Model-based mutation testing for test generation and adequacy analysis

Die Mutationsanalyse wurde in den Siebzigerjahren primär zur Beurteilung der Effizienz einer vorgegebenen Testfallmenge eingeführt. Zur systematischen Modifizierung des Quellcodes des zu prüfenden Systems (Prüfling) und somit zur Simulierung typischer Programmierfehler benutzt das Verfahren spezielle Mutationsoperatoren. Für jeden der erzeugten Mutanten wird überprüft, ob die gegebene Testfallmenge den bzw. die injizierten Fehler erkennt. Das Verhältnis aus erkannten zu nicht erkannten Fehlern wird abschließend zur Beurteilung der Effizienz der gegebenen Testfallmenge genutzt. Die vorliegende Arbeit führt eine modellbasierte Mutationsanalyse ein. Anstatt des Quellcodes des zu prüfenden Systems wird nun das gegebene, als korrekt angenommene Modell des Systems systematisch modifiziert. Für jeden so erzeugten Mutanten wird eine Testfallmenge mittels eines modellbasierten Testfallgenerators generiert, welche auf das zu prüfende System angewandt wird. Im Gegensatz zur code-basierten Mutationsanalyse ermöglicht es diese modellbasierte Sichtweise, auch "echte", d.h. nicht-injizierte Fehler im Prüfling zu erkennen. Ein weiterer Vorteil ist, dass der Quellcode des Prüflings nicht benötigt wird, weil die Mutationsanalyse auf Modellebene durchgeführt wird. Das erleichtert den Einsatz in der Praxis, weil der Quellcode des Prüflings nicht immer verfügbar ist. Der Ansatz und die benutzten elementaren Mutationsoperatoren werden zunächst syntaktisch anhand von gerichteten Graphen erläutert. Zur Berücksichtigung praktischer Belange werden exemplarisch einige bekannte Modelle herangezogen: Ereignis-Sequenz-Graphen, endliche Automaten und Statecharts. Mit Hilfe dieser Modelle identifizieren und analysieren mehrere Fallstudien die charakteristischen Eigenschaften und Vorteile des eingeführten Ansatzes.Mutation analysis was introduced in the seventies of the last century to assess the efficiency of a given test set. This approach makes use of specific mutation operators to systematically modify the source code of the system under test (SUT) to simulate typical programming errors. For each of the generated mutants, it checks whether or not the given test set detects the injected fault(s). The ratio of detected to undetected faults is finally used to assess the efficiency of the given test set. This thesis introduces a model-based mutation analysis. Instead of the source code of the SUT, its given model, which is assumed to be correct, is systematically modified. A test set is generated for each mutant using a model-based test generation algorithm, which is then applied to the SUT. In contrast to code-based mutation analysis the present model-based approach also enables the detection of "real," non-injected faults in the SUT. Another advantage is that the source code of the SUT is not needed, as the mutation analysis is performed on the model level. This eases the use in practice since the source code of the SUT is not always available. The beginning of the thesis explains the approach and syntactically introduces the basic mutation operators by means of directed graphs. To take the practical issues into account, well-known models are considered: event sequence graphs, finite-state machines, and statecharts. Using these models, several case studies identify and analyze the characteristics and advantages of the approach.Tag der Verteidigung: 07.06.2011Paderborn, Univ., Diss., 201

