A hyperelastic model for simulating cells in flow.

In the emerging field of 3D bioprinting, cell damage due to large deformations is considered a main cause for cell death and loss of functionality inside the printed construct. Those deformations, in turn, strongly depend on the mechano-elastic response of the cell to the hydrodynamic stresses experienced during printing. In this work, we present a numerical model to simulate the deformation of biological cells in arbitrary three-dimensional flows. We consider cells as an elastic continuum according to the hyperelastic Mooney-Rivlin model. We then employ force calculations on a tetrahedralized volume mesh. To calibrate our model, we perform a series of FluidFM[Formula: see text] compression experiments with REF52 cells demonstrating that all three parameters of the Mooney-Rivlin model are required for a good description of the experimental data at very large deformations up to 80%. In addition, we validate the model by comparing to previous AFM experiments on bovine endothelial cells and artificial hydrogel particles. To investigate cell deformation in flow, we incorporate our model into Lattice Boltzmann simulations via an Immersed-Boundary algorithm. In linear shear flows, our model shows excellent agreement with analytical calculations and previous simulation data.

Flow and hydrodynamic shear stress inside a printing needle during biofabrication.

We present a simple but accurate algorithm to calculate the flow and shear rate profile of shear thinning fluids, as typically used in biofabrication applications, with an arbitrary viscosity-shear rate relationship in a cylindrical nozzle. By interpolating the viscosity with a set of power-law functions, we obtain a mathematically exact piecewise solution to the incompressible Navier-Stokes equation. The algorithm is validated with known solutions for a simplified Carreau-Yasuda fluid, full numerical simulations for a realistic chitosan hydrogel as well as experimental velocity profiles of alginate and chitosan solutions in a microfluidic channel. We implement the algorithm in an easy-to-use Python tool, included as Supplementary Material, to calculate the velocity and shear rate profile during the printing process, depending on the shear thinning behavior of the bioink and printing parameters such as pressure and nozzle size. We confirm that the shear stress varies in an exactly linear fashion, starting from zero at the nozzle center to the maximum shear stress at the wall, independent of the shear thinning properties of the bioink. Finally, we demonstrate how our method can be inverted to obtain rheological bioink parameters in-situ directly before or even during printing from experimentally measured flow rate versus pressure data.

Flow-accelerated platelet biogenesis is due to an elasto-hydrodynamic instability.

Blood platelets are formed by fragmentation of long membrane extensions from bone marrow megakaryocytes in the blood flow. Using lattice-Boltzmann/immersed boundary simulations we propose a biological Rayleigh-Plateau instability as the biophysical mechanism behind this fragmentation process. This instability is akin to the surface tension-induced breakup of a liquid jet but is driven by active cortical processes including actomyosin contractility and microtubule sliding. Our fully three-dimensional simulations highlight the crucial role of actomyosin contractility, which is required to trigger the instability, and illustrate how the wavelength of the instability determines the size of the final platelets. The elasto-hydrodynamic origin of the fragmentation explains the strong acceleration of platelet biogenesis in the presence of an external flow, which we observe in agreement with experiments. Our simulations then allow us to disentangle the influence of specific flow conditions: While a homogeneous flow with uniform velocity leads to the strongest acceleration, a shear flow with a linear velocity gradient can cause fusion events of two developing platelet-sized swellings during fragmentation. A fusion event may lead to the release of larger structures which are observable as preplatelets in experiments. Together, our findings strongly indicate a mainly physical origin of fragmentation and regulation of platelet size in flow-accelerated platelet biogenesis.

Discharge electrode geometry and energy efficiency in a one-stage wire-tube electrostatic precipitator operating at high concentrations of submicron liquid aerosol.

In order to help answering the question 'Which electrode design is the best?', the precipitation performance of a tubular electrostatic precipitator was determined for different electrode designs with special attention to specific energy consumption. Experiments were conducted in a wire-tube arrangement with a high loading of liquid submicron particles causing strong space charge effects, also known as corona quenching. The experiments are supplemented by numerical simulation results showing the effect of residence time and power consumption on separation efficiency.

Computational modeling of active deformable membranes embedded in three-dimensional flows.

Active gel theory has recently been very successful in describing biologically active materials such as actin filaments or moving bacteria in temporally fixed and simple geometries such as cubes or spheres. Here we develop a computational algorithm to compute the dynamic evolution of an arbitrarily shaped, deformable thin membrane of active material embedded in a three-dimensional flowing liquid. For this, our algorithm combines active gel theory with the classical theory of thin elastic shells. To compute the actual forces resulting from active stresses, we apply a parabolic fitting procedure to the triangulated membrane surface. Active forces are then dynamically coupled via an immersed-boundary method to the surrounding fluid whose dynamics can be solved by any standard, e.g., Lattice-Boltzmann, flow solver. We validate our algorithm using the Green's functions of Berthoumieux et al. [New J. Phys. 16, 065005 (2014)10.1088/1367-2630/16/6/065005] for an active cylindrical membrane subjected (i) to a locally increased active stress and (ii) to a homogeneous active stress. For the latter scenario, we predict in addition a nonaxisymmetric instability. We highlight the versatility of our method by analyzing the flow field inside an actively deforming cell embedded in external shear flow. Further applications may be cytoplasmic streaming or active membranes in blood flows.

