Lipid Clustering Correlates with Membrane Curvature as Revealed by Molecular Simulations of Complex Lipid Bilayers

Cell membranes are complex multicomponent systems, which are highly heterogeneous in the lipid distribution and composition. To date, most molecular simulations have focussed on relatively simple lipid compositions, helping to inform our understanding of in vitro experimental studies. Here we describe on simulations of complex asymmetric plasma membrane model, which contains seven different lipids species including the glycolipid GM3 in the outer leaflet and the anionic lipid, phosphatidylinositol 4,5-bisphophate (PIP₂), in the inner leaflet. Plasma membrane models consisting of 1500 lipids and resembling the in vivo composition were constructed and simulations were run for 5 µs. In these simulations the most striking feature was the formation of nano-clusters of GM3 within the outer leaflet. In simulations of protein interactions within a plasma membrane model, GM3, PIP₂, and cholesterol all formed favorable interactions with the model α-helical protein. A larger scale simulation of a model plasma membrane containing 6000 lipid molecules revealed correlations between curvature of the bilayer surface and clustering of lipid molecules. In particular, the concave (when viewed from the extracellular side) regions of the bilayer surface were locally enriched in GM3. In summary, these simulations explore the nanoscale dynamics of model bilayers which mimic the in vivo lipid composition of mammalian plasma membranes, revealing emergent nanoscale membrane organization which may be coupled both to fluctuations in local membrane geometry and to interactions with proteins.     

The intrinsic resolution limit in the atomic force microscope: implications for heights of nano-scale features

Background

Accurate mechanical characterization by the atomic force microscope at the highest spatial resolution requires that topography is deconvoluted from indentation. The measured height of nanoscale features in the atomic force microscope (AFM) is almost always smaller than the true value, which is often explained away as sample deformation, the formation of salt deposits and/or dehydration. We show that the real height of nano-objects cannot be obtained directly: a result arising as a consequence of the local probe-sample geometry.

Methods and Findings

We have modeled the tip-surface-sample interaction as the sum of the interaction between the tip and the surface and the tip and the sample. We find that the dynamics of the AFM cannot differentiate between differences in force resulting from 1) the chemical and/or mechanical characteristics of the surface or 2) a step in topography due to the size of the sample; once the size of a feature becomes smaller than the effective area of interaction between the AFM tip and sample, the measured height is compromised. This general result is a major contributor to loss of height and can amount to up to ∼90% for nanoscale features. In particular, these very large values in height loss may occur even when there is no sample deformation, and, more generally, height loss does not correlate with sample deformation. DNA and IgG antibodies have been used as model samples where experimental height measurements are shown to closely match the predicted phenomena.

Conclusions

Being able to measure the true height of single nanoscale features is paramount in many nanotechnology applications since phenomena and properties in the nanoscale critically depend on dimensions. Our approach allows accurate predictions for the true height of nanoscale objects and will lead to reliable mechanical characterization at the highest spatial resolution.     

Nanoindentation response of cortical bone: dependency of subsurface voids

Nanoindentation test results in the axial direction of mouse femurs were the basis for the current study. Although the majority of the nanoindentation curves showed a reasonable consistency, some curves showed a significantly softer response. Detailed investigation, using focused ion beam-scanning electron microscopy, provided that the softer response is due to subsurface cavities such as lacunae. Finite element models were developed to simulate the nanoindentation of mice femur cortical bone samples with and without the incorporation of a single lacuna underneath the bone surface. Based on the material parameters determined for the cavity-free tissue, numerical simulations were run for different cases of cavity size, shape, and location. Spherical cavities with different size were considered at different distances from the surface. The results showed that subsurface cavities can lead to 50% higher indentation compared to an indentation in cavity-free material. Continuing with ellipsoidal cavities with the center located on the load axis, the results showed a nonlinear dependency of ellipsoid shape. Hence, the shape of the cavity is important for the nanoindentation response. The influence of horizontal and vertical offsets of spherical cavities was studied, thereby the results showed that an increasing horizontal offset caused a decreasing influence of the vertical distance from the surface. In perspective, the present study provides information that may help to get deeper knowledge of nanoindentation load–displacement mechanism taking place in samples with subsurface cavities.     