25-hydroxycholesterol effect on membrane structure and mechanical properties

© 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).Cholesterol is responsible for the plasticity of plasma membranes and is involved in physiological and pathophysiological responses. Cholesterol homeostasis is regulated by oxysterols, such as 25-hydroxycholesterol. The presence of 25-hydroxycholesterol at the membrane level has been shown to interfere with several viruses' entry into their target cells. We used atomic force microscopy to assess the effect of 25-hydroxycholesterol on different properties of supported lipid bilayers with controlled lipid compositions. In particular, we showed that 25-hydroxycholesterol inhibits the lipid-condensing effects of cholesterol, rendering the bilayers less rigid. This study indicates that the inclusion of 25-hydroxycholesterol in plasma membranes or the conversion of part of their cholesterol content into 25-hydroxycholesterol leads to morphological alterations of the sphingomyelin (SM)-enriched domains and promotes lipid packing inhomogeneities. These changes culminate in membrane stiffness variations.This work was supported by Fundação para a Ciência e a Tecnologia—Ministério da Ciência, Tecnologia e Ensino Superior (FCT-MCTES, Portugal) project PTDC/BBB-BQB/3494/2014, as well as by the FCT-MCTES program of “stimulus of scientific employment” CEECIND/02961/2017 to MMD.info:eu-repo/semantics/publishedVersio

Functional role of water in membranes updated: A tribute to Träuble

The classical view of a cell membrane is as a hydrophobic slab in which only nonpolar solutes can dissolve and permeate. However, water-soluble non-electrolytes such as glycerol, erythritol, urea and others can permeate lipid membranes in the liquid crystalline state. Moreover, recently polar amino acid's penetration has been explained by means of molecular dynamics in which appearance of water pockets is postulated. According to Träuble (1971), water diffuses across the lipid membranes by occupying holes formed in the lipid matrix due to fluctuations of the acyl chain trans-gauche isomers. These holes, named "kinks" have the molecular dimension of CH2 vacancies. The condensation of kinks may form aqueous spaces into which molecular species of the size of low molecular weight can dissolve. This molecular view can explain permeability properties considering that water may be distributed along the hydrocarbon chains in the lipid matrix. The purpose of this review is to consolidate the mechanism anticipated by Träuble by discussing recent data in literature that directly correlates the molecular state of methylene groups of the lipids with the state of water in each of them. In addition, the structural properties of water near the lipid residues can be related with the water activity triggering kink formation by changes in the head group conformation that induces the propagation along the acyl chains and hence to the diffusion of water.Fil: Disalvo, Edgardo Anibal. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia de Santiago del Estero. Universidad Nacional de Santiago del Estero. Centro de Investigaciones y Transferencia de Santiago del Estero; ArgentinaFil: Pinto, Oscar Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia de Santiago del Estero. Universidad Nacional de Santiago del Estero. Centro de Investigaciones y Transferencia de Santiago del Estero; ArgentinaFil: Martini, María Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Metabolismo del Fármaco. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Metabolismo del Fármaco; ArgentinaFil: Bouchet, Ana María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia de Santiago del Estero. Universidad Nacional de Santiago del Estero. Centro de Investigaciones y Transferencia de Santiago del Estero; ArgentinaFil: Hollmann, Axel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia de Santiago del Estero. Universidad Nacional de Santiago del Estero. Centro de Investigaciones y Transferencia de Santiago del Estero; Argentina. Universidad Nacional de Quilmes; ArgentinaFil: Frías, María de los Ángeles. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia de Santiago del Estero. Universidad Nacional de Santiago del Estero. Centro de Investigaciones y Transferencia de Santiago del Estero; Argentin

Evaluation of the Defay-Prigogine model for the membrane interphase in relation to biological response in membrane-protein interactions

Surface water activity appears as a common factor when the interaction of several aqueous soluble and surface active proteins with lipid membranes of different composition is measured by the changes in surface pressure of a lipid monolayer. The perturbation of the lipid surface caused by aqueous soluble proteins depends on the composition of the hydrocarbon phases, either modified by unsaturated bonds in the acyl chains or by inclusion of cholesterol. The cut-off (critical) surface pressure in monolayers, at which no effect of the proteins is found, is related to the composition of the head group region. The perturbation of surface pressure is produced by proteins when the area per lipid is above just 4% larger than that corresponding to the hydration shell of the phospholipid head groups found in the cut -off. This area excess gives place to regions in which the chemical potential of water changes with respect to bulk water. According to the Defay-Prigogine relation this interfacial water activity is the reason of the surface pressure increase induced by aqueous soluble proteins injected in the subphase. As predicted by solution chemistry, the increase of surface pressure is independent of the protein nature but depends on the water surface state determined by the lipid composition.Fil: Disalvo, Edgardo Anibal. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia de Santiago del Estero; Argentina;Fil: Hollmann, Axel. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnologia. Laboratorio de Microbiologia Molecular; Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Analítica y Fisicoquímica. Cátedra de Química General e Inorgánica; Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia de Santiago del Estero; Argentina;Fil: Semorile, Liliana Carmen. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Microbiología Molecular; Argentina;Fil: Martini, María Florencia. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Tecnología Farmacéutica; Argentina

