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.     

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.     

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.     

Inter-plot competition in yield trials of potatoes (Solanum tuberosum L.) with single-drill plots

An experiment was carried out to assess the significance of inter-plot competition in a yield trial of potato cultivars. Seventeen cultivars were deliberately chosen and assessed for yield in single-drill and four-drill plots. Inter-plot competition for fresh-weight yield was a significant factor in the single-drill plots. It was modelled using a common competition coefficient with a covariate based on neighbour fresh-weight yields. In contrast, there was no statistically significant inter-plot competition for specific gravity. After adjustment for inter-plot competition, varietal ranking in estimated monoculture yield differed little from that based on unadjusted means. However, there was a reduction in the range of yield estimates, and a closer agreement with the observed pure-stand yields from the inner two drills of the four-drill plots. The adjustment for monoculture performance was most pronounced for the higher and lower yielding varieties, as expected from the assumption that the performance of high yielding varieties was enhanced in a competitive environment at the expense of low yielding ones. A general and flexible method of estimating competition coefficients in variety trials, together with a suitable algorithm, was developed and is explained in an appendix. It was used to check for inter-plot competition in a number of potato trials with single-drill plots and a large number of entries. Competition was found in some trials but not in others. Thus, where potato tubers are grown in single-drill plots for assessment of fresh-weight yield, adjustment should be made for inter-plot competition when evidence of inter-drill competition is found.     

Site-directed mutants of charged residues in the active site of tyrosine hydroxylase

The active site of tyrosine hydroxylase consists of a hydrophobic cleft with an iron atom near the bottom. Within the cleft are several charged residues which are conserved across the family of pterin-dependent hydroxylases. We have studied four of these residues, glutamates 326 and 332, aspartate 328, and arginine 316 in tyrosine hydroxylase, by site-directed substitution with alternate amino acid residues. Replacement of arginine 316 with lysine results in a protein with a Ktyr value that is at least 400-fold greater and a V/Ktyr value that is 4000-fold lower than those found in the wild-type enzyme; substitution with alanine, serine, or glutamine yields insoluble enzyme. Arginine 316 is therefore critical for the binding of tyrosine. Replacement of glutamate 326 with alanine has no effect on the KM value for tyrosine and results in a 2-fold increase in the KM value for tetrahydropterin. The Vmax for DOPA production is reduced 9-fold, and the Vmax for dihydropterin formation is reduced 4-fold. These data suggest that glutamate 326 is not directly involved in catalysis. Replacement of aspartate 328 with serine results in a 26-fold higher KM value for tyrosine, a 8-fold lower Vmax for dihydropterin formation, and a 13-fold lower Vmax for DOPA formation. These data suggest that aspartate 328 has a role in tyrosine binding. Replacement of glutamate 332 with alanine results in a 10-fold higher KM value for 6-methyltetrahydropterin with no change in the KM value for tyrosine, a 125-fold lower Vmax for DOPA formation, and an only 3.3-fold lower Vmax for tetrahydropterin oxidation. These data suggest that glutamate 332 is required for productive tetrahydropterin binding.     

Mapping quantitative trait Loci underlying fitness-related traits in a free-living sheep population

We searched for quantitative trait loci (QTL) underlying fitness-related traits in a free-living pedigree of 588 Soay sheep in which a genetic map using 251 markers with an average spacing of 15 cM had been established previously. Traits examined included birth date and weight, considered both as maternal and offspring traits, foreleg length, hindleg length, and body weight measured on animals in August and jaw length and metacarpal length measured on cleaned skeletal material. In some cases the data were split to consider different age classes separately, yielding a total of 15 traits studied. Genetic and environmental components of phenotypic variance were estimated for each trait and, for those traits showing nonzero heritability (N= 12), a QTL search was conducted by comparing a polygenic model with a model including a putative QTL. Support for a QTL at genome-wide significance was found on chromosome 11 for jaw length; suggestive QTL were found on chromosomes 2 and 5 (for birth date as a trait of the lamb), 8 (birth weight as a trait of the lamb), and 15 (adult hindleg length). We discuss the prospects for refining estimates of QTL position and effect size in the study population, and for QTL searches in free-living pedigrees in general.     

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.     

