Puncturing lipid membranes: onset of pore formation and the role of hydrogen bonding in the presence of flavonoids.

Products of lipid peroxidation induce detrimental structural changes in cell membranes, such as the formation of water pores, which occur in the presence of lipids with partially oxidized chains. However, the influence of another class of products, dicarboxylic acids, is still unclear. These products have greater mobility in the lipid bilayer, which enables their aggregation and the formation of favorable sites for the appearance of pores. Therefore, dodecanedioic acid (DDA) was selected as a model product. Additionally, the influence of several structurally different flavonoids on DDA aggregation via formation of hydrogen bonds with carboxyl groups was investigated. The molecular dynamics of DDA in DOPC lipid bilayer revealed the formation of aggregates extending over the hydrophobic region of the bilayer and increasing its polarity. Consequently, water penetration and the appearance of water wires was observed, representing a new step in the mechanism of pore formation. Furthermore, DDA molecules were found to interact with lipid polar groups, causing them to be buried in the bilayer. The addition of flavonoids to the system disrupted aggregate formation, resulting in the displacement of DDA molecules from the center of the bilayer. The placement of DDA and flavonoids in the lipid bilayer was confirmed by small-angle X-ray scattering. Atomic force microscopy and electron paramagnetic resonance were used to characterize the structural properties. The presence of DDA increased bilayer roughness and decreased the ordering of lipid chains, confirming its detrimental effects on the membrane surface, while flavonoids were found to reduce or reverse these changes.

Oxygen Is an Ambivalent Factor for the Differentiation of Human Pluripotent Stem Cells in Cardiac 2D Monolayer and 3D Cardiac Spheroids.

Numerous protocols of cardiac differentiation have been established by essentially focusing on specific growth factors on human pluripotent stem cell (hPSC) differentiation efficiency. However, the optimal environmental factors to obtain cardiac myocytes in network are still unclear. The mesoderm germ layer differentiation is known to be enhanced by low oxygen exposure. Here, we hypothesized that low oxygen exposure enhances the molecular and functional maturity of the cardiomyocytes. We aimed at comparing the molecular and functional consequences of low (5% O2 or LOE) and high oxygen exposure (21% O2 or HOE) on cardiac differentiation of hPSCs in 2D- and 3D-based protocols. hPSC-CMs were differentiated through both the 2D (monolayer) and 3D (embryoid body) protocols using several lines. Cardiac marker expression and cell morphology were assessed. The mitochondrial localization and metabolic properties were evaluated. The intracellular Ca2+ handling and contractile properties were also monitored. The 2D cardiac monolayer can only be differentiated in HOE. The 3D cardiac spheroids containing hPSC-CMs in LOE further exhibited cardiac markers, hypertrophy, steadier SR Ca2+ release properties revealing a better SR Ca2+ handling, and enhanced contractile force. Preserved distribution of mitochondria and similar oxygen consumption by the mitochondrial respiratory chain complexes were also observed. Our results brought evidences that LOE is moderately beneficial for the 3D cardiac spheroids with hPSC-CMs exhibiting further maturity. In contrast, the 2D cardiac monolayers strictly require HOE.

Molecularly imprinted polymers and capillary electrophoresis for sensing phytoestrogens in milk.

Dairy cow feed contains, among other ingredients, soybeans, legumes, and clover, plants that are rich in phytoestrogens. Several publications have reported a positive influence of phytoestrogens on human health; however, several unfavorable effects have also been reported. In this work, a simple, selective, and eco-friendly method of phytoestrogen isolation based on the technique of noncovalent molecular imprinting was developed. Genistein was used as a template, and dopamine was chosen as a functional monomer. A layer of molecularly imprinted polymers was created in a microtitration well plate. The binding capability and selective properties of obtained molecularly imprinted polymers were investigated. The imprinted polymers exhibited higher binding affinity toward chosen phytoestrogen than did the nonimprinted polymers. A selectivity factor of 6.94 was calculated, confirming satisfactory selectivity of the polymeric layer. The applicability of the proposed sensing method was tested by isolation of genistein from a real sample of bovine milk and combined with micellar electrokinetic capillary chromatography with UV-visible detection.

