RNA-based liposomes for oral cancer: From biophysical characterization to biological evaluation.

Oral cancer incidence and mortality are increasing over time. The most common therapies for oral cancers are surgery and radiotherapy, either used alone or combined, and immunotherapy can be also an option. Although there are several therapeutic options, none of them are completely effective, and in addition, there are numerous associated side effects. To overcome these limitations, researchers have been trying to reduce these drawbacks by using drug delivery systems that carry drugs for specific delivery to cancer cells. For that purpose, RNA-coated liposomes to selectively deliver the ligands C8 (acridine orange derivative) and dexamethasone to oral cancer cells were produced, characterized, and biologically evaluated. Firstly, the RNA structure and binding interaction with ligands (C8 and dexamethasone) were evaluated by circular dichroism (CD), thermal difference spectroscopy (TDS), nuclear magnetic resonance (NMR) and fluorescence titrations. The biophysical assays evidenced the formation of an RNA hairpin and duplex structure. Moreover, steady-state and time-resolved fluorescence intensity and anisotropy experiments show that C8 forms a complex with RNA and adopts an open conformation upon RNA binding. Then, RNA-coated liposomes were characterized by dynamic light scattering, and diameters near 160 nm were observed. Time-resolved anisotropy measurements of C8 loaded in RNA-functionalized liposomes indicate the co-existence of free C8 in solution (inside the liposome) and C8 bound to RNA at the external liposome surface. The RNA-functionalized liposomes loaded with C8 or dexamethasone mediated a significant reduction in the cell viability of malignant UPCI-SCC-154 cells while maintaining viable non-malignant NHDF cells. Additionally, the liposomes were able to internalize the cells, with higher uptake by the malignant cell line. Overall, the results obtained in this work can contribute to the development of new drug delivery systems based on RNA-coated liposomes.

Developing a Cell-Microcarrier Tissue-Engineered Product for Muscle Repair Using a Bioreactor System.

Fecal incontinence, although not life-threatening, has a high impact on the economy and patient quality of life. So far, available treatments are based on both surgical and nonsurgical approaches. These can range from changes in diet, to bowel training, or sacral nerve stimulation, but none of which provides a long-term solution. New regenerative medicine-based therapies are emerging, which aim at regenerating the sphincter muscle and restoring continence. Usually, these consist of the administration of a suspension of expanded skeletal-derived muscle cells (SkMDCs) to the damaged site. However, this strategy often results in a reduced cell viability due to the need for cell harvesting from the expansion platform, as well as the non-native use of a cell suspension to deliver the anchorage-dependent cells. In this study, we propose the proof-of-concept for the bioprocessing of a new cell delivery method for the treatment of fecal incontinence, obtained by a scalable two-step process. First, patient-isolated SkMDCs were expanded using planar static culture systems. Second, by using a single-use PBS-MINI Vertical-Wheel® bioreactor, the expanded SkMDCs were combined with biocompatible and biodegradable (i.e., directly implantable) poly(lactic-co-glycolic acid) microcarriers prepared by thermally induced phase separation. This process allowed for up to 80% efficiency of SkMDCs to attach to the microcarriers. Importantly, SkMDCs were viable during all the process and maintained their myogenic features (e.g., expression of the CD56 marker) after adhesion and culture on the microcarriers. When SkMDC-containing microcarriers were placed on a culture dish, cells were able to migrate from the microcarriers onto the culture surface and differentiate into multinucleated myotubes, which highlights their potential to regenerate the damaged sphincter muscle after administration into the patient. Overall, this study proposes an innovative method to attach SkMDCs to biodegradable microcarriers, which can provide a new treatment for fecal incontinence.

Gallic Acid-Triethylene Glycol Aptadendrimers Synthesis, Biophysical Characterization and Cellular Evaluation.

