Molecular modeling and simulation of pH effects in lipid bilayers

Tese de doutoramento, Bioquímica (Bioquímica Teórica), Universidade de Lisboa, Faculdade de Ciências, 2015Biological membranes are the first barrier between cell and the extracellular environment, and also their first via of interaction. These biological structures are complex and diverse but share a common feature: they are all supported by a lipid bilayer. This bilayer is often negative and sensitive to pH and ionic strength. Computational / theoretical methods have been used to understand how these two properties inuence the bilayer structure. However, the methods available at the beginning of our work had two significant bottlenecks that we attempted to get rid of. First, none of the available Poisson{Boltzmann (PB) solvers were able to deal with both periodicity and pKa calculations. The first project was developed to overcome this technical limitation: we used an available PB solver in a new approach to perform pKa calculations taking into account the system periodicity. Secondly, to simulate highly charged membranes, a proper treatment of the ionic strength is crucial and a full neutralization of the system is probably too rough an approximation. Hence, we developed a PB-based method to determine the number of ions that should be added to the simulations. With these two problems solved, we developed a new constant-pH molecular dynamics method to deal with charged lipid bilayers (CpHMD-L). This method allows a proper treatment of the periodicity and ionic strength in model membranes. Finally, we applied our methods to three model systems to illustrate the importance of taking into account protonation / conformation coupling in molecular dynamics simulations, in particular when looking at pH dependent phenomena. To the best of our knowledge, this is the only available method that can deal simultaneously with pH considering the protonation / conformation coupling, periodic boundary conditions in protonation free energy calculations, and a careful treatment of ionic strength. This represents a significant improvement in simulations of model biological membranes. It is now possible to move a step forward in the direction of biological membranes since we are now able to simulate a lipid mixture with several different lipids

Reversibility of prion misfolding by constant-pH molecular dynamics simulations

Tese de mestrado, Bioquímica, Universidade de Lisboa, Faculdade de Ciências, 2010The prion protein (PrP) is the cause of a group of diseases known as Transmissible Spongiform Encephalopathies (TSEs). Creutzfeldt-Jakob and Bovine Spongiform Encephalopathy are examples of TSEs. The normal form of PrP (PrPC) is monomeric and soluble, however, it can misfold into a pathogenic form (PrPSc). This last form has a high content of β-structures and can aggregate forming amyloid fibrils. The mechanism of conversion between PrPC and PrPSc is not completely elucidated but it can be catalyzed by a PrPSc sample (protein-only hypothesis) or it can be induced by an external factor. The pH seems to be a factor that can induce the misfolding transition and it may occur in the endocytic pathway. The pH effect in the structure of PrP was studied recently in Molecular Simulation Group at ITQB [1] and an evident misfolding transition was observed in one simulation at pH 2. The main goal of the present work was to study the effects of a change in pH to 7 in several transient conformations of this simulation. To address this problem, we performed a total of 47 simulations, using our own Constant-pH MD methodology, accounting for a total of 1:25μs. The most significant effect caused by the change to pH 7 is a global stabilization of the protein structure. We observed that some conformational transitions induced by pH 2 were possible to be reverted in many of our simulations, but only in those started from the early moments of the misfolding transition. In other words, if we stop the misfolding process before a major conformational transition takes place, we can revert it. It was not possible to observe a complete reversibility event from a misfolded conformation. Nevertheless, we can not conclude that the transition is irreversible because we can only sample reversible phenomena that happen at sub μs timescale

A Simulated Intermediate State for Folding and Aggregation Provides Insights into ΔN6 β2-Microglobulin Amyloidogenic Behavior

A major component of ex vivo amyloid plaques of patients with dialysis-related amyloidosis (DRA) is a cleaved variant of β2-microglobulin (ΔN6) lacking the first six N-terminal residues. Here we perform a computational study on ΔN6, which provides clues to understand the amyloidogenicity of the full-length β2-microglobulin. Contrary to the wild-type form, ΔN6 is able to efficiently nucleate fibrillogenesis in vitro at physiological pH. This behavior is enhanced by a mild acidification of the medium such as that occurring in the synovial fluid of DRA patients. Results reported in this work, based on molecular simulations, indicate that deletion of the N-terminal hexapeptide triggers the formation of an intermediate state for folding and aggregation with an unstructured strand A and a native-like core. Strand A plays a pivotal role in aggregation by acting as a sticky hook in dimer assembly. This study further predicts that the detachment of strand A from the core is maximized at pH 6.2 resulting into higher aggregation efficiency. The structural mapping of the dimerization interface suggests that Tyr10, His13, Phe30 and His84 are hot-spot residues in ΔN6 amyloidogenesis

Feasibility of a mobile-based system for unsupervised monitoring in Parkinson’s disease

