Bioorthogonal Strategy for Bioprocessing of Specific-Site-Functionalized Enveloped Influenza-Virus-Like Particles

Virus-like particles (VLPs) constitute a promising platform in vaccine development and targeted drug delivery. To date, most applications use simple nonenveloped VLPs as human papillomavirus or hepatitis B vaccines, even though the envelope is known to be critical to retain the native protein folding and biological function. Here, we present tagged enveloped VLPs (TagE-VLPs) as a valuable strategy for the downstream processing and monitoring of the in vivo production of specific-site-functionalized enveloped influenza VLPs. This two-step procedure allows bioorthogonal functionalization of azide-tagged nascent influenza type A hemagglutinin proteins in the envelope of VLPs through a strain-promoted [3 + 2] alkyne-azide cycloaddition reaction. Importantly, labeling does not influence VLP production and allows for construction of functionalized VLPs without deleterious effects on their biological function. Refined discrimination and separation between VLP and baculovirus, the major impurity of the process, is achieved when this technique is combined with flow cytometry analysis, as demonstrated by atomic force microscopy. TagE-VLPs is a versatile tool broadly applicable to the production, monitoring, and purification of functionalized enveloped VLPs for vaccine design trial runs, targeted drug delivery, and molecular imaging.The authors acknowledge funding from the European Union (EDUFLUVAC project FP7-HEALTH-2013-INNOVATION), the Fundação para a Ciência e Tecnologia (FCT, Portugal; project HIVERA/0002/2013 and FCT Investigator to G.J.L.B.), EPSRC (to G.J.L.B.), the European Commission, Marie Skłodowska-Curie Actions (MSCA), and RISE project grant 644167. S. B. C., J. M. F., F. M., and D. G. acknowledge FCT for fellowships SFRH/BD/52302/2013, SFRH/BD/70423/2010, SFRH/BD/70139/2010, and SFRH/BPD/73500/2010, respectively. The authors acknowledge Ricardo Silva for all his help in fluorescence analysis implementation and fruitful discussions. The authors also acknowledge Patrícia Gomes-Alves for her help for mass spectrometry analysis. Mass spectrometry data was obtained by the Mass Spectrometry Unit (UniMS), ITQB/iBET, Oeiras, Portugal. G. J. L. B. is a Royal Society University Research Fellow and the recipient of a European Research Council Starting Grant (TagIt)

Using zeta-potential measurements to quantify peptide partition to lipid membranes

© The Author(s) 2011. This article is published with open access at Springerlink.com.Open Access: This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.Many cellular phenomena occur on the biomembranes. There are plenty of molecules (natural or xenobiotics) that interact directly or partially with the cell membrane. Biomolecules, such as several peptides (e.g., antimicrobial peptides) and proteins, exert their effects at the cell membrane level. This feature makes necessary investigating their interactions with lipids to clarify their mechanisms of action and side effects necessary. The determination of molecular lipid/water partition constants (Kp) is frequently used to quantify the extension of the interaction. The determination of this parameter has been achieved by using different methodologies, such as UV-Vis absorption spectrophotometry, fluorescence spectroscopy and ζ-potential measurements. In this work, we derived and tested a mathematical model to determine the Kp from ζ-potential data. The values obtained with this method were compared with those obtained by fluorescence spectroscopy, which is a regular technique used to quantify the interaction of intrinsically fluorescent peptides with selected biomembrane model systems. Two antimicrobial peptides (BP100 and pepR) were evaluated by this new method. The results obtained by this new methodology show that ζ-potential is a powerful technique to quantify peptide/lipid interactions of a wide variety of charged molecules, overcoming some of the limitations inherent to other techniques, such as the need for fluorescent labeling.This work was partially supported by project PTDC/QUI/ 69937/2006 from Fundação para a Ciência e Tecnologia-Ministério da Ciência, Tecnologia e Ensino Superior (FCT-MCTES, Portugal), and by Fundação Calouste Gulbenkian (Portugal). JMF and MMD also thank FCT-MCTES for grants IMM/BT/37-2010 and SFRH/BD/41750/2007, respectively