Herein, we describe the synthesis of an aptadendrimer by covalent bioconjugation of a gallic acid-triethylene glycol (GATG) dendrimer with the G-quadruplex (G4) AT11 aptamer (a modified version of AS1411) at the surface. We evaluated the loading and interaction of an acridine orange ligand, termed C8, that acts as an anticancer drug and binder/stabilizer of the G4 structure of AT11. Dynamic light scattering experiments demonstrated that the aptadendrimer was approximately 3.1 nm in diameter. Both steady-state and time-resolved fluorescence anisotropy evidenced the interaction between the aptadendrimer and C8. Additionally, we demonstrated that the iodine atom of the C8 ligand acts as an effective intramolecular quencher in solution, while upon complexation with the aptadendrimer, it adopts a more extended conformation. Docking studies support this conclusion. Release experiments show a delivery of C8 after 4 h. The aptadendrimers tend to localize in the cytoplasm of various cell lines studied as demonstrated by confocal microscopy. The internalization of the aptadendrimers is not nucleolin-mediated or by passive diffusion, but via endocytosis. MTT studies with prostate cancer cells and non-malignant cells evidenced high cytotoxicity mainly due to the C8 ligand. The rapid internalization of the aptadendrimers and the fluorescence properties make them attractive for the development of potential nanocarriers.

Making European performance and impact assessment frameworks for research infrastructures glocal.

Sustainability of research infrastructures (RIs) is a big challenge for funders, stakeholders and operators, and the development and adoption of adequate management tools is a major concern, namely tools for monitoring and evaluating their performance and impact. BioData.pt is the Portuguese Infrastructure of Biological and Portuguese node of the European Strategy Forum on Research Infrastructures "Landmark" ELIXIR. The foundations of this national research infrastructure were laid under the "Building BioData.pt" project, for four years. During this period, performance and impact indicators were collected and analysed under the light of international guidelines for assessing the performance and impact of European research infrastructures produced by the European Strategy Forum on Research Infrastructures, the Organisation for Economic Co-operation and Development and the EU-funded RI-PATHS project. The exercise shared herein showed that these frameworks can be adopted by national RIs, with the necessary adaptations, namely to reflect the national landscape and specificity of activities, and can be powerful tools in supporting the management of RIs. "Not everything that counts can be counted, and not everything that can be counted, counts". Albert Einstein, Theoretical physicist and Nobel Prize winner.

Vitamin D-related polymorphisms and vitamin D levels as risk biomarkers of COVID-19 disease severity.

Vitamin D is a fundamental regulator of host defences by activating genes related to innate and adaptive immunity. Previous research shows a correlation between the levels of vitamin D in patients infected with SARS-CoV-2 and the degree of disease severity. This work investigates the impact of the genetic background related to vitamin D pathways on COVID-19 severity. For the first time, the Portuguese population was characterized regarding the prevalence of high impact variants in genes associated with the vitamin D pathways. This study enrolled 517 patients admitted to two tertiary Portuguese hospitals. The serum concentration of 25 (OH)D, was measured in the hospital at the time of patient admission. Genetic variants, 18 variants, in the genes AMDHD1, CYP2R1, CYP24A1, DHCR7, GC, SEC23A, and VDR were analysed. The results show that polymorphisms in the vitamin D binding protein encoded by the GC gene are related to the infection severity (p = 0.005). There is an association between vitamin D polygenic risk score and the serum concentration of 25 (OH)D (p = 0.04). There is an association between 25 (OH)D levels and the survival and fatal outcomes (p = 1.5e-4). The Portuguese population has a higher prevalence of the DHCR7 RS12785878 variant when compared with its prevalence in the European population (19% versus 10%). This study shows a genetic susceptibility for vitamin D deficiency that might explain higher severity degrees in COVID-19 patients. These results reinforce the relevance of personalized strategies in the context of viral diseases.Trial registration: NCT04370808.

Lipid Hydroperoxide Compromises the Membrane Structure Organization and Softens Bending Rigidity.