Relationship between thermodynamic driving force and one-way fluxes in reversible processes

Chemical reaction systems operating in nonequilibrium open-system states arise in a great number of contexts, including the study of living organisms, in which chemical reactions, in general, are far from equilibrium. Here we introduce a theorem that relates forward and reverse fluxes and free energy for any chemical process operating in a steady state. This relationship, which is a generalization of equilibrium conditions to the case of a chemical process occurring in a nonequilibrium steady state in dilute solution, provides a novel equivalent definition for chemical reaction free energy. In addition, it is shown that previously unrelated theories introduced by Ussing and Hodgkin and Huxley for transport of ions across membranes, Hill for catalytic cycle fluxes, and Crooks for entropy production in microscopically reversible systems, are united in a common framework based on this relationship.     

Estimates of time and propensity for dental treatment among institutionalised elders

The oral health and concerns of elderly residents were surveyed in a stratified random selection of 41 long-term care facilities in Vancouver. The residents who could participate were examined and interviewed to determine their oral health and concerns about dental treatment. The need and time required for treatment were estimated in six groups to reflect the propensity for treatment in dentate and edentate subjects. The propensity for treatment was high in about one-third of the participants whereas it was unlikely that one-tenth of those examined would ever use a dental service. In general, there was a widespread need for a diagnostic service because so few had been examined by a dentist in the previous year. Prosthodontics accounted for most of the treatment in all of the propensity groups, with substantially more time required by the dentate residents. There was also a substantial need for management of mucosal pathoses and oral hygiene and, to a lesser extent, for dental restorations and endodontics. Overall, our estimates indicate a substantial need for dental treatment among residents of long-term care facilities, although the need is reduced by half if the propensity for treatment is considered.     

Rational engineering of Escherichia coli strains for plasmid biopharmaceutical manufacturing

Plasmid DNA (pDNA) has become very attractive as a biopharmaceutical, especially for gene therapy and DNA vaccination. Currently, there are a few products licensed for veterinary applications and numerous plasmids in clinical trials for use in humans. Recent work in both academia and industry demonstrates a need for technological and economical improvement in pDNA manufacturing. Significant progress has been achieved in plasmid design and downstream processing, but there is still a demand for improved production strains. This review focuses on engineering of Escherichia coli strains for plasmid DNA production, understanding the differences between the traditional use of pDNA for recombinant protein production and its role as a biopharmaceutical. We will present recent developments in engineering of E. coli strains, highlight essential genes for improvement of pDNA yield and quality, and analyze the impact of various process strategies on gene expression in pDNA production strains.     

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.     

Re-entry programs, from an anti-unconventional ex-offender

The purpose of this article seeks to question the relationship of higher levels of educational attainment and the ability for successful reintegration into mainstream society for the formerly incarcerated. Specifically this article highlights societal negligence for individual success of the formerly incarcerated, opting for a corporatized exploitation of those in the reentry process premised on the socially constructed label “ex-con.” Research surrounding labeling and reentry suggests once individuals are released from prison the social stigma experienced in society is a recipe for failure and reoffending. One way this emerges is in the form of hiring policies in corporate occupations, prohibiting the hiring of individual s convicted of a felony. Critical criminology suggests the class structure is in place for those in power to stay in power and keep those who are inferior in inferior social positions. Using both points of view as a framework, with the idea of corporate monopoly as a lens, allows for an untraditional critical perspective of the reentry process as a capitalist, for-profit industry. Questioning the role and intentions of reentry initiatives provides a general discussion for increasing success rates of those in the reentry process and reducing capital costs spent for incarceration. The specific focus of this article presents my experience of the reentry process.     

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.     

Which objectively assessed limitations in health status, activities of daily living, or in psychological and social spheres are decisive for referral to a nursing home or to a home for the aged

Which type of limitation is most decisive for a positive indication for a residential home or a somatic or psycho-geriatric nursing home, and to what extent do data on living situation (independent, adapted or intramural) and the social situation (alone or living together) influence this qualification? To obtain an answer to this question three experiments with an integrated, objective and independent assessment-for-care system were set up in the Netherlands. We studied one of them, in the town of Meppel, started in 1996. The study population consisted of all older people living in and around Meppel who had a positive indication for either a residential home, or a somatic or psycho-geriatric nursing home (N = 206 in 1998). Differences in limitations were computed by means of analysis of variance and the types of limitation and their impact on the type of allocated institutions were computed by logistic regression. The limitations only partially explain the types of indication. Indication for a residential home is based on physical limitations. When these increase they become a contra-indication for a residential home and a somatic nursing home indication is given. Mental limitations primarily yield indications for psycho-geriatric nursing homes. Social limitations have a higher incidence among persons with an indication for a somatic nursing home but cannot fully explain this indication. Persons with a residential home indication were significantly more often from an adapted living situation background. The living situation -alone or together-does not have an impact on the indication. We may not conclude that the assessment took place objectively, for, based on the registration it is impossible to conclude for what criteria a person was indicated for a specific type of institution. The objective of assessment, the allocation and distribution of collectively financed care in residential homes and nursing homes based on verifiable assessment methodology could not be realised.     