© 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 (https://creativecommons.org/licenses/by/4.0/).Mobile health (mHealth) has emerged as a potential solution to providing valuable ecological information about the severity and burden of Parkinson's disease (PD) symptoms in real-life conditions. Objective: The objective of our study was to explore the feasibility and usability of an mHealth system for continuous and objective real-life measures of patients' health and functional mobility, in unsupervised settings. Methods: Patients with a clinical diagnosis of PD, who were able to walk unassisted, and had an Android smartphone were included. Patients were asked to answer a daily survey, to perform three weekly active tests, and to perform a monthly in-person clinical assessment. Feasibility and usability were explored as primary and secondary outcomes. An exploratory analysis was performed to investigate the correlation between data from the mKinetikos app and clinical assessments. Results: Seventeen participants (85%) completed the study. Sixteen participants (94.1%) showed a medium-to-high level of compliance with the mKinetikos system. A 6-point drop in the total score of the Post-Study System Usability Questionnaire was observed. Conclusions: Our results support the feasibility of the mKinetikos system for continuous and objective real-life measures of a patient's health and functional mobility. The observed correlations of mKinetikos metrics with clinical data seem to suggest that this mHealth solution is a promising tool to support clinical decisions.info:eu-repo/semantics/publishedVersio

Sugar-based bactericides targeting phosphatidylethanolamine-enriched membranes

Free PMC Article: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6242839/Anthrax is an infectious disease caused by Bacillus anthracis, a bioterrorism agent that develops resistance to clinically used antibiotics. Therefore, alternative mechanisms of action remain a challenge. Herein, we disclose deoxy glycosides responsible for specific carbohydrate-phospholipid interactions, causing phosphatidylethanolamine lamellar-to-inverted hexagonal phase transition and acting over B. anthracis and Bacillus cereus as potent and selective bactericides. Biological studies of the synthesized compound series differing in the anomeric atom, glycone configuration and deoxygenation pattern show that the latter is indeed a key modulator of efficacy and selectivity. Biomolecular simulations show no tendency to pore formation, whereas differential metabolomics and genomics rule out proteins as targets. Complete bacteria cell death in 10 min and cellular envelope disruption corroborate an effect over lipid polymorphism. Biophysical approaches show monolayer and bilayer reorganization with fast and high permeabilizing activity toward phosphatidylethanolamine membranes. Absence of bacterial resistance further supports this mechanism, triggering innovation on membrane-targeting antimicrobials.The European Union is gratefully acknowledged for the support of the project “Diagnostic and Drug Discovery Initiative for Alzheimer’s Disease” (D3i4AD), FP7-PEOPLE-2013-IAPP, GA 612347. We thank the Management Authorities of the European Regional Development Fund and the National Strategic Reference Framework for the support of the Incentive System - Research and Technological Development Co-Promotion FACIB Project number 21457. Fundação para a Ciência e a Tecnologia is also acknowledged for the support of projects UID/Multi/00612/2013, FCT/UID/ Multi/04046/2013, IF/00808/2013/CP1159/CT0003, PTDC/BBBBQB/6071/2014, as well as for the post-doc grant SFRH/BPD/42567/2007 (A.M.), the Ph.D. grants SFRH/BDE/51998/2012 (C.D.), and SFRH/BDE/51957/2012 (J.P.P.), both co-sponsored by CIPAN, and also for the Ph.D. grant SFRH/BD/116614/2016 (R.N.).info:eu-repo/semantics/publishedVersio

Parasitas Pulmonares em Pequenos Ruminantes: Mais Conhecimento, Melhor Diagnóstico

A pneumonia parasitária por nematodes da superfamília Trichostrongyloidea e Metastrongyloidea, também conhecida como estrongilose pulmonar, apresenta elevada prevalência nalgumas regiões geográficas, mas tem merecido pouca atenção por parte dos Médicos Veterinários e investigadores [1,2]. Os estudos de prevalência disponíveis assentam predominantemente na deteção de lesões pulmonares em matadouro e no diagnóstico in vivo, com recurso à técnica de Baermann. No entanto, a velocidade de processamento das carcaças no matadouro limita substancialmente a probabilidade de deteção de lesões características de estrongilose e a técnica de Baermann, apesar de ser considerada a gold standard para diagnóstico in vivo, apresenta uma sensibilidade que não ultrapassa os 90%, é demorada e exige conhecimentos técnicos para a sua correta execução [3,4,5]. Em Portugal, do ponto de vista clínico, estas parasitoses são subdiagnosticadas, na medida em que a pesquisa de parasitas pulmonares raramente é incluída nos testes parasitológicos de rotina, requisitados pelos Médicos Veterinários aos laboratórios [6], o que, associado à escassez de investigação científica na área, cria uma lacuna no conhecimento da distribuição geográfica e de outros aspetos da epidemiologia destas infeções, comprometendo o seu tratamento e controlo. Apesar da desparasitação regular (anual ou bianual) de pequenos ruminantes estar amplamente instituída, alguns princípios ativos frequentemente utilizados para o controlo de parasitas gastrointestinais são pouco eficazes nos nematodes pulmonares, particularmente nalgumas espécies da família Protostrongylidae, pelo que a abordagem terapêutica deve ser integrada e assente no diagnóstico parasitológico prévio. Assim, este trabalho, assente na revisão da bibliografia disponível e na experiência prática dos técnicos e investigadores do Laboratório de Anatomia Patológica da Escola Superior Agrária de Viseu (LAP, ESAV) e do Laboratório de Parasitologia Victor Caeiro da Universidade de Évora (LPVC, UE), pretende sensibilizar os profissionais de saúde animal e investigadores dedicados aos pequenos ruminantes para as infeções por nematodes pulmonares, fornecendo ainda ferramentas de diagnóstico laboratorial acessíveis e orientações para a abordagem terapêutica e profilática.info:eu-repo/semantics/publishedVersio

