Quantitative Attribution of the Protective Effects of Aminosterols against Protein Aggregates to Their Chemical Structures and Ability to Modulate Biological Membranes.

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-ß 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.

Insights in Cell Biomechanics through Atomic Force Microscopy.

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.

Adsorption of the rhNGF Protein on Polypropylene with Different Grades of Copolymerization.

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.

α-Synuclein interacts differently with membranes mimicking the inner and outer leaflets of neuronal membranes.

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.

Cholesterol Hinders the Passive Uptake of Amphiphilic Nanoparticles into Fluid Lipid Membranes.

Plasma membranes represent pharmacokinetic barriers for the passive transport of site-specific drugs within cells. When engineered nanoparticles (NPs) are considered as transmembrane drug carriers, the plasma membrane composition can affect passive NP internalization in many ways. Among these, cholesterol-regulated membrane fluidity is probably one of the most biologically relevant. Herein, we consider small (2-5 nm in core diameter) amphiphilic gold NPs capable of spontaneously and nondisruptively entering the lipid bilayer of plasma membranes. We study their incorporation into model 1,2-dioleoyl-sn-glycero-3-phosphocholine membranes with increasing cholesterol content. We combine dissipative quartz crystal microbalance experiments, atomic force microscopy, and molecular dynamics simulations to show that membrane cholesterol, at biologically relevant concentrations, hinders the molecular mechanism for passive NP penetration within fluid bilayers, resulting in a dramatic reduction in the amount of NP incorporated.

Quantitative Measurement of the Affinity of Toxic and Nontoxic Misfolded Protein Oligomers for Lipid Bilayers and of its Modulation by Lipid Composition and Trodusquemine.

Many neurodegenerative diseases are associated with the self-assembly of peptides and proteins into fibrillar aggregates. Soluble misfolded oligomers formed during the aggregation process, or released by mature fibrils, play a relevant role in neurodegenerative processes through their interactions with neuronal membranes. However, the determinants of the cytotoxicity of these oligomers are still unclear. Here we used liposomes and toxic and nontoxic oligomers formed by the same protein to measure quantitatively the affinity of the two oligomeric species for lipid membranes. To this aim, we quantified the perturbation to the lipid membranes caused by the two oligomers by using the fluorescence quenching of two probes embedded in the polar and apolar regions of the lipid membranes and a well-defined protein-oligomer binding assay using fluorescently labeled oligomers to determine the Stern-Volmer and dissociation constants, respectively. With both approaches, we found that the toxic oligomers have a membrane affinity 20-25 times higher than that of nontoxic oligomers. Circular dichroism, intrinsic fluorescence, and FRET indicated that neither oligomer type changes its structure upon membrane interaction. Using liposomes enriched with trodusquemine, a potential small molecule drug known to penetrate lipid membranes and make them refractory to toxic oligomers, we found that the membrane affinity of the oligomers was remarkably lower. At protective concentrations of the small molecule, the binding of the oligomers to the lipid membranes was fully prevented. Furthermore, the affinity of the toxic oligomers for the lipid membranes was found to increase and slightly decrease with GM1 ganglioside and cholesterol content, respectively, indicating that physicochemical properties of lipid membranes modulate their affinity for misfolded oligomeric species.

Correlative nanoscopy: A multimodal approach to molecular resolution.

Atomic force microscopy (AFM) is a nano-mechanical tool uniquely suited for biological studies at the molecular scale. AFM operation is based on mechanical interaction between the tip and the sample, a mechanism of contrast capable of measuring different information, including surface topography, mechanical, and electrical properties. However, the lack of specificity highlights the need to integrate AFM data with other techniques providing compositional hints. In particular, optical microscopes based on fluorescence as a mechanism of contrast can access the local distribution of specific molecular species. The coupling between AFM and super-resolved fluorescence microscopy solves the resolution mismatch between AFM and conventional fluorescence optical microscopy. Recent advances showed that also the inherently label-free imaging capabilities of the AFM are fundamental to complement the fluorescence images. In this review, we have presented a brief historical view on correlative microscopy, and, finally, we have summarized the progress of correlative AFM-super-resolution microscopy in biological research.

