Small Cellular Particles from European Spruce Needle Homogenate.

Small cellular particles (SCPs) are being considered for their role in cell-to-cell communication. We harvested and characterized SCPs from spruce needle homogenate. SCPs were isolated by differential ultracentrifugation. They were imaged by scanning electron microscope (SEM) and cryogenic transmission electron microscope (cryo TEM), assessed for their number density and hydrodynamic diameter by interferometric light microscopy (ILM) and flow cytometry (FCM), total phenolic content (TPC) by UV-vis spectroscopy, and terpene content by gas chromatography-mass spectrometry (GC-MS). The supernatant after ultracentrifugation at 50,000× g contained bilayer-enclosed vesicles whereas in the isolate we observed small particles of other types and only a few vesicles. The number density of cell-sized particles (CSPs) (larger than 2 µm) and meso-sized particles (MSPs) (cca 400 nm-2 µm) was about four orders of magnitude lower than the number density of SCPs (sized below 500 nm). The average hydrodynamic diameter of SCPs measured in 10,029 SCPs was 161 ± 133 nm. TCP decreased considerably due to 5-day aging. Volatile terpenoid content was found in the pellet after 300× g. The above results indicate that spruce needle homogenate is a source of vesicles to be explored for potential delivery use.

Autologous Platelet and Extracellular Vesicle-Rich Plasma as Therapeutic Fluid: A Review.

The preparation of autologous platelet and extracellular vesicle-rich plasma (PVRP) has been explored in many medical fields with the aim to benefit from its healing potential. In parallel, efforts are being invested to understand the function and dynamics of PVRP that is complex in its composition and interactions. Some clinical evidence reveals beneficial effects of PVRP, while some report that there were no effects. To optimize the preparation methods, functions and mechanisms of PVRP, its constituents should be better understood. With the intention to promote further studies of autologous therapeutic PVRP, we performed a review on some topics regarding PVRP composition, harvesting, assessment and preservation, and also on clinical experience following PVRP application in humans and animals. Besides the acknowledged actions of platelets, leukocytes and different molecules, we focus on extracellular vesicles that were found abundant in PVRP.

Cell confinement reveals a branched-actin independent circuit for neutrophil polarity.

Migratory cells use distinct motility modes to navigate different microenvironments, but it is unclear whether these modes rely on the same core set of polarity components. To investigate this, we disrupted actin-related protein 2/3 (Arp2/3) and the WASP-family verprolin homologous protein (WAVE) complex, which assemble branched actin networks that are essential for neutrophil polarity and motility in standard adherent conditions. Surprisingly, confinement rescues polarity and movement of neutrophils lacking these components, revealing a processive bleb-based protrusion program that is mechanistically distinct from the branched actin-based protrusion program but shares some of the same core components and underlying molecular logic. We further find that the restriction of protrusion growth to one site does not always respond to membrane tension directly, as previously thought, but may rely on closely linked properties such as local membrane curvature. Our work reveals a hidden circuit for neutrophil polarity and indicates that cells have distinct molecular mechanisms for polarization that dominate in different microenvironments.

Solitons in the Heimburg-Jackson model of sound propagation in lipid bilayers are enabled by dispersion of a stiff membrane.

Experiments show that elastic constants of lipid bilayers vary greatly during the liquid-to-gel phase transition. This fact forms the cornerstone of the Heimburg-Jackson model of soliton propagation along membranes of axons, in which the action potential is accompanied by a traveling phase transition. However, the dispersion term, which is crucial for the existence of solitons, is added to the Heimburg-Jackson model ad hoc and set to fit experimental observations. In the present paper, we aim to consolidate this view with continuous membrane mechanics. Using literature data, we show that the compression modulus of a DPPC membrane is smaller by approximately an order of magnitude during phase transition. With a series expansion of the compression modulus, we write the action of a membrane and solve the corresponding wave equation analytically using an Exp-function method. We confirm that membrane solitons with speeds around 200 m/s are possible with amplitudes inversely proportional to their speed. We conclude that dispersion necessary for existence of solitons is directly related to a membrane's bending properties, offering a possible explanation for h. Our findings are in general agreement with existing literature and give insight into a general mechanism of wave propagation in membranes close to transition.

Enrichment of plasma in platelets and extracellular vesicles by the counterflow to erythrocyte settling.

