Novel Loss-of-Function KCNA5 Variants in Pulmonary Arterial Hypertension.

Reduced expression and/or activity of Kv1.5 channels (encoded by KCNA5) is a common hallmark in human or experimental pulmonary arterial hypertension (PAH). Likewise, genetic variants in KCNA5 have been found in patients with PAH, but their functional consequences and potential impact on the disease are largely unknown. Herein, this study aimed to characterize the functional consequences of seven KCNA5 variants found in a cohort of patients with PAH. Potassium currents were recorded by patch-clamp technique in HEK293 cells transfected with wild-type or mutant Kv1.5 cDNA. Flow cytometry, Western blot, and confocal microscopy techniques were used for measuring protein expression and cell apoptosis in HEK293 and human pulmonary artery smooth muscle cells. KCNA5 variants (namely, Arg184Pro and Gly384Arg) found in patients with PAH resulted in a clear loss of potassium channel function as assessed by electrophysiological and molecular modeling analyses. The Arg184Pro variant also resulted in a pronounced reduction of Kv1.5 expression. Transfection with Arg184Pro or Gly384Arg variants decreased apoptosis of human pulmonary artery smooth muscle cells compared with the wild-type cells, demonstrating that KCNA5 dysfunction in both variants affects cell viability. Thus, in addition to affecting channel activity, both variants were associated with impaired apoptosis, a crucial process linked to the disease. The estimated prevalence of dysfunctional KCNA5 variants in the PAH population analyzed was around 1%. The data indicate that some KCNA5 variants found in patients with PAH have critical consequences for channel function, supporting the idea that KCNA5 pathogenic variants may be a causative or contributing factor for PAH.

Lipid-A-dependent and cholesterol-dependent dynamics properties of liposomes from gram-negative bacteria in ESKAPE.

AntiMicrobial Resistance (AMR) is a worldwide health emergency. ESKAPE pathogens include the most relevant AMR bacterial families. In particular, Gram-negative bacteria stand out due to their cell envelope complexity which exhibits strong resistance to antimicrobials. A key element for AMR is the chemical structure of lipid A, modulating the physico-chemical properties of the membrane and permeability to antibiotics. Liposomes are used as models of bacterial membrane infective vesicles. In this work, coarse-grained molecular dynamics simulations were used to model liposomes from ESKAPE Gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa). We captured the role of lipid A, cardiolipin and cholesterol on liposome morphology and physico-chemical properties. Additionally, the reported antimicrobial peptides Cecropin B1, JB95, and PTCDA1-kf, were used to unveil their implications on membrane disruption. This study opens a promising starting point to understand molecular keys of bacterial membranes and to promote the discovery of new antimicrobials to overcome AMR.

Full-Atom Model of the Agonist LPS-Bound Toll-like Receptor 4 Dimer in a Membrane Environment.

The Toll-like receptor 4 (TLR4)/myeloid differentiation factor 2 (MD-2) innate immunity system is a membrane receptor of paramount importance as therapeutic target. Its assembly, upon binding of Gram-negative bacteria lipopolysaccharide (LPS), and also dependent on the membrane composition, finally triggers the immune response cascade. We have combined ab-initio calculations, molecular docking, all-atom molecular dynamics simulations, and thermodynamics calculations to provide the most realistic and complete 3D models of the active full TLR4 complex embedded into a realistic membrane to date. Our studies give functional and structural insights into the transmembrane domain behavior in different membrane environments, the ectodomain bouncing movement, and the dimerization patterns of the intracellular Toll/Interleukin-1 receptor domain. Our work provides TLR4 models as reasonable 3D structures for the (TLR4/MD-2/LPS)2 architecture accounting for the active (agonist) state of the TLR4, and pointing to a signal transduction mechanism across cell membrane. These observations unveil relevant molecular aspects involved in the TLR4 innate immune pathways and will promote the discovery of new TLR4 modulators.

How Long are Polymer Chains in Poly(3,4-ethylenedioxythiophene):Tosylate Films? An Insight from Molecular Dynamics Simulations.

Poly(3,4-ethylenedioxythiophene) (PEDOT) is one of the most important conductive polymers utilized in a variety of applications in organic electronics and bioelectronics and energy storage. PEDOT chains are believed to be rather short, but detailed knowledge of their length is missing because of the challenges in its experimental determination due to insolubility of PEDOT films. Here, we report a molecular dynamics (MD) study of in situ oxidative chemical polymerization and simultaneous crystallization of molecularly doped PEDOT focusing on the determination of its chain lengths at different polymerization temperatures. We find the average chain length to be 6, 7, and 11 monomers for 298, 323 and 373 K, respectively. At the same time, the length distribution is rather broad, for example, between 2 and 16 monomer units for T = 323 K. We demonstrate that the limiting factor determining the chain length is the diffusivity of the reactants (PEDOT monomers and oligomers). We also study the polymer film formation during solvent evaporation, and we find that although crystallization starts and proceeds already during the polymerization and doping phases, it mostly occurs during the evaporation phase. Finally, we believe that our results providing the oligomer chain length and polymerization and crystallization mechanisms obtained by means of MD "computational microscopy" provide an important insight into the morphology of PEDOT that cannot be obtained by other means.