Simultaneous study of mechanobiology and calcium dynamics on hESC-derived cardiomyocytes clusters.

Calcium ions act like ubiquitous second messengers in a wide amount of cellular processes. In cardiac myocytes, Ca2+ handling regulates the mechanical contraction necessary to the heart pump function. The field of intracellular and intercellular Ca2+ handling, employing in vitro models of cardiomyocytes, has become a cornerstone to understand the role and adaptation of calcium signalling in healthy and diseased hearts. Comprehensive in vitro systems and cell-based biosensors are powerful tools to enrich and speed up cardiac phenotypic and drug response evaluation. We have implemented a combined setup to measure contractility and calcium waves in human embryonic stem cells-derived cardiomyocyte 3D clusters, obtained from embryoid body differentiation. A combination of atomic force microscopy to monitor cardiac contractility, and sensitive fast scientific complementary metal-oxide-semiconductor camera for epifluorescence video recording, provided correlated signals in real time. To speed up the integrated data processing, we tested several post-processing algorithms, to improve the automatic detection of relevant functional parameters. The validation of our proposed method was assessed by caffeine stimulation (10mM) and detection/characterization of the induced cardiac response. We successfully report the first simultaneous recording of cardiac contractility and calcium waves on the described cardiac 3D models. The drug stimulation confirmed the automatic detection capabilities of the used algorithms, measuring expected physiological response, such as elongation of contraction time and Ca2+ cytosolic persistence, increased calcium basal fluorescence, and transient peaks. These results contribute to the implementation of novel, integrated, high-information, and reliable experimental systems for cardiac models and drug evaluation.

Application of the Enzymatic Electrochemical Biosensors for Monitoring Non-Competitive Inhibition of Enzyme Activity by Heavy Metals.

The inhibition effect of the selected heavy metals (Ag+, Cd2+, Cu2+, and Hg2+) on glucose oxidase (GOx) enzyme from Aspergillus niger (EC 1.1.3.4.) was studied using a new amperometric biosensor with an electrochemical transducer based on a glassy carbon electrode (GCE) covered with a thin layer of multi-wall carbon nanotubes (MWCNTs) incorporated with ruthenium(IV) oxide as a redox mediator. Direct adsorption of multi-wall carbon nanotubes (MWCNTs) and subsequent covering with Nafion® layer was used for immobilization of GOx. The analytical figures of merit of the developed glucose (Glc) biosensor are sufficient for determination of Glc in body fluids in clinical analysis. From all tested heavy metals, mercury(II) has the highest inhibition effect. However, it is necessary to remember that cadmium and silver ions also significantly inhibit the catalytic activity of GOx. Therefore, the development of GOx biosensors for selective indirect determination of each heavy metal still represents a challenge in the field of bioelectroanalysis. It can be concluded that amperometric biosensors, differing in the utilized enzyme, could find their application in the toxicity studies of various poisons.

Phenotypic assays for analyses of pluripotent stem cell-derived cardiomyocytes.

Stem cell-derived cardiomyocytes (CMs) hold great hopes for myocardium regeneration because of their ability to produce functional cardiac cells in large quantities. They also hold promise in dissecting the molecular principles involved in heart diseases and also in drug development, owing to their ability to model the diseases using patient-specific human pluripotent stem cell (hPSC)-derived CMs. The CM properties essential for the desired applications are frequently evaluated through morphologic and genotypic screenings. Even though these characterizations are necessary, they cannot in principle guarantee the CM functionality and their drug response. The CM functional characteristics can be quantified by phenotype assays, including electrophysiological, optical, and/or mechanical approaches implemented in the past decades, especially when used to investigate responses of the CMs to known stimuli (eg, adrenergic stimulation). Such methods can be used to indirectly determine the electrochemomechanics of the cardiac excitation-contraction coupling, which determines important functional properties of the hPSC-derived CMs, such as their differentiation efficacy, their maturation level, and their functionality. In this work, we aim to systematically review the techniques and methodologies implemented in the phenotype characterization of hPSC-derived CMs. Further, we introduce a novel approach combining atomic force microscopy, fluorescent microscopy, and external electrophysiology through microelectrode arrays. We demonstrate that this novel method can be used to gain unique information on the complex excitation-contraction coupling dynamics of the hPSC-derived CMs.

