Laser-light scattering approach to peptide–membrane interaction

© International University Line, 2010Membrane-active peptides are becoming widely used, mainly due to their high therapeutic potential. Although the therapeutic action is characterized, the mechanisms of interaction are often unclear or controversial. In biophysical studies, non-invasive techniques are overlooked when studying the effect of peptides on membranes. Light scattering techniques, such as dynamic light scattering and static light scattering, can be used as tools to determine whether promotion of membrane aggregation in the presence of peptides and of self-peptide aggregation in solution occurs. More recently, light scattering has been used for evaluating the alteration on membrane surface charge (ζ-potential) promoted by membrane–peptide interactions. The data obtained by these techniques (either by themselves or combined with complementary experimental approaches) therefore yield valuable elucidations of membrane-active peptides’ mechanisms of action at the molecular level.This work was partially supported by the Fundação para a Ciência e Tecnologia (FCT) of the Portuguese Ministry of Science, Technology and Higher Education. M.M.D. acknowledges the grant SFRH/BD/41750/2007 from FCT

Translocating the blood-brain barrier using electrostatics

Copyright © 2012 Ribeiro,Domingues, Freire,Santos and Castanho. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any third-party graphics etc.Mammalian cell membranes regulate homeostasis, protein activity, and cell signaling. The charge at the membrane surface has been correlated with these key events. Although mammalian cells are known to be slightly anionic, quantitative information on the membrane charge and the importance of electrostatic interactions in pharmacokinetics and pharmacodynamics remain elusive. Recently, we reported for the first time that brain endothelial cells (EC) are more negatively charged than human umbilical cord cells, using zeta-potential measurements by dynamic light scattering. Here, we hypothesize that anionicity is a key feature of the blood-brain barrier (BBB) and contributes to select which compounds cross into the brain. For the sake of comparison, we also studied the membrane surface charge of blood components—red blood cells (RBC), platelets, and peripheral blood mononuclear cells (PBMC).To further quantitatively correlate the negative zeta-potential values with membrane charge density, model membranes with different percentages of anionic lipids were also evaluated. From all the cells tested, brain cell membranes are the most anionic and those having their lipids mostly exposed, which explains why lipophilic cationic compounds are more prone to cross the blood-brain barrier.Fundação para a Ciência e Tecnologia — Ministério da Educação e Ciência (FCT-MEC, Portugal) is acknowledged for funding (including fellowships SFRH/BD/42158/2007 to Marta M.B. Ribeiro, SFRH/BD/41750/2007 to Marco M. Domingues and SFRH/BD/70423/2010 to João M. Freire) and project PTDC/QUI-BIQ/119509/2010. Marie Curie Industry-Academia Partnerships and Pathways (European Commission) is also acknowledged for funding (FP7-PEOPLE-2007-3-1-IAPP, Project 230654)

Protein-biomembrane interactions as therapeutic targets

Biological membranes are dynamic structures essential for several cellular phenomena. The scope of the work of the Institute of Molecular Medicine (IMM) Biomembranes Unit is the study of biochemical and biophysical processes occurring at the membrane level on human cells and on their viral and bacterial pathogens. On the viral context, we are primarily interested on HIV and dengue virus, and particularly on the two steps of their life cycle involving their interaction with host cell membranes: the viral entry into target cells and the assembly of new viral particles. A special focus will be given to the study of the role of biological membranes on the mechanism of action of the HIV entry (membrane fusion) inhibitors enfuvirtide and T-1249. We are also involved in assessing the molecular basis of the activity of microbicides, such as rBPI21, that bind to specific components of bacterial membranes. Additionally, our line of work on the binding of fibrinogen to erythrocytes, and its relevance as a cardiovascular risk factor will be presented. An approach to the latter problem by single-molecule force spectroscopy, using an atomic force microscope (AFM), allowed the molecular recognition, characterization and partial identification of the human erythrocyte receptor for fibrinogen.These lines of work were supported by Fundação para a Ciência e a Tecnologia – Ministério da Ciência, Tecnologia e Ensino Superior (FCT-MCTES, Portugal; projects PTDC/SAU-OSM/73449/2006 and PTDC/ QUI-BIQ/104787/2008), by the FP7-PEOPLE IRSES (International Research Staff Exchange Scheme) project MEMPEPACROSS (EU), and by Fundação Calouste Gulbenkian (Portugal). MMD and PMM also thank FCT-MCTES for the PhD fellowships SFRH/BD/41750/2007 and SFRH/BD/42205/2007, respectively

rBPI21 Promotes Lipopolysaccharide Aggregation and Exerts Its Antimicrobial Effects by (Hemi)fusion of PG-Containing Membranes

