Single amino acid substitutions in puroindoline-b mutants influence lipid binding properties

External reflectance Fourier transform infrared (ER-FTIR) spectroscopy and surface pressure measurements have been used to characterize the interaction of wild-type puroindoline-b (Pin-b) and two mutant forms featuring single residue substitutions-namely, Gly-46 to Ser-46 (Pin-bH) and Trp-44 to Arg-44 (Pin-bS)-with condensed-phase monolayers of zwitterionic (L-alpha-dipalmitoylphosphatidylcholine, DPPC) and anionic (L-alpha-dipalmitoylphosphatidyl-dl-glycerol, DPPG) phospholipids. The interaction with anionic DPPG monolayers, monitored by surface pressure isotherms, was influenced significantly by mutations in Pin-b (p < 0.05); wild-type Pin-b showed the highest surface pressure change of 10.6 +/- 1.0 mN m(-1), followed by Pin-bH (7.9 +/- 1.6 mN m(-1)) and Pin-bS (6.3 +/- 1.0 mN m(-1)), and the surface pressure isotherm kinetics were also different in each case. Integrated Amide I peak areas from corresponding ER-FTIR spectra confirmed the differences in adsorption kinetics, but also showed that differences in adsorbed amount were less significant, suggesting that mutations influence the degree of penetration into DPPG films. All Pin-b types showed evidence of interaction with DPPC films, detected as changes in surface pressure (5.6 +/- 1.1 mN m(-1)); however, no protein peaks were detected in the ER-FTIR spectra, which indicated that the interaction was via penetration with limited adsorption at the lipid/water interface. The expression of Pin-b mutants is linked to wheat endosperm hardness; therefore, the data presented here suggest that the lipid binding properties may be pivotal within the mechanism for this quality trait. In addition, the data suggest antimicrobial activities of Pin-b mutants would be lower than those of the wild-type Pin-b, because of decreased selectivity toward anionic phospholipids.

Genetic parameters and evaluations from single- and multiple-trait analysis of dairy cow fertility and milk production

Genetic parameters and breeding values for dairy cow fertility were estimated from 62 443 lactation records. Two-trait analysis of fertility and milk yield was investigated as a method to estimate fertility breeding values when culling or selection based on milk yield in early lactation determines presence or absence of fertility observations in later lactations. Fertility traits were calving interval, intervals from calving to first service, calving to conception and first to last service, conception success to first service and number of services per conception. Milk production traits were 305-day milk, fat and protein yield. For fertility traits, range of estimates of heritability (h(2)) was 0.012 to 0.028 and of permanent environmental variance (c(2)) was 0.016 to 0.032. Genetic correlations (r(g)) among fertility traits were generally high ( > 0.70). Genetic correlations of fertility with milk production traits were unfavourable (range -0.11 to 0.46). Single and two-trait analyses of fertility were compared using the same data set. The estimates of h(2) and c(2) were similar for two types of analyses. However, there were differences between estimated breeding values and rankings for the same trait from single versus multi-trait analyses. The range for rank correlation was 0.69-0.83 for all animals in the pedigree and 0.89-0.96 for sires with more than 25 daughters. As single-trait method is biased due to selection on milk yield, a multi-trait evaluation of fertility with milk yield is recommended. (C) 2002 Elsevier Science B.V. All rights reserved.

Decoupling EU farm support: Does the new single payment scheme fit within the green box?

Recent reform of the EU’s Common Agricultural Policy (CAP) has led to a further decoupling of farm support. The EU believes that the new Single Payment Scheme, which replaces the former system of area and headage payments to farmers, tied to production, will qualify for green-box status in the WTO. We examine this contention, particularly in light of the recent WTO panel report on upland cotton.

