Effect of Reduced Feedback Frequencies on Motor Learning in a Postural Control Task in Young Adults.

The effects of the use of reduced feedback frequencies on motor learning remain controversial in the scientific literature. At present, there is still controversy about the guidance hypothesis, with some works supporting it and others contradicting it. To shed light on this topic, an experiment was conducted with four groups, each with different feedback frequencies (0%, 33%, 67%, and 100%), which were evaluated three times (pre-test, post-test, and retention) during a postural control task. In addition, we tested whether there was a transfer in performance to another similar task involving postural control. As a result, only the 67% feedback group showed an improvement in their task performance in the post-test and retention evaluations. Nevertheless, neither group showed differences in motor transfer performance compared to another postural control task. In conclusion, the findings of this paper corroborate the hypothesis of guidance and suggest that the use of a reduced frequency of 67% is a better option for improving motor learning than options that offer feedback at a lower frequency, at all trials or not at all.

Nucleotide-Induced Nanoscale Changes in the Mechanical Properties of Rat Cerebellar Astrocytes: Selective Stimulation and Blocking of the Purinergic Receptor P2X7.

As members of the family of nucleotide receptors, P2X7 receptors are of particular interest due to their unique structural and pharmacological characteristics. As ATP-gated ionic channels, P2X7 receptors in their activation elicit membrane depolarization; extracellular calcium influx; and activation of several downstream intracellular signaling pathways, some of them independent of the ionic channel activity. Further interactions of P2X7 receptors and cytoskeleton-related proteins have also been confirmed, and we previously described the effects of P2X7 receptor stimulation on the morphology of rat cerebellar astrocytes. In the present work, we used time-lapse video microscopy and atomic force microscopy (AFM) to elucidate the effects of P2X7 receptor stimulation on the morphology, migratory capabilities, and mechanical properties of rat cerebellar astrocytes in vitro. Stimulation of P2X7 receptors with the selective agonist BzATP specifically caused an increase in cell size, motility, and number of membrane protrusions of the astrocytes in culture. These effects were reverted when cells were previously treated with the competitive antagonist of P2X7R, A 438079. AFM analysis also showed an increase in cell stiffness and viscosity after P2X7 receptor stimulation. Surprisingly, these effects on the mechanical properties of the cell were not blocked by the treatment with the antagonist. Fluorescence microscopy analysis of the actin cytoskeleton showed an increase in actin stress fibers after BzATP treatment, an effect that again was not blocked by previous treatment with the antagonist, further confirming that the effects of P2X7 receptors on the cytoskeleton of astrocytes are, at least in part, independent of the ionic channel activity.

Nanostructured scaffolds based on bioresorbable polymers and graphene oxide induce the aligned migration and accelerate the neuronal differentiation of neural stem cells.

Within the field of neural tissue engineering, there is a huge need for the development of materials that promote the adhesion, aligned migration and differentiation of stem cells into neuronal and supportive glial cells. In this study, we have fabricated bioresorbable elastomeric scaffolds combining an ordered nanopatterned topography together with a surface functionalization with graphene oxide (GO) in mild conditions. These scaffolds allowed the attachment of murine neural stem cells (NSCs) without the need of any further coating of its surface with extracellular matrix adhesion proteins. The NSCs were able to give rise to both immature neurons and supporting glial cells over the nanostructured scaffolds in vitro, promoting their aligned migration in cell clusters following the nanostructured grooves. This system has the potential to reestablish spatially oriented neural precursor cell connectivity, constituting a promising tool for future cellular therapy including nerve tissue regeneration.

Nucleotides-Induced Changes in the Mechanical Properties of Living Endothelial Cells and Astrocytes, Analyzed by Atomic Force Microscopy.