Osteoclastogenic differentiation of human precursor cells over micro- and nanostructured hydroxyapatite topography

BackgroundSurface topography is a key parameter in bone cells–biomaterials interactions. This study analyzed the behavior of human osteoclast precursor cells cultured over three hydroxyapatite (HA) surfaces ranging from a micro- to nanoscale topography.MethodsHA surfaces were prepared with microsized HA particles, at 1300 °C (HA1), and with nanosized HA particles at 1000 °C (HA2) and 830 °C (HA3). Human osteoclast precursors were cultured in the absence or presence of M-SCF and RANKL.ResultsHA surfaces had similar chemical composition, however, HA1 and HA3 presented typical micro- and nanostructured topographies, respectively, and HA2 profile was between those of HA1 and HA3. The decrease on the average grain diameter to the nanoscale range (HA3) was accompanied by an increase in surface area, porosity and hydrophilicity and a decrease in roughness. Compared to HA1 surface, HA3 allowed a lower osteoclastic adhesion, differentiation and function. Differences in the cell response appeared to be associated with the modulation of relevant intracellular signaling pathways.ConclusionsThe decrease in HA grain size to a biomimetic nanoscale range, appears less attractive to osteoclastic differentiation and function, compared to the HA microsized topography.General significanceThis observation emphasizes the role of surface topography in designing advanced biomaterials for tailored bone cells response in regenerative strategies.     

Structural evolution and dislocation behaviour study during nanoindentation of Mo20W20Co20Ta20Zr20 high entropy alloy coated Ni single crystal using molecular dynamic simulation

In this present study, deformation behaviour of Mo₂₀W₂₀Co₂₀Ta₂₀Zr₂₀ high entropy alloy (HEA) coated single crystal (SC) nickel (Ni) subjected to nanoindentation test have been investigated to study the mechanical properties and underlying mechanism during nanoindentation test using molecular dynamics (MD) simulation with embedded atom method (EAM) potential. Centro-Symmetry Parameter (CSP) Analysis and Radial Distribution Function (RDF) plots are obtained to get insight of structural evolution during nanoindentation and thereby determine the underlying physics of deformation. During nanoindention test Stacking faults (SFs) formation, dislocation generation, dislocation loops, Lomer–Cottrell (LC) lock and Hirth lock formation due to dislocation-dislocation interaction are observed. At higher indentation depth, formation of dislocation loops is augmented, which indicates nanoindentation deformation is found to be Stacking Fault dominated deformation. The accumulation and relaxation of shear stress near indenter tip at the time of deformation process under nanoindentation test causes the variation of dislocation density, strain hardening, and plastic deformation, which is influenced by the formation of dislocation barriers (LC and Hirth locks) and dislocation loops (shear and prismatic loops).     

A New Potential Model for Carbon Dioxide from AB Initio Calculations

Abstract

Ab initio quantum chemical calculations have been carried out for carbon dioxide dimer and the results have been used to establish potential functions usable in molecular simulations. Since the intermolecular interaction in carbon dioxide is fairly weak, careful treatment is required: this study uses 6–31G* basis set and takes electron correlations by the 2nd order Møller-Plesset theory into account. The potential energy surface is elucidated using the four representative relative configurations of the dimer. A new potential function model has been proposed on the basis of these ab initio data. In the super-critical region, this model is used to calculate the PVT relation of carbon dioxide fluid by the Monte Carlo simulations and confirmed to reproduce reasonably well the experimental isotherms.     

The nanoindentation responses of nickel surfaces with different crystal orientations

Molecular dynamics (MD) simulations are applied to elucidate the anisotropic characteristics in the material responses for crystallographic nickel substrates with (100), (110) and (111) surface orientations during nanoindentation, compensating for the experimental limitation of nanoindentation—particularly for pure nickel substrates of three crystallographic orientations. This study examines several factors under indentation: three-dimensional phases of plastic deformation which correspond to atomic stress distributions, pile-up patterns at maximum indentation depth, and extracted material properties at different crystallographic orientations. The present results reveal that the strain energy of the substrate exerted by the tip is stored by the formation of the homogeneous nucleation, and is dissipated by the dislocation sliding of the {111} plane. The steep variations of the indentation curve from the local peak to the local minimums are affected by the numbers of slip angle of {111} sliding plane. The pile-up patterns of the three nickel substrates prove that the crystalline nickel materials demonstrate the pile-up phenomenon from nanoindentation on the nano-scale. The three crystallographic nickel substrates exhibit differing amounts of pile-up dislocation spreading at different crystallographic orientations. Finally, the effects of surface orientation in material properties of FCC nickel material on the nano-scale are observable through the slip angle numbers of {111} sliding planes which influence hardness values, as well as the cohesive energy of different crystallographic surfaces that indicate Young's modulus.     

