Rosmarinic acid turned α-syn oligomers into non-toxic species preserving microtubules in Raw 264.7 cells.

Parkinson's disease (PD) is the second most prevalent neurodegenerative disorder worldwide, and the therapeutic is focused on several approaches including the inhibition of fibril formation by small compounds, avoiding the formation of cytotoxic oligomers. Thus, we decided to explore the capacity of compounds carrying catechol moieties to inhibit the progression of α-synuclein. Overall, the compounds rosmarinic acid (1), carnosic acid (2), carnosol (3), epiisorosmanol (4), and rosmanol (5) avoid the progression of fibril formation assessed by Thiofavine T (ThT), and atomic force microscopy images showed that morphology is influenced for the actions of compounds over fibrillization. Moreover, ITC experiments showed a Kd varying from 28 to 51 µM, the ΔG showed that the reaction between compounds and α-syn is spontaneous, and ΔH is associated with an exothermic reaction, suggesting the interactions of hydrogen bonds among compounds and α-syn. Docking experiments reinforce this idea showing the intermolecular interactions are mostly hydrogen bonding within the sites 2, 9, and 3/13 of α-synuclein, and compounds 1 and 5. Thus, compound 1, rosmarinic acid, interestingly interacts better with site 9 through catechol and Lysines. In cultured Raw 264. 7 cells, the presence of compounds showed that most of them can promote cell differentiation, especially rosmarinic acid, and rosmanol, both preserving tubulin cytoskeleton. However, once we evaluated whether or not the aggregates pre-treated with compounds could prevent the disruption of microtubules of Raw 264.7 cells, only pre-treated aggregates with rosmarinic acid prevented the disruption of the cytoskeleton. Altogether, we showed that especially rosmarinic acid not only inhibits α-syn but stabilizes the remaining aggregates turning them into not-toxic to Raw 264.7 cells suggesting a main role in cell survival and antigen processing in response to external α-syn aggregates.

The Cytotoxic Effect of α-Synuclein Aggregates.

Parkinson's disease is a neurodegenerative disorder involving a functional protein, α-synuclein, whose primary function is related to vesicle trafficking. However, α-synuclein is prone to form aggregates, and these inclusions, known as Lewy bodies, are the hallmark of Parkinson's disease. α-synuclein can alter its conformation and acquire aggregating capacity, forming aggregates containing ß-sheets. This protein's pathogenic importance is based on its ability to form oligomers that impair synaptic transmission and neuronal function by increasing membrane permeability and altering homeostasis, generating a deleterious effect over cells. First, we establish that oligomers interfere with the mechanical properties of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) membrane, as demonstrated by nanoindentation curves. In contrast, nanoindentation revealed that the α-synuclein monomer's presence leads to a much more resistant lipid bilayer. Moreover, the oligomers' interaction with cell membranes can promote lactate dehydrogenase (LDH) release, suggesting the activation of cytotoxic events.

Dammarane triterpenes targeting α-synuclein: biological activity and evaluation of binding sites by molecular docking.

Parkinson's disease (PD) is a neurodegenerative disorder that affects adult people whose treatment is palliative. Thus, we decided to test three dammarane triterpenes 1, 1a, 1b, and we determined that 1 and 1a inhibit ß-aggregation through thioflavine T rather than 1b. Since compound 1 was most active, we determined the interaction between α-synuclein and 1 at 50 µM (Kd) through microscale thermophoresis. Also, we observed differences in height and diameter of aggregates, and α-synuclein remains unfolded in the presence of 1. Also, aggregates treated with 1 do not provoke neurites' retraction in N2a cells previously induced by retinoic acid. Finally, we studied the potential sites of interaction between 1 with α-synuclein fibrils using molecular modelling. Docking experiments suggest that 1 preferably interact with the site 2 of α-synuclein through hydrogen bonds with residues Y39 and T44.

The Fumarprotocetraric Acid Inhibits Tau Covalently, Avoiding Cytotoxicity of Aggregates in Cells.

