The escalating number of Alzheimer’s disease (AD) cases and the limitations of current therapies pose a significant threat to human health, necessitating the discovery of novel drugs with innovative modes of action. To address this challenge, we pursued multitarget ligand strategy with the expectation of improved disease management. Continuing our efforts to discover new multitarget agents for AD, we decorated the planar 6-Cl-2-OCH3-9-aminoacridine core with basic heterocyclic or benzyl side chains as polar and hydrophobic structural features, respectively. All the compounds inhibited acetylcholinesterase, and in several cases also inhibited butyrylcholinesterase, with potencies comparable to or exceeding those of reference drugs. Exploring activity against MAO isoforms, heterocyclic derivatives 2, 5, 6, 9, 11, and 12 proved to be selective MAO-A inhibitors, while the 3,4-dichlorobenzyl derivative 20 provided balanced inhibition of both MAO-A and MAO-B enzymes. Favorable predicted blood-brain barrier permeability and low toxicity toward SH-SY5Y neuronal cells were also observed. Intriguingly, compounds 4, 12 and 20 altered the aggregation morphology of the neurotoxic Aβ42 peptide, revealing distinct inhibition profiles likely reflecting the different nature of the side chain. Based on these findings, the planar 6-Cl-2-OCH3-9-aminoacridine ring emerges as a valuable scaffold for future development of multitargeted anti-AD agents.

Protein structure can be analysed using a range of different techniques. Many of the available principal techniques for assessing protein structure are developed and optimized for natively folded proteins that are soluble. Amyloids are an alternative protein fold, often associated with protein malfunction and disease but are also found as the functional fold for some proteins. While functional amyloids, just like soluble globular proteins, commonly attain one specific and precise fold to exert its function, disease associated amyloids are often poorly soluble and structurally highly variable. This is known as structural polymorphism. To investigate to what extent molecular biophysics techniques can be standardized for studies of amyloid fibrils, five sites within the Horizon 2020 funded project MOlecular-Scale Biophysics Research Infrastructure (MOSBRI) addressed this issue via a joint research activity. For this study we selected bovine insulin as a convenient, accessible, and distributable amyloid model system. The benchmark comparable techniques for observing formed amyloid fibrils at the different sites were fluorescence spectroscopy of two amyloid ligands (ThT and pFTAA) and negative stain transmission electron microscopy. The outcome of this study yielded results with a large variability between different insulin amyloid fibril preparations, between sites, and within chemically identical preparations from one site. We mainly attribute these hard to control differences to the intrinsic polymorphic behaviour of insulin amyloid fibrils. We also present a range of experimental measurement techniques to highlight their potential use for studying amyloid structure and amyloid polymorphism.

ABSTRACT In AL amyloidosis, monoclonal immunoglobulin light chains (LCs) aggregate as amyloid fibrils in tissues. In synergy with the intrinsic aggregation propensity of specific LC sequences, microenvironment factors may be involved in tuning the disease pathophysiology, in particular proteolytic LC remodelling and heterotypic interactions in the extracellular milieu. Accounting for extrinsic modulators is critical for understanding the phenotypic variability of AL, usually imputed mainly to the LC diversity. We investigated the effects of apolipoprotein E (allele 3, apoE3) and clusterin (CLU), two amyloid-signature proteins involved in extracellular proteostasis, on the fibrillogenesis kinetics of amyloidogenic LC fragments from patient-derived sequences, as well as on aggregate composition, fibril morphology and thermodynamic stability. We show that apoE3 and CLU act as heterotypic interactors of prefibrillar and fibrillar LCs, significantly modulating LC amyloidogenesis, with complex and non-monotypic effects that range from anti- to pro-amyloidogenic depending on their concentration and on the LC’s intrinsic amyloidogenicity. ApoE3 and CLU also influence fibril morphology, possibly by modifying protofilament association, and alter their thermodynamic properties. LC interactors may play a significant and insofar underappreciated role in the AL pathophysiology in vivo , likely contributing to phenotypic variability and structural polymorphisms and, possibly, to fibril resilience to amyloid reabsorption strategies.