Decomposition of Random Errors Inherent to HOAPS-3.2 Near-Surface Humidity Estimates Using Multiple Triple Collocation Analysis

Latent heat fluxes (LHF) play an essential role in the global energy budget and are thus important for understanding the climate system. Satellite-based remote sensing permits a large-scale determination of LHF, which, amongst others, are based on near-surface specific humidity qa. However, the qa random retrieval error (Etot) remains unknown. Here, a novel approach is presented to quantify the error contributions to pixel-level qa of the Hamburg Ocean Atmosphere Parameters and Fluxes from Satellite (HOAPS, version 3.2) dataset. The methodology makes use of multiple triple collocation (MTC) analysis between 1995-2008 over the global ice-free oceans. Apart from satellite records, these datasets include selected ship records extracted from the Seewetteramt Hamburg (SWA) archive and the International Comprehensive Ocean-Atmosphere Data Set (ICOADS), serving as the in-situ ground reference. The MTC approach permits the derivation of Etot as the sum of model uncertainty EM and sensor noise EN, while random uncertainties due to in-situ measurement errors (Eins) and collocation (EC) are isolated concurrently. Results show an Etot average of 1.1 ± 0.3 g kg-1, whereas the mean EC (Eins) is in the order of 0.5 ± 0.1 g kg-1 (0.5 ± 0.3 g kg-1). Regional analyses indicate a maximum of Etot exceeding 1.5 g kg-1 within humidity regimes of 12-17 g kg-1, associated with the single-parameter, multilinear qa retrieval applied in HOAPS. Multi-dimensional bias analysis reveals that global maxima are located off the Arabian Peninsula

The rigid amphipathic fusion Inhibitor dUY11 acts through photosensitization of viruses

Copyright © 2014, American Society for Microbiology. All Rights Reserved.Supplemental material for this article may be found at http://dx.doi.org/10.1128 /JVI.02907-13.Rigid amphipathic fusion inhibitors (RAFIs) are lipophilic inverted-cone-shaped molecules thought to antagonize the membrane curvature transitions that occur during virus-cell fusion and are broad-spectrum antivirals against enveloped viruses (Broad-SAVE). Here, we show that RAFIs act like membrane-binding photosensitizers: their antiviral effect is dependent on light and the generation of singlet oxygen (1O2), similar to the mechanistic paradigm established for LJ001, a chemically unrelated class of Broad-SAVE. Photosensitization of viral membranes is a common mechanism that underlies these Broad-SAVE.This work was supported by NIH grants U01 AI070495, U01 AI082100, R01 AI069317, and U54 AI065359 (PSWRCE) (to B.L.) and by Fundação para a Ciência e a Tecnologia-Ministério da Educação e Ciência (Portugal) project DELIN-HIVERA/0002/2013 and fellowship SFRH/BPD/72037/2010 (to N.C.S. and A.H., respectively

Conjugation of cholesterol to HIV-1 fusion inhibitor C34 increases peptide-membrane interactions potentiating its action