Endothelial cells and astrocytes preferentially express metabotropic P2Y nucleotide receptors, which are involved in the maintenance of vascular and neural function. Among these, P2Y1 and P2Y2 receptors appear as main actors, since their stimulation induces intracellular calcium mobilization and activates signaling cascades linked to cytoskeletal reorganization. In the present work, we have analyzed, by means of atomic force microscopy (AFM) in force spectroscopy mode, the mechanical response of human umbilical vein endothelial cells (HUVEC) and astrocytes upon 2MeSADP and UTP stimulation. This approach allows for simultaneous measurement of variations in factors such as Young's modulus, maximum adhesion force and rupture event formation, which reflect the potential changes in both the stiffness and adhesiveness of the plasma membrane. The largest effect was observed in both endothelial cells and astrocytes after P2Y2 receptor stimulation with UTP. Such exposure to UTP doubled the Young's modulus and reduced both the adhesion force and the number of rupture events. In astrocytes, 2MeSADP stimulation also had a remarkable effect on AFM parameters. Additional studies performed with the selective P2Y1 and P2Y13 receptor antagonists revealed that the 2MeSADP-induced mechanical changes were mediated by the P2Y13 receptor, although they were negatively modulated by P2Y1 receptor stimulation. Hence, our results demonstrate that AFM can be a very useful tool to evaluate functional native nucleotide receptors in living cells.

Analysis of Binding Interactions of Ramipril and Quercetin on Human Serum Albumin: A Novel Method in Affinity Evaluation.

The aim of this study was to analyze the binding interactions between a common antihypertensive drug (ramipril, R) and the widely distributed plant flavonoid quercetin (Q), in the presence of human serum albumin (HSA). From the observed fluorescence spectra of the (HSA + R) system we can assume that ramipril is also one of the Site 3 ligands-similar to fusidic acid-the binding of which has been proven by RTG crystallography. Our claim is supported by near-UV CD spectroscopy, microscale themophoresis and molecular modeling. The presence of R slightly inhibited the subsequent binding of Q to HSA and, on the contrary, the pre-incubation of HSA with Q caused a stronger binding of R, most likely due to allosteric interactions. At high concentrations, R is also able to displace Q from its binding site. The dissociation constant KD for the binding of R is more than hundredfold larger than for Q, which means that R is a very weak binder to HSA. The knowledge of qualitative and quantitative parameters of R, as well as the methods used in this study, are important for future research into HSA binding. This study shows the importance of implementing other methods for KD determination. Microscale thermophoresis has proved to be a novel, practical and accurate method for KD determination on HSA, especially in cases when fluorescence spectroscopy is unable to produce usable results.

Resveratrol-Induced Temporal Variation in the Mechanical Properties of MCF-7 Breast Cancer Cells Investigated by Atomic Force Microscopy.

Atomic force microscopy (AFM) combined with fluorescence microscopy has been used to quantify cytomechanical modifications induced by resveratrol (at a fixed concentration of 50 µM) in a breast cancer cell line (MCF-7) upon temporal variation. Cell indentation methodology has been utilized to determine simultaneous variations of Young's modulus, the maximum adhesion force, and tether formation, thereby determining cell motility and adhesiveness. Effects of treatment were measured at several time-points (0-6 h, 24 h, and 48 h); longer exposures resulted in cell death. Our results demonstrated that AFM can be efficiently used as a diagnostic tool to monitor irreversible morpho/nano-mechanical changes in cancer cells during the early steps of drug treatment.

Study of Interactions between Amlodipine and Quercetin on Human Serum Albumin: Spectroscopic and Modeling Approaches.

The aim of this study was to analyze the binding interactions between a common antihypertensive drug (amlodipine besylate-AML) and the widely distributed plant flavonoid quercetin (Q), in the presence of human serum albumin (HSA). Fluorescence analysis was implemented to investigate the effect of ligands on albumin intrinsic fluorescence and to define the binding and quenching properties. Further methods, such as circular dichroism and FT-IR, were used to obtain more details. The data show that both of these compounds bind to Sudlow's Site 1 on HSA and that there exists a competitive interaction between them. Q is able to displace AML from its binding site and the presence of AML makes it easier for Q to bind. AML binds with the lower affinity and if the binding site is already occupied by Q, it binds to the secondary binding site inside the same hydrophobic pocket of Sudlow's Site 1, with exactly the same affinity. Experimental data were complemented with molecular docking studies. The obtained results provide useful information about possible pharmacokinetic interactions upon simultaneous co-administration of the food/dietary supplement and the antihypertensive drug.