Polydopamine nanoparticles (PDNPs) are a class of nanomaterials formed by the self-polymerization of dopamine. They exhibit high biocompatibility, biodegradability, antioxidant properties, and ease of functionalization and can serve as photothermal agents when exposed to near-infrared (NIR) light. Another notable feature of PDNPs is their potential to act as contrast agents in photoacoustic imaging (PAI). In this technique, light absorption by endogenous chromophores or nanostructures induces thermal expansion, which generates sound waves that can be exploited to create images. Although PDNPs have shown promise as PAI contrast agents, their capabilities remain underexplored and insufficiently characterized in biological systems. This study presents the first comprehensive evaluation of PDNPs as PAI contrast agents. We investigated PDNPs of various sizes (∼150-1000 nm) and assessed their photoacoustic performance in diverse environments, including aqueous dispersions, ex vivo tissues, U87 cancer cell spheroids, fertilized quail eggs, and zebrafish embryos. Additionally, experimental results supported the development of a computational model to predict PDNP photoacoustic properties. Overall, this work highlights the significant, yet largely unexplored, potential of PDNPs as label-free PAI contrast agents, contributing to their future exploitation in clinical imaging.

Mesenchymal stem cells (MSCs) hold significant promise for tissue engineering and regenerative medicine thanks to their combination of accessibility and multilineage differentiation potential. Among the several factors that influence MSC function, mechanical properties are increasingly recognized as key regulators of cell fate decisions. Despite their importance, the variability of these properties across biologically relevant conditions remains poorly characterized. In this study, we used atomic force microscopy nanoindentation to quantify two mechanical parameters in human adipose tissue-derived MSCs: the Young's modulus (E) and, for the first time in this cell type, the poroelastic diffusion constant (D-p). Measurements were performed on 120 cells derived from six human subjects (three female and three male), at two passages (P4 and P7), and in two subcellular compartments (above the nucleus and above the cytoplasm). Results revealed statistically significant mechanical heterogeneity across subjects, higher values with increasing passages for both E and D-p, and a sex-based difference in E, with female-derived cells being stiffer, but no differences in D-p. These findings preliminarily reveal and quantify mechanical heterogeneity in MSCs, and underscore the need for considering biological variables when designing MSC-based therapies for regenerative medicine and tissue engineering. Our dataset, based on a relatively small sample size, is not intended to capture the full variability of these factors. Instead, it provides preliminary data collected under consistent conditions, intended to inform the estimation of optimal sample sizes for future large-scale studies investigating mechanical heterogeneity.

The measurement of the Young's modulus of thin, soft films on solid support is affected by the presence of the so-called bottom effect artefact, which arises from the presence of a rigid substrate and increases the observed value of the elastic modulus of the sample. Here we measured the Young's modulus of a multicomponent, phase-separated supported lipid bilayer mimicking the biological membrane in the absence and presence of various concentrations of trodusquemine, which is a molecule with potential applications against neurodegenerative diseases. Applying the Garcia and Garcia theory for bottom effect correction on a multiphase system enabled the evaluation of the intrinsic Young's modulus of the lipid bilayer, decoupling it from the substrate contribution. We found that at all trodusquemine concentrations tested both the ordered and disordered phases of the lipid bilayer increase their rigidity. The highest increase was measured at the lowest concentration tested of 1 μM, supporting the relevance and therapeutic potential of this class of molecules at physiologically achievable doses. Overall, this study provides a methodological framework for investigating the mechanical properties of supported thin films endowed of a complex texture.

In this Comment, we direct attention to initial efforts to establish a high-quality databank of atomic force microscopy (AFM) data: bioAFM-DB. We outline the state of this endeavor, its challenges, and potential courses of action.

In this study, we explore the potential of ten quantitative (radiofrequency-based) ultrasound parameters to assess the progressive loss of collagen and proteoglycans, mimicking an osteoarthritis condition in ex-vivo bovine cartilage samples. Most analyzed metrics showed significant changes as the degradation progressed, especially with collagenase treatment. We propose for the first time a combination of these ultrasound parameters through machine learning models aimed at automatically identifying healthy and degraded cartilage samples. The random forest model showed good performance in distinguishing healthy cartilage from trypsin-treated samples, with an accuracy of 60%. The support vector machine demonstrated excellent accuracy (96%) in differentiating healthy cartilage from collagenase-degraded samples. Histological and mechanical analyses further confirmed these findings, with collagenase having a more pronounced impact on both mechanical and histological properties, compared to trypsin. These metrics were obtained using an ultrasound probe having a transmission frequency of 15 MHz, typically used for the diagnosis of musculoskeletal diseases, enabling a fully non-invasive procedure without requiring arthroscopic probes. As a perspective, the proposed quantitative ultrasound assessment has the potential to become a new standard for monitoring cartilage health, enabling the early detection of cartilage pathologies and timely interventions.