© 2013 Hollmann et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Recently, the covalent binding of a cholesterol moiety to a classical HIV-1 fusion inhibitor peptide, C34, was shown to potentiate its antiviral activity. Our purpose was to evaluate the interaction of cholesterol-conjugated and native C34 with membrane model systems and human blood cells to understand the effects of this derivatization. Lipid vesicles and monolayers with defined compositions were used as model membranes. C34-cholesterol partitions more to fluid phase membranes that mimic biological membranes. Importantly, there is a preference of the conjugate for liquid ordered membranes, rich in cholesterol and/or sphingomyelin, as observed both from partition and surface pressure studies. In human erythrocytes and peripheral blood mononuclear cells (PBMC), C34-cholesterol significantly decreases the membrane dipole potential. In PBMC, the conjugate was 14- and 115-fold more membranotropic than T-1249 and enfuvirtide, respectively. C34 or cholesterol alone did not show significant membrane activity. The enhanced interaction of C34-cholesterol with biological membranes correlates with its higher antiviral potency. Higher partitions for lipid-raft like compositions direct the drug to the receptor-rich domains where membrane fusion is likely to occur. This intermediary membrane binding step may facilitate the drug delivery to gp41 in its pre-fusion state.This work was funded by Fundação para a Ciência e Tecnologia, Portugal (fellowships SFRH/BPD/72037/2010 and SFRH/BD/42205/2007 to A.H. and P.M.M., respectively, and projects PTDC/QUI-BIQ/104787/2008, PTDC/QUI-BIQ/112929/2009 and VIH/SAU/0047/2011)

Antimicrobial peptides: Interaction with model and biological membranes and synergism with chemical antibiotics

Antimicrobial peptides (AMPs) are promising novel antibiotics since they have shown antimicrobial activity against a wide range of bacterial species, including multiresistant bacteria; however, toxicity is the major barrier to convert antimicrobial peptides into active drugs. A profound and proper understanding of the complex interactions between these peptides and biological membranes using biophysical tools and model membranes seems to be a key factor in the race to develop a suitable antimicrobial peptide therapy for clinical use. In the search for such therapy, different combined approaches with conventional antibiotics have been evaluated in recent years and demonstrated to improve the therapeutic potential of AMPs. Some of these approaches have revealed promising additive or synergistic activity between AMPs and chemical antibiotics. This review will give an insight into the possibilities that physicochemical tools can give in the AMPs research and also address the state of the art on the current promising combined therapies between AMPs and conventional antibiotics, which appear to be a plausible future opportunity for AMPs treatment.Fil: Hollmann, Axel. Universidad Nacional de Santiago del Estero; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes; ArgentinaFil: Martínez, Melina María Belén. Universidad Nacional de Quilmes; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Maturana, Patricia del Valle. Universidad Nacional de Santiago del Estero; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Semorile, Liliana Carmen. Universidad Nacional de Quilmes; ArgentinaFil: Maffia, Paulo Cesar. Universidad Nacional de Quilmes; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Zeta potential beyond materials science: Applications to bacterial systems and to the development of novel antimicrobials