Forced aggregation and defined factors allow highly uniform-sized embryoid bodies and functional cardiomyocytes from human embryonic and induced pluripotent stem cells.

In vitro human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) can differentiate into functional cardiomyocytes (CMs). Protocols for cardiac differentiation of hESCs and hiPSCs include formation of the three-dimensional cell aggregates called embryoid bodies (EBs). The traditional suspension method for EB formation from clumps of cells results in an EB population heterogeneous in size and shape. In this study we show that forced aggregation of a defined number of single cells on AggreWell plates gives a high number of homogeneous EBs that can be efficiently differentiated into functional CMs by application of defined growth factors in the media. For cardiac differentiation, we used three hESC lines and one hiPSC line. Our contracting EBs and the resulting CMs express cardiac markers, namely myosin heavy chain α and ß, cardiac ryanodine receptor/calcium release channel, and cardiac troponin T, shown by real-time polymerase chain reaction and immunocytochemistry. Using Ca(2+) imaging and atomic force microscopy, we demonstrate the functionality of RyR2 to release Ca(2+) from the sarcoplasmic reticulum as well as reliability in contractile and beating properties of hESC-EBs and hiPSC-EBs upon the stimulation or inhibition of the ß-adrenergic pathway.

Hyaluronic acid-based hydrogels with tunable mechanics improved structural and contractile properties of cells.

Extracellular matrix (ECM) regulates cellular responses through mechanotransduction. The standard approach of in vitro culturing on plastic surfaces overlooks this phenomenon, so there is a need for biocompatible materials that exhibit adjustable mechanical and structural properties, promote cell adhesion and proliferation at low cost and for use in 2D or 3D cell cultures. This study presents a new tunable hydrogel system prepared from high-molecular hyaluronic acid (HA), Bovine serum albumin (BSA), and gelatin cross-linked using EDC/NHS. Hydrogels with Young's moduli (E) ranging from subunit to units of kilopascals were prepared by gradually increasing HA and BSA concentrations. Concentrated high-molecular HA network led to stiffer hydrogel with lower cluster size and swelling capacity. Medium and oxygen diffusion capability of all hydrogels showed they are suitable for 3D cell cultures. Mechanical and structural changes of mouse embryonic fibroblasts (MEFs) on hydrogels were compared with cells on standard cultivation surfaces. Experiments showed that hydrogels have suitable mechanical and cell adhesion capabilities, resulting in structural changes of actin filaments. Lastly, applying hydrogel for a more complex HL-1 cell line revealed improved mechanical and electrophysiological contractile properties.

YAP Signaling Regulates the Cellular Uptake and Therapeutic Effect of Nanoparticles.

Interactions between living cells and nanoparticles are extensively studied to enhance the delivery of therapeutics. Nanoparticles size, shape, stiffness, and surface charge are regarded as the main features able to control the fate of cell-nanoparticle interactions. However, the clinical translation of nanotherapies has so far been limited, and there is a need to better understand the biology of cell-nanoparticle interactions. This study investigates the role of cellular mechanosensitive components in cell-nanoparticle interactions. It is demonstrated that the genetic and pharmacologic inhibition of yes-associated protein (YAP), a key component of cancer cell mechanosensing apparatus and Hippo pathway effector, improves nanoparticle internalization in triple-negative breast cancer cells regardless of nanoparticle properties or substrate characteristics. This process occurs through YAP-dependent regulation of endocytic pathways, cell mechanics, and membrane organization. Hence, the study proposes targeting YAP may sensitize triple-negative breast cancer cells to chemotherapy and increase the selectivity of nanotherapy.