The structure of sensors on the antennae of Oeceticus omnivorus (Lepidoptera: Psychidae)

The antennae of male bagworm moths were examined with the scanning electron microscope. More than 90% of the sensilla present are long, thin trichodea regularly arrayed along the anterior face of each pectination and presumably involved in locating females. Two shorter types of trichodea occur mostly along the anterior ridge of each pectination. At the tip of each pectination is a single chaeticum; a small number or similar chaetica are dispersed on the posterior, almost sensor‐free surface of each pectination. Sensors resembling both the styloconica and coeloconica of Noctuidae are present, mostly near the tip of each pectination. A single short, blunt peg set in an unusual bulbous base is closely associated with the chaeticum at the tip of each pectination.     

Shielding disk position in intra-operative electron radiotherapy (IOERT): A Monte Carlo study

PurposeIn IOERT breast treatments, a shielding disk is frequently used to protect the underlying healthy structures. The disk is usually composed of two materials, a low-Z material intended to be oriented towards the beam and a high-Z material. As tissues are repositioned around the shield before treatment, the disk is no longer visible and its correct alignment with respect to the beam is guaranteed. This paper studies the dosimetric characteristics of four possible clinical positioning scenarios of the shielding disk. A new alignment method for the shielding disk in the beam is introduced. Finally, it suggests a new design for the shielding disk.MethodsAs the first step, the IOERT machine “Mobetron 1000” was modeled by using Monte Carlo simulation, tuning the MC model until an excellent match with the measured PDDs and profiles was achieved. Four possible shielding disk positioning scenarios were considered, determining the dosimetric impact. Furthermore, in our center, to prevent beam misalignment, we have developed a shielding disk equipped with guiding rods. Having ascertained a correct alignment between the disk and the beam, we can propose a new internal design of the shielding disk that can improve the dose distribution with a better coverage of the treated area.ResultsAll MC simulations were performed with a 12 MeV beam, the maximum energy of Mobetron 1000 and a 5.5 cm diameter flat tip applicator, this applicator being the most clinically used. The simulations were compared with measurements performed in a water phantom and showed good results within 2.2% of root mean square difference (RMSD). The misplacement positions of the shielding disk have dosimetric impacts in the treatment volume and a small translation could have a significant influence on healthy tissues. The D-scenario is the worst which could happens when the shielding disk is flipped upside down, giving up to 144% dose instead of 90% at the surface of the Pb/Al shielding disk. A new shielding design used, together with our alignment tool, is able to give a more homogeneous dose in the target area.ConclusionsThe accuracy of shielding disk position can still be problematic in IOERT dosimetry. Any method that can ascertain the good alignment between the shielding disk and the beam is beneficial for the dose distribution and is a prerequisite for an optimized shield internal design that could improve the coverage of the treated area and the protection of healthy tissues.     

Fracture Toughness Properties of Three Different Biomaterials Measured by Nanoindentation

The fracture toughness of hard biomaterials, such as nacre, bovine hoof wall and beetle cuticle, is associated with fibrous or lamellar structures that deflect or stop growing cracks. Their hardness and reduced modulus were measured by using a nanoindenter in this paper. Micro/nanoscale cracks were generated by nanoindentation using a Berkovich tip. Nanoindentation of nacre and bovine hoof wall resulted in pile-up around the indent. It was found that the fracture toughness （Kc） of bovine hoof wall is the maximum, the second is nacre, and the elytra cuticle of dung beetle is the least one.     