Neurodegenerative disorders, including Tauopathies that involve tau protein, base their pathological mechanism on forming proteinaceous aggregates, which has a deleterious effect on cells triggering an inflammatory response. Moreover, tau inhibitors can exert their mechanism of action through noncovalent and covalent interactions. Thus, Michael's addition appears as a feasible type of interaction involving an α, ß unsaturated carbonyl moiety to avoid pathological confirmation and further cytotoxicity. Moreover, we isolated three compounds from Antarctic lichens Cladonia cariosa and Himantormia lugubris: protolichesterinic acid (1), fumarprotocetraric acid (2), and lichesterinic acid (3). The maleimide cysteine labeling assay showed that compounds 1, 2, and 3 inhibit at 50 µM, but compounds 2 and 3 are statistically significant. Based on its inhibition capacity, we decided to test compound 2 further. Thus, our results suggest that compound 2 remodel soluble oligomers and diminish ß sheet content, as demonstrated through ThT experiments. Hence, we added externally treated oligomers with compound 2 to demonstrate that they are harmless in cell culture. First, the morphology of cells in the presence of aggregates does not suffer evident changes compared to the control. Additionally, the externally added aggregates do not provoke a substantial LDH release compared to the control, indicating that treated oligomers do not provoke membrane damage in cell culture compared with aggregates alone. Thus, in the present work, we demonstrated that Michael's acceptors found in lichens could serve as a scaffold to explore different mechanisms of action to turn tau aggregates into harmless species.

Gel-Like Calcium Carbonate Precursors Observed by in situ AFM.

The debate about crystallization processes is still ongoing and nonclassical crystallization mechanisms attract more and more attention. This work indicates that polymer induced liquid precursor (PILP) phases play a role for nonclassical calcium carbonate crystallization and growth processes. Here we report the observation of gel-like precursors for the crystal growth on a calcite surface by means of an in situ AFM study. These precursors spread out on the surface with time supporting their liquid character. This study will give new insights into biomineralization and crystallization processes in general.

Rosmarinic acid prevents fibrillization and diminishes vibrational modes associated to ß sheet in tau protein linked to Alzheimer's disease.

Alzheimer's disease is a common tauopathy where fibril formation and aggregates are the hallmark of the disease. Efforts targeting amyloid-ß plaques have succeeded to remove plaques but failed in clinical trials to improve cognition; thus, the current therapeutic strategy is at preventing tau aggregation. Here, we demonstrated that four phenolic diterpenoids and rosmarinic acid inhibit fibrillization. Since, rosmarinic acid was the most active compound, we observe morphological changes in atomic force microscopy images after treatment. Hence, rosmarinic acid leads to a decrease in amide regions I and III, indicating that rosmarinic acid prevents ß-sheet assembly. Molecular docking study inside the steric zipper model of the hexapeptide 306VQIVYK311 involved in fibrillization and ß sheet formation, suggests that rosmarinic acid binds to the steric zipper with similar chemical interactions with respect to those observed for orange G, a known pharmacofore for amyloid.

Assessment of the nature interactions of ß-amyloid protein by a nanoprobe method.

We present a method based on atomic force microscopy (AFM) to assess the work of adhesion between the interfaces of gold AFM tips functionalized with three peptides derived from ß-sheet breaker LPFFD [CLPFFD-NH2 (i0) and their isomers CDLPFF-NH2 (i1) and CLPDFF-NH2 (i2)], and the beta-amyloid protein (Aß1-42). ß-Amyloid protein was deposited onto a highly oriented graphite (HOPG) surface as protofibrils and fibrils. The presence of the residues Leu (L), Phe (F), and Phe (F), which are also present in the native sequence, confirm that the peptides are able to bind to the aggregates of Aß1-42 fibrils and protofibrils. Force of adhesion data were directly obtained from the maximum force on retraction, and the work of adhesion was calculated from the Jhonson-Kendall-Roberts model (JKR-Model). Both the polar and dispersive contributions to the surface energy of the peptides i0, i1, and i2, as well as Aß1-42 fibrils and protofibrils, were determined by means of measuring the contact angle and using the two-fluid method. The macroscopic energies of the functionalized gold surfaces do not differ significantly between isomers, which confirms the similar nature of the peptides i0, i1, and i2 but suggests that the macroscopic measurements are not able to distinguish specific sequences. The nanoprobe reveals a typical adhesion work value associated with the interaction of protofibrils with i0 and i2; this value is three times higher than that of i1. The difference is attributed to the hydrophobic nature of protofibrils, the predominant exposition of hydrophobic residues of the peptides i0 and i2, with respect to i1, and the degree of functionalization. i0 and i2 presented a slight adhesion with Aß fibrils, which is associated with the exposed hydrophilic groups of these fibrils (onto HOPG) compared to the protofibrils. However, i1 showed interaction with both Aß fibrils and protofibrils. For this, we propose an explanation based on the fact that the peptide i1 locates itself adjacent to the gold surface of the probe, concealing their hydrophobic groups and therefore decreasing the probability of interaction with Aß fibrils and protofibrils. The peptide-gold nano probe represents a useful tool to study the nanobiointeractions of functionalized nanoparticles with amyloid aggregates.

Mechanical force activates the light-dependent channels TRP and TRPL in excised patches from the rhabdomere of Drosophila photoreceptors.