Antimicrobial peptides (AMPs) are important potential alternatives to conventional therapies against bacterial infections. rBPI21 is a 21 kDa peptide based on the N-terminal region of the neutrophil bactericidal/permeability-increasing protein (BPI). This AMP possesses highly selective bactericidal effects on Gram-negative bacteria and have affinity for lipopolysaccharide (LPS) which is believed to be at the origin of its neutralizing effect of the LPS segregated into the bloodstream. We aim at understanding the molecular bases of rBPI21 bactericidal and LPS neutralization actions, using biomembrane model systems. Using dynamic light scattering spectroscopy we demonstrate that rBPI21 promotes aggregation of negatively charged large unilamellar vesicles (LUV), even in the absence of LPS, and LPS aggregates, while for zwitterionic phosphatidylcholine (POPC) LUV the size remains unchanged. The peptide also promotes the fusion (or hemifusion) of membranes containing phosphatidylglycerol (POPG). The aggregation and fusion of negatively charged LUV are peptide concentration-dependent until massive aggregation is reached, followed by sample flocculation/precipitation. Concomitantly, there is a progressive change in the zeta-potential of the LUV systems and LPS aggregates. LUV systems composed of phosphatidylglycerol (POPG) and lipid mixtures with POPG have higher zeta-potential variations than in the absence of POPG. The interaction of rBPI21 with lipid vesicles is followed by leakage, with higher effect in POPG-containing membranes. LPS aggregation can be related with a decreased toxicity, possibly by facilitating its clearance by macrophage phagocytosis and/or blocking of LPS specific receptor recognition. Our data indicate that rBPI21 mechanism of action at the molecular level involves the interaction with the LPS of the outer membrane of Gram-negative bacteria, followed by internalization and leakage induction through the (hemi)fusion of the bacterial outer and inner membranes, both enriched in phosphatidylglycerol

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

Perfil Ictiofaunístico de duas Lagoas no Rio Paraná, Região do Parque Nacional de Ilha Grande - PR

O Rio Paraná é o décimo maior do mundo em descarga e nele se encontra a região do Parque Nacional de Ilha Grande, que é formada por um conjunto de ilhas, lagoas e várzeas periodicamente alagadas, sendo estas de extrema importância para os peixes, servindo de refúgio contra predação, berçário natural e áreas de alimentação para muitas espécies de peixes. Este artigo apresenta um levantamento icitiofaunístico nas lagoas Saraiva e São João, situadas no Parque Nacional de Ilha Grande, com enfoque nas variações espaço-temporais, na composição específica e estrutura etária das assembléias de peixes, sendo que estes foram amostrados em coletas trimestrais, utilizando-se redes de espera de diferentes malhagens. Em termos de número de indivíduos capturados e proporção, tem-se 54% das espécies pertencentes a ordem Characiformes, 42% a Siruliformes, 3% a Perciformes. Rajiformes e Gymnotiformes contribuíram com menos de 1,5% do total das capturas. Na lagoa Saraiva foram capturadas 34 espécies pertencentes a cinco ordens e 17 famílias, destacando-se numericamente Loricariichthys platymetopon, Raphiodon vulpinus, Serrasalmus marginatus e Plagioscion squamosissimus. As maiores contribuições em peso foram proporcionadas por R. vulpinus, Potanotrygon motoro, P. squamosissimus e S. marginatus. Na lagoa São João ocorreram 46 espécies pertencentes a cinco ordens e 17 famílias, destacando-se em número L. platymetopon, Acestrohynchus lacustris, Serrasalmus spilopleura e S. marginatus. Em peso, Prochilodus lineatus, A. lacustris, L. platymetopon e Serrasalmus spilopleura foram as mais representativas. Destaca-se que a maioria das espécies registradas em ambas as lagoas são típicas de ambientes lênticos, utilizando esses locais para seu desenvolvimento e crescimento

Influence of the electrolyte salt concentration on DNA detection with graphene transistors