Lipid hydroperoxides are key mediators of diseases and cell death. In this work, the structural and dynamic perturbations induced by the hydroperoxidized POPC lipid (POPC-OOH) in fluid POPC membranes, at both 23 and 37 °C, were addressed using advanced small-angle X-ray scattering (SAXS) and fluorescence methodologies. Notably, SAXS reveals that the hydroperoxide group decreases the lipid bilayer bending rigidity. This alteration disfavors the bilayer stacking and increases the swelling in-between stacked bilayers. We further investigated the changes in the apolar/polar interface of hydroperoxide-containing membranes through time-resolved fluorescence/anisotropy experiments of the probe TMA-DPH and time-dependent fluorescence shifts of Laurdan. A shorter mean fluorescence lifetime for TMA-DPH was obtained in enriched POPC-OOH membranes, revealing a higher degree of hydration near the membrane interface. Moreover, a higher microviscosity near TMA-DPH and lower order are predicted for these oxidized membranes, at variance with the usual trend of variation of these two parameters. Finally, the complex relaxation process of Laurdan in pure POPC-OOH membranes also indicates a higher membrane hydration and viscosity in the close vicinity of the -OOH moiety. Altogether, our combined approach reveals that the hydroperoxide group promotes alterations in the membrane structure organization, namely, at the level of membrane order, viscosity, and bending rigidity.

Schistosomicidal effect of divaricatic acid from Canoparmelia texana (Lichen): In vitro evaluation and ultrastructural analysis against adult worms of Schistosoma mansoni.

In this study we evaluated the in vitro effect of divaricatic acid against coupled worms of Schistosoma mansoni. The schistosomicidal effect was evaluated through the bioassay of motility and mortality, cellular viability of the worms and ultrastructural analysis through Scanning Electron Microscopy. To evaluate the cytotoxicity of divaricatic acid, a cell viability assay was performed with human peripheral blood mononuclear cells. Divaricatic acid proved effect against S. mansoni after 3 hours of exposure. At the end of 24 h the concentrations of 100 - 200 µM presented lethality to the worms. Motility changes were observed at sublethal concentrations. The IC50 obtained by the cell viability assay for S. mansoni was 100.6 µM (96.24 - 105.2 µM). Extensive damage to the worm's tegument was observed such as peeling, erosion, bubbles, edema, damage and loss of tubercles and spines, fissures and tissue ruptures. No cytotoxicity was observed in human peripheral blood mononuclear cells. This report provides data showing the schistosomicidal effect of divaricatic acid on S. mansoni, causing death, motile changes and ultrastructural damage to worms. In addition, divaricatic acid was shown to be non-toxic to human peripheral blood mononuclear cells at concentrations effective on S. mansoni.

Sublethal concentrations of usnic acid potassium salt impairs physiological parameters of Biomphalaria glabrata (Say, 1818) (Pulmonata: Planorbidae) infected and not infected with Schistosoma mansoni.

Schistosomiasis is a public health problem in many developing countries. The mollusc Biomphalaria glabrata is the most important vector of Schistosoma mansoni in South America. The population control of this vector to prevent the spread of schistosomiasis is currently done with the application of highly toxic molluscicide to the environment. The screening of substances in sublethal concentrations that have deleterious effects on physiological parameters is very relevant for the control of schistosomiasis, since the effectiveness of disease prevention increases if it acts on population control of the vector and on reproduction and elimination in S. mansoni cercariae. The objective of this study was to evaluate the reproductive parameters (fecundity and fertility), intra-mollusk effect (sporocysts I (72 h) and II (14 days after)) on the development of cercariae of S. mansoni and the immune cell profile of B. glabrata exposed to sublethal concentrations (LC25 - 0.5 µg/mL and LC50 - 0.92 µg/mL) of the usnic acid potassium salt (potassium usnate). LC 25 and LC 50 significantly reduced (p < 0.05) the fecundity of B. glabrata when treated infected and/or not exposed to infection, while unviable embryos were not observed in sporocyst stage I, being only significant (p < 0.05) for mollusks infected and treated with LC50 on sporocyst II. LC25 and LC50 of the potassium usnate caused significant reductions (p < 0.05) in the production and cercarial shedding when evaluated on sporocysts I and II. In addition, the mortality of infected and treated B. glabrata in the sporocyst II phase was quite marked after the 9th week of infection. Regarding the immunological cell profile of uninfected B. glabrata, both concentrations led to immunomodulatory responses, with significant morphological changes predominant of hemocytes that entered programmed cell death (apoptosis). It was concluded that the application of LC25 and LC50 from the potassium usnate could be useful in the population control of B. glabrata, since it interferes both in their biology and physiology and in the reproduction of the infectious agent of schistosomiasis mansoni.