Effect of titanium nanostructured surface on fibroblast behavior.

As the consumption of implants increases, so do the requirements for individual types of implants, for example, improved biocompatibility or longevity. Therefore, the nano-modification of the titanium surface is often chosen. The aim was to characterize the modified surface with a focus on medical applications. The titanium surface was modified by the anodic oxidation method to form nanotubes. Subsequently, the material was characterized and analyzed for medical applications-surface morphology, surface wettability, chemical composition, and release of ions into biological fluids. A human gingival fibroblasts (HGFb) cell line was used in the viability study. A homogeneous layer of nanotubes of defined dimensions was formed on the titanium surface, ensuring the material's biocompatibility-the preparation conditions influence the resulting properties of the nanostructured surface. Nanostructured titanium exhibited more suitable characteristics (e.g., wettability, roughness, ion release) for biological applications than compared to pure titanium. It was possible to understand the behavior of the modified layer on the titanium surface and its effect on cell behavior. Another contribution of this work is the combination of material characterization (ion release) with the study of cytocompatibility (direct contact of cells with metals).

Unveiling the nanotoxicological aspects of Se nanomaterials differing in size and morphology.

Although the general concept of nanotechnology relies on exploitation of size-dependent properties of nanoscaled materials, the relation between the size/morphology of nanoparticles with their biological activity remains not well understood. Therefore, we aimed at investigating the biological activity of Se nanoparticles, one of the most promising candidates of nanomaterials for biomedicine, possessing the same crystal structure, but differing in morphology (nanorods vs. spherical particles) and aspect ratios (AR, 11.5 vs. 22.3 vs. 1.0) in human cells and BALB/c mice. Herein, we report that in case of nanorod-shaped Se nanomaterials, AR is a critical factor describing their cytotoxicity and biocompatibility. However, spherical nanoparticles (AR 1.0) do not fit this statement and exhibit markedly higher cytotoxicity than lower-AR Se nanorods. Beside of cytotoxicity, we also show that morphology and size substantially affect the uptake and intracellular fate of Se nanomaterials. In line with in vitro data, in vivo i.v. administration of Se nanomaterials revealed the highest toxicity for higher-AR nanorods followed by spherical nanoparticles and lower-AR nanorods. Moreover, we revealed that Se nanomaterials are able to alter intracellular redox homeostasis, and affect the acidic intracellular vesicles and cytoskeletal architecture in a size- and morphology-dependent manner. Although the tested nanoparticles were produced from the similar sources, their behavior differs markedly, since each type is promising for several various application scenarios, and the presented testing protocol could serve as a concept standardizing the biological relevance of the size and morphology of the various types of nanomaterials and nanoparticles.

New insight into the biocompatibility/toxicity of graphene oxides and their reduced forms on Chlamydomonas reinhardtii.

Graphene oxides (GOs) and their reduced forms are often discussed both positively and negatively due to the lack of information about their chemistry and structure. This study utilized GOs with two sheet sizes that were further reduced by two reducing agents (sodium borohydride and hydrazine) to obtain two different degrees of reduction. The synthesized nanomaterials were characterized using scanning electron microscopy (SEM), atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), elemental analysis (EA), Fourier transform infrared (FTIR) spectroscopy, and Raman spectroscopy (RA) to understand their chemistry and structure. The second focus of our investigation included in vitro testing of the biocompatibility/toxicity of these materials on a model organism, the freshwater microalga Chlamydomonas reinhardtii. The effects were studied on the basis of biological endpoints complemented by biomass investigation (FTIR spectroscopy, EA, and atomic absorption spectrometry (AAS)). The results showed that the biocompatibility/toxicity of GOs is dependent on their chemistry and structure and that it is impossible to generalize the toxicity of graphene-based nanomaterials.