Establishing paediatric diagnostic reference levels using reference curves – A feasibility study including conventional and CT examinations

PurposeTo derive Regional Diagnostic Reference Levels (RDRL) for paediatric conventional and CT examinations using weight-based DRL curves and compare the outcome with DRL derived using the weight groups.MethodsData from 1722 examinations performed at 29 hospitals in four countries were included. DRL was derived for four conventional x-ray (chest, abdomen, pelvis, hips/joints) and two types of CT examinations (thorax, abdomen). DRL curves were derived using an exponential fit to the data using weight as an independent variable and the respective radiation dose indices (P_KA, CTDI_vol, DLP) as dependent variables. DRL was also derived for weight groups for comparison. The result was compared with national diagnostic reference level (NDRL) curves.ResultsThe derived curves show similarities with the NDRL curves available and corresponded sufficiently well with DRL for weight groups using the same data set, if sufficient number of data was available.ConclusionsWe conclude that weight-based DRL curves are a feasible approach and could be used together with DRL for weight groups. The main advantage of DRL curves is its application in the clinic. When the examination frequency is low, time to collect enough data to establish typical values for one or several weight groups may be unreasonably long. The curve provides the means to compare dose level faster and with fewer data points.     

Mechanisms of extracellular vesicle uptake in stressed retinal pigment epithelial cell monolayers

PurposeExtracellular vesicles (EVs) can mediate long-distance communication in polarized RPE monolayers. Specifically, EVs from oxidatively stressed donor cells (stress EVs) rapidly reduced barrier function (transepithelial resistance, TER) in naïve recipient monolayers, when compared to control EVs. This effect on TER was dependent on dynamin-mediated EV uptake, which occurred rapidly with EVs from oxidatively stressed donor cells. Here, we further determined molecular mechanisms involved in uptake of EVs by naïve RPE cells.MethodsRPE cells were grown as monolayers in media supplemented with 1% FBS followed by transfer to FBS-free media. Cultures were used to collect control or stress EVs upon treatment with H₂O₂, others served as naïve recipient cells. In recipient monolayers, TER was used to monitor EV-uptake-based activity, live-cell imaging confirmed uptake. EV surface proteins were quantified by protein chemistry.ResultsClathrin-independent, lipid raft-mediated internalization was excluded as an uptake mechanism. Known ligand-receptor interactions involved in clathrin-dependent endocytosis include integrins and proteoglycans. Desialylated glycans and integrin-receptors on recipient cells were necessary for EV uptake and subsequent reduction of TER in recipient cells. Protein quantifications confirmed elevated levels of ligands and neuraminidase on stress EVs. However, control EVs could confer activity in the TER assay if exogenous neuraminidase or additional ligand was provided.ConclusionsIn summary, while EVs from both stressed cells and control contain cargo to communicate stress messages to naive RPE cells, stress EVs contain surface ligands that confer rapid uptake by recipient cells. We propose that EVs potentially contribute to RPE dysfunction in aging and disease.     

The sweeter aspects of platelet activation: A lectin-based assay reveals agonist-specific glycosylation patterns

BackgroundThe diversity of platelet functions implies multiple activation states arising in response to different stimuli. Distinguishing between these states has been challenging.MethodsWe used fluorescently labelled carbohydrate binding proteins lectins to investigate agonist-induced changes in platelet surface glycosylation.ResultsEach of the seven agonists we used caused a unique set of changes in platelet surface glycosylation, eliciting a unique functional state. Some of these changes could be correlated with the expression of granule-specific markers CD62P and CD63, but lectins proved much more sensitive to differences between agonists than antibodies against those markers. This sensitivity appears to arise from the relation between the surface glycosylation changes and the signalling pathways through which various agonists act. In this context it is interesting that the effects of calcium ionophore were significantly different from those of other agonists. We also found that that P-selectin (CD62P) contains haptens for lectins VFA and PTII, because these lectins compete with the anti-CD62P antibody binding and vice a versa.ConclusionsWe report for the first time that changes in platelet surface glycosylation are agonist-specific and can be distinguished using lectin-binding assays. Lectin fingerprinting represents a new research and diagnostic tool for studying platelet activation.General significanceThe observation of agonist-specific platelet surface glycosylation changes is interesting in the context of the diversity of platelet function, because surface glycans mediate contact interactions between platelets and other cells and serve as binding sites for some of the agonists (galectins).     