Interactions of Meibum and Tears with Mucomimetic Polymers: A Hint towards the Interplay between the Layers of the Tear Film.

Recent clinical findings suggest that mucomimetic polymers (MMP) can alter not only the texture of the aqueous tear but also the spreading and structure of the tear film (TF) lipid layer, thereby allowing for their synchronized performance in vivo. Thus, we aimed to evaluate in vitro (i) the capability of pharmaceutically applicable MMP to ensure the formation of post-evaporative ferning patterns (a characteristic feature of the "healthy" tear colloid) and (ii) the MMP interactions with human meibum films accessed in the course of blink-like deformations via Langmuir surface balance and Brewster angle microscopy (BAM). Four MMP were used- hyaluronic acid (HA), cross-linked hyaluronic acid (CHA), carboxymethyl cellulose (CMC) and gellan gum (GG)- at the concentrations of 0.0001%, 0.001%, 0.01%, 0.05% and 0.1%. Significant differences were observed in the MMP fern formation capability: CHA (≥0.001%) > HA (≥0.01%) = CMC (≥0.01%) > GG (≥0.05%). All MMP affected the spreading of meibum, with BAM micrographs revealing thickening of the films. CHA was particularly efficient, showing concentration-dependent enhancement of tear ferning and of meibomian layer structure, surfactant properties and viscoelasticity. Thus, endogenous and exogenous MMP may play key roles for the concerted action of the TF layers at the ocular surface, revealing novel routes for TF-oriented therapeutic applications.

Comparative study of micronucleus assays and dicentric plus ring chromosomes for dose assessment in particular cases of partial-body exposure.

Purpose: The goal was to compare the micronucleus (MN) and dicentric plus ring chromosomes (D + R) assays for dose assessment in cases of partial body irradiations (PBI). Materials and methods: We constructed calibration curves for each assay at doses ranging from 0 to 5 Gy of X-rays at dose rate of 0.275 Gy/min. To simulate partial-body exposures, blood samples from two donors were irradiated with 0.5, 1, 2 and 4 Gy and the ratios of irradiated to unirradiated blood were 25, 50, and 100%. Different tests were used to confirm if all samples were overdispersed or zero-inflated and for partial-body dose assessment we used the Qdr, Dolphin and Bayesian model. Results: In our samples for D + R calibration curve, practically all doses agreed with Poisson assumption, but MN exhibited overdispersed and zero-inflated cellular distributions. The exact Poisson tests and zero-inflated tests demonstrate that virtually all samples of D + R from PBI simulation fit the Poisson distribution and were not zero-inflated, but the MN samples were also overdispersed and zero-inflated. In the partial-body estimation, when Qdr and Dolphin methods were used the D + R results were better than MN, but the doses estimation defined by the Bayesian methodology were more accurate than the classical methods. Conclusions: Dicentric chromosomes continue to prove to be the best biological marker for dose assessment. However exposure scenarios of partial-body estimation, overdispersion and zero-inflation may not occur, it being a critical point not only for dose assessment, but also to confirm partial-body exposure. MN could be used as alternative assay for partial-body dose estimation, but in case of an accident without any information, the MN assay could not define whether the accident was a whole-body irradiation (WBI) or a PBI.

Untangling the Conformational Polymorphism of Disordered Proteins Associated With Neurodegeneration at the Single-Molecule Level.

