The role of phadiatop in the screening of respiratory allergy

Phadiatop is a new in vitro screening test for respiratory allergy. This test, based on the RAST procedure, detects in serum, the presence of specific IgE to a mixture of common inhalent allergens. Among 70 patients (26 children and 44 adults) consulting for respiratory syndrome, Phadiatop was positive in 31 cases. There were a good correlation between this new test and skin tests (59% for adults and 92% for children), total IgE (70% for adults and 65% for children) and RAST (93% for adults and 96% for children). Phadiatop, with a specificity of 100%, a sensitivity of 82% (76% for adults and 92% for children) and an efficiency of 90% (86% for adults and 96% for children), is a more accurate test than total IgE and could be an excellent in vitro screening test for respiratory allergy.     

Screening for functional expression and overexpression of a family of diiron-containing interfacial membrane proteins using the univector recombination system

The large number of uncharacterized genes emerging from genome sequencing projects has resulted in a need for quick and reliable screening methods for protein expression parameters. We have utilized the univector plasmid recombination system (as previously reported) to develop a series of vectors for rapid screening for expression in Escherichia coli. A high level of recombinant protein expression is a requirement for purification of protein for structural determination and other purposes. In other applications, successful complementation of a missing enzyme activity in E. coli, as well as directed evolution studies and metabolic engineering, often require a much lower level of protein expression. In this report we describe the construction of a number of new pHOST vectors that can be screened for both low- and high-level expression. We isolated a mutant vector for MBP fusions that exhibited a more optimal level of expression for complementation of aerobic respiration in hemA(-) E. coli, our functional assay for the alternative oxidase. We then demonstrated the use of our system to rapidly screen for both optimal functional expression and optimal overexpression of the alternative oxidase as well as two other members of a family of membrane-bound diiron carboxylate proteins, the plastid terminal oxidase and 5-demethoxyquinone hydroxylase.     

Hexanoylation of a VPAC2 receptor-preferring ligand markedly increased its selectivity and potency

We synthesized a VIP analog that combines mutations that decrease the affinity for the VPAC1 receptor but maintain a high affinity for the VPAC2 receptor with an amino-terminal hexanoylation that increases the affinity for the VPAC2 receptor with a limited decrease in the affinity of the VPAC1 receptor. The resulting Hexanoyl[A19,K(27,28)]VIP had the expected properties of a high affinity for the VPAC2 receptor and a low affinity for the VPAC1 receptor and also a low affinity for the PAC1 and secretin receptors. With a 1000-fold preference for the VPAC2 receptor and a IC50 value of binding of 1 nM, this compound is the most potent and the most selective agonist presently described.     

Integrated allele-specific polymerase chain reaction-capillary electrophoresis microdevice for single nucleotide polymorphism genotyping

An integrated allele-specific (AS) polymerase chain reaction (PCR) and capillary electrophoresis (CE) microdevice has been developed for multiplex single nucleotide polymorphism (SNP) genotyping on a portable instrumentation, which was applied for on-site identification of HANWOO (Korean indigenous beef cattle). Twelve sets of primers were designed for targeting beef cattle's eleven SNP loci for HANWOO verification and one primer set for a positive PCR control, and the success rate for identification of HANWOO was demonstrated statistically. The AS PCR and CE separation for multiplex SNP typing was carried out on a glass-based microchip consisting of four layers: a microchannel plate for microfluidic control, a Pt-electrode plate for a resistance temperature detector (RTD), a poly(dimethylsiloxane) (PDMS) membrane and a manifold glass for microvalve function. The operation of the sample loading, AS PCR, microvalve, and CE on a chip was automated with a portable genetic analyzer, and the laser-induced fluorescence detection was performed on a miniaturized fluorescence detector. The blind samples were correctly identified as a HANWOO by showing one or two amplicon peaks in the electropherogram, while the imported beef cattle revealed more than five peaks. Our genetic analysis platform provides rapid, accurate, and on-site multiplex SNP typing.     