Clustering of Major Histocompatibility Complex-Class I Molecules in Healthy and Cancer Colon Cells Revealed from Their Nanomechanical Properties.

The activation of the T cell mediated immune response relies on the fine interaction between the T cell receptor on the immune cell and the antigen-presenting major histocompatibility complex (MHC) molecules on the membrane surface of antigen-presenting cells. Both the distribution and quantity of MHC/peptide complexes and their adequate morphological presentation affect the activation of the immune cells. In several types of cancer the immune response is down-regulated due to the low expression of MHC-class I (MHC-I) molecules on the cell's surface, and in addition, the mechanical properties of the membrane seem to play a role. Herein, we investigate the distribution of MHC-I molecules and the related nanoscale mechanical environment on the cell surface of two cell lines derived from colon adenocarcinoma and a healthy epithelial colon reference cell line. Atomic force microscopy (AFM) force spectroscopy analysis using an antibody-tagged pyramidal probe specific for MHC-I molecules and a formula that relates the elasticity of the cell to the energy of adhesion revealed the different population distributions of MHC-I molecules in healthy cells compared to cancer cells. We found that MHC-I molecules are significantly less expressed in cancer cells. Moreover, the local elastic modulus is significantly reduced in cancer cells. We speculate that these results might be related to the proven ability of cancer cells to evade the immune system, not only by reducing MHC-I cell surface expression but also by modifying the local mechanical properties affecting the overall morphology of MHC-I synapse presentation to immune cells.

Impact of Experimental Parameters on Cell-Cell Force Spectroscopy Signature.

Atomic force microscopy is an extremely versatile technique, featuring atomic-scale imaging resolution, and also offering the possibility to probe interaction forces down to few pN. Recently, this technique has been specialized to study the interaction between single living cells, one on the substrate, and a second being adhered on the cantilever. Cell-cell force spectroscopy offers a unique tool to investigate in fine detail intra-cellular interactions, and it holds great promise to elucidate elusive phenomena in physiology and pathology. Here we present a systematic study of the effect of the main measurement parameters on cell-cell curves, showing the importance of controlling the experimental conditions. Moreover, a simple theoretical interpretation is proposed, based on the number of contacts formed between the two interacting cells. The results show that single cell-cell force spectroscopy experiments carry a wealth of information that can be exploited to understand the inner dynamics of the interaction of living cells at the molecular level.

Multitarget Biological Profiling of New Naphthoquinone and Anthraquinone-Based Derivatives for the Treatment of Alzheimer's Disease.

Two series of naphthoquinone and anthraquinone derivatives decorated with an aromatic/heteroaromatic chain have been synthesized and evaluated as potential promiscuous agents capable of targeting different factors playing a key role in Alzheimer's disease (AD) pathogenesis. On the basis of the in vitro biological profiling, most of them exhibited a significant ability to inhibit amyloid aggregation, PHF6 tau sequence aggregation, acetylcholinesterase (AChE), and monoamine oxidase (MAO) B. In particular, naphthoquinone 2 resulted as one of the best performing multitarget-directed ligand (MTDL) experiencing a high potency profile in inhibiting ß-amyloid (Aß40) aggregation (IC50 = 3.2 µM), PHF6 tau fragment (91% at 10 µM), AChE enzyme (IC50 = 9.2 µM) jointly with a remarkable inhibitory activity against MAO B (IC50 = 7.7 nM). Molecular modeling studies explained the structure-activity relationship (SAR) around the binding modes of representative compound 2 in complex with hMAO B and hAChE enzymes, revealing inhibitor/protein key contacts and the likely molecular rationale for enzyme selectivity. Compound 2 was also demonstrated to be a strong inhibitor of Aß42 aggregation, with potency comparable to quercetin. Accordingly, atomic force microscopy (AFM) revealed that the most promising naphthoquinones 2 and 5 and anthraquinones 11 and 12 were able to impair Aß42 fibrillation, deconstructing the morphologies of its fibrillar aggregates. Moreover, the same compounds exerted a moderate neuroprotective effect against Aß42 toxicity in primary cultures of cerebellar granule cells. Therefore, our findings demonstrate that these molecules may represent valuable chemotypes toward the development of promising candidates for AD therapy.

Full-length TDP-43 and its C-terminal domain form filaments in vitro having non-amyloid properties.

