Strongly Accelerated Margination of Active Particles in Blood Flow

Synthetic nanoparticles and other stiff objects injected into a blood vessel filled with red blood cells are known to marginate toward the vessel walls. By means of hydrodynamic lattice-Boltzmann simulations, we show that active particles can strongly accelerate their margination by moving against the flow direction: particles located initially in the channel center migrate much faster to their final position near the wall than in the nonactive case. We explain our findings by an enhanced rate of collisions between the stiff particles and the deformable red blood cells. Our results imply that a significantly faster margination can be achieved either technically by the application of an external magnetic field (if the particles are magnetic) or biologically by self-propulsion (if the particles are, e.g., swimming bacteria).     

Morphofunctional study of the statocyst of the cuban crayfishprocambarus cubensis

After replacing sand, a natural component of the endogenous otolithic apparatus of the Cuban crayfishProcamabrus cubensis by particles of reducible iron and subsequent exposure of the animals to a constant magnetic field, the following changes were noted in their behavior: during the first 24 h the motor activity of the animals increased and was accompanied by frequent strong beats of antennulae on water; animals tried to go away from the magnet attached to the aquarium wall and to hide in a shelter; by the day 10–12, there appeared signs of adaptation to the action of magnet; the animals spent most time near the wall with the magnet and were clinging close to it. If the magnet was moved the animals not only slipped to their side but also rotated around their longitudinal axis. The eyestalks also started moving. After molt and repeated introduction of reduced iron particles into the statocysts, a negative reaction to magnet was practically absent, and the crayfish preferred staying near the magnet. Study of structural organization of the statocysts under a scanning electron microscope showed that the iron particles in the otolithic apparatus of the crayfish are covered with a thinner layer of glycocalyx as compared to control animals whose otolithic apparatus is composed of sand grains. Besides, the otolithic apparatus of the experimental animals is a loose formation, unlike the dense glycocalyx rich in conglomerate in control animals.     

Morphofunctional study of the statocyst of the Cuban crayfish Procambarus cubensis

After replacing sand, a natural component of the endogenous otolithic apparatus of the Cuban crayfishProcamabrus cubensis by particles of reducible iron and subsequent exposure of the animals to a constant magnetic field, the following changes were noted in their behavior: during the first 24 h the motor activity of the animals increased and was accompanied by frequent strong beats of antennulae on water; animals tried to go away from the magnet attached to the aquarium wall and to hide in a shelter; by the day 10—12, there appeared signs of adaptation to the action of magnet; the animals spent most time near the wall with the magnet and were clinging close to it. If the magnet was moved the animals not only slipped to their side but also rotated around their longitudinal axis. The eyestalks also started moving. After molt and repeated introduction of reduced iron particles into the statocysts, a negative reaction to magnet was practically absent, and the crayfish preferred staying near the magnet. Study of structural organization of the statocysts under a scanning electron microscope showed that the iron particles in the otolithic apparatus of the crayfish are covered with a thinner layer of glycocalyx as compared to control animals whose otolithic apparatus is composed of sand grains. Besides, the otolithic apparatus of the experimental animals is a loose formation, unlike the dense glycocalyx rich in conglomerate in control animals.     

In?Vitro Measurement of Particle Margination in the Microchannel Flow: Effect of Varying Hematocrit

It has long been known that platelets undergo margination when flowing in blood vessels, such that there is an excess concentration near the vessel wall. We conduct experiments and three-dimensional boundary integral simulations of platelet-sized spherical particles in a microchannel 30 μm in height to measure the particle-concentration distribution profile and observe its margination at 10%, 20%, and 30% red blood cell hematocrit. The experiments involved adding 2.15-μm-diameter spheres into a solution of red blood cells, plasma, and water and flowing this mixture down a microfluidic channel at a wall shear rate of 1000 s⁻¹. Fluorescence imaging was used to determine the height and velocity of particles in the channel. Experimental results indicate that margination has largely occurred before particles travel 1 cm downstream and that hematocrit plays a role in the degree of margination. With simulations, we can track the trajectories of the particles with higher resolution. These simulations also confirm that margination from an initially uniform distribution of spheres and red blood cells occurs over the length scale of O(1 cm), with higher hematocrit showing faster margination. The results presented here, from both experiments and 3D simulations, may help explain the relationship between bleeding time in vessel trauma and red blood cell hematocrit as platelets move to a vessel wall.     