Atomic force spectroscopy is a promising tool to study contractile properties of cardiac cells.

Atomic Force Microscopy (AFM) is a rather new method with increasing potential in analyzing various biosamples. Moreover, it can serve as a multi-functional device in the studies of biological specimens under physiological conditions. However, it is becoming increasingly popular among biochemists and biologists, it is not often used in cardiology. Heart disease causes millions of deaths every year. A common point in all heart diseases is the inferior function of cardiomyocytes, which are the contracting unit of the heart. Therefore, these cells are a frequent target of scientific studies. However, few of them use innovative techniques such as AFM and related methods or parallel combinations with complementary techniques such as cell potential measurements. The aim of this review is to illustrate the potential of AFM microscopy in the study of cardiac cells, comparing it with related methods and other techniques used to study the biomechanics and electrophysiology of this cell type. A better understanding of these methods may lead to a better description of the pathophysiology of the heart disease and an improved understanding of the effect of selected drugs.

hESC derived cardiomyocyte biosensor to detect the different types of arrhythmogenic properties of drugs.

In the present work, we introduce a new cell-based biosensor for detecting arrhythmias based on a novel utilization of the combination of the Atomic Force Microscope (AFM) lateral force measurement as a nanosensor with a dual 3D cardiomyocyte syncytium. Two spontaneously coupled clusters of cardiomyocytes form this. The syncytium's functional contraction behavior was assessed using video sequences analyzed with Musclemotion ImageJ/Fiji software, and immunocytochemistry evaluated phenotype composition. The application of caffeine solution induced arrhythmia as a model drug, and its spontaneous resolution was monitored by AFM lateral force recording and interpretation and calcium fluorescence imaging as a reference method describing non-synchronized contractions of cardiomyocytes. The phenotypic analysis revealed the syncytium as a functional contractile and conduction cardiac behavior model. Calcium fluorescence imaging was used to validate that AFM fully enabled to discriminate cardiac arrhythmias in this in vitro cellular model. The described novel 3D hESCs-based cellular biosensor is suitable to detect arrhythmic events on the level of cardiac contractile and conduction tissue cellular model. The resulting biosensor allows for screening of arrhythmogenic properties of tailored drugs enabling its use in precision medicine.

Measurement of Liver Stiffness using Atomic Force Microscopy Coupled with Polarization Microscopy.

Matrix stiffening has been recognized as one of the key drivers of the progression of liver fibrosis. It has profound effects on various aspects of cell behavior such as cell function, differentiation, and motility. However, as these processes are not homogeneous throughout the whole organ, it has become increasingly important to understand changes in the mechanical properties of tissues on the cellular level. To be able to monitor the stiffening of collagen-rich areas within the liver lobes, this paper presents a protocol for measuring liver tissue elastic moduli with high spatial precision by atomic force microscopy (AFM). AFM is a sensitive method with the potential to characterize local mechanical properties, calculated as Young's (also referred to as elastic) modulus. AFM coupled with polarization microscopy can be used to specifically locate the areas of fibrosis development based on the birefringence of collagen fibers in tissues. Using the presented protocol, we characterized the stiffness of collagen-rich areas from fibrotic mouse livers and corresponding areas in the livers of control mice. A prominent increase in the stiffness of collagen-positive areas was observed with fibrosis development. The presented protocol allows for a highly reproducible method of AFM measurement, due to the use of mildly fixed liver tissue, that can be used to further the understanding of disease-initiated changes in local tissue mechanical properties and their effect on the fate of neighboring cells.

Passive Diffusion vs Active pH-Dependent Encapsulation of Tyrosine Kinase Inhibitors Vandetanib and Lenvatinib into Folate-Targeted Ferritin Delivery System.