A large fraction of the human genome encodes intrinsically disordered proteins/regions (IDPs/IDRs) that are involved in diverse cellular functions/regulation and dysfunctions. Moreover, several neurodegenerative disorders are associated with the pathological self-assembly of neuronal IDPs, including tau [Alzheimer's disease (AD)], α-synuclein [Parkinson's disease (PD)], and huntingtin exon 1 [Huntington's disease (HD)]. Therefore, there is an urgent and emerging clinical interest in understanding the physical and structural features of their functional and disease states. However, their biophysical characterization is inherently challenging by traditional ensemble techniques. First, unlike globular proteins, IDPs lack stable secondary/tertiary structures under physiological conditions and may interact with multiple and distinct biological partners, subsequently folding differentially, thus contributing to the conformational polymorphism. Second, amyloidogenic IDPs display a high aggregation propensity, undergoing complex heterogeneous self-assembly mechanisms. In this review article, we discuss the advantages of employing cutting-edge single-molecule fluorescence (SMF) techniques to characterize the conformational ensemble of three selected neuronal IDPs (huntingtin exon 1, tau, and α-synuclein). Specifically, we survey the versatility of these powerful approaches to describe their monomeric conformational ensemble under functional and aggregation-prone conditions, and binding to biological partners. Together, the information gained from these studies provides unique insights into the role of gain or loss of function of these disordered proteins in neurodegeneration, which may assist the development of new therapeutic molecules to prevent and treat these devastating human disorders.

Potassium usnate toxicity against embryonic stages of the snail Biomphalaria glabrata and Schistosoma mansoni cercariae.

The snail Biomphalaria glabrata is the most important vector for Schistosoma mansoni. Control of this vector to prevent the spread of schistosomiasis is currently performed with the application of a niclosamide molluscicide, which is highly toxic to the environment. Screening of substances that show embryotoxic molluscicidal potential as well as have detrimental effects on cercariae is very relevant for the control of schistosomiasis, as the efficacy of prevention of the disease is increased if it acts as a molluscicide as well as on the cercariae of S. mansoni. The aim of this work was to evaluate the effect of potassium usnate derived from usnic acid on different stages of embryonic development of B. glabrata and on S. mansoni cercariae. After 24 h of exposure, potassium usnate showed embryotoxic activity across all embryonic stages. The values obtained from the LC50 for the embryonic stages were the following: blastula 5.22 µg/mL, gastrula 3.21 µg/mL, trochophore 3.58 µg/mL, veliger 2.79, and hippo stage 2.52 µg/mL. Against S. mansoni cercariae, it had LC90 and 100% mortality at concentrations of 2.5 and 5 µg/mL in 2 h of exposure. In conclusion, this is the first report of potassium usnate toxicity on the embryonic stages of B. glabrata and cercariae of S. mansoni, and this study shows the potassium usnate as a promising agent for the control of mansoni schistosomiasis.

Toxicity of Usnic Acid from Cladonia substellata (Lichen) to embryos and adults of Biomphalaria glabrata.

This study reports the molluscicidal activity of usnic acid isolated from Cladonia substellata Vanio (lichen) on embryos at various stages of development and in adult mollusks of Biomphalaria glabrata. The toxicity of usnic acid was also evaluated through Artemia salina larvae mortality. Usnic acid was extracted with diethyl ether, isolated, purified, and its structure confirmed by analyzing the spectra of proton nuclear magnetic resonance. LC90 for 24 h of exposure were 1.62, 4.45, 5.36, and 4.49 µg mL-1 for blastula, gastrula, trocophore, and veliger embryonic stages, respectively, and 3.45 µg mL-1 for adult snails; LC50 of usnic acid against A. salina was 2.46 µg mL-1. LC90 assessed 7 days after exposure was 2.56 µg mL-1 for adult mollusks. In conclusion, these findings demonstrate that under laboratory conditions usnic acid has teratogenic and molluscicide potential to control the aquatic snail B. glabrata and may prove to be a promising candidate in the search for new molluscicide agents, but further detailed studies on its molluscicidal effect and possible environmental effects are needed.

Insights into tau function and dysfunction through single-molecule fluorescence.

Fluorescence correlation spectroscopy and single-molecule Förster resonance energy transfer are powerful and versatile techniques to quantify and describe molecular interactions. They are particularly well suited to the study of dynamic proteins and assemblies, as they can overcome some of the challenges that stymie more conventional ensemble approaches. In this chapter, we describe the application of these methods to study the interaction of tau with the molecular aggregation inducer, heparin, and the functional binding partner, soluble tubulin. Specifically, we outline the practical aspects of both techniques to characterize the critical first steps of tau aggregation and tau-mediated microtubule polymerization. The information gained from these measurements provides unique insight into tau function and its role in disease.