Bacteriophage T4D Receptor and the Escherichia coli Cell Wall Structure: Binding of Endotoxin-Like Particles to the Cell Wall

A variety of degradative treatments have been used to investigate the nature of the structure and components of the cell walls of Escherichia coli B. The binding and localization of the endotoxin-like particles found on the cell walls were of special interest because some of them are associated with the site where the inner tail tube of bacteriophage T4D penetrates the cell wall. Modified cell walls were obtained by heating a suspension of bacterial cells originally in 0.1 M phosphate, pH 7.0, after the addition of 12.5 M NaOH to a final concentration of 0.25 M. With regard to the endotoxin-like particles, it was found that: (i) at least part of them still remained bound to the modified cell wall after the alkali treatment; (ii) the subsequent incubation of alkali-treated cell walls with lysozyme destroyed the bacterial form and released a complex of endotoxin-like particles together with a fibrous material; (iii) on the other hand, treatment with 45% phenol at 70°C removed the endotoxin-like particles from the surface of the alkali-treated cell walls, but most of the fibrous material was left on the cell wall; and (iv) incubation of alkali-treated cell walls with 5 mM ethylenediaminetetraacetic acid at 20°C also removed the endotoxin-like particles, but did not disrupt the rodlike bacterial form. However, if the ethylenediaminetetraacetic acid treatment was performed at 55°C, the bacterium-like form was destroyed. These differential sensitivities to ethylenediaminetetraacetic acid suggested that loosely bound divalent metal ions normally hold these endotoxin-like particles on the cell wall surface, but that probably more tightly bound metal ions are involved in the determination of cell shape. Analysis of the protein components of the alkalitreated cell walls showed that only one protein was present in significant amounts, and this protein had an electrophoretic mobility similar to that of the Braun lipoprotein. This protein was released from the alkali-treated cell walls upon heating with 2% sodium dodecyl sulfate at 100°C. Phospholipids were also absent from this structure. The distribution of the remaining cell wall components on the alkali-treated cell walls is discussed.     

Hydrodynamics of Sperm Cells near Surfaces

Sperm are propelled by an actively beating tail, and display a wide variety of swimming patterns. When confined between two parallel walls, sperm swim either in circles or on curvilinear trajectories close to the walls. We employ mesoscale hydrodynamics simulations in combination with a mechanical sperm model to study the swimming behavior near walls. The simulations show that sperm become captured at the wall due to the hydrodynamic flow fields which are generated by the flagellar beat. The circular trajectories are determined by the chiral asymmetry of the sperm shape. For strong (weak) chirality, sperm swim in tight (wide) circles, with the beating plane of the flagellum oriented perpendicular (parallel) to the wall. For comparison, we also perform simulations based on a local anisotropic friction of the flagellum. In this resistive force approximation, surface adhesion and circular swimming patterns are obtained as well. However, the adhesion mechanism is now due to steric repulsion, and the orientation of the beating plane is different. Our model provides a theoretical framework that explains several distinct swimming behaviors of sperm near and far from a wall. Moreover, the model suggests a mechanism by which sperm navigate in a chemical gradient via a change of their shape.     