How large a managed area is needed to protect a threatened animal species? Combining simple dispersal and population models

The size and shape of the managed area for a threatened species to have a stable or growing population is a central issue for conservation management, for example for kiwi, Apteryx sp. Combining geometric probability results for retention of juveniles to breed in the protected area with a standard matrix population model allows the creation of an explicit relationship between the minimum area needed and how far juveniles of the species disperse to establish breeding territories. For a given set of demographic parameters for the population, and a rectangular protected area, there is a quadratic relationship between area and dispersal distance. Extensions for a circular protected area, and for a probability distribution on dispersal distance, are considered. The results are applied to kiwi, using established population parameters, giving results that match closely those from a previously published simulation. This approach can provide simple tools, readily implemented in a spreadsheet, for assessment of the size and shape of protected areas needed in conservation management of animals that disperse before breeding.     

A population model based on a life table that includes marriage and parity.

This paper presents a method for constructing a one-sex life table that incorporates age, marriage and parity. The life table is the basis for a generalized population model, with immediate extension to a stable population differentiated by age, marriage and parity status. The method is quite general and could be extended, without major modification, to more complex life tables.Computation of intrinsic rates of increase for a number of populations adjusted for age, for age and parity, for age and marriage, and for age, marriage and parity shows that adjustment for marriage accounts for most of the difference between the age-adjusted rate and the age-, marriage-, and parity-adjusted rate. Adjustment for parity without adjustment for marriage may be misleading.     

Mechanism of Agrobacterium beta-glucosidase: kinetic studies.

The beta-glucosidase from Agrobacterium faecalis (previously Alcaligenes faecalis) has been subjected to a detailed kinetic investigation with a range of substrates to probe its specificity and mechanism. It has a relatively broad specificity for the substrate sugar moiety and exhibits a classical pH dependence for its kinetic parameters with three different substrates and an identical pH dependence for its inactivation by a mechanism-based inactivator, cyclophellitol. Measurement of kcat and Km values for a series of aryl glucoside substrates has allowed construction of a Bronsted plot, the concave-downward shape of which is consistent with the anticipated two-step mechanism involving a glucosyl-enzyme intermediate which is formed and hydrolyzed via oxocarbonium ion-like transition states. The slope of the leaving group-dependent portion of the Bronsted plot (beta 1g = -0.7) indicates a large degree of bond cleavage at the transition state. Secondary deuterium kinetic isotope effects measured for five different aryl glucosides are also consistent with this mechanism and further suggest that the transition state for formation of the glucosyl-enzyme intermediate, probed with the slower substrates for which kH/kD = 1.06, is more SN2-like than that for its hydrolysis (for which kH/kD = 1.11). Reasons for this difference are proposed, and values of Ki for several ground-state and transition-state analogue inhibitors are presented which support the concept of sp2-hybridized transition states.     

Tools for comparative protein structure modeling and analysis

The following resources for comparative protein structure modeling and analysis are described (http://salilab.org): MODELLER, a program for comparative modeling by satisfaction of spatial restraints; MODWEB, a web server for automated comparative modeling that relies on PSI-BLAST, IMPALA and MODELLER; MODLOOP, a web server for automated loop modeling that relies on MODELLER; MOULDER, a CPU intensive protocol of MODWEB for building comparative models based on distant known structures; MODBASE, a comprehensive database of annotated comparative models for all sequences detectably related to a known structure; MODVIEW, a Netscape plugin for Linux that integrates viewing of multiple sequences and structures; and SNPWEB, a web server for structure-based prediction of the functional impact of a single amino acid substitution.     

Evaluation of Finnish Diabetes Risk Score in Screening Undiagnosed Diabetes and Prediabetes among U.S. Adults by Gender and Race: NHANES 1999-2010

Objective

To evaluate the performance of Finnish Diabetes Risk Score (FINDRISC) in detecting undiagnosed diabetes and prediabetes among U.S. adults by gender and race.

Methods

This cross-sectional analysis included participants (aged ≥20 years) from the National Health and Nutrition Examination Survey (NHANES) 1999–2010. Sensitivity, specificity, area under the receiver operating characteristic (ROC) curve and the optimal cutoff points for identifying undiagnosed diabetes and prediabetes were calculated for FINDRISC by gender and race/ethnicity.