A functional role for intrinsic disorder in the tau-tubulin complex.

Tau is an intrinsically disordered protein with an important role in maintaining the dynamic instability of neuronal microtubules. Despite intensive study, a detailed understanding of the functional mechanism of tau is lacking. Here, we address this deficiency by using intramolecular single-molecule Förster Resonance Energy Transfer (smFRET) to characterize the conformational ensemble of tau bound to soluble tubulin heterodimers. Tau adopts an open conformation on binding tubulin, in which the long-range contacts between both termini and the microtubule binding region that characterize its compact solution structure are diminished. Moreover, the individual repeats within the microtubule binding region that directly interface with tubulin expand to accommodate tubulin binding, despite a lack of extension in the overall dimensions of this region. These results suggest that the disordered nature of tau provides the significant flexibility required to allow for local changes in conformation while preserving global features. The tubulin-associated conformational ensemble is distinct from its aggregation-prone one, highlighting differences between functional and dysfunctional states of tau. Using constraints derived from our measurements, we construct a model of tubulin-bound tau, which draws attention to the importance of the role of tau's conformational plasticity in function.

Supramolecular organization of bacterial aerobic respiratory chains: From cells and back.

Aerobic respiratory chains from all life kingdoms are composed by several complexes that have been deeply characterized in their isolated form. These membranous complexes link the oxidation of reducing substrates to the reduction of molecular oxygen, in a process that conserves energy by ion translocation between both sides of the mitochondrial or prokaryotic cytoplasmatic membranes. In recent years there has been increasing evidence that those complexes are organized as supramolecular structures, the so-called supercomplexes and respirasomes, being available for eukaryotes strong data namely obtained by electron microscopy and single particle analysis. A parallel study has been developed for prokaryotes, based on blue native gels and mass spectrometry analysis, showing that in these more simple unicellular organisms such supercomplexes also exist, involving not only typical aerobic-respiration associated complexes, but also anaerobic-linked enzymes. After a short overview of the data on eukaryotic supercomplexes, we will analyse comprehensively the different types of prokaryotic aerobic respiratory supercomplexes that have been thus far suggested, in both bacteria and archaea. This article is part of a Special Issue entitled Organization and dynamics of bioenergetic systems in bacteria, edited by Prof Conrad Mullineaux.

Cross-Scale Integrin Regulation Organizes ECM and Tissue Topology.

The diverse morphologies of animal tissues are underlain by different configurations of adherent cells and extracellular matrix (ECM). Here, we elucidate a cross-scale mechanism for tissue assembly and ECM remodeling involving Cadherin 2, the ECM protein Fibronectin, and its receptor Integrin α5. Fluorescence cross-correlation spectroscopy within the zebrafish paraxial mesoderm mesenchyme reveals a physical association between Integrin α5 on adjacent cell membranes. This Integrin-Integrin complex correlates with conformationally inactive Integrin. Cadherin 2 stabilizes both the Integrin association and inactive Integrin conformation. Thus, Integrin repression within the adherent mesenchymal interior of the tissue biases Fibronectin fibrillogenesis to the tissue surface lacking cell-cell adhesions. Along nascent somite boundaries, Cadherin 2 levels decrease, becoming anti-correlated with levels of Integrin α5. Simultaneously, Integrin α5 clusters and adopts the active conformation and then commences ECM assembly. This cross-scale regulation of Integrin activation organizes a stereotypic pattern of ECM necessary for vertebrate body elongation and segmentation.

Usnic acid potassium salt: an alternative for the control of Biomphalaria glabrata (Say, 1818).