Multi-scale mechanical characterization of prostate cancer cell lines: Relevant biological markers to evaluate the cell metastatic potential

BackgroundConsidering the importance of cellular mechanics in the birth and evolution of cancer towards increasingly aggressive stages, we compared nano-mechanical properties of non-tumoral (WPMY-1) and highly aggressive metastatic (PC-3) prostate cell lines both on cell aggregates, single cells, and membrane lipids.MethodsCell aggregate rheological properties were analyzed during dynamic compression stress performed on a homemade rheometer. Single cell visco-elasticity measurements were performed by Atomic Force Microscopy using a cantilever with round tip on surface-attached cells. At a molecular level, the lateral diffusion coefficient of total extracted lipids deposited as a Langmuir monolayer on an air-water interface was measured by the FRAP technique.ResultsAt cellular pellet scale, and at single cell scale, PC-3 cells were less stiff, less viscous, and thus more prone to deformation than the WPMY-1 control. Interestingly, stress-relaxation curves indicated a two-step response, which we attributed to a differential response coming from two cell elements, successively stressed. Both responses are faster for PC-3 cells. At a molecular scale, the dynamics of the PC-3 lipid extracts are also faster than that of WPMY-1 lipid extracts.ConclusionsAs the evolution of cancer towards increasingly aggressive stages is accompanied by alterations both in membrane composition and in cytoskeleton dynamical properties, we attribute differences in viscoelasticity between PC-3 and WPMY-1 cells to modifications of both elements.General significanceA decrease in stiffness and a less viscous behavior may be one of the diverse mechanisms that cancer cells adopt to cope with the various physiological conditions that they encounter.     

Can forearm muscle activity be selectively recorded using conventional surface EMG-electrodes in transcranial magnetic stimulation? A feasibility study

ObjectivesThis feasibility study evaluates the effect of varying the position of conventional surface EMG-electrodes on the forearm when using Transcranial Magnetic Stimulation (TMS). The aim was to find optimal bipolar electrode positions for forearm extensor muscles, which would be clinically relevant to predict motor recovery after stroke.MethodsIn a healthy female subject, three rings of surface EMG-electrodes were placed around the dominant forearm, leading to 200 different electrode pairs. Both peripheral electrical stimulation and TMS were applied at suprathreshold intensities.ResultsWith electrical stimulation of the median and radial nerve, similar waveform morphology was found for all electrode pairs, covering both flexors and extensors. Also with TMS, remarkable similarities between all electrode pairs were found, suggesting minimal selectivity. In both peripheral electrical stimulation and TMS, the curves became more irregular with decreasing inter-electrode distances.ConclusionNeither with peripheral electrical stimulation nor with TMS it was possible to selectively record extensor or flexor forearm muscle activity using conventional surface EMG-electrodes.SignificanceDespite this negative result, the important role of the forearm extensor muscles in the prognosis of motor recovery after stroke warrants further research into novel methods for selectively recording muscle activity in TMS other than by conventional surface EMG.     

Women with stress urinary incontinence demonstrate motor control differences during coughing

IntroductionThis study compared the patterns of pelvic floor muscle (PFM) activity during coughing between women with stress urinary incontinence (SUI) and continent women, using surface electromyography (EMG) and posterior vaginal wall (PVW) pressure.MethodsTwenty-four women participated: eight continent, eight with mild SUI and eight with severe SUI. Volunteers performed three maximum coughs in supine and standing. Maximum PFM EMG and PVW pressure amplitudes and the timing of the EMG peak relative to the PVW pressure peak were determined. Ensemble average PVW pressure versus EMG curves were created.ResultsThere were no significant differences among the groups in the maximum EMG or PVW pressure amplitudes. The EMG and PVW pressure peaked simultaneously in both positions in the continent group. In the mild SUI group, the EMG and PVW pressure peaked simultaneously in supine, but the EMG peaked before the PVW pressure in standing. In the severe SUI group, the EMG peaked before the PVW pressure in both positions. The shapes of the PVW pressure versus EMG curves were similar among the groups and positions, however the SUI groups displayed higher EMG-intercepts than the continent women. Conclusion: These findings suggest that urine leakage during coughing in women with SUI may be related to delays in force generation rather than PFM weakness.     