Liquid-gated Graphene Field-Effect Transistors (GFET) are ultrasensitive bio-detection platforms carrying out the graphene’s exceptional intrinsic functionalities. Buffer and dilution factor are prevalent strategies towards the optimum performance of the GFETs. However, beyond the Debye length (λD), the role of the graphene-electrolytes’ ionic species interactions on the DNA behavior at the nanoscale interface is complicated. We studied the characteristics of the GFETs under different ionic strength, pH, and electrolyte type, e.g., phosphate buffer (PB), and phosphate buffer saline (PBS), in an automatic portable built-in system. The electrostatic gating and charge transfer phenomena were inferred from the field-effect measurements of the Dirac point position in single-layer graphene (SLG) transistors transfer curves. Results denote that λD is not the main factor governing the effective nanoscale screening environment. We observed that the longer λD was not the determining characteristic for sensitivity increment and limit of detection (LoD) as demonstrated by different types and ionic strengths of measuring buffers. In the DNA hybridization study, our findings show the role of the additional salts present in PBS, as compared to PB, in increasing graphene electron mobility, electrostatic shielding, intermolecular forces and DNA adsorption kinetics leading to an improved sensitivity.This research is supported by PORTGRAPHE-Control of Port and DouroWines authenticity using graphene DNA sensors project co-funded by FCT (PTDC/BIA-MOL/31069/2017) and the ERDF through COMPETE2020 (POCI-01-0145-FEDER-031069)

Application of Light Scattering Techniques to Nanoparticle Characterization and Development

Over the years, the scientific importance of nanoparticles for biomedical applications has increased. The high stability and biocompatibility, together with the low toxicity of the nanoparticles developed lead to their use as targeted drug delivery systems, bioimaging systems, and biosensors. The wide range of nanoparticles size, from 10 nm to 1 μm, as well as their optical properties, allow them to be studied using microscopy and spectroscopy techniques. In order to be effectively used, the physicochemical properties of nanoparticle formulations need to be taken into account, namely, particle size, surface charge distribution, surface derivatization and/or loading capacity, and related interactions. These properties need to be optimized considering the final nanoparticle intended biodistribution and target. In this review, we cover light scattering based techniques, namely dynamic light scattering and zeta-potential, used for the physicochemical characterization of nanoparticles. Dynamic light scattering is used to measure nanoparticles size, but also to evaluate their stability over time in suspension, at different pH and temperature conditions. Zeta-potential is used to characterize nanoparticles surface charge, obtaining information about their stability and surface interaction with other molecules. In this review, we focus on nanoparticle characterization and application in infection, cancer and cardiovascular diseases

Sequential application of soil vapor extraction and bioremediation processes for the remediation of ethylbenzene-contaminated soils

Soil vapor extraction (SVE) is an efficient, well-known and widely applied soil remediation technology. However, under certain conditions it cannot achieve the defined cleanup goals, requiring further treatment, for example, through bioremediation (BR). The sequential application of these technologies is presented as a valid option but is not yet entirely studied. This work presents the study of the remediation of ethylbenzene (EB)-contaminated soils, with different soil water and natural organic matter (NOMC) contents, using sequential SVE and BR. The obtained results allow the conclusion that: (1) SVE was sufficient to reach the cleanup goals in 63% of the experiments (all the soils with NOMC below 4%), (2) higher NOMCs led to longer SVE remediation times, (3) BR showed to be a possible and cost-effective option when EB concentrations were lower than 335 mg kgsoil −1, and (4) concentrations of EB above 438 mg kgsoil −1 showed to be inhibitory for microbial activity

Epigenetic reprogramming by TET enzymes impacts co-transcriptional R-loops

PTDC/BIA-MOL/30438/2017 PTDC/MED-OUT/4301/2020 RiboMed 857119 PD/BD/128292/2017 LCF/PR/HP21/52310016 PTDC/BIA-MOL/6624/2020 PTDC/MED-ONC/7864/2020DNA oxidation by ten-eleven translocation (TET) family enzymes is essential for epigenetic reprogramming. The conversion of 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) initiates developmental and cell-type-specific transcriptional programs through mechanisms that include changes in the chromatin structure. Here, we show that the presence of 5hmC in the transcribed gene promotes the annealing of the nascent RNA to the template DNA strand, leading to the formation of an R-loop. Depletion of TET enzymes reduced global R-loops in the absence of gene expression changes, whereas CRISPR-mediated tethering of TET to an active gene promoted the formation of R-loops. The genome-wide distribution of 5hmC and R-loops shows a positive correlation in mouse and human stem cells and overlap in half of all active genes. Moreover, R-loop resolution leads to differential expression of a subset of genes that are involved in crucial events during stem cell proliferation. Altogether, our data reveal that epigenetic reprogramming via TET activity promotes co-transcriptional R-loop formation, disclosing new mechanisms of gene expression regulation.publishersversionpublishe