Design of adsorptive columns for specific pathogen removal: application to staphylococcal enterotoxin B

The removal of pathogens such as toxins, viruses, bacteria, and prions in human blood, mammalian cell culture media, fermentation broths, food items, and water streams has gained increasing importance in ensuring product safety and in combatting acts of terrorism. Adsorption processes can play an important role in removing such pathogens from solution without affecting other desirable components. Adsorptive columns that can remove specific families of pathogens would need to achieve a reduction of several logs in pathogen concentration. This requirement is much more stringent than the normal yield requirements associated with adsorptive separations aimed at product recovery and purification in a process stream. This paper considers the design of an adsorptive column aimed at reducing the concentration of infectious agents from a known volume of solution by several logs in a fixed amount of time. The general rate (GR) model of chromatography is used in the analysis, including all major transport and kinetic steps in the adsorption process. The theory, with no adjustable parameters, is shown to predict with great accuracy the effect of residence time on the log removal of staphylococcal enterotoxin B (SEB) from solution using an affinity resin with a small peptide (YYWLHH) that has been found to bind specifically to this toxin.     

Mutational screening of the CYP26A1 gene in patients with caudal regression syndrome

BACKGROUND: The retinoic acid (RA)-catabolizing enzyme Cyp26a1 plays an important role in protecting tailbud tissues from inappropriate exposure to RA. Cyp26a1-null animals exhibit caudal agenesis and spina bifida, imperforate anus, agenesis of the caudal portions of the digestive and urogenital tracts, and malformed lumbosacral skeletal elements. This phenotype closely resembles the most severe form of caudal agenesis in humans. In view of these findings, we investigated a potential involvement of the human CYP26A1 gene in the pathogenesis of caudal regression syndrome (CRS). METHODS: Mutational screening of 49 CRS patients and 132 controls was performed using denaturing high-performance liquid chromatography and sequencing. Differences in the genotype and allele frequency of each SNP were evaluated by chi(2) analysis. The biological significance of the intronic variants was investigated by transfection assays of mutant constructs and by analysis of the splicing patterns with RT-PCR. RESULTS: Mutational screening allowed us to identify 6 SNPs, 4 of which (447 C>G, 1134 G>A, IVS 1+10 G>C, and IVS 4+8 AG>GA) are new. In addition, we describe a novel 2-site haplotype consisting of the 2 intronic SNPs. Both single-locus and haplotype analyses revealed no association with increased risk for CRS. The consequences of the 2 intronic polymorphisms on the mRNA splicing process were also investigated. Moreover, using functional and computational methods we demonstrated that both of these intronic polymorphisms affect the intron splicing efficiency. CONCLUSIONS: Our research did not provide evidence that CYP26A1 has implications for the pathogenesis of human CRS. However, the relationship between CRS risk and the CYP26A1 genotype requires further study with a larger number of genotyped subjects.     

High survival rate of a critically endangered species, the Azores Bullfinch Pyrrhula murina, as a contribution to population recovery

This paper reports analyses of a capture–mark–recapture (CMR) dataset of 149 Azores Bullfinches ringed on S?o Miguel island (Azores) between 2005 and 2007, and recaptured–resighted on a monthly basis over a 4-year period (2005–2008) throughout their breeding range. We examined the effect of time, age (adults vs. juveniles), gender (adult males and females), and environmental covariates (temperature, rainfall, NAO index) on survival probabilities. The modelling found a high and constant monthly survival probability (mean ± SE) estimated at 0.96 ± 0.01, similar between both adults and juveniles and independent of environmental conditions and gender. These findings agree with expectations from island-based life-history theory where relatively mild conditions and lack of predators should favour high survival rates to compensate for the low reproductive output. The annual survival rate was estimated at 0.62, which was also consistent with this pattern when compared with survival estimates of mainland bullfinch and passerine species on other subtropical islands obtained in similar CMR studies. Based on a canonical estimator, the size of the studied population (mean ± SE) was estimated at 1608 ± 326 individuals. Given that the population size was only around 120–400 individuals in the early 1990s, we suggest that the high survival probabilities currently applying to this critically endangered species may have substantially contributed to the recent recovery of this population. Future research studies on the species’ demography should continue to monitor survival in order to measure the effect of management interventions currently taking place within the range of the Azores Bullfinch, including the restoration of the biodiversity rich laurel forest, but also focusing on nest success, which is important for understanding population dynamics.     