A new type of two-color fluorescence staining for cytology specimens.

A new two-color fluorescence staining technique for cervical cytology specimens is described. To permit application of this staining in automated cytology, techniques for specimen collection and cell preparation giving a sufficient number of well-separated cells on slides were used. The staining consists of a combination of a modified Feulgen-acriflavine procedure for DNA and a primulin or stilbene isothiocyanate staining for protein. This results in a bright yellow nuclear fluorescence and a blue cytoplasmic fluorescence. The staining procedure can be completed in about 90 min and is therefore suitable for routine application. Sequential inspection of the yellow nuclear and blue cytoplasmic fluorescence can be done with the two-wavelength excitation method used in fluorescence microscopy. For the application of this method, special vertical illuminators are now available. These illuminators are provided with quickly interchangeable filter sets permitting consecutive visualization of, for example, only the nuclei in the first image and the whole cell in the second image. This procedure opens new possibilities for rapid image-analysis systems.     

Kinetic analyses and structure-activity relationship studies of synthetic lysine acetylation catalysts

Lysine acylation of proteins is a crucial chemical reaction, both as a post-translational modification and as a method for bioconjugation. We previously developed a chemical catalyst, DSH, which activates a chemically stable thioester including acyl-CoA, allowing the site-selective lysine acylation of histones under physiological conditions. However, a more active catalyst is required for efficient lysine acylation in more complex biological milieu, such as in living cells, but there are no rational guidelines for developing efficient lysine acylation catalysts for use under physiological conditions as opposed to in organic solvents. We, herein, conducted a kinetic analysis of the ability of DSH and several derivatives to mediate lysine acetylation to better understand the structural elements essential for high acetylation activity under physiological conditions. Interestingly, the obtained trend in reactivity was different from that observed in organic solvents, suggesting that a different principle is necessary for designing chemical catalysts specifically for use under physiological conditions compared to catalysts for use in organic solvents. Based on the obtained information, we identified a new catalyst scaffold with high activity and structural flexibility for further modification to improve this catalyst system.     

Screening for aneuploidy in twin pregnancies: maternal age- and race-specific risk assessment between 9-14 weeks.

The aim of this study was to calculate the risk for aneuploidy in twin pregnancies between 9-14 weeks utilizing maternal age, race and dizygotic twinning rates. Using previously published risks for aneuploidy in singletons and twins at the time of amniocentesis and at term, we calculated new risk estimates for twins at 9-14 weeks gestation or at the time of chorionic villus sampling. Using these tables, the risk for trisomy 21 in at least one fetus of a twin gestation in a 32-year-old at 9-14 weeks is 1/285 for Whites and for African-Americans. This is equivalent to the risk for trisomy 21 (1/265) in a 35-year-old woman with a singleton at the same gestational age. The risks for trisomies 18 and 13 also follow similar trends. In counseling women with twin pregnancies at the time of first trimester nuchal translucency screening or chorionic villus sampling, it should be noted that the maternal age-related risk for aneuploidy for a 32-year-old is equivalent to that of a 35-year-old woman with a singleton gestation.     

A complete graphical criterion for the adjustment formula in mediation analysis

Various assumptions have been used in the literature to identify natural direct and indirect effects in mediation analysis. These effects are of interest because they allow for effect decomposition of a total effect into a direct and indirect effect even in the presence of interactions or non-linear models. In this paper, we consider the relation and interpretation of various identification assumptions in terms of causal diagrams interpreted as a set of non-parametric structural equations. We show that for such causal diagrams, two sets of assumptions for identification that have been described in the literature are in fact equivalent in the sense that if either set of assumptions holds for all models inducing a particular causal diagram, then the other set of assumptions will also hold for all models inducing that diagram. We moreover build on prior work concerning a complete graphical identification criterion for covariate adjustment for total effects to provide a complete graphical criterion for using covariate adjustment to identify natural direct and indirect effects. Finally, we show that this criterion is equivalent to the two sets of independence assumptions used previously for mediation analysis.