In order to prepare optimal platelet and extracellular vesicle (EV)-rich plasma for the treatment of chronic temporal bone inflammation, we studied effects of centrifugation parameters on redistribution of blood constituents in blood samples of 23 patients and 20 volunteers with no record of disease. Concentrations of blood cells and EVs were measured by flow cytometry. Sample content was inspected by scanning electron microscopy. A mathematical model was constructed to interpret the experimental results. The observed enrichment of plasma in platelets and EVs after a single spin of blood depended on the erythrocyte sedimentation rate, thereby indicating the presence of a flow of plasma that carried platelets and EVs in the direction opposite to settling of erythrocytes. Prolonged handling time correlated with the decrease of concentration of platelets and larger EVs in platelet and EV-rich plasma (PVRP), R = -0.538, p = 0.003, indicating cell fragmentation during the processing of samples. In further centrifugation of the obtained plasma, platelet and EV enrichment depended on the average distance of the sample from the centrifuge rotor axis. Based on the agreement of the model predictions with observations, we propose the centrifugation protocol optimal for platelet and EV enrichment and recovery in an individual sample, adjusted to the dimensions of the centrifuge rotor, volume of blood and erythrocyte sedimentation rate.[Figure: see text].

Interaction between Microalgae P. tricornutum and Bacteria Thalassospira sp. for Removal of Bisphenols from Conditioned Media.

We studied the efficiency of three culture series of the microalgae Phaeodactylum tricornutum (P. tricornutum) and bacteria Thalassospira sp. (axenic microalgae, bacterial culture and co-culture of the two) in removing bisphenols (BPs) from their growth medium. Bacteria were identified by 16S ribosomal RNA polymerase chain reaction (16S rRNA PCR). The microorganism growth rate was determined by flow cytometry. Cultures and isolates of their small cellular particles (SCPs) were imaged by scanning electron microscopy (SEM) and cryogenic transmission electron microscopy (Cryo-TEM). BPs were analyzed by gas chromatography coupled with tandem mass spectrometry (GC-MS/MS). Our results indicate that some organisms may have the ability to remove a specific pollutant with high efficiency. P. tricornutum in axenic culture and in mixed culture removed almost all (more than 99%) of BPC2. Notable differences in the removal of 8 out of 18 BPs between the axenic, mixed and bacterial cultures were found. The overall removals of BPs in axenic P. tricornutum, mixed and bacterial cultures were 11%, 18% and 10%, respectively. Finding the respective organisms and creating microbe societies seems to be key for the improvement of wastewater treatment. As a possible mediating factor, numerous small cellular particles from all three cultures were detected by electron microscopy. Further research on the mechanisms of interspecies communication is needed to advance the understanding of microbial communities at the nano-level.

Assessment of Small Cellular Particles from Four Different Natural Sources and Liposomes by Interferometric Light Microscopy.

Small particles in natural sources are a subject of interest for their potential role in intercellular, inter-organism, and inter-species interactions, but their harvesting and assessment present a challenge due to their small size and transient identity. We applied a recently developed interferometric light microscopy (ILM) to assess the number density and hydrodynamic radius (Rh) of isolated small cellular particles (SCPs) from blood preparations (plasma and washed erythrocytes) (B), spruce needle homogenate (S), suspension of flagellae of microalgae Tetraselmis chuii (T), conditioned culture media of microalgae Phaeodactylum tricornutum (P), and liposomes (L). The aliquots were also assessed by flow cytometry (FCM), dynamic light scattering (DLS), ultraviolet-visible spectrometry (UV-vis), and imaging by cryogenic transmission electron microscopy (cryo-TEM). In Rh, ILM showed agreement with DLS within the measurement error in 10 out of 13 samples and was the only method used here that yielded particle density. Cryo-TEM revealed that representative SCPs from Tetraselmis chuii flagella (T) did not have a globular shape, so the interpretation by Rh of the batch methods was biased. Cryo-TEM showed the presence of thin filaments in isolates from Phaeodactylum tricornutum conditioned culture media (P), which provides an explanation for the considerably larger Rh obtained by batch methods than the sizes of particles observed by cryo-TEM images. ILM proved convenient for assessment of number density and Rh of SCPs in blood preparations (e.g., plasma); therefore, its use in population and clinical studies is indicated.

A Monte Carlo study of giant vesicle morphologies in nonequilibrium environments.

It is known that giant vesicles undergo dynamic morphological changes when exposed to a detergent. The solubilization process may take multiple pathways. In this work, we identify lipid vesicle shape dynamics before the solubilization of 1,2-dioleoyl-sn-glycero-3-phosphocholine giant vesicles with Triton X-100 (TR) detergent. The violent lipid vesicle dynamics was observed with laser confocal scanning microscopy and was qualitatively explained via a numerical simulation. A three-dimensional Monte Carlo scheme was constructed that emulated the nonequilibrium conditions at the beginning stages of solubilization, accounting for a gradual addition of TR detergent molecules into the lipid bilayers. We suggest that the main driving factor for morphology change in lipid vesicles is the associative tendency of the TR molecules, which induces spontaneous curvature of the detergent inclusions, an intrinsic consequence of their molecular shape. The majority of the observed lipid vesicle shapes in the experiments were found to correspond very well to the numerically calculated shapes in the phase space of possible solutions. The results give an insight into the early stages of lipid vesicle solubilization by amphiphilic molecules, which is nonequilibrium in nature and very difficult to study.