Influence of quercetin on the interaction of gliclazide with human serum albumin - spectroscopic and docking approaches.

Protein-binding interactions are displacement reactions which have been implicated as the causative mechanisms in many drug-drug interactions. Thus, the aim of presented study was to analyse human serum albumin-binding displacement interaction between two ligands, hypoglycaemic drug gliclazide and widely distributed plant flavonoid quercetin. Fluorescence analysis was used in order to investigate the effect of substances on intrinsic fluorescence of human serum albumin (HSA) and to define binding and quenching properties of ligand-albumin complexes in binary and ternary systems, respectively. Both ligands showed the ability to bind to HSA, although to a different extent. The displacement effect of one ligand from HSA by the other one has been described on the basis of the quenching curves and binding constants comparison for the binary and ternary systems. According to the fluorescence data analysis, gliclazide presents a substance with a lower binding capacity towards HSA compared with quercetin. Results also showed that the presence of quercetin hindered the interaction between HSA and gliclazide, as the binding constant for gliclazide in the ternary system was remarkably lower compared with the binary system. This finding indicates a possibility for an increase in the non-bound fraction of gliclazide which can lead to its more significant hypoglycaemic effect. Additionally, secondary and tertiary structure conformational alterations of HSA upon binding of both ligands were investigated using synchronous fluorescence, circular dichroism and FT-IR. Experimental data were complemented with molecular docking studies. Obtained results provide beneficial information about possible interference upon simultaneous co-administration of the food/dietary supplement and drug.

In Vitro Characterization of the Two-Stage Non-Classical Reassembly Pathway of S-Layers.

The recombinant bacterial surface layer (S-layer) protein rSbpA of Lysinibacillus sphaericus CCM 2177 is an ideal model system to study non-classical nucleation and growth of protein crystals at surfaces since the recrystallization process may be separated into two distinct steps: (i) adsorption of S-layer protein monomers on silicon surfaces is completed within 5 min and the amount of bound S-layer protein sufficient for the subsequent formation of a closed crystalline monolayer; (ii) the recrystallization process is triggered-after washing away the unbound S-layer protein-by the addition of a CaCl2 containing buffer solution, and completed after approximately 2 h. The entire self-assembly process including the formation of amorphous clusters, the subsequent transformation into crystalline monomolecular arrays, and finally crystal growth into extended lattices was investigated by quartz crystal microbalance with dissipation (QCM-D) and atomic force microscopy (AFM). Moreover, contact angle measurements showed that the surface properties of S-layers change from hydrophilic to hydrophobic as the crystallization proceeds. This two-step approach is new in basic and application driven S-layer research and, most likely, will have advantages for functionalizing surfaces (e.g., by spray-coating) with tailor-made biological sensing layers.

Physical activity, physical fitness and academic achievement in adolescents: a self-organizing maps approach.

The relationship among physical activity, physical fitness and academic achievement in adolescents has been widely studied; however, controversy concerning this topic persists. The methods used thus far to analyse the relationship between these variables have included mostly traditional lineal analysis according to the available literature. The aim of this study was to perform a visual analysis of this relationship with self-organizing maps and to monitor the subject's evolution during the 4 years of secondary school. Four hundred and forty-four students participated in the study. The physical activity and physical fitness of the participants were measured, and the participants' grade point averages were obtained from the five participant institutions. Four main clusters representing two primary student profiles with few differences between boys and girls were observed. The clustering demonstrated that students with higher energy expenditure and better physical fitness exhibited lower body mass index (BMI) and higher academic performance, whereas those adolescents with lower energy expenditure exhibited worse physical fitness, higher BMI and lower academic performance. With respect to the evolution of the students during the 4 years, ∼25% of the students originally clustered in a negative profile moved to a positive profile, and there was no movement in the opposite direction.