Implications for food production, land use and rural development of the European Union's Single Farm Payment: Indications from a survey of farmers' intentions in Germany, Portugal and the UK

The 2003 reform of the European Union's (EU) Common Agricultural Policy introduced a decoupled income support for farmers called the Single Farm Payment (SFP). Concerns were raised about possible future land use and production changes and their impact on rural communities. Here, such concerns are considered against the workings of the SFP in three EU Member States. Various quantitative studies that have determined the likely impact of the SFP within the EU and the study countries are reviewed. We present the results of a farm survey conducted in the study countries in which farmers' responses to a decoupling scenario similar to the SFP were sought. We found that little short-term change was proposed in the three, rather different, study countries with only 30% of the farmers stating that they would alter their mix of farm activities. Furthermore, less than 30% of all respondents in each country would idle any land under decoupling. Of those who would adopt a new activity, the most popular choices were forestry, woodland and non-food crops. (c) 2007 Elsevier Ltd. All rights reserved.

The isolation of a single spore isolate of Pasteuria penetrans and its pathogenicity on Meloidogyne javanica

We have obtained a single spore isolate of Pasteuria penetrans, derived by allowing a single spore to attach to a second-stage juvenile (J2) of the root-knot nematode Meloidogyne javanica. By analysing DNA sequences at three different loci we have obtained evidence that the isolate is, indeed, genetically pure. We compared the ability of the single spore isolate and the parent population from which it was selected to attach to and parasitise both the original population of M. javanica on which it was isolated and a single egg mass line derived from it. There was no difference in the attachment of spores of the single spore isolate to juveniles compared to the parental population, although there were higher numbers of both attaching to J2 of the single egg mass line compared to its parental population. Judging from the numbers of egg masses and Pasteuria-infected females, the single spore isolate was less pathogenic to the parental population of M. javanica than was the parental spore population.

An adaptive group sequential design for phase II/III clinical trials that select a single treatment from several

There is increasing interest in combining Phases II and III of clinical development into a single trial in which one of a small number of competing experimental treatments is ultimately selected and where a valid comparison is made between this treatment and the control treatment. Such a trial usually proceeds in stages, with the least promising experimental treatments dropped as soon as possible. In this paper we present a highly flexible design that uses adaptive group sequential methodology to monitor an order statistic. By using this approach, it is possible to design a trial which can have any number of stages, begins with any number of experimental treatments, and permits any number of these to continue at any stage. The test statistic used is based upon efficient scores, so the method can be easily applied to binary, ordinal, failure time, or normally distributed outcomes. The method is illustrated with an example, and simulations are conducted to investigate its type I error rate and power under a range of scenarios.

Overview of mathematical approaches used to model bacterial chemotaxis I: the single cell

Mathematical modeling of bacterial chemotaxis systems has been influential and insightful in helping to understand experimental observations. We provide here a comprehensive overview of the range of mathematical approaches used for modeling, within a single bacterium, chemotactic processes caused by changes to external gradients in its environment. Specific areas of the bacterial system which have been studied and modeled are discussed in detail, including the modeling of adaptation in response to attractant gradients, the intracellular phosphorylation cascade, membrane receptor clustering, and spatial modeling of intracellular protein signal transduction. The importance of producing robust models that address adaptation, gain, and sensitivity are also discussed. This review highlights that while mathematical modeling has aided in understanding bacterial chemotaxis on the individual cell scale and guiding experimental design, no single model succeeds in robustly describing all of the basic elements of the cell. We conclude by discussing the importance of this and the future of modeling in this area.

Bayesian design and conduct of phase II single-arm clinical trials with binary outcomes: a tutorial

The aim of phase II single-arm clinical trials of a new drug is to determine whether it has sufficient promising activity to warrant its further development. For the last several years Bayesian statistical methods have been proposed and used. Bayesian approaches are ideal for earlier phase trials as they take into account information that accrues during a trial. Predictive probabilities are then updated and so become more accurate as the trial progresses. Suitable priors can act as pseudo samples, which make small sample clinical trials more informative. Thus patients have better chances to receive better treatments. The goal of this paper is to provide a tutorial for statisticians who use Bayesian methods for the first time or investigators who have some statistical background. In addition, real data from three clinical trials are presented as examples to illustrate how to conduct a Bayesian approach for phase II single-arm clinical trials with binary outcomes.