Smartphone-Based Body Location-Independent Functional Mobility Analysis in Patients with Parkinson’s Disease: A Step towards Precise Medicine

Ecological evaluation of gait using mobile technologies provides crucial information regarding the evolution of symptoms in Parkinson’s disease (PD). However, the reliability and validity of such information may be influenced by the smartphone’s location on the body. This study analyzed how the smartphone location affects the assessment of PD patients’ gait in a free-living environment. Twenty PD patients (mean ± SD age, 64.3 ± 10.6 years; 9 women (45%) performed 3 trials of a 250 m outdoor walk using smartphones in 5 different body locations (pants pocket, belt, hand, shirt pocket, and a shoulder bag). A method to derive gait-related metrics from smartphone sensors is presented, and its reliability is evaluated between different trials as well as its concurrent validity against optoelectronic and smartphone criteria. Excellent relative reliability was found with all intraclass correlation coefficient values above or equal to 0.85. High absolute reliability was observed in 21 out of 30 comparisons. Bland-Altman analysis revealed a high level of agreement (LoA between 4.4 and 17.5%), supporting the use of the presented method. This study advances the use of mobile technology to accurately and reliably quantify gait-related metrics from PD patients in free-living walking regardless of the smartphone’s location on the body

Halogen bonding: an underestimated player in membrane-ligand interactions

Halogen bonds (XBs) are noncovalent interactions where halogen atoms act as electrophilic species interacting with Lewis bases. These interactions are relevant in biochemical systems being increasingly explored in drug discovery, mainly to modulate protein–ligand interactions, but are also found in engineered protein or nucleic acid systems. In this work, we report direct evidence for the existence of XBs in the context of biological membrane systems thus expanding the scope of application of these interactions. Indeed, our molecular dynamics simulations show the presence of favorable interactions between halobenzene derivatives and both phosphate or ester oxygen acceptors from model phospholipid bilayers, thus supporting the existence of XB mediated phospholipid–halogen recognition phenomena influencing the membrane insertion profile of the ligands and their orientational preferences. This represents a relevant interaction, previously overlooked, eventually determining the pharmacological or toxicological activity of halogenated compounds and hence with potential implications in drug discovery and development, a place where such species account for a significant part of the chemical space. We also provide insights into a potential role for XBs in water-to membrane insertion of halogenated ligands as XBs are systematically observed during this process. Therefore, our data strongly suggests that, as the ubiquitous hydrogen bond, XBs should be accounted for in the development of membrane partition models.</div

Tackling Halogenated Species with PBSA: Effect of Emulating the σ-Hole

To model halogen bond phenomena using classical force fields, an extra-point (EP) of charge is frequently introduced at a given distance from the halogen (X) to emulate the σ-hole. The resulting molecular dynamics (MD) trajectories can be used in subsequent molecular mechanics (MM) combined with Poisson–Boltzmann and surface area calculations (MM PBSA) to estimate protein–ligand binding free energies (∆Gbind). While EP addition improves the MM/MD description of halogen-containing systems, its effect on the calculation of solvation free energies (∆Gsolv) using the PBSA approach is yet to be assessed. As the PBSA calculations depend, among other parameters, on the empirical assignment of radii (PB radii), a problematic issue arises since standard halogen radii are smaller than the typical X· · · EP distances (usually corresponding to Rmin), thus placing the EP within the solvent dielectric. Herein, we performed a comprehensive study on the performance of PBSA (using three different setups) in the calculation of ∆Gsolv values for 142 halogenated compounds (bearing Cl, Br, or I) for which the experimental values are known. By conducting an optimization (minimizing the error against experimental values), we provide a new optimized set of halogen PB radii, for each PBSA setup, that should be used when the EP is located at R min in the context of GAFF. A simultaneous optimization of PB radii and X· · · EP distances shows that a wide range of distance/radius pairs can be used without significant loss of accuracy, therefore laying the basis for expanding this halogen radii optimization strategy to other force fields and EP implementations. As ligand ∆Gsolv estimation is an important term in the determination of protein–ligand ∆Gbind , this work is particularly relevant in the framework of structure-based virtual screening and related computer-aided drug design routines.</div

Dinuclear Zinc-N-Heterocyclic Carbene Complexes for Either the Controlled Ring-Opening Polymerization of Lactide or the Controlled Degradation of Polylactide Under Mild Conditions

International audienc