that the statistical techniques they use are appropriate for the design and sampling methods of their studies. If the techniques they employ are not appropriate, they run the risk of misinterpreting findings by using inappropriate, unrepresentative and biased samples.     

Phenotypic and genetic heterogeneity of familial hyperkalaemic hypertension (Gordon syndrome)

1. Familial hyperkalaemic hypertension (FHH), also called pseudohypoaldosteronism type II (PHA2) or Gordon syndrome, is a rare Mendelian-form of low-renin hypertension. The first cases of FHH were reported approximately 30 years ago and they described the peculiar biochemical abnormalities (i.e. hyperkalaemia and hyperchloraemic acidosis despite a normal glomerular filtration rate). 2. Since then, more than 90 single cases and families have been reported in the literature. These various reports show marked differences in phenotype. 3. Our group has now collected 14 unrelated pedigrees originating from different parts of France and Europe. We confirm the large variations in the age of discovery and in the severity of the biochemical abnormalities from one individual to another and from one family to another one. 4. Blood pressure levels have no significant relationship with hyperkalaemia or hyperchloraemia, but there is a positive relationship with age, as in the normal population. 5. Analyses of clinical features and Mendelian segregation in our families demonstrate autosomal-dominant inheritance, as expected from the literature. 6. Efforts have been made in the past years to unravel the gene responsible for the disease. Until now, a primary responsibility of the gene encoding the thiazide-sensitive Na-Cl cotransporter (SLC12A3) has been excluded in PHA2 families. Three loci have been identified on chromosomes 1 (PHA2A), 17 (PHA2B) and 12 (PHA2C). 7. More recently, analysis of three additional pedigrees, including 10 affected subjects, with over 25 members allowed us to demonstrate further genetic heterogeneity and the existence of at least a fourth locus. 8. The genetic heterogeneity of this syndrome, and thus the variety of molecular defects, suggests the role of either several new components of the same pathway, multiple aldosterone- regulated effectors or direct or indirect partners of the Na-Cl cotransporter.