Phasor-FLIM for a direct investigation of Transportan 10 interactions with model membranes

Transportan 10 (TP10), a short and positive charged peptide, belonging to the family of the cell penetrating peptides has gained increasing attention for its antimicrobial and anticancer activity but also for its applications in drug delivery as it is able to translocate therapeutic molecules in cellular environment. Due to the complexity of the phenomena involved in cellular uptake and following processes, which strongly depend on the membrane lipid composition, structural details of the peptide (e.g., charge, hydrophobicity, steric hindrance) and environmental conditions, it is not easy to understand the general rules governing them. Here, we combine spectroscopic techniques and fluorescence lifetime imaging microscopy (FLIM) to investigate (i) the fate of the TP10 in the presence of model membranes, analyzing its conformational changes occurring at membrane interface and distinguishing peptide adsorption from insertion into the lipid bilayer (ii) the changes of the fluidity of the membrane and the formation of pores into the latter induced by TP10 interaction. In addition, thanks to the use of the environment sensitive fluorescence dyes, Laurdan and di-4-ANEPPDHQ, and of the phasor approach to analyze FLIM data, we were able to monitor in real time fine events at different depths of phospholipid bilayers

Different a-casein association states and their interaction with lipid vesicles to study antibacterial activity

The interactions between caseins and lipid membranes are fundamental for the physiological function of these proteins. Moreover, the understanding of the underlying molecular mechanisms is of great interest for the development of new casein derived antimicrobial peptides. Indeed, it was already shown that peptides derived from caseins possess antibacterial activity but their mechanisms of action is still debated. Here, we present an experimental study on the interaction between model lipid membranes and a-casein by means of spectroscopy and fluorescence microscopy techniques. a-casein is an unfolded protein that due to its amphiphilic nature is known to self-assembly into non-stable micellar structures whose presence, diameter and compactness depend on environmental conditions. Presented experiments are aimed at assessing the effects of this protein in different states (monomeric, micellar and aggregated) on the membranes highlighting the role of micelles.The association state of a-casein at different pH and temperatures was analysed by fluorescence spectroscopy, circular dichroism and dynamic light scattering. Then, a-casein in different states was added to giant lipid vesicles and fluorescence microscopy and spectroscopy techniques were used to map and quantify induced modifications on the membrane. Our results indicate that, depending on the specific properties of the added protein state, different membrane structure and morphology changes occur. Interestingly, the most effective species in altering membranes is constituted by highly hydrophobic oligomers originating from larger aggregates disassembly

Oxidation effects in antiaggregogenic properties of Epigallocatechingallate

Epigallocatechin-gallate (EGCG), the most abundant flavonoid in green tea, has been extensively studied for its potential in the treatment of amyloid related disorders. This molecule was found to modulate abnormal protein self-assembly, reducing resulting cellular toxicity. EGCG is known to suppress or to slow down the aggregation processes of several proteins, thus supporting the idea that general mechanisms regulate its anti-aggregogenic effects and, interestingly, in the oxidised form it demonstrated an higher efficiency in reducing protein aggregation with respect to intact molecule. We here investigate the effects of intact and oxidized EGCG the thermal aggregation pathway of Bovine Serum Albumin (BSA), a well-known model protein whose aggregation processes are known in details. By means of different spectroscopic methods, we evaluate similarities and differences of the two molecules during protein aggregation. Different solution conditions are investigated, close and away from the isoelectric point of the protein, with the aim of eliciting the role of electrostatics in the observed EGCG-Protein interaction and in the supramolecular assembly which are dramatically dependent on solution conditions

Electrostatics regulate Epigallocatechin-Gallate effects on Bovine Serum Albumin aggregation

Protein aggregation processes are complex phenomena often involved in the etiology of several pathologies. It is now assessed that all proteins, in suitable conditions, may undergo supramolecular assembly. Aggregation pathways are known to be controlled by solution conditions which regulate protein-protein and protein-solvent interactions affecting binding mechanisms, morphology and inherent toxicity of the aggregate species. In this context, the presence of small molecules was indicated as a promising method to modulate protein-protein interactions reducing pathogenic aggregation. In the light of the idea that common mechanisms regulate anti-aggregogenic properties of small molecules, we here investigate Epigallocatechin-Gallate (EGCG) effects on the thermal aggregation pathway of Bovine Serum Albumin (BSA), a well-known model protein. EGCG is a small molecule extracted from green tea, which is known to reduce aggregation of key proteins involved in neurodegenerative diseases [1]. Fundamental mechanisms which regulate EGCG effectiveness as therapeutic molecule are still not clearly elucidated. The interaction of EGCG with BSA and its effects on thermal aggregation pathway were investigated by means of spectroscopic methods and Isothermal Titration calorimetry as a function of solution conditions. Results show that electrostatic forces modulated by pH play a key role in regulating EGCG interactions with BSA. Data shows that close to the isoelectric point of the protein, EGCG is found to promote the supramolecular assembly, whilst away from the isoelectric point, EGCG is found to reduce aggregation mechanisms increasing protein conformational stability. These results reveal the large impact of electrostatics in small molecules effects on the protein aggregation phenomena requiring larger investigation aimed at rationalizing their effects on related pathogenic mechanisms

