Mechanisms for covalent immobilization of horseradish peroxi-dase on ion beam treated polyethylene

The mechanism that provides the observed strong binding of biomolecules to polymer sur-faces modified by ion beams is investigated. The surface of polyethylene (PE) was modified by plasma immersion ion implantation with nitrogen ions. Structure changes including car-bonization and oxidation were observed in the modified surface layer of PE by Raman spec-troscopy, FTIR ATR spectroscopy, atomic force microscopy, surface energy measurement and XPS spectroscopy. An observed high surface energy of the modified polyethylene was attributed to the presence of free radicals on the surface. The surface energy decay with stor-age time after PIII treatment was explained by a decay of the free radical concentration while the concentration of oxygen-containing groups increased with storage time. Horseradish per-oxidase was covalently attached onto the modified PE surface. The enzymatic activity of co-valently attached protein remained high. A mechanism based on the covalent attachment by the reaction of protein with free radicals in the modified surface is proposed. Appropriate blocking agents can block this reaction. All aminoacid residues can take part in the covalent attachment process, providing a universal mechanism of attachment for all proteins. The long-term activity of the modified layer to attach protein (at least 2 years) is explained by stabilisa-tion of unpaired electrons in sp2 carbon structures. The native conformation of attached pro-tein is retained due to hydrophilic interactions in the interface region. A high concentration of free radicals on the surface can give multiple covalent bonds to the protein molecule and de-stroy the native conformation and with it the catalytic activity. The universal mechanism of protein attachment to free radicals could be extended to various methods of radiation damage of polymers

Deriving accurate microbiota profiles from human samples with low bacterial content through post-sequencing processing of Illumina MiSeq data

BackgroundThe rapid expansion of 16S rRNA gene sequencing in challenging clinical contexts has resulted in a growing body of literature of variable quality. To a large extent, this is due to a failure to address spurious signal that is characteristic of samples with low levels of bacteria and high levels of non-bacterial DNA. We have developed a workflow based on the paired-end read Illumina MiSeq-based approach, which enables significant improvement in data quality, post-sequencing. We demonstrate the efficacy of this methodology through its application to paediatric upper-respiratory samples from several anatomical sites.ResultsA workflow for processing sequence data was developed based on commonly available tools. Data generated from different sample types showed a marked variation in levels of non-bacterial signal and ‘contaminant’ bacterial reads. Significant differences in the ability of reference databases to accurately assign identity to operational taxonomic units (OTU) were observed. Three OTU-picking strategies were trialled as follows: de novo, open-reference and closed-reference, with open-reference performing substantially better. Relative abundance of OTUs identified as potential reagent contamination showed a strong inverse correlation with amplicon concentration allowing their objective removal. The removal of the spurious signal showed the greatest improvement in sample types typically containing low levels of bacteria and high levels of human DNA. A substantial impact of pre-filtering data and spurious signal removal was demonstrated by principal coordinate and co-occurrence analysis. For example, analysis of taxon co-occurrence in adenoid swab and middle ear fluid samples indicated that failure to remove the spurious signal resulted in the inclusion of six out of eleven bacterial genera that accounted for 80% of similarity between the sample types.ConclusionsThe application of the presented workflow to a set of challenging clinical samples demonstrates its utility in removing the spurious signal from the dataset, allowing clinical insight to be derived from what would otherwise be highly misleading output. While other approaches could potentially achieve similar improvements, the methodology employed here represents an accessible means to exclude the signal from contamination and other artefacts

Stellar occultations enable milliarcsecond astrometry for Trans-Neptunian objects and Centaurs

Trans-Neptunian objects (TNOs) and Centaurs are remnants of our planetary system formation, and their physical properties have invaluable information for evolutionary theories. Stellar occultation is a ground-based method for studying these small bodies and has presented exciting results. These observations can provide precise profiles of the involved body, allowing an accurate determination of its size and shape. The goal is to show that even single-chord detections of TNOs allow us to measure their milliarcsecond astrometric positions in the reference frame of the Gaia second data release (DR2). Accurated ephemerides can then be generated, allowing predictions of stellar occultations with much higher reliability. We analyzed data from stellar occultations to obtain astrometric positions of the involved bodies. The events published before the Gaia era were updated so that the Gaia DR2 catalog is the reference. Previously determined sizes were used to calculate the position of the object center and its corresponding error with respect to the detected chord and the International Celestial Reference System (ICRS) propagated Gaia DR2 star position. We derive 37 precise astrometric positions for 19 TNOs and 4 Centaurs. Twenty-one of these events are presented here for the first time. Although about 68\% of our results are based on single-chord detection, most have intrinsic precision at the submilliarcsecond level. Lower limits on the diameter and shape constraints for a few bodies are also presented as valuable byproducts. Using the Gaia DR2 catalog, we show that even a single detection of a stellar occultation allows improving the object ephemeris significantly, which in turn enables predicting a future stellar occultation with high accuracy. Observational campaigns can be efficiently organized with this help, and may provide a full physical characterization of the involved object.Comment: 16 pages, 28 figures. The manuscript was accepted and is to be publishe

Evidence of an Antimicrobial-Immunomodulatory Role of Atlantic Salmon Cathelicidins during Infection with Yersinia ruckeri

Cathelicidins are a family of antimicrobial peptides that act as effector molecules of the innate immune system with broad-spectrum antimicrobial properties. These evolutionary conserved cationic host-defence peptides are integral components of the immune response of fish, which are generally believed to rely heavily on innate immune defences to invading pathogens. In this study we showed that Atlantic salmon cathelicidin 1 and 2 (asCATH1 and asCATH2) stimulated peripheral blood leukocytes increasing the transcription of the chemokine interleukin-8. Further, functional differences were identified between the two cathelicidins. In the presence of serum, asCATH1 displayed greatly diminished host haemolytic activity, while the constitutively expressed asCATH2 had no haemolytic activity with or without serum. These findings support our hypothesis that fish cathelicidins exert their primary antimicrobial action at the site of pathogen invasion such as epithelial surfaces. Further, we hypothesise that like their mammalian counterparts in the presence of serum they act as mediators of the innate and adaptive immune response via the release of cytokines thus indirectly protecting against a variety of pathogens. We highlight the importance of this immunomodulatory role from the involvement of asCATHs during an infection with the fish pathogen Yersinia ruckeri. While we were able to demonstrate in vitro that asCATH1 and 2, possessed direct microbicidal activity against the fish pathogen, Vibrio anguillarum, and a common gram negative bacterium, Escherichia coli, little or no bactericidal activity was found against Y. ruckeri. The contribution of either asCATH in the immune response or as a potential virulence factor during yersiniosis is highlighted from the increased expression of asCATH1 and 2 mRNA during an in vivo challenge with Y. ruckeri . We propose that Atlantic salmon cathelicidins participate in the interplay between the innate and adaptive immune systems via the release of cytokines enabling a more effective response to invading pathogens