Numerous neurodegenerative disorders, including Alzheimer's and Parkinson's diseases, are associated with the misfolding and aggregation of proteins. In this study, we investigate the protective role of the natural alkaloid berberine against protein misfolded aggregates, given its potential as a therapeutic agent. Our findings demonstrate that berberine binds to the lipid membranes of reconstituted liposomes with diverse lipid compositions and can cross lipid membranes via diffusion. Correspondingly, in cultured human neuroblastoma cells, berberine interacts with the plasma membrane and is rapidly internalized into the cytoplasm. Furthermore, berberine exhibited rapid and dose-dependent protective effects upon exposing LUVs or cells to misfolded protein aggregates able to induce lipid membrane damage. Specifically, in human neuroblastoma cells pre-treated with berberine, the molecule reduced plasma membrane alterations and the associated Ca2+ ion influx, diminished the production of reactive oxygen species, and reduced mitochondrial metabolism dysfunction, induced by the aggregates. Concomitant administration of berberine and aggregates was not found to be protective against aggregates and, moreover, berberine does not significantly modify the structure of the protein aggregates, suggesting that its protective effect against these aberrant species in cells is due to its ability to rapidly interact with and cross the cell membrane. Together, these findings provide insight into the mechanism of protective action of berberine and support its therapeutic potential in mitigating cellular damage associated with protein aggregation.

The surface properties of drug containers should reduce the adsorption of the drug and avoid packaging surface/drug interactions, especially in the case of biologically-derived products. Here, we developed a multi-technique approach that combined Differential Scanning Calorimetry (DSC), Atomic Force Microscopy (AFM), Contact Angle (CA), Quartz Crystal Microbalance with Dissipation monitoring (QCM-D), and X-ray Photoemission Spectroscopy (XPS) to investigate the interactions of rhNGF on different pharma grade polymeric materials. Polypropylene (PP)/polyethylene (PE) copolymers and PP homopolymers, both as spin-coated films and injected molded samples, were evaluated for their degree of crystallinity and adsorption of protein. Our analyses showed that copolymers are characterized by a lower degree of crystallinity and lower roughness compared to PP homopolymers. In line with this, PP/PE copolymers also show higher contact angle values, indicating a lower surface wettability for the rhNGF solution on copolymers than PP homopolymers. Thus, we demonstrated that the chemical composition of the polymeric material and, in turn, its surface roughness determine the interaction with the protein and identified that copolymers may offer an advantage in terms of protein interaction/adsorption. The combined QCM-D and XPS data indicated that protein adsorption is a self-limiting process that passivates the surface after the deposition of roughly one molecular layer, preventing any further protein adsorption in the long term.

We review the advances obtained by using Atomic Force Microscopy (AFM)-based approaches in the field of cell/tissue mechanics and adhesion, comparing the solutions proposed and critically discussing them. AFM offers a wide range of detectable forces with a high force sensitivity, thus allowing a broad class of biological issues to be addressed. Furthermore, it allows for the accurate control of the probe position during the experiments, providing spatially resolved mechanical maps of the biological samples with subcellular resolution. Nowadays, mechanobiology is recognized as a subject of great relevance in biotechnological and biomedical fields. Focusing on the past decade, we discuss the intriguing issues of cellular mechanosensing, i.e., how cells sense and adapt to their mechanical environment. Next, we examine the relationship between cell mechanical properties and pathological states, focusing on cancer and neurodegenerative diseases. We show how AFM has contributed to the characterization of pathological mechanisms and discuss its role in the development of a new class of diagnostic tools that consider cell mechanics as new tumor biomarkers. Finally, we describe the unique ability of AFM to study cell adhesion, working quantitatively and at the single-cell level. Again, we relate cell adhesion experiments to the study of mechanisms directly or secondarily involved in pathologies.