INTRODUCTION: The present study reports on examination of the effects of encapsulating the tyrosine kinase inhibitors (TKIs) vandetanib and lenvatinib into a biomacromolecular ferritin-based delivery system. METHODS: The encapsulation of TKIs was performed via two strategies: i) using an active reversible pH-dependent reassembly of ferritin´s quaternary structure and ii) passive loading of hydrophobic TKIs through the hydrophobic channels at the junctions of ferritin subunits. After encapsulation, ferritins were surface-functionalized with folic acid promoting active-targeting capabilities. RESULTS: The physico-chemical and nanomechanical analyses revealed that despite the comparable encapsulation efficiencies of both protocols, the active loading affects stability and rigidity of ferritins, plausibly due to their imperfect reassembly. Biological experiments with hormone-responsive breast cancer cells (T47-D and MCF-7) confirmed the cytotoxicity of encapsulated and folate-targeted TKIs to folate-receptor positive cancer cells, but only limited cytotoxic effects to healthy breast epithelium. Importantly, the long-term cytotoxic experiments revealed that compared to the pH-dependent encapsulation, the passively-loaded TKIs exert markedly higher anticancer activity, most likely due to undesired influence of harsh acidic environment used for the pH-dependent encapsulation on the TKIs' structural and functional properties. CONCLUSION: Since the passive loading does not require a reassembly step for which acids are needed, the presented investigation serves as a solid basis for future studies focused on encapsulation of small hydrophobic molecules.

Aminophylline Induces Two Types of Arrhythmic Events in Human Pluripotent Stem Cell-Derived Cardiomyocytes.

Cardiac side effects of some pulmonary drugs are observed in clinical practice. Aminophylline, a methylxanthine bronchodilator with documented proarrhythmic action, may serve as an example. Data on the action of aminophylline on cardiac cell electrophysiology and contractility are not available. Hence, this study was focused on the analysis of changes in the beat rate and contraction force of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) and HL-1 cardiomyocytes in the presence of increasing concentrations of aminophylline (10 µM-10 mM in hPSC-CM and 8-512 µM in HL-1 cardiomyocytes). Basic biomedical parameters, namely, the beat rate (BR) and contraction force, were assessed in hPSC-CMs using an atomic force microscope (AFM). The beat rate changes under aminophylline were also examined on the HL-1 cardiac muscle cell line via a multielectrode array (MEA). Additionally, calcium imaging was used to evaluate the effect of aminophylline on intracellular Ca2+ dynamics in HL-1 cardiomyocytes. The BR was significantly increased after the application of aminophylline both in hPSC-CMs (with 10 mM aminophylline) and in HL-1 cardiomyocytes (with 256 and 512 µM aminophylline) in comparison with controls. A significant increase in the contraction force was also observed in hPSC-CMs with 10 µM aminophylline (a similar trend was visible at higher concentrations as well). We demonstrated that all aminophylline concentrations significantly increased the frequency of rhythm irregularities (extreme interbeat intervals) both in hPSC-CMs and HL-1 cells. The occurrence of the calcium sparks in HL-1 cardiomyocytes was significantly increased with the presence of 512 µM aminophylline. We conclude that the observed aberrant cardiomyocyte response to aminophylline suggests an arrhythmogenic potential of the drug. The acquired data represent a missing link between the arrhythmic events related to the aminophylline/theophylline treatment in clinical practice and describe cellular mechanisms of methylxanthine arrhythmogenesis. An AFM combined with hPSC-CMs may serve as a robust platform for direct drug effect screening.

Atomic force microscopy and surface plasmon resonance for real-time single-cell monitoring of bacteriophage-mediated lysis of bacteria.