Understanding the Antibacterial Resistance: Computational Explorations in Bacterial Membranes.

Antimicrobial resistance (AMR) represents a major threat to global public health in the 21st century, dramatically increasing the pandemic expectations in the coming years. The ongoing need to develop new antimicrobial treatments that are effective against multi-drug-resistant pathogens has led the research community to investigate innovative strategies to tackle AMR. The bacterial cell envelope has been identified as one of the key molecular players responsible for antibiotic resistance, attracting considerable interest as a potential target for novel antimicrobials effective against AMR, to be used alone or in combination with other drugs. However, the multicomponent complexity of bacterial membranes provides a heterogeneous morphology, which is typically difficult to study at the molecular level by experimental techniques, in spite of the significant development of fast and efficient experimental protocols. In recent years, computational modeling, in particular, molecular dynamics simulations, has proven to be an effective tool to reveal key aspects in the architecture and membrane organization of bacterial cell walls. Here, after a general overview about bacterial membranes, AMR mechanisms, and experimental approaches to study AMR, we review the state-of-the-art computational approaches to investigate bacterial AMR envelopes, including their limitations and challenges ahead. Representative examples illustrate how these techniques improve our understanding of bacterial membrane resistance mechanisms, hopefully leading to the development of novel antimicrobial drugs escaping from bacterial resistance strategies.

Tuning melatonin receptor subtype selectivity in oxadiazolone-based analogues: Discovery of QR2 ligands and NRF2 activators with neurogenic properties.

New multi-target indole and naphthalene derivatives containing the oxadiazolone scaffold as a bioisostere of the melatonin acetamido group have been developed. The novel compounds were characterized at melatonin receptors MT1R and MT2R, quinone reductase 2 (QR2), lipoxygenase-5 (LOX-5), and monoamine oxidases (MAO-A and MAO-B), and also as radical scavengers. We found that selectivity within the oxadiazolone series can be modulated by modifying the side chain functionality and co-planarity with the indole or naphthalene ring. In phenotypic assays, several oxadiazolone-based derivatives induced signalling mediated by the transcription factor NRF2 and promoted the maturation of neural stem-cells into a neuronal phenotype. Activation of NRF2 could be due to the binding of indole derivatives to KEAP1, as deduced from surface plasmon resonance (SPR) experiments. Molecular modelling studies using the crystal structures of QR2 and the KEAP1 Kelch-domain, as well as the recently described X-ray free-electron laser (XFEL) structures of chimeric MT1R and MT2R, provided a rationale for the experimental data and afforded valuable insights for future drug design endeavours.

Computational microscopy study of the granular structure and pH dependence of PEDOT:PSS.

Computational microscopy based on Martini coarse grained molecular dynamics (MD) simulations of a doped conducting polymer poly(3,4-ethylenedioxythiophene)polystyrene sulfonate (best known as PEDOT:PSS) was performed focussing on the formation of the granular structure and PEDOT crystallites, and the effect of pH on the material morphology. The PEDOT:PSS morphology is shown to be sensitive to the initial distribution of PEDOT and PSS in the solution, and the results of the modelling suggest that the experimentally observed granular structure of PEDOT:PSS can be only obtained if the PEDOT/PSS solution is in the dispersive state in the initial crystallization stages. Variation of the pH is demonstrated to strongly affect the morphology of PEDOT:PSS films, altering their structure between granular-type and homogeneous. It also affects the size of crystallites and the relative arrangement of PEDOT and PSS chains. It is shown that the crystallites in PEDOT:PSS are smaller than those in PEDOT with molecular counterions such as PEDOT:tosylate, which is consistent with the available experimental data. The predicted changes of the PEDOT:PSS morphology with variation of the pH can be tested experimentally, and the calculated atomistic picture of PEDOT:PSS films (not accessible by conventional experimental techniques) is instrumental for understanding the material structure and building realistic models of PEDOT:PSS morphology.

Substrate-Dependent Morphology and Its Effect on Electrical Mobility of Doped Poly(3,4-ethylenedioxythiophene) (PEDOT) Thin Films.