This review summarizes the theory of zeta potential (ZP) and the most relevant data about how it has been used for studying bacteria. We have especially focused on the discovery and characterization of novel antimicrobial compounds. The ZP technique may be considered an indirect tool to estimate the surface potential of bacteria, a physical characteristic that is key to maintaining optimal cell function. For this reason, targeting the bacterial surface is of paramount interest in the development of new antimicrobials. Surface-acting agents have been found to display a remarkable bactericidal effect and have simultaneously revealed a low tendency to trigger resistance. Changes in the bacterial surface as a result of various processes can also be followed by ZP measurements. However, due to the complexity of the bacterial surface, some considerations regarding the assessment of ZP must first be taken into account. Evidence on the application of ZP measurements to the characterization of bacteria and biofilm formation is presented next. We finally discuss the feasibility of using the ZP technique to assess antimicrobial-induced changes in the bacterial surface. Among these changes are those related to the interaction of the agent with different components of the cell envelope, membrane permeabilization, and loss of viability.Fil: Ferreyra Maillard, Anike Paula Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet Noa Sur. Centro de Investigación en Biofísica Aplicada y Alimentos. - Universidad Nacional de Santiago del Estero. Centro de Investigación en Biofísica Aplicada y Alimentos; ArgentinaFil: Espeche, Juan Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet Noa Sur. Centro de Investigación en Biofísica Aplicada y Alimentos. - Universidad Nacional de Santiago del Estero. Centro de Investigación en Biofísica Aplicada y Alimentos; ArgentinaFil: Maturana, Patricia del Valle. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet Noa Sur. Centro de Investigación en Biofísica Aplicada y Alimentos. - Universidad Nacional de Santiago del Estero. Centro de Investigación en Biofísica Aplicada y Alimentos; ArgentinaFil: Cutró, Andrea Carmen. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet Noa Sur. Centro de Investigación en Biofísica Aplicada y Alimentos. - Universidad Nacional de Santiago del Estero. Centro de Investigación en Biofísica Aplicada y Alimentos; Argentina. Universidad Nacional de Santiago del Estero. Facultad de Ciencias Médicas; ArgentinaFil: Hollmann, Axel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet Noa Sur. Centro de Investigación en Biofísica Aplicada y Alimentos. - Universidad Nacional de Santiago del Estero. Centro de Investigación en Biofísica Aplicada y Alimentos; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Microbiología Molecular; Argentin

Studies on interaction of green silver nanoparticles with whole bacteria by surface characterization techniques

The use of silver nanoparticles (AgNPs) with their novel and distinct physical, chemical, and biological properties, has proven to be an alternative for the development of new antibacterial agents. In particular, the possibility to generate AgNPs coated with novel capping agents, such as phytomolecules obtained via a green synthesis (G-AgNPs), is attracting great attention in scientific research. Recently, we showed that membrane interactions seem to be involved in the antibacterial activity of AgNPs obtained via a green chemical synthesis using the aqueous leaf extract of chicory (Cichorium intybus L.). Furthermore, we observed that these G-AgNPs exhibited higher antibacterial activity than those obtained by chemical synthesis. In order to achieve the green AgNPs mode of action as well as their cellular target, we aimed to study the antibacterial activity of this novel green AgNPs against Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria. The effect of the G-AgNPs on the bacterial surface was first evaluated by zeta potential measurements and correlated with direct plate count agar method. Afterwards, atomic force microscopy was applied to directly unravel the effects of these G-AgNPs on bacterial envelopes. Overall, the data obtained in this study seems correlate with a multi-step mechanism by which G-AgNPs-lipid membrane interactions is the first step prior to membrane disruption, resulting in antibacterial activity.Fil: Ferreyra Maillard, Anike Paula Virginia. Universidad Nacional de Santiago del Estero. Instituto de Bionanotecnología del Noa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto de Bionanotecnología del Noa; ArgentinaFil: Gonçalves, Sónia. Universidade Nova de Lisboa. Faculdade de Ciencias Medicas; PortugalFil: Santos, Nuno C.. Universidade Nova de Lisboa. Faculdade de Ciencias Medicas; PortugalFil: López de Mishima, Beatriz A.. Universidad Nacional de Santiago del Estero. Instituto de Bionanotecnología del Noa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto de Bionanotecnología del Noa; ArgentinaFil: Dalmasso, Pablo Roberto. Universidad Tecnologica Nacional. Facultad Regional Cordoba. Departamento de Ingenieria Quimica. Centro de Investigacion y Transferencia En Ingenieria Quimica.; ArgentinaFil: Hollmann, Axel. Universidad Nacional de Quilmes; Argentina. Universidade Nova de Lisboa. Faculdade de Ciencias Medicas; Portugal. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet Noa Sur. Centro de Investigación en Biofísica Aplicada y Alimentos. - Universidad Nacional de Santiago del Estero. Centro de Investigación en Biofísica Aplicada y Alimentos; Argentin