Deposition of glucuronoxylans on the secondary cell wall of Japanese beech as observed by immuno-scanning electron microscopy

Summary Glucuronoxylans (GXs), the main hemicellulosic component of hardwoods, are localized exclusively in the secondary wall of Japanese beech and gradually increase during the course of fiber differentiation. To reveal where GXs deposit within secondary wall and how they affect cell wall ultrastructure, immuno-scanning electron microscopy using anti-GXs antiserum was applied in this study. In fibers forming the outer layer of the secondary wall (S₁), cellulose fibrils were small in diameter and deposited sparsely on the inner surface of the cell wall. Fine fibrils with approximately 5 nm width aggregated and formed thick fibrils with 12 nm width. Some of these thick fibrils further aggregated to form bundles which labelled positively for GXs. In fibers forming the middle layer of the secondary wall (S₂), fibrils were thicker than those found in S₁ forming fibers and were densely deposited. The S₂ layer labelled intensely for GXs with no preferential distribution recognized. Compared with newly formed secondary walls, previously formed secondary walls were composed of thick and highly packed microfibrils. Labels against GXs were much more prevalent on mature secondary walls than on newly deposited secondary walls. This result implies that the deposition of GXs into the cell wall may occur continuously after cellulose microfibril deposition and may be responsible for the increase in diameter of the microfibrils.Abbreviations GXs glucuronoxylans - PBS phosphate-buffered saline - RFDE rapid-freeze and deep-etching technique - FE-SEM field emission scanning electron microscope - TEM transmission electron microscope     

Theoretical comparison of wall-derived and erythrocyte-derived mechanisms for metabolic flow regulation in heterogeneous microvascular networks

The objective of this study is to compare the effectiveness of metabolic signals derived from erythrocytes and derived from the vessel wall for regulating blood flow in heterogeneous microvascular networks. A theoretical model is used to simulate blood flow, mass transport, and vascular responses. The model accounts for myogenic, shear-dependent, and metabolic flow regulation. Metabolic signals are assumed to be propagated upstream along vessel walls via a conducted response. Arteriolar tone is assumed to depend on the conducted metabolic signal as well as local wall shear stress and wall tension, and arteriolar diameters are calculated based on vascular smooth muscle mechanics. The model shows that under certain conditions metabolic regulation based on wall-derived signals can be more effective in matching perfusion to local oxygen demand relative to regulation based on erythrocyte-derived signals, resulting in higher extraction and lower oxygen deficit. The lower effectiveness of the erythrocyte-derived signal is shown to result in part from the unequal partition of hematocrit at diverging bifurcations, such that low-flow vessels tend to receive a reduced hematocrit and thereby experience a reduced erythrocyte-derived metabolic signal. The model simulations predict that metabolic signals independent of erythrocytes may play an important role in local metabolic regulation of vascular tone and flow distribution in heterogeneous microvessel networks.     

Extracellular hydrogen peroxide,produced through a respiratory burst oxidase/superoxide dismutase pathway,directs ingrowth wall formation in epidermal transfer cells of Vicia faba cotyledons

The intricate, and often polarized, ingrowth walls of transfer cells (TCs) amplify their plasma membrane surface areas to confer a transport function of supporting high rates of nutrient exchange across apo-/symplasmic interfaces. The TC ingrowth wall comprises a uniform wall layer on which wall ingrowths are deposited. Signals and signal cascades inducing trans-differentiation events leading to formation of TC ingrowth walls are poorly understood. Vicia faba cotyledons offer a robust experimental model to examine TC induction as, when placed into culture, their adaxial epidermal cells rapidly (h) and synchronously form polarized ingrowth walls accessible for experimental observations. Using this model, we recently reported findings consistent with extracellular hydrogen peroxide, produced through a respiratory burst oxidase homolog/superoxide dismutase pathway, initiating cell wall biosynthetic activity and providing directional information guiding deposition of the polarized uniform wall. Our conclusions rested on observations derived from pharmacological manipulations of hydrogen peroxide production and correlative gene expression data sets. A series of additional studies were undertaken, the results of which verify that extracellular hydrogen peroxide contributes to regulating ingrowth wall formation and is generated by a respiratory burst oxidase homolog/superoxide dismutase pathway.     