Arsenic release and speciation in a degraded fen as affected by soil redox potential at varied moisture regime

Soil surveys have demonstrated arsenic (As) contents of up to 1600 mg kg(-1) in surface horizons of degraded fens in the Bavarian Molasse basin, Germany. Ground water from the Tertiary aquifer seems to be the primary source of As. Yet, the cause of its accumulation in the topsoil is unclear. Focussing the influence of redox processes on As redistribution, we conducted soil column experiments with the A (716 mg As kg(-1)), Ag (293 mg As kg(-1)) and 2Ag (37 mg As kg(-1)) horizons of a Mollic Gleysol (pH 7.2). The fixed beds were equipped with redox electrodes and suction cups and subjected to a saturation-drainage-saturation cycle. Water table fluctuations were simulated by defined pressure heads applied to the lower column boundaries via a digitally controlled vacuum system. After water saturation, the redox potential (E(H)) dropped to minimum values of around 0 mV in the A/Ag and-400 mV in the 2Ag horizon. Soil drainage resulted in a quick return to oxidising conditions. Both in the A and Ag horizons total aqueous As concentrations were low (up to 20 mu g l(-1)) and not related to E(H). In contrast, aqueous As concentrations of the 2Ag horizon were between 5 and 140 mu g l(-1) and increased as the E(H) decreased. However, the As species distribution showed no clear trend with E(H) since both As(III) and As(V) were detected under reducing conditions. High release of As in the 2Ag horizon is consistent with low contents in Fe (hydr)oxides. In the A and Ag horizons, pedogenetic enrichment of sesquioxides contributes to a comparably lower sensitivity of E(H) to water saturation and favours As retention. Thereby, As released under saturated conditions in the 2Ag horizon may be stabilised in the topsoil following capillary rise. Thus, surface horizons may act both as historic and as recent sinks for geogenic As at the site. (C) 2010 Elsevier B.V. All rights reserved

Elektrokinetische Reinigung kontaminierter Boeden Zwischenbericht zum 01.10.1998

Available from TIB Hannover: F99B904 / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekSIGLEDEGerman

Obesogenic eating behaviour and dietary intake in German children and adolescents: Results from the GINIplus and LISA birth cohort studies.

Background/objectives: The transition to adolescence is characterised by considerable behavioural changes, including diet. This study describes the level of obesogenic eating behaviours in 10- and 15-year-olds, and their association with dietary intake. Subjects/methods: Participants of the 10- and 15-year follow-ups of the German GINIplus and LISA birth cohort studies were included (N10 = 2257; N15 = 1880). Eating behaviours and dietary intake were assessed via self-report questionnaires. Sex-stratified, cross-sectional associations of “external eating”, “emotional eating” and “dietary restraint” (the latter at age 15 years only) with dietary intake (17 food groups—categorised into tertiles, macronutrients, and total energy) were assessed using multinomial logistic or multiple linear regression as required, adjusting for covariates and correcting for multiple testing. Results: Reported levels of eating behaviours were low in both age-groups. External eating was higher in 10-year-old males than females, while all eating behaviours were most pronounced in 15-year-old females. At 10 years, emotional eating was associated with medium vegetable intake in females (Relative Risk Ratio (RRR) = 1.84, p = 0.0017). At 15 years, external eating was associated with total energy (kJ) in females (β = 718, p = 0.0002) and high butter intake in males (RRR = 1.96, p = 0.0019). Dietary restraint in females was inversely associated with total energy (β = −967, p < 0.0001) and omega-3 fatty acids (Means Ratio (MR) = 0.94, p = 0.0017), and positively associated with high fruit (RRR = 2.20, p = 0.0003) and whole grains (RRR = 1.94, p = 0.0013). Conclusion: Obesogenic eating behaviour scores are low among children and adolescents of a predominantly high socioeconomic status population and present only few associations with specific aspects of diet, mainly among adolescent females