Structure and phospholipase function of peroxiredoxin 6: identification of the catalytic triad and its role in phospholipid substrate binding

Peroxiredoxin 6 (Prdx6) is a bifunctional protein with glutathione peroxidase and phospholipase A(2) (PLA(2)) activities, and it alone among mammalian peroxiredoxins can hydrolyze phospholipids. After identifying a potential catalytic triad (S32, H26, D140) from the crystal structure, site-specific mutations were used to evaluate the role of these residues in protein structure and function. The S32A mutation increased Prdx6 alpha-helical content, whereas secondary structure was unchanged by mutation to H26A and D140A. Lipid binding by wild-type Prdx6 to negatively charged unilamellar liposomes showed an apparent rate constant of 11.2 x 10(6) M(-1) s(-1) and a dissociation constant of 0.36 microM. Both binding and PLA(2) activity were abolished in S32A and H26A; in D140A, activity was abolished but binding was unaffected. Overoxidation of the peroxidatic C47 had no effect on lipid binding or PLA(2) activity. Fluorescence resonance energy transfer from endogenous tryptophanyls to lipid probes showed binding of the phospholipid polar head in close proximity to S32. Thus, H26 is a site for interfacial binding to the liposomal surface, S32 has a key role in maintaining Prdx6 structure and for phospholipid substrate binding, and D140 is involved in catalysis. This putative catalytic triad plays an essential role for interactions of Prdx6 with phospholipid substrate to optimize the protein-substrate complex for hydrolysis.     

Binding patterns of human norovirus-like particles to buccal and intestinal tissues of gnotobiotic pigs in relation to A/H histo-blood group antigen expression

Histo-blood group antigen (HBGA) phenotypes have been associated with susceptibility to human noroviruses (HuNoVs). Our aims were: (i) to determine the patterns of A/H HBGA expression in buccal and intestinal tissues of gnotobiotic (Gn) pigs; (ii) to determine if virus-like particles (VLPs) of HuNoV genogroup I (GI) and GII bind to A- or H-type tissues; (iii) to compare A/H expression and VLP binding patterns and confirm their binding specificities by blocking assays; (iv) to develop a hemagglutination inhibition test using buccal cells from live pigs to determine the Gn pig's A/H phenotype and to match viral strains with previously determined HuNoV VLP binding specificities; and (v) to determine the A/H phenotypes and compare these data to the infection outcomes of a previous study of 65 Gn pigs inoculated with HuNoV GII/4 strain HS66 and expressing A and/or H or neither antigen on their buccal and intestinal tissues (S. Cheetham, M. Souza, T. Meulia, S. Grimes, M. G. Han, and L. J. Saif, J. Virol. 80:10372-10381, 2006). We found that the HuNoV GI/GII VLPs of different clusters bound to tissues from four pigs tested (two A+ and two H+). The GI/1 and GII/4 VLPs bound extensively to duodenal and buccal tissues from either A+ or H+ pigs, but surprisingly, GII/1 and GII/3 VLPs bound minimally to the duodenum of an A+ pig. The VLP binding was partially inhibited by A-, H1-, or H2-specific monoclonal antibodies, but was completely blocked by porcine mucin. Comparing the A/H phenotypes of 65 HS66-inoculated Gn pigs from our previous study, we found that significantly more A+ and H(+) pigs (51%) than non-A+ and non-H+ pigs (12.5%) shed virus. From the 22 convalescent pigs, significantly more A+ or H+ pigs (66%) than non-A+ or H+ pigs (25%) seroconverted.     

Growth media effects on morphology and 17β-HSD activity in the fungusCurvularia lunata

The filamentous fungusCurvularia lunata has enzymatic activities of biotechnological significance. Its 11β-hydroxylase actvity produces hydrocortisone, and its 17-ketosteroid reductase (17β-HSD) activity is important in the production of 17β-hydroxysteroids. We have examined the effects of different media on the 17β-HSD activity, and on the morphology and ultrastructure ofC. lunata in the search for a correlation between high enzymatic activity, morphology and ultrastructure. The highest 17β-HSD activity per dry weight ofC. lunata was seen in Czapek medium, while more product per volume was formed in malt extract broth. In all growth media tested, the highest 17β-HSD activity coincided with the exponential phase of growth. In Czapek broth medium, fungal hyphae formed pellets, while in malt extract and yeast nitrogen base broth media, the fungus formed branched hyphae dispersed throughout the media. Scanning electron microscopy revealed differences in the thickness of hyphal cells; those of the fungus grown in Czapek and yeast nitrogen base media were much thicker than those in malt extract medium. Transmission electron microscopy showed melanized cell walls in the fungus grown in malt extract and Czapek media, but not in yeast nitrogen base medium. Our results show that theC. lunata with the highest 17β-HSD activity grow as dispersed, melanized mycelia with thin hyphae.     

Systematic Development of Sandwich Immunoassays for the Plasma Secretome

The plasma proteome offers a clinically useful window into human health. Recent advances from highly multiplexed assays now call for appropriate pipelines to validate individual candidates. Here, a workflow is developed to build dual binder sandwich immunoassays (SIA) and for proteins predicted to be secreted into plasma. Utilizing suspension bead arrays, ≈1800 unique antibody pairs are first screened against 209 proteins with recombinant proteins as well as EDTA plasma. Employing 624 unique antibodies, dilution‐dependent curves in plasma and concentration‐dependent curves of full‐length proteins for 102 (49%) of the targets are obtained. For 22 protein assays, the longitudinal, interindividual, and technical performance is determined in a set of plasma samples collected from 18 healthy subjects every third month over 1 year. Finally, 14 of these assays are compared with with SIAs composed of other binders, proximity extension assays, and affinity‐free targeted mass spectrometry. The workflow provides a multiplexed approach to screen for SIA pairs that suggests using at least three antibodies per target. This design is applicable for a wider range of targets of the plasma proteome, and the assays can be applied for discovery but also to validate emerging candidates derived from other platforms.     