Accumulation of ubiquitin-positive, tau- and α-synuclein-negative intracellular inclusions of TDP-43 in the central nervous system represents the major hallmark correlated to amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U). Such inclusions have variably been described as amorphous aggregates or more structured deposits having amyloid properties. Here we have purified full-length TDP-43 (FL TDP-43) and its C-terminal domain (Ct TDP-43) to investigate the morphological, structural and tinctorial features of aggregates formed in vitro by them at pH 7.4 and 37 °C. AFM images indicate that both protein variants show a tendency to form filaments. Moreover, we show that both FL TDP-43 and Ct TDP-43 filaments possess a largely disordered secondary structure, as ascertained by far-UV circular dichroism and Fourier transform infra-red spectroscopy, do not bind Congo red and induce a very weak increase of thioflavin T fluorescence, indicating the absence of a clear amyloid-like signature.

Making biological membrane resistant to the toxicity of misfolded protein oligomers: a lesson from trodusquemine.

Trodusquemine is an aminosterol known to prevent the binding of misfolded protein oligomers to cell membranes and to reduce their toxicity in a wide range of neurodegenerative diseases. Its precise mechanism of action, however, remains unclear. To investigate this mechanism, we performed confocal microscopy, fluorescence resonance energy transfer (FRET) and nuclear magnetic resonance (NMR) measurements, which revealed a strong binding of trodusquemine to large unilamellar vesicles (LUVs) and neuroblastoma cell membranes. Then, by combining quartz crystal microbalance (QCM), fluorescence quenching and anisotropy, and molecular dynamics (MD) simulations, we found that trodusquemine localises within, and penetrates, the polar region of lipid bilayer. This binding behaviour causes a decrease of the negative charge of the bilayer, as observed through ζ potential measurements, an increment in the mechanical resistance of the bilayer, as revealed by measurements of the breakthrough force applied with AFM and ζ potential measurements at high temperature, and a rearrangement of the spatial distances between ganglioside and cholesterol molecules in the LUVs, as determined by FRET measurements. These physicochemical changes are all known to impair the interaction of misfolded oligomers with cell membranes, protecting them from their toxicity. Taken together, our results illustrate how the incorporation in cell membranes of sterol molecules modified by the addition of polyamine tails leads to the modulation of physicochemical properties of the cell membranes themselves, making them more resistant to protein aggregates associated with neurodegeneration. More generally, they suggest that therapeutic strategies can be developed to reinforce cell membranes against protein misfolded assemblies.

Amphiphilic gold nanoparticles perturb phase separation in multidomain lipid membranes.

Amphiphilic gold nanoparticles with diameters in the 2-4 nm range are promising as theranostic agents thanks to their spontaneous translocation through cell membranes. This study addresses the effects that these nanoparticles may have on a distinct feature of plasma membranes: lipid lateral phase separation. Atomic force microscopy, quartz crystal microbalance, and molecular dynamics are combined to study the interaction between model neuronal membranes, which spontaneously form ordered and disordered lipid domains, and amphiphilic gold nanoparticles having negatively charged surface functionalization. Nanoparticles are found to interact with the bilayer and form bilayer-embedded ordered aggregates. Nanoparticles also suppress lipid phase separation, in a concentration-dependent fashion. A general, yet simple thermodynamic model is developed to show that the change of lipid-lipid enthalpy is the dominant driving force towards the nanoparticle-induced destabilization of phase separation.

Morphological and Mechanical Characterization of DNA SAMs Combining Nanolithography with AFM and Optical Methods.

The morphological and mechanical properties of thiolated ssDNA films self-assembled at different ionic strength on flat gold surfaces have been investigated using Atomic Force Microscopy (AFM). AFM nanoshaving experiments, performed in hard tapping mode, allowed selectively removing molecules from micro-sized regions. To image the shaved areas, in addition to the soft contact mode, we explored the use of the Quantitative Imaging (QI) mode. QI is a less perturbative imaging mode that allows obtaining quantitative information on both sample topography and mechanical properties. AFM analysis showed that DNA SAMs assembled at high ionic strength are thicker and less deformable than films prepared at low ionic strength. In the case of thicker films, the difference between film and substrate Young's moduli could be assessed from the analysis of QI data. The AFM finding of thicker and denser films was confirmed by X-Ray Photoelectron Spectroscopy (XPS) and Spectroscopic Ellipsometry (SE) analysis. SE data allowed detecting the DNA UV absorption on dense monomolecular films. Moreover, feeding the SE analysis with the thickness data obtained by AFM, we could estimate the refractive index of dense DNA films.