On the Role of Curved Membrane Nanodomains, and Passive and Active Skeleton Forces in the Determination of Cell Shape and Membrane Budding.

Biological membranes are composed of isotropic and anisotropic curved nanodomains. Anisotropic membrane components, such as Bin/Amphiphysin/Rvs (BAR) superfamily protein domains, could trigger/facilitate the growth of membrane tubular protrusions, while isotropic curved nanodomains may induce undulated (necklace-like) membrane protrusions. We review the role of isotropic and anisotropic membrane nanodomains in stability of tubular and undulated membrane structures generated or stabilized by cyto- or membrane-skeleton. We also describe the theory of spontaneous self-assembly of isotropic curved membrane nanodomains and derive the critical concentration above which the spontaneous necklace-like membrane protrusion growth is favorable. We show that the actin cytoskeleton growth inside the vesicle or cell can change its equilibrium shape, induce higher degree of segregation of membrane nanodomains or even alter the average orientation angle of anisotropic nanodomains such as BAR domains. These effects may indicate whether the actin cytoskeleton role is only to stabilize membrane protrusions or to generate them by stretching the vesicle membrane. Furthermore, we demonstrate that by taking into account the in-plane orientational ordering of anisotropic membrane nanodomains, direct interactions between them and the extrinsic (deviatoric) curvature elasticity, it is possible to explain the experimentally observed stability of oblate (discocyte) shapes of red blood cells in a broad interval of cell reduced volume. Finally, we present results of numerical calculations and Monte-Carlo simulations which indicate that the active forces of membrane skeleton and cytoskeleton applied to plasma membrane may considerably influence cell shape and membrane budding.

Stability of Erythrocyte-Derived Nanovesicles Assessed by Light Scattering and Electron Microscopy.

Extracellular vesicles (EVs) are gaining increasing amounts of attention due to their potential use in diagnostics and therapy, but the poor reproducibility of the studies that have been conducted on these structures hinders their breakthrough into routine practice. We believe that a better understanding of EVs stability and methods to control their integrity are the key to resolving this issue. In this work, erythrocyte EVs (hbEVs) were isolated by centrifugation from suspensions of human erythrocytes that had been aged in vitro. The isolate was characterised by scanning (SEM) and cryo-transmission electron microscopy (cryo-TEM), flow cytometry (FCM), dynamic/static light scattering (LS), protein electrophoresis, and UV-V spectrometry. The hbEVs were exposed to various conditions (pH (4-10), osmolarity (50-1000 mOsm/L), temperature (15-60 °C), and surfactant Triton X-100 (10-500 µM)). Their stability was evaluated by LS by considering the hydrodynamic radius (Rh), intensity of scattered light (I), and the shape parameter (ρ). The morphology of the hbEVs that had been stored in phosphate-buffered saline with citrate (PBS-citrate) at 4 °C remained consistent for more than 6 months. A change in the media properties (50-1000 mOsm/L, pH 4-10) had no significant effect on the Rh (=100-130 nm). At pH values below 6 and above 8, at temperatures above 45 °C, and in the presence of Triton X-100, hbEVs degradation was indicated by a decrease in I of more than 20%. Due to the simple preparation, homogeneous morphology, and stability of hbEVs under a wide range of conditions, they are considered to be a suitable option for EV reference material.

Electric Double Layer and Orientational Ordering of Water Dipoles in Narrow Channels within a Modified Langevin Poisson-Boltzmann Model.

The electric double layer (EDL) is an important phenomenon that arises in systems where a charged surface comes into contact with an electrolyte solution. In this work we describe the generalization of classic Poisson-Boltzmann (PB) theory for point-like ions by taking into account orientational ordering of water molecules. The modified Langevin Poisson-Boltzmann (LPB) model of EDL is derived by minimizing the corresponding Helmholtz free energy functional, which includes also orientational entropy contribution of water dipoles. The formation of EDL is important in many artificial and biological systems bound by a cylindrical geometry. We therefore numerically solve the modified LPB equation in cylindrical coordinates, determining the spatial dependencies of electric potential, relative permittivity and average orientations of water dipoles within charged tubes of different radii. Results show that for tubes of a large radius, macroscopic (net) volume charge density of coions and counterions is zero at the geometrical axis. This is attributed to effective electrolyte charge screening in the vicinity of the inner charged surface of the tube. For tubes of small radii, the screening region extends into the whole inner space of the tube, leading to non-zero net volume charge density and non-zero orientational ordering of water dipoles near the axis.