RADIOAUTOGRAPHIC STUDY OF CELL WALL DEPOSITION IN GROWING PLANT CELLS

Segments cut from growing oat coleoptiles and pea stems were fed glucose-³H in presence and absence of the growth hormone indoleacetic acid (IAA). By means of electron microscope radioautography it was demonstrated that new cell wall material is deposited both at the wall surface (apposition) and within the preexisting wall structure (internally). Quantitative profiles for the distribution of incorporation with position through the thickness of the wall were obtained for the thick outer wall of epidermal cells. With both oat coleoptile and pea stem epidermal outer walls, it was found that a larger proportion of the newly synthesized wall material appeared to become incorporated within the wall in the presence of IAA. Extraction experiments on coleoptile tissue showed that activity that had been incorporated into the cell wall interior represented noncellulosic constituents, mainly hemicelluloses, whereas cellulose was deposited largely or entirely by apposition. It seems possible that internal incorporation of hemicelluloses plays a role in the cell wall expansion process that is involved in cell growth.     

Changes in the arrangement of cellulose microfibrils associated with the cessation of cell expansion in tracheids

 The relationship between the cessation of cell expansion and formation of the secondary wall was investigated in the early-wood tracheids of Abies sachalinensis Masters by image analysis and field emission scanning electron microscopy. The area of the lumen and the length of the perimeter of the lumen of differentiating tracheids increased from the cambium towards the xylem. These increases had just ceased in the case of tracheids closest to the cambium in which birefringence was first detected by observations with a polarizing light microscope. Cellulose microfibrils (MFs) deposited on the innermost surfaces of radial walls were not well ordered during the expansion of cells, but well ordered MFs were deposited at the subsequent stage of cell wall formation. The first well ordered MFs were oriented in an S-helix. The well ordered MFs had already been deposited at the tracheids where birefringence was first detected under the polarizing light microscope. These results indicate that the deposition of the well ordered MFs, namely, the formation of the secondary wall, begins before the cessation of cell expansion of tracheids. Therefore, it seems that the expansion of tracheids is restricted by the deposition of the secondary wall because the cell walls become rigid simultaneously with the development of the secondary wall and, therefore, the yield point of cell walls exceeds the turgor pressure of the cell. Received: 3 July 1996 / Accepted: 24 September 1996     

Evidence for a net-like organization of lipopolysaccharide particles in the Escherichia coli outer membrane

Cell wall LPS of Escherichia coli are organized as particles which are visible in the electron microscope, after treatment of the wall with alkali. We now describe alkali treated walls of three E. coli strains with differences in susceptibility to the T4 phage infection. Strain CR63, a usual host for the T4 phage, shows the LPS particles on the murein layer. These particles are absent in alkali treated cell walls of the strain W. Walls of this strain are broken during T4 infection and phages can be seen bearing pieces of membrane attached to their long as well as their short tail fibers. Strain AS19 which is hypersensitive to the lysis from without caused by T4 shows murein layers with no LPS particles on their surface, and networks of LPS particles with bacterial shape. This suggested that LPS are organized in a network of particles which may serve as the skeleton of the cell wall.     

Rapid removal of magnetic particles from large volumes of suspensions

Summary Two simple and rapid procedures for removal of fine magnetic particles from large volumes of suspensions are described. One of them is based on the flow of magnetic suspension through the modified glass pipette placed on a flat magnet, in the second one the magnetic suspension is poured on a plastic film covering the magnet.     