Results

Among the 20,633 adults (≥20 years), 49.8% were women and 53.0% were non-Hispanic White. The prevalence of undiagnosed diabetes and prediabetes was 4.1% and 35.6%, respectively. FINDRISC was positively associated with the prevalence of diabetes (OR = 1.48 for 1 unit increase, p<0.001) and prediabetes (OR = 1.15 for 1 unit increase, p<0.001). The area under ROC for detecting undiagnosed diabetes was 0.75 for total population, 0.74 for men and 0.78 for women (p = 0.04); 0.76 for White, 0.76 for Black and 0.72 for Hispanics (p = 0.03 for White vs. Hispanics). The area under ROC for detecting prediabetes was 0.67 for total population, 0.66 for men and 0.70 for women (p<0.001); 0.68 for White, 0.67 for Black and 0.65 for Hispanics (p<0.001 for White vs. Hispanics). The optimal cutoff point was 10 (sensitivity = 0.75) for men and 12 (sensitivity = 0.72) for women for detecting undiagnosed diabetes; 9 (sensitivity = 0.61) for men and 10 (sensitivity = 0.69) for women for detecting prediabetes.

Conclusions

FINDRISC is a simple and non-invasive screening tool to identify individuals at high risk for diabetes in the U.S. adults.     

A conceptual framework for mapping quantitative trait Loci regulating ontogenetic allometry

Although ontogenetic changes in body shape and its associated allometry has been studied for over a century, essentially nothing is known about their underlying genetic and developmental mechanisms. One of the reasons for this ignorance is the unavailability of a conceptual framework to formulate the experimental design for data collection and statistical models for data analyses. We developed a framework model for unraveling the genetic machinery for ontogenetic changes of allometry. The model incorporates the mathematical aspects of ontogenetic growth and allometry into a maximum likelihood framework for quantitative trait locus (QTL) mapping. As a quantitative platform, the model allows for the testing of a number of biologically meaningful hypotheses to explore the pleiotropic basis of the QTL that regulate ontogeny and allometry. Simulation studies and real data analysis of a live example in soybean have been performed to investigate the statistical behavior of the model and validate its practical utilization. The statistical model proposed will help to study the genetic architecture of complex phenotypes and, therefore, gain better insights into the mechanistic regulation for developmental patterns and processes in organisms.     

The Efficacy of an Immunoisolating Membrane System for Islet Xenotransplantation in Minipigs

Developing a device that protects xenogeneic islets to allow treatment and potentially cure of diabetes in large mammals has been a major challenge in the past decade. Using xenogeneic islets for transplantation is required in light of donor shortage and the large number of diabetic patients that qualify for islet transplantation. Until now, however, host immunoreactivity against the xenogeneic graft has been a major drawback for the use of porcine islets. Our study demonstrates the applicability of a novel immunoprotective membrane that allows successful xenotransplantation of rat islets in diabetic minipigs without immunosuppressive therapy. Rat pancreatic islets were encapsulated in highly purified alginate and integrated into a plastic macrochamber covered by a poly-membrane for subcutaneous transplantation. Diabetic Sinclair pigs were transplanted and followed for up to 90 days. We demonstrated a persistent graft function and restoration of normoglycemia without the need for immunosuppressive therapy. This concept could potentially offer an attractive strategy for a more widespread islet replacement therapy that would restore endogenous insulin secretion in diabetic patients without the need for immunosuppressive drugs and may even open up an avenue for safe utilization of xenogeneic islet donors.     

A test of transmission/disequilibrium for quantitative traits in pedigree data, by multiple regression.

