1,721,092 research outputs found
alpha-casein-membrane interactions: quantitative fluorescence analysis of the effects of protein aggregation state
No full textNile Red lifetime reveals microplastic identity
No full textMicroplastic pollution is recognized as a worldwide environmental problem. The increasing daily use and release of plastics into the environment have led to the accumulation of fragmented microplastics, with potentially awful consequences for the environment, and animal and human health. The detection and identification of microplastics are of utmost importance, but available methods are still limited. In this work, a new approach is presented for the analysis of microplastics based on hydrophobic fluorescence staining with Nile Red, using spectrally resolved confocal fluorescence microscopy and fluorescence lifetime imaging microscopy (FLIM). Significant differences were observed in the emission spectra and fluorescence lifetimes of the analyzed microplastics. Nile Red fluorescence shows determinable behavior based on the polymer matrix and provides a fingerprint for the identification of fragments from different types of plastics. Lifetime imaging coupled with phasor analysis constitutes a fast, robust, and straightforward method for mapping and identifying different microplastics within the same sample in an aquatic environment
Two-Photon Imaging
No full textThis chapter will provide an overview of two-photon microscopy from elements of the theory underpinning fluorescence phenomena to functioning principles of a two-photon microscope including step-by-step practical advice on how to conduct an experiment using a two-photon microscope. In this context multi-photon excitation is also taken into consideration. By reading this chapter, you will have a synopsis of the basic principles of two-photon excitation, optical sectioning, and 3D microscopy. Furthermore, fundamentals of promising advanced methods for tissue imaging available for two-photon imaging as second harmonic generation (SHG) and fluorescence lifetime imaging microscopy (FLIM) are briefly described together with classical applications on deep tissue imaging and functional brain imaging
Protein-membrane interaction: insights from advanced microscopy
Full text linkThe interaction between proteins and membranes is sub ject of renewed interest in biomedical and biotechnologi cal research for its implication in many functional and dys functional processes and for its pharmaceutical applications.
It has been shown that the interaction between amyloido genic proteins and membranes results in mutually destruc tive structural perturbations. The study we present is focused on the interaction between synthetic model membranes
and alpha-lactalbumin (α-La), widely studied for its biolog ical function since it can induce apoptosis in tumor cells.
Upon α-La addition to giant vesicles (GVs) samples, the sys tem has been characterized by means of spectroscopy meth ods and advanced microscopy techniques. Using Raster Image Correlation Spectroscopy (RICS) and Fluorescence Life time Imaging Microscopy (FLIM), the interaction has been investigated at different protein:lipid ratios. Starting from the molten globule conformation, a quick insertion of α-La into the lipid bilayer takes place, with evident changes in
GVs morphology as well as in protein structure. The pro cess, ruled by a combination of electrostatic and hydrophobic interactions, ends with the formation of heterogeneous struc tures containing both protein and lipids.
Eur Biophys J (2017) 46 (Suppl 1):S43–S402 S351 1
Unveiling water ordering in liquid–liquid phase separation using bovine serum albumin-polyethylene glycol systems
No full textLiquid–liquid phase separation (LLPS) is a fundamental physicochemical process where a homogeneous liquid solution spontaneously separates into two distinct liquid phases. Initially studied in polymer science, LLPS has emerged as a crucial mechanism in various biological processes, particularly in the formation of membrane-less organelles within cells. These organelles are biomolecular condensates that compartmentalise biochemical reactions without relying on traditional lipid membranes. LLPS is driven by a balance of enthalpic and entropic contributions, with protein–protein and protein-solvent interactions playing a pivotal role. Environmental factors, including temperature, pH, and solute concentrations, critically influence these interactions and thus the phase separation process. In this study, we investigate the role of water dynamics in the regulation of LLPS processes using a binary system of Bovine Serum Albumin (BSA) and Polyethylene Glycol (PEG). Classical spectroscopic methods and fluorescence lifetime imaging microscopy (FLIM), combined with phasor plot analysis, are employed to probe the local environment within protein condensates. Central to this approach is the use of 6-acetyl-2-dimethylaminonaphthalene (ACDAN), a fluorescent dye renowned for its sensitivity to water dipolar relaxation and changes in solvent polarity. This research aims to deepen our understanding of LLPS, particularly the role of water, offering insights into cellular processes and potential therapeutic strategies for LLPS-related diseases
α-casein micelles-membranes interaction: Flower-like lipid protein coaggregates formation
No full textBackground: Environmental conditions regulate the association/aggregation states of proteins and their action in cellular compartments. Analysing protein behaviour in presence of lipid membranes is fundamental for the comprehension of many functional and dysfunctional processes. Here, we present an experimental study on the interaction between model membranes and α-casein. α-casein is the major component of milk proteins and it is recognised to play a key role in performing biological functions. The conformational properties of this protein and its capability to form supramolecular structures, like micelles or irreversible aggregates, are key effectors in functional and pathological effects. Methods: By means of quantitative fluorescence imaging and complementary spectroscopic methods, we were able to characterise α-casein association state and the course of events induced by pH changes, which regulate the interaction of this molecule with membranes. Results: The study of these complex dynamic events revealed that the initial conformation of the protein critically regulates the fate of α-casein, size and structure of the newly formed aggregates and their effect on membrane structures. Disassembly of micelles due to modification in electrostatic interactions results in increased membrane structure rigidity which accompanies the formation of protein lipid flower-like co-aggregates with protein molecules localised in the external part. General significance: These results may contribute to the comprehension of how the initial state of a protein establishes the course of events that occur upon changes in the molecular environment. These events which may occur in cells may be essential to functional, pathological or therapeutical properties specifically associated to casein proteins
Image Mean Square Displacement analysis: a new method to study protein diffusion in cell membranes
No full textThe image mean square displacement..