Drosophila phototransduction in light-sensitive microvilli involves a metabotropic signaling cascade. Photoisomerized rhodopsin couples to G-protein, activating phospholipase C, which cleaves phosphatidylinositol bisphosphate (PIP2) into inositol trisphosphate, diacylglycerol (DAG) and a proton. DAG is converted into phosphatidic acid by DAG-kinase and metabolized to L-linoleoyl glycerol (2-LG) by DAG-lipase. This complex enzyme cascade ultimately opens the light-dependent transient receptor potential channels, TRP and TRPL. PIP2, DAG, H+ and 2-LG are possible channel activators, either individually or combined, but their direct participation in channel-gating remains unresolved. Molecular interaction with the channels, modification of the channels' lipid moiety and mechanical force on the channels by changes in the membrane structure derived from light-dependent changes in lipid composition are possible gating agents. In this regard, mechanical activation was suggested, based on a rapid light-dependent contraction of the photoreceptors mediated by the phototransduction cascade. Here, we further examined this possibility by applying force to inside-out patches from the microvilli membrane by changing the pressure in the pipette or pulling the membrane with a magnet through superparamagnetic nanospheres. The channels were opened by mechanical force, while mutant lacking both channels was insensitive to mechanical stimulation. Atomic Force Microscopy showed that the stiffness of an artificial phospholipid bilayer was increased by arachidonic acid and diacylglycerol whereas elaidic acid was ineffective, mirroring their relative effects in channel activity previously observed electrophysiologically. Together, the results are consistent with the notion that light-induced changes in lipid composition alter the membrane structure, generating mechanical force on the channels leading to channel opening.

α-Synuclein Drives Tau's Cytotoxic Aggregates Formation through Hydrophobic Interactions.

Tau and α-synuclein are proteins involved in pathologies known as tauopathies and synucleinopathies, respectively. Moreover, evidence shows that there is a crosstalk between them as is seen in the brains of individuals with sporadic neurodegenerative disorders. Based on that, we present data showing that the hydrophobic α-peptide 71 VTGVTAVAQKTV82 induces the aggregation of the full-length tau fragment in the absence of heparin assessed by ThT. Moreover, AFM images reveal the presence of straight filaments and amorphous aggregates of full-length tau in the presence of the α-peptide. Additionally, ITC experiments showed the interaction of the α-peptide with tau full-length (441 amino acids),4R (amino acids from 244 to 369), and both hexapeptides 275 VQIINK280 and 306 VQIVYK311 through hydrophobic interactions. The Raman spectroscopy spectra showed conformational changes in the Amide region in the aggregates formed with full-length tau and α-syn peptide. Furthermore, the incubation of extracellular aggregates with N2a cells showed morphological differences in the cellular body and the nucleus suggesting cell death. Moreover,, the incubation of different types of aggregates in cell culture provokes the release of Lactate dehydrogenase (LDH). Altogether, we found that α-synuclein peptide can drive the aggregation of full-length tau-provoking morphological and structural changes evoking cytotoxic effects.

Adhesion, stretching, and electrical charge assessment of dermatan sulfate molecules by colloidal probes.

Electrical and mechanical properties of dermatan sulfate (DS) molecules are studied in an aqueous environment as a function of pH. DS molecules linked at various points distributed on the surface of mica previously silanizated along with a suitable functionalized microsphere, attached to the cantilever of an atomic force microscope (AFM), provided suitable surfaces for testing interactions through the colloidal probe methodology. The repulsive force between the surfaces indicated that the charge of DS increases with pH as a result of the gradual deprotonation of acidic groups. Pulling experiments revealed increasing adhesion of DS to the monolayer as a function of pH, presumably due both to the electrical nature of the interaction between these molecules and the progressive increase of the charge of DS with pH. Serrations exhibited by the force in pulling experiments indicate that more than a single DS molecule is stretched at the same time. In addition, pulling force remained significant even at extensions that went beyond the average contour length of a single DS molecule, which suggests the existence of a significant link between DS molecules.

Exploring the surface charge on peptide-gold nanoparticle conjugates by force spectroscopy.