Pectin methylesterase and its proteinaceous inhibitor: a review

Pectin methylesterase (PME) catalyses the demethoxylation of pectin, a major plant cell wall polysaccharide. Through modification of the number and distribution of methyl-esters on the pectin backbone, PME affects the susceptibility of pectin towards subsequent (non-) enzymatic conversion reactions (e.g., pectin depolymerisation) and gel formation, and, hence, its functionality in both plant cell wall and pectin-containing food products. The enzyme plays a key role in vegetative and reproductive plant development in addition to plant-pathogen interactions. In addition, PME action can impact favourably or deleteriously on the structural quality of plant-derived food products. Consequently, PME and also the proteinaceous PME inhibitor (PMEI) found in several plant species and specifically inhibiting plant PMEs are highly relevant for plant biologists as well as for food technologists and are intensively studied in both fields. This review paper provides a structured, comprehensive overview of the knowledge accumulated over the years with regard to PME and PMEI. Attention is paid to both well-established and novel data concerning (i) their occurrence, polymorphism and physicochemical properties, (ii) primary and three-dimensional protein structures, (iii) catalytic and inhibitory activities, (iv) physiological roles in vivo and (v) relevance of (endogenous and exogenous) enzyme and inhibitor in the (food) industry. Remaining research challenges are indicated.     

Ab initio prediction of peptide-MHC binding geometry for diverse class I MHC allotypes

Since determining the crystallographic structure of all peptide-MHC complexes is infeasible, an accurate prediction of the conformation is a critical computational problem. These models can be useful for determining binding energetics, predicting the structures of specific ternary complexes with T-cell receptors, and designing new molecules interacting with these complexes. The main difficulties are (1) adequate sampling of the large number of conformational degrees of freedom for the flexible peptide, (2) predicting subtle changes in the MHC interface geometry upon binding, and (3) building models for numerous MHC allotypes without known structures. Whereas previous studies have approached the sampling problem by dividing the conformational variables into different sets and predicting them separately, we have refined the Biased-Probability Monte Carlo docking protocol in internal coordinates to optimize a physical energy function for all peptide variables simultaneously. We also imitated the induced fit by docking into a more permissive smooth grid representation of the MHC followed by refinement and reranking using an all-atom MHC model. Our method was tested by a comparison of the results of cross-docking 14 peptides into HLA-A*0201 and 9 peptides into H-2K(b) as well as docking peptides into homology models for five different HLA allotypes with a comprehensive set of experimental structures. The surprisingly accurate prediction (0.75 A backbone RMSD) for cross-docking of a highly flexible decapeptide, dissimilar to the original bound peptide, as well as docking predictions using homology models for two allotypes with low average backbone RMSDs of less than 1.0 A illustrate the method's effectiveness. Finally, energy terms calculated using the predicted structures were combined with supervised learning on a large data set to classify peptides as either HLA-A*0201 binders or nonbinders. In contrast with sequence-based prediction methods, this model was also able to predict the binding affinity for peptides to a different MHC allotype (H-2K(b)), not used for training, with comparable prediction accuracy.     