S-layer protein self-assembly.

Crystalline S(urface)-layers are the most commonly observed cell surface structures in prokaryotic organisms (bacteria and archaea). S-layers are highly porous protein meshworks with unit cell sizes in the range of 3 to 30 nm, and thicknesses of ~10 nm. One of the key features of S-layer proteins is their intrinsic capability to form self-assembled mono- or double layers in solution, and at interfaces. Basic research on S-layer proteins laid foundation to make use of the unique self-assembly properties of native and, in particular, genetically functionalized S-layer protein lattices, in a broad range of applications in the life and non-life sciences. This contribution briefly summarizes the knowledge about structure, genetics, chemistry, morphogenesis, and function of S-layer proteins and pays particular attention to the self-assembly in solution, and at differently functionalized solid supports.

A stereochemical switch in the aDrs model system, a candidate for a functional amyloid.

Amyloid fibrils are commonly observed to adopt multiple distinct morphologies, which eventually can have significantly different neurotoxicities, as e.g. demonstrated in case of the Alzheimer peptide. The architecture of amyloid deposits is apparently also determined by the stereochemistry of amino acids. Post-translational changes of the chirality of certain residues may thus be a factor in controlling the formation of functional or disease-related amyloids. Anionic dermaseptin (aDrs), an unusual peptide from the skin secretions of the frog Pachymedusa dacnicolor, assembles to amyloid-like fibrils in a pH-dependent manner, which could play a functional role in defense. aDrs can be enzymatically converted into the diastereomer [d-Leu2]-aDrs by an l/d-isomerase. EM and AFM on fibrils formed by these isomers have shown that their predominant morphology is controlled by the stereochemistry of residue 2, whereas kinetic and thermodynamic parameters of aggregation are barely affected. When fibrils were grown from preformed seeds, backbone stereochemistry rather than templating-effects apparently dominated the superstructural organization of the isomers. Interestingly, MD indicated small differences in the conformational propensities between the isomers. Our results demonstrate how d-amino acid substitutions could take active part in the formation of functional or disease-related amyloid. Moreover, these findings contribute to the development of amyloid-based nanomaterials.

Rolling Circle Amplification Tailored for Plasmonic Biosensors: From Ensemble to Single-Molecule Detection.

We report on the tailoring of rolling circle amplification (RCA) for affinity biosensors relying on the optical probing of their surface with confined surface plasmon field. Affinity capture of the target analyte at the metallic sensor surface (e.g., by using immunoassays) is followed by the RCA step for subsequent readout based on increased refractive index (surface plasmon resonance, SPR) or RCA-incorporated high number of fluorophores (in surface plasmon-enhanced fluorescence, PEF). By combining SPR and PEF methods, this work investigates the impact of the conformation of long RCA-generated single-stranded DNA (ssDNA) chains to the plasmonic sensor response enhancement. In order to confine the RCA reaction within the evanescent surface plasmon field and hence maximize the sensor response, an interface carrying analyte-capturing molecules and additional guiding ssDNA strands (complementary to the repeating segments of RCA-generated chains) is developed. When using the circular padlock probe as a model target analyte, the PEF readout shows that the reported RCA implementation improves the limit of detection (LOD) from 13 pM to high femtomolar concentration when compared to direct labeling. The respective enhancement factor is of about 2 orders of magnitude, which agrees with the maximum number of fluorophore emitters attached to the RCA chain that is folded in the evanescent surface plasmon field by the developed biointerface. Moreover, the RCA allows facile visualizing of individual binding events by fluorescence microscopy, which enables direct counting of captured molecules. This approach offers a versatile route toward a fast digital readout format of single-molecule detection with further reduced LOD.

Stress relaxation and creep on living cells with the atomic force microscope: a means to calculate elastic moduli and viscosities of cell components.