The growing incidence of multidrug-resistant bacterial strains presents a major challenge in modern medicine. Antibiotic resistance is often exhibited by Staphylococcus aureus, which causes severe infections in human and animal hosts and leads to significant economic losses. Antimicrobial agents with enzymatic activity (enzybiotics) and phage therapy represent promising and effective alternatives to classic antibiotics. However, new tools are needed to study phage-bacteria interactions and bacterial lysis with high resolution and in real-time. Here, we introduce a method for studying the lysis of S. aureus at the single-cell level in real-time using atomic force microscopy (AFM) in liquid. We demonstrate the ability of the method to monitor the effect of the enzyme lysostaphin on S. aureus and the lytic action of the Podoviridae phage P68. AFM allowed the topographic and biomechanical properties of individual bacterial cells to be monitored at high resolution over the course of their lysis, under near-physiological conditions. Changes in the stiffness of S. aureus cells during lysis were studied by analyzing force-distance curves to determine Young's modulus. This allowed observing a progressive decline in cellular stiffness corresponding to the disintegration of the cell envelope. The AFM experiments were complemented by surface plasmon resonance (SPR) experiments that provided information on the kinetics of phage-bacterium binding and the subsequent lytic processes. This approach forms the foundation of an innovative framework for studying the lysis of individual bacteria that may facilitate the further development of phage therapy.

Myeloperoxidase mediated alteration of endothelial function is dependent on its cationic charge.

Endothelial cell (EC) glycocalyx (GLX) comprise a multicomponent layer of proteoglycans and glycoproteins. Alteration of its integrity contributes to chronic vascular inflammation and leads to the development of cardiovascular diseases. Myeloperoxidase (MPO), a highly abundant enzyme released by polymorphonuclear neutrophils, binds to the GLX and deleteriously affects vascular EC functions. The focus of this study was to elucidate the mechanisms of MPO-mediated alteration of GLX molecules, and to unravel subsequent changes in endothelial integrity and function. MPO binding to GLX of human ECs and subsequent internalization was mediated by cell surface heparan sulfate chains. Moreover, interaction of MPO, which is carrying a cationic charge, with anionic glycosaminoglycans (GAGs) resulted in reduction of their relative charge. By means of micro-viscometry and atomic force microscopy, we disclosed that MPO can crosslink GAG chains. MPO-dependent modulation of GLX structure was further supported by alteration of wheat germ agglutinin staining. Increased expression of ICAM-1 documented endothelial cell activation by both catalytically active and also inactive MPO. Furthermore, MPO increased vascular permeability connected with reorganization of intracellular junctions, however, this was dependent on MPO's catalytic activity. Novel proteins interacting with MPO during transcytosis were identified by proteomic analysis. Altogether, these findings provide evidence that MPO through interaction with GAGs modulates overall charge of the GLX, causing modification of its structure and thus affecting EC function. Importantly, our results also suggest a number of proteins interacting with MPO that possess a variety of cellular localizations and functions.

The Structural Integrity of the Model Lipid Membrane during Induced Lipid Peroxidation: The Role of Flavonols in the Inhibition of Lipid Peroxidation.

The structural integrity, elasticity, and fluidity of lipid membranes are critical for cellular activities such as communication between cells, exocytosis, and endocytosis. Unsaturated lipids, the main components of biological membranes, are particularly susceptible to the oxidative attack of reactive oxygen species. The peroxidation of unsaturated lipids, in our case 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), induces the structural reorganization of the membrane. We have employed a multi-technique approach to analyze typical properties of lipid bilayers, i.e., roughness, thickness, elasticity, and fluidity. We compared the alteration of the membrane properties upon initiated lipid peroxidation and examined the ability of flavonols, namely quercetin (QUE), myricetin (MCE), and myricitrin (MCI) at different molar fractions, to inhibit this change. Using Mass Spectrometry (MS) and Fourier Transform Infrared Spectroscopy (FTIR), we identified various carbonyl products and examined the extent of the reaction. From Atomic Force Microscopy (AFM), Force Spectroscopy (FS), Small Angle X-Ray Scattering (SAXS), and Electron Paramagnetic Resonance (EPR) experiments, we concluded that the membranes with inserted flavonols exhibit resistance against the structural changes induced by the oxidative attack, which is a finding with multiple biological implications. Our approach reveals the interplay between the flavonol molecular structure and the crucial membrane properties under oxidative attack and provides insight into the pathophysiology of cellular oxidative injury.