Cell wall dimensions reign supreme: cell wall composition is irrelevant for the temperature signal of latewood density/blue intensity in Scots pine

Many microdensitometric techniques are available for deriving maximum latewood density (MXD), which is the state-of-the-art proxy parameter for local to hemispheric-scale temperature reconstructions of the last millennium. Techniques based on X-ray radiation and visible light reflection, such as “blue intensity” (BI), integrate both the density/composition and the dimensions of the cell walls to derive microdensitometric data. In contrast, the dendroanatomical technique relies only on the dimensions of the cell walls. It is therefore possible to isolate cell wall variables by subtracting data derived using the dendroanatomical technique from data derived using X-ray and BI-based techniques.In this study, we explore differences in well-replicated data from parallel X-ray, BI, and dendroanatomical measurements of temperature-sensitive Pinus sylvestris trees from northern Finland. We aim to determine whether cell wall density is critical to the success of X-ray-based MXD, and whether the BI-based parameter counterpart, here termed MXBI, contains useful information about the composition of the cell wall (specifically the lignin).Our results indicate that cell wall density and cell wall BI have no relevant influence on MXD and MXBI measurements. Even in years with severely reduced lignification, identified as so-called “blue rings”, dendroanatomical MXD (aMXD) measurements do not deviate significantly from their MXD or MXBI counterparts. Moreover, derived chronologies of cell wall density and cell wall BI contain no significant climate signals when correlated with local climate. Maximum latewood density of conifers can thus be obtained without bias using the dendroanatomical technique. Because lignin content appears to play a negligible role for cell wall BI, the cell wall BI likely presents the biggest challenge when producing unbiased MXBI data. This is because BI data is notorious for cell wall color distortion across the heartwood and sapwood, and between living wood and dead wood, and may therefore distort the otherwise strong link with wood density on multidecadal scales.     

Immunomagnetic isolation of Xanthomonas campestris pv. pelargonii

Immunomagnetic fishing was developed as an improved procedure for increasing the bacterial target to non-target recovery ratio in suspensions containing mixtures of target and non-target organisms. A cell suspension containing the target Xanthomonas campestris pv. pelargonii and non-target organisms, is treated with rabbit polyclonal antiserum against X.c. pv. pelargonii and incubated for 1 h. The suspension is then mixed with paramagnetic iron oxide particles coated with goat anti-rabbit antibodies (immunomagnetic particles). After incubation, the polished surface of a 14 mm diameter neodymium supermagnet is placed at the air-water interace and the magnetic particles are attracted to the magnet. After all visible magnetic particles have attached to the bottom of the magnet, the magnet is dipped in sterile buffer to remove non-target organisms. The magnet with attached magnetic particles is rubbed evenly over an agar surface to dislodge the particles and attached bacteria. Conventional immunomagnetic isolation (immunomagnetic attraction) and immunomagnetic fishing were compared, for the recovery of the target organism in geranium leaf washings spiked with X.c. pv. pelargonii. With immunomagnetic attraction and immunomagnetic fishing, bacterial non-target organisms were reduced to 11.4 and 1.5% of the initial population, respectively, whereas the target was only reduced to 63.7 and 53.8%.     

Multinucleate transfer cells induced in coleus roots by the root-knot nematode,Meloidogyne arenaria

Summary The occurrence and position of wall protuberances in giant cells induced in coleus roots by the root-knot nematodeMeloidogyne arenaria is described, and the structure and function of giant cells is compared with that of syncytia induced by cyst-nematodes.Extensive protuberance development occurs on walls of giant cells adjacent to xylem vessels. Protuberances are less well developed next to sieve elements, and almost absent next to parenchyma cells. On walls between giant cells they occur on both sides or only one side. The formation of protuberances indicates that giant cells are multinucleate transfer cells. The position of protuberances marks the wall area where solutes enter the cell. Solutes are obtained from xylem and phloem elements, and the position of protuberances at the junction between giant cells and vascular elements indicates an extensive flow of solutes along cell walls. The observations support the hypothesis that wall protuberances form as a result of selective solute flow across the plasmalemma.No cell wall dissolution was observed, although wall gaps may occur between giant cells as a result of breakage during rapid cell expansion.     