Deposition dynamics, crystallization, molecular packing, and electronic mobility of poly(3,4-ethylenedioxythiophene) (PEDOT) thin films are affected by the nature of the substrate. Computational microscopy has been carried out to reveal the morphology-substrate dependence for PEDOT thin films doped with molecular tosylate deposited on different substrates including graphite, Si3N4, silicon, and amorphous SiO2. It is shown that the substrate is instrumental in formation of the lamellar structure. PEDOT films on the ordered substrates (graphite, Si3N4, and silicon) exhibit preferential face-on orientation, with graphite showing the most ordered and pronounced face-on packing. In contrast, PEDOT on amorphous SiO2 exhibits the dominant edge-on orientation, except in the dry state where both packings are equally presented. The role of water and the porosity of the substrate in formation of the edge-on structure on SiO2 is outlined. On the basis of the calculated morphology, the multiscale calculations of the electronic transport and percolative analysis are performed outlining how the character of the substrate affects the electron mobility. It is demonstrated that good crystallinity (PEDOT on graphite substrate) and high content of edge-on (PEDOT on SiO2 substrate) are not enough to achieve the highest electrical in-plane mobility. Instead, the least ordered material with lower degree of the edge-on content (PEDOT on silicon substrate) provides the highest mobility because it exhibits an efficient network of π-π stacked chain extending throughout the entire sample.

Morphology and ion diffusion in PEDOT:Tos. A coarse grained molecular dynamics simulation.

A Martini coarse-grained Molecular Dynamics (MD) model for the doped conducting polymer poly(3,4-ethylenedioxythiophene) (PEDOT) is developed. The morphology of PEDOT:Tos (i.e. PEDOT doped with molecular tosylate) and its crystallization in aqueous solution for different oxidation levels were calculated using the developed method and compared with corresponding all atomistic MD simulations. The diffusion coefficients of Na+ and Cl- ions in PEDOT:Tos are studied using the developed coarse-grained MD approach. It is shown that the diffusion coefficients decrease exponentially as the hydration level is reduced. It is also predicted that the diffusion coefficients decrease when the doping level of PEDOT is increased. The observed behavior is related to the evolution of water clusters and trapping of ions around the polymer matrix as the hydration level changes. The predicted behavior of the ionic diffusion coefficients can be tested experimentally, and we believe that molecular picture of ionic diffusion in PEDOT unraveled in the present study is instrumental for the design of polymeric materials and devices for better and enhanced performance.

Morphology of a self-doped conducting oligomer for green energy applications.

A recently synthesized self-doped conducting oligomer, salt of bis[3,4-ethylenedioxythiophene]3thiophene butyric acid, ETE-S, is a novel promising material for green energy applications. Recently, it has been demonstrated that it can polymerize in vivo, in plant systems, leading to a formation of long-range conducting wires, charge storage and supercapacitive behaviour of living plants. Here we investigate the morphology of ETE-S combining the experimental characterisation using Grazing Incidence Wide Angle X-ray Scattering (GIWAXS) and atomistic molecular dynamics (MD) simulations. The GIWAXS measurements reveal a formation of small crystallites consisting of π-π stacked oligomers (with the staking distance 3.5 Å) that are further organized in h00 lamellae. These experimental results are confirmed by MD calculations, where we calculated the X-ray diffraction pattern and the radial distribution function for the distance between ETE-S chains. Our MD simulations also demonstrate the formation of the percolative paths for charge carriers that extend throughout the whole structure, despite the fact that the oligomers are short (6-9 rings) and crystallites are thin along the π-π stacking direction, consisting of only two or three π-π stacked oligomers. The existence of the percolative paths explains the previously observed high conductivity in in vivo polymerized ETE-S. We also explored the geometrical conformation of ETE-S oligomers and the bending of their aliphatic chains as a function of the oligomer lengths.

pH Dependence of γ-Aminobutyric Acid Iontronic Transport.

The organic electronic ion pump (OEIP) has been developed as an "iontronic" tool for delivery of biological signaling compounds. OEIPs rely on electrophoretically "pumping" charged compounds, either at neutral or shifted pH, through an ion-selective channel. Significant shifts in pH lead to an abundance of H+ or OH-, which are delivered along with the intended substance. While this method has been used to transport various neurotransmitters, the role of pH has not been explored. Here we present an investigation of the role of pH on OEIP transport efficiency using the neurotransmitter γ-aminobutyric acid (GABA) as the model cationic delivery substance. GABA transport is evaluated at various pHs using electrical and chemical characterization and compared to molecular dynamics simulations, all of which agree that pH 3 is ideal for GABA transport. These results demonstrate a useful method for optimizing transport of other substances and thus broadening OEIP applications.

Molecular Dynamics Study of Morphology of Doped PEDOT: From Solution to Dry Phase.

