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

Análisis intercultural del trastorno por uso de alcohol: criterios diagnósticos en universitarios

This study aimed to examine the clinical manifestations of Alcohol Use Disorder (AUD) in university students from countries with different drinking practices, and the potential of a drinking measure as an AUD marker. University students from Argentina (N = 2157), Spain (N = 320), and Brazil (N = 149) participated in the study. The TUA criteria and the cut off-points were compatible with the DSM 5, and a measure of binge drinking was evaluated. Descriptive and latent class analyses were performed. In general, the severe AUD is characterized by the endorsement of all criteria; the moderate-mild, by tolerance, loss of control (quit/cut), interpersonal problems, and role impairment. In countries with a wet drinking pattern (Argentina and Spain), the criteria of hazardous use and psychological or physical problems due to use were characteristic of different severity levels. The binge drinking measure showed potential as a marker of TUA.El objetivo de este estudio fue examinar las manifestaciones clínicas del trastorno por uso de alcohol (TUA) en universitarios de países con distintas prácticas de consumo, y estimar el potencial de una medida de consumo de alcohol como marcador de TUA. Participaron estudiantes de universidades en Argentina (N = 2157), España (N = 320) y Brasil (N = 149). Se evaluaron criterios diagnósticos de TUA, puntos de corte compatibles con el DSM 5, y una medida de consumo excesivo episódico de alcohol. Se realizaron análisis descriptivos y de clases latentes. Generalmente el TUA grave se caracterizó por endorsar todos los criterios; el moderado-leve por tolerancia, pérdida de control, problemas interpersonales y negligencia de obligaciones. En los países con un patrón húmedo de consumo (Argentina y España) los criterios uso en situaciones peligrosas y consumo a pesar de problemas mentales/físicos se manifestaron en categorías distintas. La medida de consumo presentó potencial como marcador de TUA

Antimicrobial and cell-penetrating peptides induce lipid vesicle fusion by folding and aggregation

According to their distinct biological functions, membrane-active peptides are generally classified as antimicrobial (AMP), cell-penetrating (CPP), or fusion peptides (FP). The former two classes are known to have some structural and physicochemical similarities, but fusogenic peptides tend to have rather different features and sequences. Nevertheless, we found that many CPPs and some AMPs exhibit a pronounced fusogenic activity, as measured by a lipid mixing assay with vesicles composed of typical eukaryotic lipids. Compared to the HIV fusion peptide (FP23) as a representative standard, all designer-made peptides showed much higher lipid-mixing activities (MSI-103, MAP, transportan, penetratin, Pep1). Native sequences, on the other hand, were less fusogenic (magainin 2, PGLa, gramicidin S), and pre-aggregated ones were inactive (alamethicin, SAP). The peptide structures were characterized by circular dichroism before and after interacting with the lipid vesicles. A striking correlation between the extent of conformational change and the respective fusion activities was found for the series of peptides investigated here. At the same time, the CD data show that lipid mixing can be triggered by any type of conformation acquired upon binding, whether α-helical, β-stranded, or other. These observations suggest that lipid vesicle fusion can simply be driven by the energy released upon membrane binding, peptide folding, and possibly further aggregation. This comparative study of AMPs, CPPs, and FPs emphasizes the multifunctional aspects of membrane-active peptides, and it suggests that the origin of a peptide (native sequence or designer-made) may be more relevant to define its functional range than any given name

Trajectory/Path-Following Controller Based on Nonlinear Jerk-Level Error Dynamics

This study proposes a novel, nonlinear trajectory/path-following controller based on jerk-level error dynamics. Therefore, at first the nonlinear acceleration-based kinematic equations of motion of a dynamic system are differentiated with respect to time to obtain a representation connecting the translation jerk with the (specific) force derivative. Furthermore, the path deviation, i.e., the difference between the planned and the actual path, is formulated as nonlinear error dynamics based on the jerk error. Combining the derived equations of motion with the nonlinear error dynamics as well as employing nonlinear dynamic inversion, a control law can be derived that provides force derivative commands, which may be commanded to an inner loop for trajectory control. This command ensures an increased smoothness and faster reaction time compared to traditional approaches based on a force directly. Furthermore, the nonlinear parts in the error dynamic are feedforward components that improve the general performance due to their physical connection with the real dynamics. The validity and performance of the proposed trajectory/path-following controller are shown in an aircraft-related application example