Novel Ultrathin Films Based on a Blend of PEG-b-PCL and PLLA and Doped with ZnO Nanoparticles.

In this paper, a novel nanofilm type is proposed based on a blend of poly(ethylene glycol)-block-poly(ε-caprolactone) methyl ether (PEG-b-PCL) and poly(l-lactic acid), doped with zinc oxide nanoparticles (ZnO NPs) at different concentrations (0.1, 1, and 10 mg/mL). All nanofilm types were featured by a thickness value of ∼500 nm. Increasing ZnO NP concentrations implied larger roughness values (∼22 nm for the bare nanofilm and ∼67 nm for the films with 10 mg/mL of NPs), larger piezoelectricity (average d33 coefficient for the film up to ∼1.98 pm/V), and elastic modulus: the nanofilms doped with 1 and 10 mg/mL of NPs were much stiffer than the nondoped controls and nanofilms doped with 0.1 mg/mL of NPs. The ZnO NP content was also directly proportional to the material melting point and crystallinity and inversely proportional to the material degradation rate, thus highlighting the stabilization role of ZnO particles. In vitro tests were carried out with cells of the musculoskeletal apparatus (fibroblasts, osteoblasts, chondrocytes, and myoblasts). All cell types showed good adhesion and viability on all substrate formulations. Interestingly, a higher content of ZnO NPs in the matrix demonstrated higher bioactivity, boosting the metabolic activity of fibroblasts, myoblasts, and chondrocytes and enhancing the osteogenic and myogenic differentiation. These findings demonstrated the potential of these nanocomposite matrices for regenerative medicine applications, such as tissue engineering.

AFM-STED correlative nanoscopy reveals a dark side in fluorescence microscopy imaging.

It is known that the presence of fluorophores can influence the dynamics of molecular processes. Despite this, an affordable technique to control the fluorophore distribution within the sample, as well as the rise of unpredictable anomalous processes induced by the fluorophore itself, is missing. We coupled a stimulated emission depletion (STED) microscope with an atomic force microscope to investigate the formation of amyloid aggregates. In particular, we studied the in vitro aggregation of insulin and two alloforms of ß amyloid peptides. We followed standard methods to induce the aggregation and to label the molecules at different dye-to-protein ratios. Only a fraction of the fibrillar aggregates was displayed in STED images, indicating that the labeled molecules did not participate indistinctly to the aggregation process. This finding demonstrates that labeled molecules follow only selected pathways of aggregation, among the multiple that are present in the aggregation reaction.

Poly(furfuryl alcohol)-Polycaprolactone Blends.

Poly(furfuryl alcohol) (PFA) is a bioresin synthesized from furfuryl alcohol (FA) that is derived from renewable saccharide-rich biomass. In this study, we compounded this bioresin with polycaprolactone (PCL) for the first time, introducing new functional polymer blends. Although PCL is biodegradable, its production relies on petroleum precursors such as cyclohexanone oils. With the method proposed herein, this dependence on petroleum-derived precursors/monomers is reduced by using PFA without significantly modifying some important properties of the PCL. Polymer blend films were produced by simple solvent casting. The blends were characterized in terms of surface topography by atomic force microscopy (AFM), chemical interactions between PCL and PFA by attenuated total reflection-Fourier transform infrared (ATR-FTIR), crystallinity by XRD, thermal properties by differential scanning calorimetry (DSC), and mechanical properties by tensile tests and biocompatibility by direct and indirect toxicity tests. PFA was found to improve the gas barrier properties of PCL without compromising its mechanical properties, and it demonstrated sustained antioxidant effect with excellent biocompatibility. Our results indicate that these new blends can be potentially used in diverse applications ranging from food packing to biomedical devices.

Soft Hydrogel Zwitterionic Coatings Minimize Fibroblast and Macrophage Adhesion on Polyimide Substrates.