Novel single chain antibodies to the prion protein identified by phage display

A well defined structure is available for the carboxyl half of the cellular prion protein (PrPc), while the structure of the amino terminal half of the molecule remains ill defined. The unstructured nature of the polypeptide has meant that relatively few of the many antibodies generated against PrPc recognise this region. To circumvent this problem, we have used a previously characterised and well expressed fragment derived from the amino terminus of PrPc as bait for panning a single chain antibody phage (scFv-P) library. Using this approach, we identified and characterised I predominant and 3 additional scFv-Ps that contained different V-H and V-L sequences and that bound specifically to the PrPc target. Epitope mapping revealed that all scFv-Ps recognised linear epitopes between PrPc residues 76 and 156. When compared with existing monoclonal antibodies (MAb), the binding of the scFvs was significantly different in that high level binding was evident on truncated forms of PrPc that reacted poorly or not at all with several pre-existing MAbs. These data suggest that the isolated scFv-Ps bind to novel epitopes within the aminocentral region of PrPc. In addition, the binding of MAbs to known linear epitopes within PrPc depends strongly on the endpoints of the target PrPc fragment used. (c) 2006 Elsevier Inc. All rights reserved.

The tagged microarray marker (TAM) method for high-throughput detection of single nucleotide and indel polymorphisms

The tagged microarray marker (TAM) method allows high-throughput differentiation between predicted alternative PCR products. Typically, the method is used as a molecular marker approach to determining the allelic states of single nucleotide polymorphisms (SNPs) or insertion-deletion (indel) alleles at genomic loci in multiple individuals. Biotin-labeled PCR products are spotted, unpurified, onto a streptavidin-coated glass slide and the alternative products are differentiated by hybridization to fluorescent detector oligonucleotides that recognize corresponding allele-specific tags on the PCR primers. The main attractions of this method are its high throughput (thousands of PCRs are analyzed per slide), flexibility of scoring (any combination, from a single marker in thousands of samples to thousands of markers in a single sample, can be analyzed) and flexibility of scale (any experimental scale, from a small lab setting up to a large project). This protocol describes an experiment involving 3,072 PCRs scored on a slide. The whole process from the start of PCR setup to receiving the data spreadsheet takes 2 d.

The expected performance of single nucleotide polymorphism loci in paternity testing

We discuss the utility of single nucleotide polymorphism loci for full trio and mother-unavailable paternity testing cases, in the presence of population substructure and relatedness of putative and actual fathers. We focus primarily on the expected number of loci required to gain specified probabilities of mismatches, and report the expected proportion of paternity indices greater than three threshold values for these loci. (c) 2004 Elsevier Ireland Ltd. All rights reserved.

Specificity grafting of human antibody frameworks selected from a phage display library: generation of a highly stable humanized anti-CD22 single-chain Fv fragment

A prerequisite for the enrichment of antibodies screened from phage display libraries is their stable expression on a phage during multiple selection rounds. Thus, if stringent panning procedures are employed, selection is simultaneously driven by antigen affinity, stability and solubility. To take advantage of robust pre-selected scaffolds of such molecules, we grafted single-chain Fv (scFv) antibodies, previously isolated from a human phage display library after multiple rounds of in vitro panning on tumor cells, with the specificity of the clinically established murine monoclonal anti-CD22 antibody RFB4. We show that a panel of grafted scFvs retained the specificity of the murine monoclonal antibody, bound to the target antigen with high affinity (6.4-9.6 nM), and exhibited exceptional biophysical stability with retention of 89-93% of the initial binding activity after 6 days of incubation in human serum at 37degreesC. Selection of stable human scaffolds with high sequence identity to both the human germline and the rodent frameworks required only a small number of murine residues to be retained within the human frameworks in order to maintain the structural integrity of the antigen binding site. We expect this approach may be applicable for the rapid generation of highly stable humanized antibodies with low immunogenic potential.