Direct observation of alpha-lactalbumin, adsorption and incorporation into lipid membrane and formation of lipid/protein hybrid structures
No full textThe interaction between proteins and membranes is of great interest in biomedical and biotechnological research for its implication in many functional and dysfunctional processes. We present an experimental study on the interaction between model membranes and alpha-lactalbumin (α-La). α-La is widely studied for both its biological function and its anti-tumoral properties. We use advanced fluorescence microscopy and spectroscopy techniques to characterize α-La-membrane mechanisms of interaction and α-La-induced modifications of membranes when insertion of partially disordered regions of protein chains in the lipid bilayer is favored. Moreover, using fluorescence lifetime imaging, we are able to distinguish between protein adsorption and insertion in the membranes. Our results indicate that, upon addition of α-La to giant vesicles samples, protein is inserted into the lipid bilayer with rates that are concentration-dependent. The formation of heterogeneous hybrid protein-lipid co-aggregates, paralleled with protein conformational and structural changes, alters the membrane structure and morphology, leading to an increase in membrane fluidity
Transportan 10 Induces Perturbation and Pores Formation in Giant Plasma Membrane Vesicles Derived from Cancer Liver Cells
No full textContinuous progress has been made in the development of new molecules for therapeutic purposes. This is driven by the need to address several challenges such as molecular instability and biocompatibility, difficulties in crossing the plasma membrane, and the development of host resistance. In this context, cell-penetrating peptides (CPPs) constitute a promising tool for the development of new therapies due to their intrinsic ability to deliver therapeutic molecules to cells and tissues. These short peptides have gained increasing attention for applications in drug delivery as well as for their antimicrobial and anticancer activity but the general rules regulating the events involved in cellular uptake and in the following processes are still unclear. Here, we use fluorescence microscopy methods to analyze the interactions between the multifunctional peptide Transportan 10 (TP10) and the giant plasma membrane vesicles (GPMVs) derived from cancer cells. This aims to highlight the molecular mechanisms underlying functional interactions which bring its translocation across the membrane or cytotoxic mechanisms leading to membrane collapse and disruption. The Fluorescence Lifetime Imaging Microscopy (FLIM) method coupled with the phasor approach analysis proved to be the winning choice for following highly dynamic spatially heterogeneous events in real-time and highlighting aspects of such complex phenomena. Thanks to the presented approach, we were able to identify and monitor TP10 translocation into the lumen, internalization, and membrane-induced modifications depending on the peptide concentration regime
Pea Protein Isolate-Based Nanofibers for Delivery of Clotrimazole
No full textA large part of the global population, including both immunocompromised and healthy individuals, suffers from fungal infections. The majority of current drug candidates for treating fungal infections exhibit poor water solubility, which hampers their permeability through biological barriers and limits their bioavailability. Here, we fabricated pea protein isolate (PPI)/poly(ethylene oxide) nanofibers (PPI/PEO nanofibers), with a high protein content [65% (w/w)] by waterborne electrospinning as an eco-friendly drug delivery system. X-ray diffraction results demonstrated that the solid-state properties of the individual components (PPI and PEO) were retained in the PPI/PEO nanofibers. We then encapsulated the poorly water-soluble drug, clotrimazole (CTZ), in the nanofibers (PPI/PEO/CTZ nanofibers), without heat treatment and/or use of an organic solvent or surfactant to presolubilize CTZ, generating an antifungal delivery system for topical administration. An in vitro study demonstrated that CTZ was successfully loaded in and released from the nanofibers. Additionally, the nanofibers were not toxic to HeLa cells. Finally, based on an antifungal disc agar diffusion study, CTZ-loaded nanofibers were shown to be effective against Candida albicans. The overall results demonstrate the potential of PPI-based nanofibers as a green platform for the generation of CTZ-loaded efficient drug delivery systems for antifungal treatment
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