Theoretical study of vesicle shapes driven by coupling curved proteins and active cytoskeletal forces.

Eukaryote cells have a flexible shape, which dynamically changes according to the function performed by the cell. One mechanism for deforming the cell membrane into the desired shape is through the expression of curved membrane proteins. Furthermore, these curved membrane proteins are often associated with the recruitment of the cytoskeleton, which then applies active forces that deform the membrane. This coupling between curvature and activity was previously explored theoretically in the linear limit of small deformations, and low dimensionality. Here we explore the unrestricted shapes of vesicles that contain active curved membrane proteins, in three-dimensions, using Monte-Carlo numerical simulations. The activity of the proteins is in the form of protrusive forces that push the membrane outwards, as may arise from the cytoskeleton of the cell due to actin or microtubule polymerization occurring near the membrane. For proteins that have an isotropic convex shape, the additional protrusive force enhances their tendency to aggregate and form membrane protrusions (buds). In addition, we find another transition from deformed spheres with necklace type aggregates, to flat pancake-shaped vesicles, where the curved proteins line the outer rim. This second transition is driven by the active forces, coupled to the spontaneous curvature, and the resulting configurations may shed light on the formation of sheet-like protrusions and lamellipodia of adhered and motile cells.

Spermidine-Induced Attraction of Like-Charged Surfaces Is Correlated with the pH-Dependent Spermidine Charge: Force Spectroscopy Characterization.

The ubiquitous molecule spermidine is known for its pivotal roles in the contact mediation, fusion, and reorganization of biological membranes and DNA. In our model system, borosilicate beads were attached to atomic force microscopy cantilevers and used to probe mica surfaces to study the details of the spermidine-induced attractions. The negative surface charges of both materials were largely constant over the measured pH range of pH 7.8 to 12. The repulsion observed between the surfaces turned into attraction after the addition of spermidine. The attractive force was correlated with the degree of spermidine protonation, which changed from +3 to +1 over the measured pH range. The force was maximal at pH 7.8. To explain the observed pH and spermidine concentration dependence, two different theoretical approaches were used: a chemical model of the charge equilibrium of spermidine and Monte-Carlo simulations of the orientation of the rodlike spermidine molecules in the gap between the borosilicate and mica surfaces. Monte-Carlo simulations of the orientational ordering of the rodlike spermidine molecules suggested the induction of attractive interactions between the surfaces if the gap was bridged by the molecules. For larger gaps, the orientational distribution function of the spermidine molecules predicted a considerable degree of parallel attachment of the molecules to the surfaces, resulting in reduced effective surface charge densities of both surfaces, which reduced their electrostatic repulsion.

Multifunctional Gadolinium-Doped Mesoporous TiO2 Nanobeads: Photoluminescence, Enhanced Spin Relaxation, and Reactive Oxygen Species Photogeneration, Beneficial for Cancer Diagnosis and Treatment.

Materials with controllable multifunctional abilities for optical imaging (OI) and magnetic resonant imaging (MRI) that also can be used in photodynamic therapy are very interesting for future applications. Mesoporous TiO2 sub-micrometer particles are doped with gadolinium to improve photoluminescence functionality and spin relaxation for MRI, with the added benefit of enhanced generation of reactive oxygen species (ROS). The Gd-doped TiO2 exhibits red emission at 637 nm that is beneficial for OI and significantly improves MRI relaxation times, with a beneficial decrease in spin-lattice and spin-spin relaxation times. Density functional theory calculations show that Gd3+ ions introduce impurity energy levels inside the bandgap of anatase TiO2 , and also create dipoles that are beneficial for charge separation and decreased electron-hole recombination in the doped lattice. The Gd-doped TiO2 nanobeads (NBs) show enhanced ability for ROS monitored via • OH radical photogeneration, in comparison with undoped TiO2 nanobeads and TiO2 P25, for Gd-doping up to 10%. Cellular internalization and biocompatibility of TiO2 @xGd NBs are tested in vitro on MG-63 human osteosarcoma cells, showing full biocompatibility. After photoactivation of the particles, anticancer trace by means of ROS photogeneration is observed just after 3 min irradiation.

Applicability of extracellular vesicles in clinical studies.