In Brazil, the snail Biomphalaria glabrata is the most important vector of schistosomiasis due to its wide geographical distribution, high infection rate and efficient disease transmission. Among the methods of schistosomiasis control, the World Health Organization recommends the use of synthetic molluscicides, such as niclosamide. However, different substances of natural origin have been tested as alternatives for the control or eradication of mollusks. The literature describes the antitumor, antimicrobial and antiviral properties of usnic acid as well as other important activities of common interest between medicine and the environment. However, usnic acid has a low degree of water solubility, which can be a limiting factor for its use, especially in aquatic environments, since the organic solvents commonly used to solubilize this substance can have toxic effects on aquatic biota. Thus, the aim of the present study was to test the potassium salt of usnic acid (potassium usnate) with regard to molluscicidal activity and toxicity to brine shrimp (Artemia salina). To obtain potassium usnate, usnic acid was extracted with diethyl ether isolated and purified from the lichen Cladonia substellata. Biological assays were performed with embryos and adult snails of B. glabrata exposed for 24 h to the usnate solution solubilized in dechlorinated water at 2.5; 5 and 10 µg/ml for embryos, 0.5; 0.9; 1;5 and 10 µg/ml for mollusks and 0.5; 1; 5; 10 µg/ml for A. salina. The lowest lethal concentration for the embryos and adult snails was 10 and 1 µg/ml, respectively. No toxicity to A. salina was found. The results show that modified usnic acid has increased solubility (100%) without losing its biological activity and may be a viable alternative for the control of B. glabrata.

Exploring homo-FRET to quantify the oligomer stoichiometry of membrane-bound proteins involved in a cooperative partition equilibrium.

The establishment of protein-protein interactions between membrane-bound proteins is associated with several biological functions and dysfunctions. Here, an analytical framework that uses energy homo transfer to directly probe quantitatively the oligomerization state of membrane-bound proteins engaged in a three-state cooperative partition is presented. Briefly, this model assumes that monomeric protein molecules partition into the bilayer surface and reversibly assemble into oligomers with k subunits. A general equation relating the overall steady-state fluorescence anisotropy of the sample to its fractional labeling was derived by considering explicitly that the anisotropy of mixed oligomers containing i-labeled monomers is inversely proportional to the number of labeled subunits per oligomer (Runnels and Scarlata limit). This method was very robust in describing the electrostatic interaction of Alexa Fluor 488 fluorescently labeled lysozyme (Lz-A488) with phosphatidylserine-containing membranes. The pronounced decrease detected in the fluorescence anisotropy of Lz-A488 always correlated with the system reaching a high membrane surface density of the protein (at a low lipid-to-protein (L/P) molar ratio). The occurrence of energy homo transfer-induced fluorescence depolarization was further confirmed by measuring the anisotropy decays of Lz-A488 under these conditions. A global analysis of the steady-state anisotropy data obtained under a wide range of experimental conditions (variable anionic lipid content of the liposomes, L/P molar ratios and protein fractional labeling) confirmed that membrane-bound Lz-A488 assembled into oligomeric complexes, possibly with a stoichiometry of k = 6 ± 1. This study illustrates that even in the presence of a coupled partition-oligomerization equilibrium, steady-state anisotropy measurements provide a simple and reliable tool to monitor the self-assembly of membrane-bound proteins.

Electrostatically driven lipid-lysozyme mixed fibers display a multilamellar structure without amyloid features.

Understanding the interactions between anionic lipid membranes and amyloidogenic proteins/peptides is key to elucidate the molecular mechanisms underlying the membrane-driven amyloid fiber formation. Here, hen egg-white lysozyme was used as a model protein to test whether this same process also occurs with non-amyloidogenic lipid-binding proteins/peptides. A complementary set of biophysical techniques was employed to study the structure and dynamics of the lipid-lysozyme mixed fibers produced at a low lipid/protein molar ratio that have been proposed earlier to present "amyloid-like" characteristics. The multilamellar architecture of these elongated mesoscopic structures was established by performing time-resolved Förster resonance energy transfer measurements, at both bulk (ensemble) and single-fiber level. The predominantly oligomeric lysozyme and phospholipids were both found to display significantly decreased lateral mobility when embedded in these mixed fibers. Notably, two-photon microscopy of Laurdan revealed that a pronounced membrane surface dehydration/increased molecular interfacial packing was produced exclusively in these elongated mixed supramolecular fibers present in the highly polymorphic samples. Infrared spectroscopic studies of lysozyme in these samples further showed that this protein did not exhibit a rich ß-sheet structure characteristic of amyloid fibrils. These results support the conclusion that negatively charged lipid membranes do not have the general ability to trigger amyloid fibril formation of non-amyloidogenic proteins.