The transmission/disequilibrium (TD) test (TDT), proposed, by Spielman et al., for binary traits is a powerful method for detection of linkage between a marker locus and a disease locus, in the presence of allelic association. As a test for linkage disequilibrium, the TDT makes the assumption that any allelic association present is due to linkage. Allison proposed a series of TD-type tests for quantitative traits and calculated their power, assuming that the marker locus is the disease locus. All these tests assume that the observations are independent, and therefore they are applicable, as a test for linkage, only for nuclear-family data. In this report, we propose a regression-based TD-type test for linkage between a marker locus and a quantitative trait locus, using information on the parent-to-offspring transmission status of the associated allele at the marker locus. This method does not require independence of observations, thus allowing for analysis of pedigree data as well, and allows adjustment for covariates. We investigate the statistical power and validity of the test by simulating markers at various recombination fractions from the disease locus.     

A profiling platform for the identification of selective metalloprotease inhibitors

Although proteases represent an estimated 5% to 10% of potential drug targets, inhibitors for metalloproteases (MPs) account for only a small proportion of all approved drugs, failures of which have typically been associated with lack of selectivity. In this study, the authors describe a novel and universal binding assay based on an actinonin derivative and show its binding activities for several MPs and its lack of activity toward all the non-MPs tested. This newly developed assay would allow for the rapid screening for inhibitors of a given MP and for the selectivity profiling of the resulting hits. The assay has successfully enabled for the first time simultaneous profiling of 8 well-known inhibitors against a panel of selected MPs. Previously published activities for these inhibitors were confirmed, and the authors have also discovered new molecular targets for some of them. The authors conclude that their profiling platform provides a generic assay solution for the identification of novel metalloprotease inhibitors as well as their selectivity profiling using a simple and homogeneous assay.     

Identification by a multiplex PCR-based assay of Salmonella Typhimurium and Salmonella Enteritidis strains from environmental swabs of poultry houses

A multiplex-PCR-based assay (m-PCR) was developed for the detection of Salmonella and for the identification of the two serotypes Enteritidis and Typhimurium. Three sets of primers selected from different genomic sequences amplified a 429 bp fragment specific for the genus Salmonella within a randomly cloned sequence, a 559 bp target specific for Salmonella Typhimurium within the fliC gene and a 312 bp fragment specific for Salmonella Enteritidis within the sefA gene. The m-PCR-based assay was used for detecting Salmonella from 1078 environmental swabs of poultry houses. Prior to PCR, these swabs were pre-enriched in phosphate-buffered peptone water for 18-20 h and then sub-cultured on a Modified Semi-solid Rappaport Vassiliadis medium (MSRV) for 18-20 h. The m-PCR combined with MSRV had a better sensitivity (95%) than the bacteriological method (92.5%). The MSRV-m-PCR assay and the bacteriological method had an agreement rate of 95.6%.     

Estimation and confidence regions for multi-dimensional effective dose

The problem of finding confidence regions for multiple predictor variables corresponding to given expected values of a response variable has not been adequately resolved. Motivated by an example from a study on hyperbaric exposure using a logistic regression model, we develop a conceptual framework for the estimation of the multi-dimensional effective dose for binary outcomes. The k -dimensional effective dose can be determined by conditioning on k - 1 components and solving for the last component as a conditional univariate effective dose. We consider various approaches for calculating confidence regions for the multi-dimensional effective dose and compare them via a simulation study for a range of possible designs. We analyze data related to decompression sickness to illustrate our procedure. Our results provide a practical approach to finding confidence regions for predictor variables for a given response value.     