Natural aminosterols are promising drug candidates against neurodegenerative diseases, like Alzheimer and Parkinson, and one relevant protective mechanism occurs via their binding to biological membranes and displacement or binding inhibition of amyloidogenic proteins and their cytotoxic oligomers. We compared three chemically different aminosterols, finding that they exhibited different (i) binding affinities, (ii) charge neutralizations, (iii) mechanical reinforcements, and (iv) key lipid redistributions within membranes of reconstituted liposomes. They also had different potencies (EC50) in protecting cultured cell membranes against amyloid-β oligomers. A global fitting analysis led to an analytical equation describing quantitatively the protective effects of aminosterols as a function of their concentration and relevant membrane effects. The analysis correlates aminosterol-mediated protection with well-defined chemical moieties, including the polyamine group inducing a partial membrane-neutralizing effect (79 ± 7%) and the cholestane-like tail causing lipid redistribution and bilayer mechanical resistance (21 ± 7%), linking quantitatively their chemistry to their protective effects on biological membranes.

In a previous study, the coexistence of different aggregation pathways of insulin and β-amyloid (Aβ) peptides was demonstrated by correlative stimulated emission depletion (STED) microscopy and atomic force microscopy (AFM). This had been explained by suboptimal proteins labeling strategies that generate heterogeneous populations of aggregating species. However, because of the limited number of proteins considered, the failure of the fluorescent labeling that occurs in a large portion of the aggregating fibrils observed for insulin and Aβ peptides, could not be considered a general phenomenon valid for all molecular systems. Here, we investigated the aggregation process of α-synuclein (α-syn), an amyloidogenic peptide involved in Parkinson's disease, which is significantly larger (MW ∼14 kDa) than insulin and Aβ, previously investigated. The results showed that an unspecific labeling procedure, such as that previously adopted for shorter proteins, reproduced the coexistence of labeled/unlabeled fibers. Therefore, a site-specific labeling method was developed to target a domain of the peptide scarcely involved in the aggregation process. Correlative STED-AFM illustrated that all fibrillar aggregates derived from the aggregation of α-syn at the dye-to-protein ratio of 1 : 22 were fluorescent. These results, demonstrated here for the specific case of α-syn, highlight that the labeling artifacts can be avoided by careful designing the labeling strategy for the molecular system under investigation. The use of a label-free correlative microscopy technique would play a crucial role in the control of the setting of these conditions.

The aggregation process of amyloidogenic peptides can be characterized by advanced correlative techniques to understand the molecular mechanisms of misfolded protein diseases. We performed correlative AFM-STED measurements to study amyloid aggregates formation starting from monomeric peptides. A previous study, performed by our group on insulin and β-amyloid peptides (MW∼ 4kDa) suggested that the presence of fluorophores not only decreased the kinetics of the aggregation, a very well-known effect, but also favored the coexistence of labeled and unlabeled fibrils. The results suggested the coexistence of different aggregation pathways. Labeled molecules can follow just some of these pathways. However, clear structural diversity between fluorescent, or dark fibrils was not detected. Particularly, we investigated the in vitro aggregation of α-synuclein (α-syn), the amyloidogenic protein responsible for Parkinson's disease (PD), which is significantly larger (MW∼ 14kDa) with respect to insulin, previously investigated. The fluorophore was covalently bind at the C-terminal group of the peptide, an area of the polypeptide chain not or scarcely involved in fibrillation. We employed ATTO488 with a functional maleimide group that covalently bind the free cysteine at the C-terminal domain. We used dye-to-protein ratio 1:20 and 1:10. We demonstrated that the fibrillary aggregates of α-syn are all fluorescent, or at least just very minor portions of the sample are not labeled. These results show that labeling artifacts can be avoided, but also that the labeling strategies must be carefully designed to the molecular system under investigation. This work provides evidence of a fundamental drawback in fluorescence microscopy, generating a warning for all the fluorescence users: a deeper control for possible artifacts induced by the fluorophore is needed. The use of a correlative microscopy technique would play a key role in the control of the setting of these conditions.