An interspecific functional complementation test in Drosophila for introductory genetics laboratory courses

Introductory genetics courses often include evolutionary genetics concepts such as sequence homology and functional conservation. It is usually assumed that two sequences showing homology (i.e., sharing a common ancestral sequence) perform the same molecular function. The correlation, however, does not always hold true, and evidence for functional conservation must come from functional studies. In this study we describe a genetics laboratory class that demonstrates functional conservation between the Drosophila protein Muscleblind (Mbl) and its human ortholog MBNL1. We use the Gal4/UAS system to express MBNL1 in a Drosophila mutant background and measure the in vivo activity of the human protein by rescue of mbl mutant phenotype in embryos. As a control, ubiquitous expression of Drosophila MblC, one of the four protein isoforms encoded by the gene, increased by 71% the viability of mbl mutant embryos and greatly reduced the hypercontracted abdomen of mutant larvae. In a parallel experiment, human MBNL1 provided a robust rescue of the embryonic lethality (78%) and improved abdomen hypercontraction as well. Under both conditions, rescued larvae die as first instars, probably due to overexpression effects, lack of alternative protein isoforms, or incomplete expression in critical tissues such as the nervous system. The use of two constructs in the rescue experiment (UAS-mblC and UAS-MBNL1) and the incomplete rescue prompt several questions for students. The fact that a human protein works in a Drosophila cellular context illustrates the use of an in vivo test to prove functional conservation.     

IL-1 beta breaks tolerance through expansion of CD25+ effector T cells

IL-1 is a key proinflammatory driver of several autoimmune diseases including juvenile inflammatory arthritis, diseases with mutations in the NALP/cryopyrin complex and Crohn's disease, and is genetically or clinically associated with many others. IL-1 is a pleiotropic proinflammatory cytokine; however the mechanisms by which increased IL-1 signaling promotes autoreactive T cell activity are not clear. Here we show that autoimmune-prone NOD and IL-1 receptor antagonist-deficient C57BL/6 mice both produce high levels of IL-1, which drives autoreactive effector cell expansion. IL-1beta drives proliferation and cytokine production by CD4(+)CD25(+)FoxP3(-) effector/memory T cells, attenuates CD4(+)CD25(+)FoxP3(+) regulatory T cell function, and allows escape of CD4(+)CD25(-) autoreactive effectors from suppression. Thus, inflammation or constitutive overexpression of IL-1beta in a genetically predisposed host can promote autoreactive effector T cell expansion and function, which attenuates the ability of regulatory T cells to maintain tolerance to self.     

Focal adhesion kinase protein levels in gut epithelial motility

Mucosal healing requires migration and proliferation. Most studies of focal adhesion kinase (FAK), a protein that regulates motility, proliferation, and apoptosis, have focused on rapid phosphorylation. We reported lower FAK protein levels in motile Caco-2 colon cancer cells and postulated that this reduction in FAK available for activation might impact cell migration and mucosal healing. Therefore, total and active FAK (FAK(397)) immunoreactivity was assessed at the migrating fronts of human Caco-2 and rat IEC-6 intestinal epithelial cells. Caco-2 and IEC-6 motility, quantitated as migration into linear or circular wounds, was examined following FAK protein inhibition by small interfering RNA (siRNA). FAK protein stability and mRNA expression were ascertained by cycloheximide decay, RT-PCR, and in situ hybridization in static and migrating Caco-2 cells. Cells at the migrating front of Caco-2 and IEC-6 monolayers exhibited lower immunostaining for both total and activated FAK than cells immediately behind the front. Western blot analysis also demonstrated diminished FAK protein levels in motile cells by >/=30% in both the differential density seeding and multiple scrape models. siRNA FAK protein inhibition enhanced motility in both the linear scrape (20% in Caco-2) and circular wound (16% in Caco-2 and 19% in IEC-6 cells) models. FAK protein degradation did not differ in motile and static Caco-2 cells and was unaffected by FAK(397) phosphorylation, but FAK mRNA was lower in migrating Caco-2 cells. Thus FAK protein abundance appears regulated at the mRNA level during gut epithelial cell motility and may influence epithelial cell migration coordinately with signals that modify FAK phosphorylation.