The conformation and charge exposure of peptides attached to colloidal gold nanoparticles (AuNPs) are critical for both the colloidal stability and for the recognition of biological targets in biomedical applications such as diagnostics and therapy. We prepared conjugates of AuNPs and three isomer peptides capable of recognizing toxic aggregates of the amyloid beta protein (Abeta) involved in Alzheimer's disease, namely, CLPFFD-CONH(2) (i0), CDLPFF-CONH(2) (i1), and CLPDFF-CONH(2) (i2), where D is the amino acid aspartic acid that is negatively charged at pH = 7.4. We then studied the effect of peptide sequence on the charge exposure through force spectroscopy measurements. The peptide-AuNPs conjugates were fixed on glass surfaces, and their interactions with peptide-functionalized tips were determined. Our results show a higher density of surface charge in the conjugates of the isomers i0 and i2 and a lower density in i1, which is due to the higher degree of functionalization in the first two compared with the third. However, the charge per molecule of the peptide is higher for i1 with respect to i0 and i2, which could be related to the local conformation that the peptides adopt on the surface. The acid-base behavior of the peptide anchored to the AuNPs is different than expected in aqueous solutions of free peptides, which could be related to the low accessibility of the NH(2)-terminal group belonging to the cysteine that is located near the AuNPs surface. In contrast with other techniques, the fixation of the peptide-AuNPs conjugates to a surface allows for characterization of the local charge exposure of peptides anchored to AuNPs over a wide range of pH.

Does Bacterial Elasticity Affect Adhesion to Polymer Fibers?

The factors governing bacterial adhesion to substrates with different topographies are still not fully identified. The present work seeks to elucidate for the first time and with quantitative data the roles of bacterial elasticity and shape and substrate topography in bacterial adhesion. With this aim, populations of three bacterial species, P. aeruginosa DSM 22644, B. subtilis DSM 10, and S. aureus DSM 20231 adhered on flat substrates covered with electrospun polycaprolactone fibers of different diameters ranging from 0.4 to 5.5 µm are counted. Populations of bacterial cells are classified according to the preferred binding sites of the bacteria to the substrate. The colloidal probe technique was used to assess the stiffness of the bacteria and bacteria-polymer surface adhesion energy. A theoretical model is developed to interpret the observed populations in terms of a balance between stiffness and adhesion energy of the bacteria. The model, which also incorporates the radius of the fiber and the size and shape of the bacteria, predicts increased adhesion for a low level of stiffness and for a larger number of available bacteria-fiber contact points. Te adhesive propensity of bacteria depends in a nontrivial way on the radius of the fibers due to the random arrangement of fibers.

Stress transfer and matrix-cohesive fracture mechanism in microfibrillated cellulose-gelatin nanocomposite films.

Microfibrillated cellulose (MFC) obtained from eucalyptus was embedded in gelatin from two sources; namely bovine and salmon gelatin. Raman spectroscopy revealed that stress is transferred more efficiently from bovine gelatin to the MFC when compared to salmon gelatin. Young's modulus, tensile strength, strain at failure and work of fracture of the nanocomposite films were improved by ∼67, 131, 43 y 243% respectively when using salmon gelatin as matrix material instead of bovine gelatin. Imaging of the tensile fracture surface of the MFC-gelatin nanocomposites revealed that crack formation occurs predominantly within bovine and salmon gelatin matrices rather than within the MFC or at the MFC/gelatin interface. This suggests that the mechanical failure mechanism in these nanocomposite materials is predominantly governed by a matrix-cohesive fracture mechanism. Both strength and flexibility are desirable properties for composite coatings made from gelatin-based materials, and so the findings of this study could assist in their utilization in the food and pharmaceutical industry.

Fulvic acid inhibits aggregation and promotes disassembly of tau fibrils associated with Alzheimer's disease.

Alzheimer's disease is a neurodegenerative disorder involving extracellular plaques (amyloid-ß) and intracellular tangles of tau protein. Recently, tangle formation has been identified as a major event involved in the neurodegenerative process, due to the conversion of either soluble peptides or oligomers into insoluble filaments. At present, the current therapeutic strategies are aimed at natural phytocomplexes and polyphenolics compounds able to either inhibit the formation of tau filaments or disaggregate them. However, only a few polyphenolic molecules have emerged to prevent tau aggregation, and natural drugs targeting tau have not been approved yet. Fulvic acid, a humic substance, has several nutraceutical properties with potential activity to protect cognitive impairment. In this work we provide evidence to show that the aggregation process of tau protein, forming paired helical filaments (PHFs) in vitro, is inhibited by fulvic acid affecting the length of fibrils and their morphology. In addition, we investigated whether fulvic acid is capable of disassembling preformed PHFs. We show that the fulvic acid is an active compound against preformed fibrils affecting the whole structure by diminishing length of PHFs and probably acting at the hydrophobic level, as we observed by atomic force techniques. Thus, fulvic acid is likely to provide new insights in the development of potential treatments for Alzheimer's disease using natural products.

Droplets of fine powders running uphill by vertical vibration.

We observe "droplets" forming when an inclined surface initially covered by fine powders is vibrated vertically. Droplets move uphill in the direction of maximum local slope. The speed of droplets is nearly independent on their size whereas it is an increasing function of the plate acceleration and inclination. By evacuating the container we show that the interstitial air flow plays an important role on droplets forming and their drift.