BACKGROUND: Extracellular vesicles (EVs) are submicron cellular fragments that mediate intercellular communication. EVs have in the last decade attracted major interest as biomarkers or platforms for biomarkers of health and disease. To better understand the reasons why despite great expectations and considerable effort, EV-based methods have not yet been introduced into clinical practice, we present a systematic analysis of published results of clinical studies. MATERIALS AND METHODS: Clinical studies on populations of body fluid samples, published from 2010 to including 2015, applying centrifugation of fluid human samples with centrifuge accelerations up to about 25 000 g and flow cytometry for detection of EVs were analysed with respect to statistical significance (p), statistical power (P), clinical significance (CS), defined as the difference between the means divided by the sum of standard deviations, and size of the populations (Nmin ), defined as the number of samples in the smaller group. RESULTS: Final analysis included 65 publications with 716 comparisons reporting 308 (43%) statistically significant differences (P < 0·05), 242 (34%) had statistical power P > 0·8 and 88 (12%) had clinical importance CS > 1·96. None of comparison with CS > 1·96 included populations in which the smaller group consisted of 50 or more samples. CONCLUSIONS: To fulfil claimed expectations for EV-based methods as promising diagnostic tools, more evidence on EV-based mechanisms of diseases should be gathered. Also, the methods of EV harvesting and assessment should be improved to yield better repeatability and thus allow clinical studies with larger number of samples.

On the role of external force of actin filaments in the formation of tubular protrusions of closed membrane shapes with anisotropic membrane components.

Biological membranes are composed of different components and there is no a priori reason to assume that all components are isotropic. It was previously shown that the anisotropic properties of membrane components may explain the stability of membrane tubular protrusions even without the application of external force. Our theoretical study focuses on the role of anisotropic membrane components in the stability of membrane tubular structures generated or stabilized by actin filaments. We show that the growth of the actin cytoskeleton inside the vesicle can induce the partial lateral segregation of different membrane components. The entropy of mixing of membrane components hinders the total lateral segregation of the anisotropic and isotropic membrane components. Self-assembled aggregates formed by anisotropic membrane components facilitate the growth of long membrane tubular protrusions. Protrusive force generated by actin filaments favors strong segregation of membrane components by diminishing the opposing effect of mixing entropy.

Unequal size of ions in modified Wicke-Eigen model of electric double layer.

Numerous electric double layer models have been developed through the years to capture the complex electrode/electrolyte interface. In the present study, the Wicke-Eigen model of electric double layer is generalized to incorporate the asymmetric size of cations and anions. Analytical expressions for the spatial distribution of ions and water dipoles are derived. Asymmetric and symmetric Wicke-Eigen models are analysed. Arguments are given in favour of changing the recently adopted name of so-called Bikerman model/equation to Wicke-Eigen model/equation.

Effect of carbon black nanomaterial on biological membranes revealed by shape of human erythrocytes, platelets and phospholipid vesicles.

BACKGROUND: We studied the effect of carbon black (CB) agglomerated nanomaterial on biological membranes as revealed by shapes of human erythrocytes, platelets and giant phospholipid vesicles. Diluted human blood was incubated with CB nanomaterial and observed by different microscopic techniques. Giant unilamellar phospholipid vesicles (GUVs) created by electroformation were incubated with CB nanomaterial and observed by optical microscopy. Populations of erythrocytes and GUVs were analyzed: the effect of CB nanomaterial was assessed by the average number and distribution of erythrocyte shape types (discocytes, echinocytes, stomatocytes) and of vesicles in test suspensions, with respect to control suspensions. Ensembles of representative images were created and analyzed using computer aided image processing and statistical methods. In a population study, blood of 14 healthy human donors was incubated with CB nanomaterial. Blood cell parameters (concentration of different cell types, their volumes and distributions) were assessed. RESULTS: We found that CB nanomaterial formed micrometer-sized agglomerates in citrated and phosphate buffered saline, in diluted blood and in blood plasma. These agglomerates interacted with erythrocyte membranes but did not affect erythrocyte shape locally or globally. CB nanomaterial agglomerates were found to mediate attractive interaction between blood cells and to present seeds for formation of agglomerate - blood cells complexes. Distortion of disc shape of resting platelets due to incubation with CB nanomaterial was not observed. CB nanomaterial induced bursting of GUVs while the shape of the remaining vesicles was on the average more elongated than in control suspension, indicating indirect osmotic effects of CB nanomaterial. CONCLUSIONS: CB nanomaterial interacts with membranes of blood cells but does not have a direct effect on local or global membrane shape in physiological in vitro conditions. Blood cells and GUVs are convenient and ethically acceptable methods for the study of effects of various substances on biological membranes and therefrom derived effects on organisms.