In this work we present a unified method to study the mechanical properties of cells using the atomic force microscope. Stress relaxation and creep compliance measurements permitted us to determine, the relaxation times, the Young moduli and the viscosity of breast cancer cells (MCF-7). The results show that the mechanical behaviour of MCF-7 cells responds to a two-layered model of similar elasticity but differing viscosity. Treatment of MCF-7 cells with an actin-depolymerising agent results in an overall decrease in both cell elasticity and viscosity, however to a different extent for each layer. The layer that undergoes the smaller decrease (36-38%) is assigned to the cell membrane/cortex while the layer that experiences the larger decrease (70-80%) is attributed to the cell cytoplasm. The combination of the method presented in this work, together with the approach based on stress relaxation microscopy (Moreno-Flores et al 2010 J. Biomech. 43 349-54), constitutes a unique AFM-based experimental framework to study cell mechanics. This methodology can also be extended to study the mechanical properties of biomaterials in general.

Assessment of a long-term in vitro model to characterize the mechanical behavior and macrophage-mediated degradation of a novel, degradable, electrospun poly-urethane vascular graft.

An assessment tool to evaluate the degradation of biodegradable materials in a more physiological environment is still needed. Macrophages are critical players in host response, remodeling and degradation. In this study, a cell culture model using monocyte-derived primary macrophages was established to study the degradation, macro-/micro-mechanical behavior and inflammatory behavior of a new designed, biodegradable thermoplastic polyurethane (TPU) scaffold, over an extended period of time in vitro. For in vivo study, the scaffolds were implanted subcutaneously in a rat model for up to 36 weeks. TPU scaffolds were fabricated via the electrospinning method. This technique provided a fibrous scaffold with an average fiber diameter of 1.39 ± 0.76 µm and an average pore size of 7.5 ± 1.1 µm. The results showed that TPU scaffolds supported the attachment and migration of macrophages throughout the three-dimensional matrix. Scaffold degradation could be detected in localized areas, emphasizing the role of adherent macrophages in scaffold degradation. Weight loss, molecular weight and biomechanical strength reduction were evident in the presence of the primary macrophage cells. TPU favored the switch from initial pro-inflammatory response of macrophages to an anti-inflammatory response over time both in vitro and in vivo. Expression of MMP-2 and MMP-9 (the key enzymes in tissue remodeling based on ECM modifications) was also evident in vitro and in vivo. This study showed that the primary monocyte-derived cell culture model represents a promising tool to characterize the degradation, mechanical behavior as well as biocompatibility of the scaffolds during an extended period of observation.

UV-Laser Interference Lithography for Local Functionalization of Plasmonic Nanostructures with Responsive Hydrogel.

A novel approach to local functionalization of plasmonic hotspots at gold nanoparticles with biofunctional moieties is reported. It relies on photocrosslinking and attachment of a responsive hydrogel binding matrix by the use of a UV interference field. A thermoresponsive poly(N-isopropylacrylamide)-based (pNIPAAm) hydrogel with photocrosslinkable benzophenone groups and carboxylic groups for its postmodification was employed. UV-laser interference lithography with a phase mask configuration allowed for the generation of a high-contrast interference field that was used for the recording of periodic arrays of pNIPAAm-based hydrogel features with the size as small as 170 nm. These hydrogel arrays were overlaid and attached on the top of periodic arrays of gold nanoparticles, exhibiting a diameter of 130 nm and employed as a three-dimensional binding matrix in a plasmonic biosensor. Such a hybrid material was postmodified with ligand biomolecules and utilized for plasmon-enhanced fluorescence readout of an immunoassay. Additional enhancement of the fluorescence sensor signal by the collapse of the responsive hydrogel binding matrix that compacts the target analyte at the plasmonic hotspot is demonstrated.

Effects of electrical stimulation on muscle trophism in patients with hemophilic arthropathy.