Boomeranging around Australia: Historical biogeography and population genomics of the anti‐equatorial fish Microcanthus strigatus (Teleostei: Microcanthidae)

The geographic distributions of marine fishes have been shaped by ancient vicariance and ongoing dispersal events. Some species exhibit anti‐equatorial distributions, inhabiting temperate regions on both sides of the tropics while being absent from equatorial latitudes. The perciform fish Microcanthus strigatus (the stripey) exhibits such a distribution with disjunct populations occurring in East Asia, Hawaii, Western Australia, and the southwest Pacific. Here, we examine the historical biogeography and evolutionary history of M. strigatus, based on more than 80 specimens sampled from the four major populations. We analysed 36 morphological characters, three mitochondrial markers, and two sets of 7,120 and 12,771 single‐nucleotide polymorphisms from the nuclear genome. Our results suggest that M. strigatus represents a cryptic species complex comprising at least two genetically distinct populations worthy of species‐level recognition, with one population exhibiting strong genetic structuring but with intermittent, historical gene flow. We provide evidence for a southwest Pacific origin for the ancestral Microcanthus and explain how past connectivity between these regions might have given rise to the relationships observed in present‐day marine fauna. Our ancestral range reconstructions and molecular‐clock analyses support a southwest Pacific centre of origin for Microcanthus, with subsequent colonization of Western Australia through the Bass Strait followed by transequatorial dispersals to the Northern Hemisphere during the Pleistocene. Our results detail an anti‐tropical dispersal pattern that is highly unusual and previously undocumented, thereby emphasizing the importance of integrative systematics in the evaluation of widespread species.     

Thinking Styles and Regret in Physicians

Background

Decision-making relies on both analytical and emotional thinking. Cognitive reasoning styles (e.g. maximizing and satisficing tendencies) heavily influence analytical processes, while affective processes are often dependent on regret. The relationship between regret and cognitive reasoning styles has not been well studied in physicians, and is the focus of this paper.

Methods

A regret questionnaire and 6 scales measuring individual differences in cognitive styles (maximizing-satisficing tendencies; analytical vs. intuitive reasoning; need for cognition; intolerance toward ambiguity; objectivism; and cognitive reflection) were administered through a web-based survey to physicians of the University of South Florida. Bonferroni’s adjustment was applied to the overall correlation analysis. The correlation analysis was also performed without Bonferroni’s correction, given the strong theoretical rationale indicating the need for a separate hypothesis. We also conducted a multivariate regression analysis to identify the unique influence of predictors on regret.

Results

165 trainees and 56 attending physicians (age range 25 to 69) participated in the survey. After bivariate analysis we found that maximizing tendency positively correlated with regret with respect to both decision difficulty (r=0.673; p<0.001) and alternate search strategy (r=0.239; p=0.002). When Bonferroni’s correction was not applied, we also found a negative relationship between satisficing tendency and regret (r=-0.156; p=0.021). In trainees, but not faculty, regret negatively correlated with rational-analytical thinking (r=-0.422; p<0.001), need for cognition (r=-0.340; p<0.001), and objectivism (r=-0.309; p=0.003) and positively correlated with ambiguity intolerance (r=0.285; p=0.012). However, after conducting a multivariate regression analysis, we found that regret was positively associated with maximizing only with respect to decision difficulty (r=0.791; p<0.001), while it was negatively associated with satisficing (r=-0.257; p=0.020) and objectivism (r=-0.267; p=0.034). We found no statistically significant relationship between regret and overall accuracy on conditional inferential tasks.

Conclusion

Regret in physicians is strongly associated with their tendency to maximize; i.e. the tendency to consider more choices among abundant options leads to more regret. However, physicians who exhibit satisficing tendency – the inclination to accept a “good enough” solution – feel less regret. Our observation that objectivism is a negative predictor of regret indicates that the tendency to seek and use empirical data in decision-making leads to less regret. Therefore, promotion of evidence-based reasoning may lead to lower regret.     

Corneal thickness in pseudoexfoliative glaucoma

Measurements of central cornea thickness (CCT) have a very important value in glaucoma patients; if the central cornea is thinner than it suggests, then the intraocular pressure is falsely low. This study compares the central cornea thickness between patients with pseudoexfoliative glaucoma, open angle glaucoma, angle closure glaucoma and control group. This study included 34 patients with pseudoexfoliative glaucoma, 31 patient with open angle laucoma, 28 patients with angle closure laucoma and 36 normal subjects in a control group. Patients in all groups and also normal subjects in control group had no other corneal disorders, no history of trauma, corneal surgery and were not patients with contacts lens use. Patients with pseudoexfoliative glaucoma and also patients with open angle glaucoma had significantly lower values of central cornea thickness compared with normal subjects in control group. Tomey EM 3000 is a non contact specular microscope which was used to measure central corneal thickness in this study. Pachymetry is an important method for diagnoses of glaucoma and for examination of the intraocular pressure in glaucoma patients, because values of the central corneal thickness affect the exact intraocular pressure readings.