Minimizing the foreign body reaction to polyimide-based implanted devices plays a pivotal role in several biomedical applications. In this work, we propose materials exhibiting nonbiofouling properties and a Young's modulus reflecting that of soft human tissues. We describe the synthesis, characterization, and in vitro validation of poly(carboxybetaine) hydrogel coatings covalently attached to polyimide substrates via a photolabile 4-azidophenyl group, incorporated in poly(carboxybetaine) chains at two concentrations of 1.6 and 3.1 mol %. The presence of coatings was confirmed by attenuated total reflectance Fourier transform infrared spectroscopy. White light interferometry was used to evaluate the coating continuity and thickness (between 3 and 6 µm under dry conditions). Confocal laser scanning microscopy allowed us to quantify the thickness of the swollen hydrogel coatings that ranged between 13 and 32 µm. The different hydrogel formulations resulted in stiffness values ranging from 2 to 19 kPa and led to different fibroblast and macrophage responses in vitro. Both cell types showed a minimum adhesion on the softest hydrogel type. In addition, both the overall macrophage activation and cytotoxicity were observed to be negligible for all of the tested material formulations. These results are a promising starting point toward future advanced implantable systems. In particular, such technology paves the way for novel neural interfaces able to minimize the fibrotic reaction, once implanted in vivo, and to maximize their long-term stability and functionality.

Nanoscale Discrimination between Toxic and Nontoxic Protein Misfolded Oligomers with Tip-Enhanced Raman Spectroscopy.

Highly toxic protein misfolded oligomers associated with neurological disorders such as Alzheimer's and Parkinson's diseases are nowadays considered primarily responsible for promoting synaptic failure and neuronal death. Unraveling the relationship between structure and neurotoxicity of protein oligomers appears pivotal in understanding the causes of the pathological process, as well as in designing novel diagnostic and therapeutic strategies tuned toward the earliest and presymptomatic stages of the disease. Here, it is benefited from tip-enhanced Raman spectroscopy (TERS) as a surface-sensitive tool with spatial resolution on the nanoscale, to inspect the spatial organization and surface character of individual protein oligomers from two samples formed by the same polypeptide sequence and different toxicity levels. TERS provides direct assignment of specific amino acid residues that are exposed to a large extent on the surface of toxic species and buried in nontoxic oligomers. These residues, thanks to their outward disposition, might represent structural factors driving the pathogenic behavior exhibited by protein misfolded oligomers, including affecting cell membrane integrity and specific signaling pathways in neurodegenerative conditions.

Plasminogen activation triggers transthyretin amyloidogenesis in vitro.

Systemic amyloidosis is a usually fatal disease caused by extracellular accumulation of abnormal protein fibers, amyloid fibrils, derived by misfolding and aggregation of soluble globular plasma protein precursors. Both WT and genetic variants of the normal plasma protein transthyretin (TTR) form amyloid, but neither the misfolding leading to fibrillogenesis nor the anatomical localization of TTR amyloid deposition are understood. We have previously shown that, under physiological conditions, trypsin cleaves human TTR in a mechano-enzymatic mechanism that generates abundant amyloid fibrils in vitro In sharp contrast, the widely used in vitro model of denaturation and aggregation of TTR by prolonged exposure to pH 4.0 yields almost no clearly defined amyloid fibrils. However, the exclusive duodenal location of trypsin means that this enzyme cannot contribute to systemic extracellular TTR amyloid deposition in vivo Here, we therefore conducted a bioinformatics search for systemically active tryptic proteases with appropriate tissue distribution, which unexpectedly identified plasmin as the leading candidate. We confirmed that plasmin, just as trypsin, selectively cleaves human TTR between residues 48 and 49 under physiological conditions in vitro Truncated and full-length protomers are then released from the native homotetramer and rapidly aggregate into abundant fibrils indistinguishable from ex vivo TTR amyloid. Our findings suggest that physiological fibrinolysis is likely to play a critical role in TTR amyloid formation in vivo Identification of this surprising intersection between two hitherto unrelated pathways opens new avenues for elucidating the mechanisms of TTR amyloidosis, for seeking susceptibility risk factors, and for therapeutic innovation.