A Theoretical Study of Single-Cell Electroporation in a Microchannel

Electroporation of a single cell in a microchannel was studied. The effects of electrical (e.g., strength of the electric pulse) and geometrical (e.g., microchannel height, electrode size and position) parameters on cell membrane permeabilization were investigated. The electrodes were assumed to be embedded in the walls of the microchannel; the cell was suspended between these two electrodes. By keeping the electric pulse constant, increasing the microchannel height reduces the number and the radius of the biggest nanopores, as well as the electroporated area of the cell membrane. If the width of the electrodes is bigger than the cell diameter, the transmembrane potential will be centralized and have a sinusoidal distribution around the cell if nanopores are not generated. As the width of the electrode decreases and becomes smaller than the cell diameter, the local transmembrane potential decreases; in the nonelectroporative area, the transmembrane potential distribution deviates from the sinusoidal behavior; the induced transmembrane potential also concentrates around the poles of the cell membrane (the nearest points of the cell membrane to the electrodes). During cell membrane permeabilization, the biggest nanopores are initially created at the poles and then the nanopore population expands toward the equator. The number of the created nanopores reaches its maximal value within a few microseconds; further presence of the electric pulse may not influence the number and location of the created nanopores anymore but will develop the generated nanopores. Strengthening the electric pulse intensifies the size and number of the created nanopores as well as the electroporated area on the cell membrane.     

GEANT4 models for the secondary radiation flux in the collimation system of a 300 MeV proton microbeam

In Harbin, we are developing a 300 MeV proton microbeam for many applications in space science including upset studies in microelectronic devices, radiation hardness of materials for satellites and radiation effects in human tissues. There are also applications of this facility proposed for proton therapy. The microbeam system will employ a purpose-built proton synchrotron to provide the beam. However there are many challenges to be addressed in the design, construction and operation of this facility. Here we address two important design aspects for which we apply GEANT4 modeling. First, the high energy proton beam interacts strongly with beam line materials, especially the collimation slits, to produce showers of secondary particles which could introduce significant background signals and degrade the resolution of the proton microbeam. Second, the beam transport within the residual vacuum of the beam line may also introduce undesirable background radiation. In both cases mitigation strategies need to be incorporated during the design phase of the new system. We study the use of a dipole magnet following the aperture collimator to reduce the flux of secondary particles incident on the analysis chamber. Monte Carlo simulations are performed using GEANT4 and SRIM. By inserting the dipole magnet, we find as expected a significant reduction in the scattering of protons and other particles, such as neutrons and gamma rays, at the collimation system exit position. Secondary radiation from the residual gas pressure within the beam line vacuum system are also modelled and found to be negligible under the standard operating conditions.     

Brownian dynamics simulation of the lateral distribution of charged membrane components

Brownian dynamics simulations were performed to study the contribution of electric interactions between charged membrane components to their lateral distribution in a two-dimensional viscous liquid (bilayer lipid membrane). The electrostatic interaction potential was derived from an analytical solution of the linearized Poisson-Boltzmann equation for point charges in an electrolyte solution — membrane — electrolyte solution system. Equilibrium as well as dynamic quantities were investigated. The lateral organization of membrane particles, modelled by mobile cylinders in a homogeneous membrane separating two electrolyte solutions was described by spatial distribution functions, diffusion coefficients and cluster statistics. Disorder, local order and crystal-like arrangements were observed as a function of the particle charge, the closest possible distances between the charges and the particle density. The simulations revealed that the system is very sensitive to the position of the charges with respect to the electrolyte solution — membrane interface. Electrostatic interactions of charges placed directly on the membrane surface were almost negligible, whereas deeper charges demonstrated pronounced interaction. Biologically relevant parameters corresponded at most to local and transient ordering. It was found that lateral electric forces can give rise to a preferred formation of clusters with an even number of constituents provided that the closest possible charge-charge distances are small. It is concluded that lateral electrostatic interactions can account for local particle aggregations, but their impact on the global arrangement and movement of membrane components is limited. Correspondence to: D. Walther