Non-uniform shrinkage for obtaining computational start shape for in-vivo MRI-based plaque vulnerability assessment

BackgroundCritical mechanical conditions, such as stress within the structure and shear stress due to blood flow, predicted from in-vivo magnetic resonance image (MRI)-based computational simulations have shown to be potential in assessing carotid plaque vulnerability. Plaque contours obtained from in-vivo MRI are a result of a pressurized configuration due to physiological loading. However, in order to make accurate predictions, the computational model must be based on the loading-free geometry. A shrinkage procedure can be used to obtain the computational start shape.MethodIn this study, electrocardiograph (ECG)-gated MR-images of carotid plaques were obtained from 28 patients. The contours of each plaque were segmented manually. Additional to a uniform shrinkage procedure, a non-uniform shrinkage refinement procedure was used. This procedure was repeated until the pressurized lumen contour and fibrous cap thickness had the best match with the in-vivo image.ResultsCompared to the uniform shrinkage procedure, the non-uniform shrinkage significantly reduced the difference in lumen shape and in cap thickness at the thinnest site. Results indicate that uniform shrinkage would underestimate the critical stress in the structure by 20.5±10.7%.ConclusionFor slices with an irregular lumen shape (the ratio of the maximum width to the minimum width is more than 1.05), the non-uniform shrinkage procedure is needed to get an accurate stress profile for mechanics and MRI-based carotid plaque vulnerability assessment.     

Effect of the anisotropic permeability in the frequency dependent properties of the superficial layer of articular cartilage

Abstract

Articular cartilage is a tissue of fundamental importance for the mechanics of joints, since it provides a smooth and lubricated surface for the proper transfer of loads. From a mechanical point of view, this tissue is an anisotropic poroviscoelastic material: its characteristics at the macroscopic level depend on the complex microscopic architecture. With the ability to probe the local microscopic features, dynamic nanoindentation test is a powerful tool to investigate cartilage mechanics. In this work we focus on a length scale where the time dependent behaviour is regulated by poroelasticity more than viscoelasticity and we aim to understand the effect of the anisotropic permeability on the mechanics of the superficial layer of the articular cartilage. In a previous work, a finite element model for the dynamic nanoindentation test has been presented. In this work, we improve the model by considering the presence of an anisotropic permeability tensor that depends on the collagen fibers distribution. Our sensitivity analysis highlights that the permeability decreases with increasing indentation, thus making the tissue stiffer than the case of isotropic permeability, when solicited at the same frequency. With this improved model, a revised identification of the mechanical and physical parameters for articular cartilage is provided. To this purpose the model was used to simulate experimental data from tests performed on bovine tissue, giving a better estimation of the anisotropy in the elastic properties. A relation between the identified macroscopic anisotropic permeability properties and the microscopic rearrangement of the fiber/matrix structure during indentation is also provided.     

Simulating the approximate irregular field dose distribution in radiotherapy using an ultrasound tracking technique

PurposeThis study used an ultrasound image tracking algorithm (UITA) in combination with a proposed simulation program for the approximate irregular field dose distribution (SPAD) to assess the feasibility of performing dose distribution simulations for two-dimensional radiotherapy.MethodsThis study created five different types of multileaf collimator openings, and applied a SPAD to analyze the matrix position parameters for each regular field to generate a static program-simulation dose distribution map (PDDM), whose similarity was then compared with a static radiochromic film experimental-measurement dose distribution map (EDDM). A two-dimensional respiration motion simulation system (RMSS) was used to reproduce the respiration motion, and the UITA was used to capture the respiration signals. Respiration signals were input to the SPAD to generate two dynamic PDDMs, which were compared for similarity with the dynamic EDDM.ResultsIn order to verify the dose distribution between different dose measurement techniques, the gamma passing rate with 2%/2 mm criterion was used for the EDDM and PDDM, the passing rates were between 94.31% and 99.71% in the static field analyses, and between 84.45% and 96.09% for simulations with the UITA signal input and between 89.35% and 97.78% for simulations with the original signal input in the dynamic field analyses.ConclusionsStatic and dynamic dose distribution maps can be simulated based on the proposed matrix position parameters of various fields and by using the UITA to track respiration signals during radiation therapy. The present findings indicate that it is possible to develop a reusable and time-saving dose distribution measurement tool.