OBJECTIVE: To determine changes occurring in the cross-sectional area, electromyography (EMG) activity, and the strength of the biceps brachii after an 8-week period of bilateral training with surface muscle electrical stimulation in patients with hemophilic arthropathy. DESIGN: Controlled trial. SETTING: Coagulopathy unit, university hospital. PARTICIPANTS: Volunteer subjects (N=30) participated in this study: 15 with severe hemophilia A (hemophilic group) and 15 nonhemophilic control subjects (control group). INTERVENTIONS: The hemophilic group followed a surface electrical stimulation program (frequency 45 Hz, impulse 200 micros, 10s on/10s off) over an 8-week period on the biceps brachii of both arms. The control group did no training of any kind. MAIN OUTCOME MEASURES: The cross-sectional area, maximum voluntary isometric contraction, and EMG activity of the biceps brachii in both arms were determined before and after the 8-week-long task. RESULTS: The results of the hemophilic group showed significant increases in the diameter (15.8%, P<.001), isometric force (4.6%, P<.05), and EMG activity (37.6%, P<.05) of the biceps brachii muscles in both arms. No significant changes were observed for the control group. CONCLUSIONS: Our findings confirm the efficacy of muscle electrical stimulation in causing muscles to hypertrophy in patients with hemophilia, thereby improving their muscular strength. In addition, these results may also be clinically applicable in the rehabilitation of patients who have similar deficiencies in the locomotor system.

Novel biodegradable and non-fouling systems for controlled-release based on poly(ε-caprolactone)/Quercetin blends and biomimetic bacterial S-layer coatings.

Quercetin is a strong antioxidant with low bioavailability due to its high crystallinity. A further drawback is that Quercetin has potentially toxic effects at high concentrations. To improve this low water solubility, as well as control the concentration of the flavonoid in the body, Quercetin is incorporated into a polymeric matrix to form an amorphous solid dispersion (ASD) stable enough to resist the recrystallization of the drug. For this purpose, miscible poly(ε-caprolactone) (PCL) and Quercetin (Q) blends are prepared, provided that they have complementary interacting groups. For compositions in which the flavonoid remains in an amorphous state thanks to the interactions with polymer chains, various PCL/Q drug release platforms are fabricated: micrometric films by solvent casting, nanometric films by spin coating, and nanofibers by electrospinning. Then, the potential use of bacterial S-layer proteins as release-preventive membranes is tested on PCL-Quercetin blends, due to their ability to construct a biomimetic coating including nanometric pores. For all the platforms, the SbpA coating can maintain a stable release under the toxicity level of Quercetin. Accordingly, a PCL/Q system with an S-layer coating allows the design of versatile bioavailable Quercetin eluting devices that prevent toxicity and biofouling issues.

Correction: Novel biodegradable and non-fouling systems for controlled-release based on poly(ε-caprolactone)/Quercetin blends and biomimetic bacterial S-layer coatings.

[This corrects the article DOI: 10.1039/C9RA04398E.].

Mechanical properties of gelatin nanoparticles in dependency of crosslinking time and storage.

Mechanical properties of nanoparticles are an important characteristic for drug delivery and therefore, they have gained interest in pharmaceutical research during the last years. Among others, cellular uptake, blood circulation time and accumulation in organs are influenced by the elastic modulus of nanoparticles. Thus, by varying the stiffness of nanoparticles a more specific drug targeting might be achieved. Gelatin nanoparticles (GNPs) show advantageous characteristics in respect to encapsulation and delivery of hydrophilic drugs such as antibodies or other biologicals. Furthermore, the GNPs as hydrogel-nanoparticles offer adjustable elastic behavior. In this study, a method for GNP sample preparation and the determination of the mechanical properties by nanoindentation experiments using atomic force microscopy (AFM) was developed. The obtained force-distance curves were evaluated and fitted with the Hertzian model in order to calculate the Young's modulus. GNPs were crosslinked with glutaraldehyde (GTA) for different incubation times to investigate a possible modification of the Young's modulus. In addition, this study addresses the influence of storage on the mechanical characteristics of GNPs. The results provide first insights about the elastic properties of GNPs and their development over time. In the tested range of crosslinking times no notable differences in the mechanical properties occurred. In turn, the influence of the storage on the mechanical particle properties was observed: particle stiffness raised over time. Furthermore, it could be observed that the cellular uptake in a model cell line (A549) was increased for harder particles.