Morphology of the conducting polymer PEDOT:TOS (poly(3,4-ethylenedioxythiophene) doped with molecular tosylate) and its crystallization in aqueous solution were studied using atomistic molecular dynamics simulations. It was found that (a) PEDOT comprises crystallite aggregates consisting of 3-6 π-π stacked chains. The crystallites are linked by interpenetrating π-π stacked chains such that percolative paths in the structure are formed. (b) The size of the crystallites depends on the water content, but the π-π stacking distance is practically independent of the chain length, charge concentration and water content.

In vivo polymerization and manufacturing of wires and supercapacitors in plants.

Electronic plants, e-Plants, are an organic bioelectronic platform that allows electronic interfacing with plants. Recently we have demonstrated plants with augmented electronic functionality. Using the vascular system and organs of a plant, we manufactured organic electronic devices and circuits in vivo, leveraging the internal structure and physiology of the plant as the template, and an integral part of the devices. However, this electronic functionality was only achieved in localized regions, whereas new electronic materials that could be distributed to every part of the plant would provide versatility in device and circuit fabrication and create possibilities for new device concepts. Here we report the synthesis of such a conjugated oligomer that can be distributed and form longer oligomers and polymer in every part of the xylem vascular tissue of a Rosa floribunda cutting, forming long-range conducting wires. The plant's structure acts as a physical template, whereas the plant's biochemical response mechanism acts as the catalyst for polymerization. In addition, the oligomer can cross through the veins and enter the apoplastic space in the leaves. Finally, using the plant's natural architecture we manufacture supercapacitors along the stem. Our results are preludes to autonomous energy systems integrated within plants and distribute interconnected sensor-actuator systems for plant control and optimization.

Exploring the dynamics and interaction of a full ErbB2 receptor and Trastuzumab-Fab antibody in a lipid bilayer model using Martini coarse-grained force field.

Coarse grained (CG) modeling has been applied to study the influence of the Trastuzumab monoclonal antibody on the structure and dynamics of the full ErbB2 receptor dimer, including the lipid bilayer. The usage of CG models to study such complexes is almost mandatory, at present, due to the large size of the whole system. We will show that the Martini model performs satisfactorily well, giving results well-matched with those obtained by atomistic models as well as with the experimental information existing on homolog receptors. For example, the extra and intracellular domains approach the bilayer surface in both the monomer and dimer cases. The Trastuzumab-Fab hinders the interaction of the receptors with the lipid bilayer. Another interesting effect of the antibody is the disruption of the antiparallel arrangement of the juxtamembrane segments in the dimer case. These findings might help to understand the effect of the antibody on the receptor bioactivity.

Simulation of homology models for the extracellular domains (ECD) of ErbB3, ErbB4 and the ErbB2-ErbB3 complex in their active conformations.

Epidermal growth factor receptors (EGFR) are associated with a number of biological processes and are becoming increasingly recognized as important therapeutic targets against cancer. In this work, we provide models based on homology for the extracellular domains (ECD) of ErbB3 and ErbB4 in their active conformations, including a Heregulin ligand, followed by further refinement of the models by molecular dynamics simulations at atomistic scale. We compare the results with a model built for ErbB2 based on crystallographic information and analyze the common features observed among members of the family, namely, the periscope movement of the dimerization arm and the hinge displacement of domain IV. Finally, we refine a model for the interaction of the ECDs corresponding to a ErbB2-ErbB3 heterodimer, which is widely recognized to have a high impact in cancer development.

Conformational flexibility of the ErbB2 ectodomain and trastuzumab antibody complex as revealed by molecular dynamics and principal component analysis.

Human epidermal growth factor receptor 2 (ErbB2) is a transmembrane oncoprotein that is over expressed in breast cancer. A successful therapeutic treatment is a monoclonal antibody called trastuzumab which interacts with the ErbB2 extracellular domain (ErbB2-ECD). A better understanding of the detailed structure of the receptor-antibody interaction is indeed of prime interest for the design of more effective anticancer therapies. In order to discuss the flexibility of the complex ErbB2-ECD/trastuzumab, we present, in this study, a multi-nanosecond molecular dynamics simulation (MD) together with an analysis of fluctuations, through a principal component analysis (PCA) of this system. Previous to this step and in order to validate the simulations, we have performed a detailed analysis of the variable antibody domain interactions with the extracellular domain IV of ErbB2. This structure has been statically elucidated by x-ray studies. Indeed, the simulation results are in excellent agreement with the available experimental information during the full trajectory. The PCA shows eigenvector fluctuations resulting in a hinge motion in which domain II and C(H) domains approach each other. This move is likely stabilized by the formation of H-bonds and salt bridge interactions between residues of the dimerization arm in the domain II and trastuzumab residues located in the C(H) domain. Finally, we discuss the flexibility of the MD/PCA model in relation with the static x-ray structure. A movement of the antibody toward the dimerization domain of the ErbB2 receptor is reported for the first time. This finding could have important consequences on the biological action of the monoclonal antibody.