Parallel dose-response curves in combination experiments

A possible experimental design for combination experiments is to compare the doseresponse curve of a single agent with the corresponding curve of the same agent using either a fixed amount of a second one or a fixed dose ratio. No interaction is then often defined by a parallel shift of these curves. We have performed a systematic study for various types of doseresponse relations both for the dose-additivity (Loewe additivity) and for the independence (Bliss independence) criteria for defining zero interaction. Parallelism between doseresponse curves of a single agent and those of the same agent in the presence of a fixed amount of another one is found for the Loewe-additivity criterion for linear doseresponse relations. For nonlinear relations, one has to differentiate between effect parallelism (parallel shift on the effect scale) and dose parallelism (parallel shift on the dose scale). In the case of Loewe additivity, zero-interaction dose parallelism is found for power, Weibull, median-effect and logistic doseresponse relations, given that special parameter relationships are fulfilled. The mechanistic model of competitive interaction exhibits dose parallelism but not effect parallelism for Loewe additivity. Bliss independence and Loewe additivity lead to identical results for exponential doseresponse curves. This is the only case for which dose parallelism was found for Bliss independence. Parallelism between single-agent doseresponse relations and Loewe additivity mixture relations is found for examples with a fixed doseratio design. However, this is again not a general property of the design adopted but holds only if special conditions are fulfilled. The comparison of combination doseresponse curves with single-agent relations has to be performed taking into account both potency and shape parameters. The results of this analysis lead to the conclusion that parallelism between zero interaction combination and single-agent doseresponse relations is found only for special cases and cannot be used as a general criterion for defining zero-interaction in combined-action assessment even if the correct potency shift is taken into account.     

Experimental tests for an evolutionary trade-off between growth rate and yield in E. coli

Theoretical studies have predicted a trade-off between growth rate and yield in heterotrophic organisms. Here we test for the existence of this trade-off by analyzing the growth characteristics of 12 E. coli B populations that evolved for 20,000 generations under a constant selection regime. We performed three different tests. First, we analyzed changes in growth rate and yield over evolutionary time for each population. Second, we tested for a negative correlation between rate and yield across the 12 populations. Finally, we isolated clones from four selected populations and tested for a negative correlation between rate and yield within these populations. We did not find evidence for a trade-off based on the first two tests. However, we did observe a trade-off based on the within-population correlation of yield and rate. Our results indicate that, at least for the populations studied here, an analysis of the within-population diversity might be the most sensitive test for the existence of a trade-off. The observation of a trade-off within, but not between, populations suggests that the populations evolved different genetic solutions for growth in the selective environment, which in turn led to different physiological constraints.     

High-affinity binding of Ca2+ to bovine alpha-lactalbumin in the absence and presence of EGTA.

Literature values for the Kd for Ca2+ in bovine alpha-lactalbumin range over 3 orders of magnitude. There is a difference between two results obtained with EGTA as a metal-ion buffer, partly because different values for the Kd of Ca2+-EGTA were used in the calculations, and a much wider difference between results obtained in the presence and absence of EGTA, which has been attributed to an interaction between EGTA and the protein. Titrations in a flow-dialysis cell showed that Mn2+ competed with Ca2+ for the high-affinity site on the protein, and the results, combined with a Kd for Mn2+ of 2.1 +/- 0.1 microM, which was determined fluorimetrically, gave a Kd for Ca2+ of 1.3 +/- 0.1 nM. When alpha-lactalbumin containing 45Ca2+ was titrated with EGTA in a flow-dialysis cell, and widely accepted metal-chelation data for EGTA were used in the calculations, a Kd for Ca2+ of 1.10 +/- 0.03 nM was obtained. The results from the two methods are so similar as to indicate that the affinity for Ca2+ was unaffected by the presence of EGTA.     

Bipartite expressions for diffusional mass transport in biomembranes

Analytical expressions for solute diffusion through a membrane barrier for different initial and boundary conditions are available in the literature. The three commonest initial and boundary conditions are for a membrane without solute respectively immersed in a solution of constant concentration, immersed in such a solution for one side but with the other side isolated, and immersed in such a solution for one side and with the other side kept at zero concentration. The physical quantities for the first two initial and boundary conditions are concentration and average concentration (the total solute entering the membrane) with amperometric current (flux) and solute that permeates through the membrane (charge passed) for the third initial and boundary condition. Expressions for these methods in the literature are inconvenient for practical applications because of the infinite mathematical series required. An investigation of convergence of these expressions was therefore carried out. Simple but accurate bipartite expressions for these methods were constructed and provided theoretical support for studies on mass transport characterization of biomembranes. As a specific application, these expressions enabled a direct fit of the simulated observables to experimental values to obtain diffusion coefficients. For these initial and boundary conditions and corresponding physical quantities, simple one point methods for diffusion coefficient estimation are also suggested. These latter diffusion coefficients can be initial values for numerical fit methods.