Effect of cholesterol on the interaction between amphyphylic peptides and liposomes

With the rise of antibiotic resistance, antimicrobial peptides (AMPs) have been proposed as an alternative novel class of therapeutic agents. They are polycationic, with a net positive charge of more than +2, and they are characterized by amphipathic structures, with both a hydrophobic and a hydrophilic domain. These characteristics allow them to selectively bind to negatively charged lipids (largely present in bacteria, not in mammalian cells), via hydrophobic and electrostatic interactions. Moreover, mammalian cells are characterized by a high content of cholesterol. For this reason, here we present an experimental study on the effect of the presence of cholesterol on the capability of amphyphylic peptide Trasportant 10 (TP10) to interact with model membranes with selected composition. The study was performed by means of fluorescence spectroscopy and fluorescence confocal microscopy measurements also exploiting the advantages of phasor plot analysis of Fluorescence Lifetime Imaging (FLIM) measurements. Our results show that the presence of cholesterol inhibits TP-10 interaction with lipid vesicles, the extent of the observed effect being dependent on the cholesterol concentration in the membrane

Improved Photocatalytic Activity of Polysiloxane TiO2 Composites by Thermally Induced Nanoparticle Bulk Clustering and Dye Adsorption

Fine control of nanoparticle clustering within polymeric matrices can be tuned to enhance the physicochemical properties of the resulting composites, which are governed by the interplay of nanoparticle surface segregation and bulk clustering. To this aim, out-of-equilibrium strategies can be leveraged to program the multiscale organization of such systems. Here, we present experimental results indicating that bulk assembly of highly photoactive clusters of titanium dioxide nanoparticles within an in situ synthesized polysiloxane matrix can be thermally tuned. Remarkably, the controlled nanoparticle clustering results in improved degradation photocatalytic performances of the material under 1 sun toward methylene blue. The resulting coatings, in particular the 35 wt % TiO2-loaded composites, show a photocatalytic degradation of about 80%, which was comparable to the equivalent amount of bare TiO2 and two-fold higher with respect to the corresponding composites not subjected to thermal treatment. These findings highlight the role of thermally induced bulk clustering in enhancing photoactive nanoparticle/polymer composite properties

Flexible multi-beam light-sheet fluorescence microscope for live imaging without striping artifacts

The development of light-sheet fluorescence microscopy (LSFM) has greatly expanded the experimental capabilities in many biological and biomedical research fields, enabling for example live studies of murine and zebrafish neural activity or of cell growth and division. The key feature of the method is the selective illumination of a sample single plane, providing an intrinsic optical sectioning and allowing direct 2D image recording. On the other hand, this excitation scheme is more affected by absorption or scattering artifacts in comparison to point scanning methods, leading to un-even illumination. We present here an easily implementable method, based on acousto-optical deflectors (AOD), to overcome this obstacle. We report the advantages provided by flexible and fast AODs in generating simultaneous angled multiple beams from a single laser beam and in fast light sheet pivoting and we demonstrate the suppression of illumination artifacts

Fast whole-brain imaging of seizures in zebrafish larvae by two-photon light-sheet microscopy

Light-sheet fluorescence microscopy (LSFM) enables real-time whole-brain functional imaging in zebrafish larvae. Conventional one photon LSFM can however induce undesirable visual stimulation due to the use of visible excitation light. The use of two-photon (2P) excitation, employing near-infrared invisible light, provides unbiased investigation of neuronal circuit dynamics. However, due to the low efficiency of the 2P absorption process, the imaging speed of this technique is typically limited by the signal-to-noise-ratio. Here, we describe a 2P LSFM setup designed for non-invasive imaging that enables quintuplicating state-of-the-art volumetric acquisition rate of the larval zebrafish brain (5 Hz) while keeping low the laser intensity on the specimen. We applied our system to the study of pharmacologically-induced acute seizures, characterizing the spatial-temporal dynamics of pathological activity and describing for the first time the appearance of caudo-rostral ictal waves (CRIWs).Comment: Replacement: accepted version of the manuscript, to be published in Biomedical Optics Express. 36 pages, 15 figure

Dual-beam confocal light-sheet microscopy via flexible acousto-optic deflector

Confocal detection in digital scanned laser light-sheet fluorescence microscopy (DSLM) has been established as a gold standard method to improve image quality. The selective line detection of a complementary metal-oxide-semiconductor camera (CMOS) working in rolling shutter mode allows the rejection of out-of-focus and scattered light, thus reducing background signal during image formation. Most modern CMOS have two rolling shutters, but usually only a single illuminating beam is used, halving the maximum obtainable frame rate. We report on the capability to recover the full image acquisition rate via dual confocal DSLM by using an acousto-optic deflector. Such a simple solution enables us to independently generate, control and synchronize two beams with the two rolling slits on the camera. We show that the doubling of the imaging speed does not affect the confocal detection high contrast