During their lifespan, Red blood cells (RBC), due to their inability to self-replicate, undergo an ageing degradation phenomenon. This pathway, both in vitro and in vivo, consists of a series of chemical and morphological modifications, which include deviation from the biconcave cellular shape, oxidative stress, membrane peroxidation, lipid content decrease and uncoupling of the membrane-skeleton from the lipid bilayer. Here, we use the capabilities of atomic force microscopy based infrared nanospectroscopy (AFM-IR) to study and correlate, with nanoscale resolution, the morphological and chemical modifications that occur during the natural degradation of RBCs at the subcellular level. By using the tip of an AFM to detect the photothermal expansion of RBCs, it is possible to obtain nearly two orders of magnitude higher spatial resolution IR spectra, and absorbance images than can be obtained on diffraction-limited commercial Fourier-transform Infrared (FT-IR) microscopes. Using this approach, we demonstrate that we can identify localized sites of oxidative stress and membrane peroxidation on individual RBC, before the occurrence of neat morphological changes in the cellular shape.

The collection and analysis of Atomic Force Microscopy force curves is a well-established procedure to obtain high-resolution information of non-topographic data from any kind of sample, including biological specimens. In particular, these analyses are commonly employed to study elasticity, stiffness or adhesion properties of the samples. Furthermore, the collection of several force curves over an extended area of the specimens allows reconstructing maps, called force volume maps, of the spatial distribution of the mechanical properties. Coupling these maps with the conventional high-resolution topographic reconstruction of the sample’s surface, provides a deeper insight on the sample composition from the structural and nanomechanical point of view. In this paper we present the open source software package FC_analysis that automatically analyses single force curves or entire force volume maps to yield the corresponding elasticity and deformability images. The principal characteristic of the FC_analysis is a large adaptability to the various experimental setups and to different analysis methodologies. For instance, the user can provide custom values for the detector sensitivity, scanner-z sensitivity, cantilever’s elastic constant and map’s acquisition modality and can choose between different analysis methodologies. Furthermore, the software allows the optimization of the fitting parameters and gives direct control on each step of the analysis procedure. Notably, to overcome a limitation common to many other analysis programs, FC_analysis can be applied to a rectangular portion of the image, allowing the analysis of inhomogeneous samples. Finally, the software allows reconstructing a Young’s modulus map at different penetration depths, enabling the use of modern investigation tools such as the force tomography. The FC_analysis software aims to become a useful tool for the analysis of force curves maps collected using custom or commercial Atomic Force Microscopes, and is especially useful in those cases for which the producer doesn’t release a dedicated software.

The β-amyloid (Aβ) peptide plays a key role in the pathogenesis of Alzheimer’s disease. The methionine (Met) residue at position 35 in Aβ C-terminal domain is critical for neurotoxicity, aggregation, and free radical formation initiated by the peptide. The role of Met in modulating toxicological properties of Aβ most likely involves an oxidative event at the sulfur atom. We therefore investigated the one- or two-electron oxidation of the Met residue of Aβ_25-35 fragment and the effect of such oxidation on the behavior of the peptide. Bicarbonate promotes two-electron oxidations mediated by hydrogen peroxide after generation of peroxymonocarbonate (HCO₄⁻, PMC). The bicarbonate/carbon dioxide pair stimulates one-electron oxidations mediated by carbonate radical anion (CO₃^•−). PMC efficiently oxidizes thioether sulfur of the Met residue to sulfoxide. Interestingly, such oxidation hampers the tendency of Aβ to aggregate. Conversely, CO₃^•− causes the one-electron oxidation of methionine residue to sulfur radical cation (MetS^•+). The formation of this transient reactive intermediate during Aβ oxidation may play an important role in the process underlying amyloid neurotoxicity and free radical generation.

Autophagy plays a key role in removing protein aggregates and damaged organelles. In addition to its conventional degradative functions, autophagy machinery contributes to the release of cytosolic proteins through an unconventional secretion pathway. In this research, we analyzed autophagy-induced extracellular vesicles (EVs) in HT1080-derived human fibrosarcoma 2FTGH cells using transmission electron microscopy and atomic force microscopy (AFM). We preliminary observed that autophagy induces the formation of a subset of large heterogeneous intracellular vesicular structures. Moreover, AFM showed that autophagy triggering led to a more visible smooth cell surface with a reduced amount of plasma membrane protrusions. Next, we characterized EVs secreted by cells following autophagy induction, demonstrating that cells release both plasma membrane-derived microvesicles and exosomes. A self-forming iodixanol gradient was performed for cell subfractionation. Western blot analysis showed that endogenous LC3-II co-fractionated with CD63 and CD81. Then, we analyzed whether raft components are enriched within EV cargoes following autophagy triggering. We observed that the raft marker GD3 and ER marker ERLIN1 co-fractionated with LC3-II; dual staining by immunogold electron microscopy and coimmunoprecipitation revealed GD3-LC3-II association, indicating that autophagy promotes enrichment of raft components within EVs. Introducing a new brick in the crosstalk between autophagy and the endolysosomal system may have important implications for the knowledge of pathogenic mechanisms, suggesting alternative raft target therapies in diseases in which the generation of EV is active.