Hypericin (Hyp), a pigment extracted from Hypericum perforatum, is a naturally occurring photosensitizer (PS), i.e. a compound that sensitizes the production of singlet oxygen (1O2) upon visible photo-excitation. 1O2 is an oxidizing molecule capable of damaging biomolecules such as lipids, nucleic acids and amino acids. The combination of a PS, visible light and molecular oxygen is at the basis of photodynamic therapy (PDT), a clinically approved treatment against cancer cells, but also effective against pathogenic bacteria, fungi and viruses. Hyp represents a very interesting PS, because it shows high singlet-oxygen quantum yield (φΔ=0.33) and high fluorescence quantum yield (φF=0.35) in DMSO. Due to the hydrophobic character of the molecule, these properties are lost in aqueous solution, where Hyp tends to form photo-inactive aggregates. The photo-physical and photo-dynamic properties are recovered when Hyp is bound to apolar systems, such as phospholipidic membranes or protein hydrophobic pockets. These features are useful for localization of the PS by fluorescence microscopy. In particular, Hyp was found to effectively inactivate viruses with a phospholipidic envelope, like SARS-CoV-2 upon light exposure, and sometimes also in dark conditions.We used fluorescence spectroscopy and microscopy to explore the interaction between SARS-CoV-2 and Hyp, and correlative AFM-fluorescence microscopy on a bilayer model membrane, to study the morphological changes induced by the presence of the PS. The aim of this research project is to assess the binding and photosensitizing properties of Hyp with SARS-CoV-2 and a model membrane, in order to provide more data for a future therapeutic implementation of PDT with this antiviral agent against SARS-CoV-2 and, more generally, all enveloped viruses.

The toxicity of α-synuclein (α-syn), the amyloidogenic protein responsible for Parkinson's disease, is likely related to its interaction with the asymmetric neuronal membrane. α-Syn exists as cytoplasmatic and as extracellular protein as well. To shed light on the different interactions occurring at the different α-syn localizations, we have here modelled the external and internal membrane leaflets of the neuronal membrane with two complex lipid mixtures, characterized by phase coexistence and with negative charge confined to either the ordered or the disordered phase, respectively. To this purpose, we selected a five-component (DOPC/SM/DOPE/DOPS/chol) and a four-component (DOPC/SM/GM1/chol) lipid mixtures, which contained the main membrane lipid constituents and exhibited a phase separation with formation of ordered domains. We have compared the action of α-syn in monomeric form and at different concentrations (1 nM, 40 nM, and 200 nM) with respect to lipid systems with different composition and shape by AFM, QCM-D, and vesicle leakage experiments. The experiments coherently showed a higher stability of the membranes composed by the internal leaflet mixture to the interaction with α-syn. Damage to membranes made of the external leaflet mixture was detected in a concentration-dependent manner. Interestingly, the membrane damage was related to the fluidity of the lipid domains and not to the presence of negatively charged lipids.

SLBs are a well-established model of the plasma membrane of eukaryotic cells. It is known that α-synuclein (α-syn), a peptide involved in Parkinson's disease, is present in both the inner (cytosol) and extracellular space. Both the endogenous and exogenous components seem to have a role in the neurodegenerative process typical of the disorder. It is also demonstrated that the physiological membrane has an asymmetric structure, i.e., the lipid compositions of the inner and outer leaflet are different. As a consequence of this asymmetry, also the organization of the lipid phase in the two leaflets are different. α-syn interacts with supported lipid bilayers (SLBs) with two different compositions, mimicking the inner and the outer leaflet of the cell membrane respectively, to reveal the possible destabilization induced by the pathological peptides. It is known that the presence of the rigid substrate, that support the membrane, reduces the diffusion of the single lipid molecules within the membrane and induce a mechanical stabilization of the membrane, that increase the resistance of the bilayer toward the action of an external agent. We proposed a new approach, making the peptides interact with lipid vescicles in solution, creating a planar bilayer after this interaction. in this way, α-syn interacts with a tridimensional membrane, that is better resembling the properties of the cell membrane. Before and after the interaction with α-syn the membrane were characterized by atomic force microscopy, quartz micor-balance, and fluorescence techniques.