Antimargination of Microparticles and Platelets in the Vicinity of Branching Vessels.

We investigate the margination of microparticles/platelets in blood flow through complex geometries typical for in vivo vessel networks: a vessel confluence and a bifurcation. Using three-dimensional lattice Boltzmann simulations, we confirm that behind the confluence of two vessels, a cell-free layer devoid of red blood cells develops in the channel center. Despite its small size of roughly 1 µm, this central cell-free layer persists for up to 100 µm after the confluence. Most importantly, we show from simulations that this layer also contains a significant amount of microparticles/platelets and validate this result by in vivo microscopy in mouse venules. At bifurcations, however, a similar effect does not appear, and margination is largely unaffected by the geometry. This antimargination toward the vessel center after a confluence may explain earlier in vivo observations, which found that platelet concentrations near the vessel wall are seen to be much higher on the arteriolar side (containing bifurcations) than on the venular side (containing confluences) of the vascular system.

Transient colocalization of X-inactivation centres accompanies the initiation of X inactivation.

The initial differential treatment of the two X chromosomes during X-chromosome inactivation is controlled by the X-inactivation centre (Xic). This locus determines how many X chromosomes are present in a cell ('counting') and which X chromosome will be inactivated in female cells ('choice'). Critical control sequences in the Xic include the non-coding RNAs Xist and Tsix, and long-range chromatin elements. However, little is known about the process that ensures that X inactivation is triggered appropriately when more than one Xic is present in a cell. Using three-dimensional fluorescence in situ hybridization (FISH) analysis, we showed that the two Xics transiently colocalize, just before X inactivation, in differentiating female embryonic stem cells. Using Xic transgenes capable of imprinted but not random X inactivation, and Xic deletions that disrupt random X inactivation, we demonstrated that Xic colocalization is linked to Xic function in random X inactivation. Both long-range sequences and the Tsix element, which generates the antisense transcript to Xist, are required for the transient interaction of Xics. We propose that transient colocalization of Xics may be necessary for a cell to determine Xic number and to ensure the correct initiation of X inactivation.

A contractile nuclear actin network drives chromosome congression in oocytes.

Chromosome capture by microtubules is widely accepted as the universal mechanism of spindle assembly in dividing cells. However, the observed length of spindle microtubules and computer simulations of spindle assembly predict that chromosome capture is efficient in small cells, but may fail in cells with large nuclear volumes such as animal oocytes. Here we investigate chromosome congression during the first meiotic division in starfish oocytes. We show that microtubules are not sufficient for capturing chromosomes. Instead, chromosome congression requires actin polymerization. After nuclear envelope breakdown, we observe the formation of a filamentous actin mesh in the nuclear region, and find that contraction of this network delivers chromosomes to the microtubule spindle. We show that this mechanism is essential for preventing chromosome loss and aneuploidy of the egg--a leading cause of pregnancy loss and birth defects in humans.

4-D single particle tracking of synthetic and proteinaceous microspheres reveals preferential movement of nuclear particles along chromatin - poor tracks.

BACKGROUND: The dynamics of nuclear organization, nuclear bodies and RNPs in particular has been the focus of many studies. To understand their function, knowledge of their spatial nuclear position and temporal translocation is essential. Typically, such studies generate a wealth of data that require novel methods in image analysis and computational tools to quantitatively track particle movement on the background of moving cells and shape changing nuclei. RESULTS: We developed a novel 4-D image processing platform (TIKAL) for the work with laser scanning and wide field microscopes. TIKAL provides a registration software for correcting global movements and local deformations of cells as well as 2-D and 3-D tracking software. With this new tool, we studied the dynamics of two different types of nuclear particles, namely nuclear bodies made from GFP-NLS-vimentin and microinjected 0.1 mum - wide polystyrene beads, by live cell time-lapse microscopy combined with single particle tracking and mobility analysis. We now provide a tool for the automatic 3-D analysis of particle movement in parallel with the acquisition of chromatin density data. CONCLUSIONS: Kinetic analysis revealed 4 modes of movement: confined obstructed, normal diffusion and directed motion. Particle tracking on the background of stained chromatin revealed that particle movement is directly related to local reorganization of chromatin. Further a direct comparison of particle movement in the nucleoplasm and the cytoplasm exhibited an entirely different kinetic behaviour of vimentin particles in both compartments. The kinetics of nuclear particles were slightly affected by depletion of ATP and significantly disturbed by disruption of actin and microtubule networks. Moreover, the hydration state of the nucleus had a strong impact on the mobility of nuclear bodies since both normal diffusion and directed motion were entirely abolished when cells were challenged with 0.6 M sorbitol. This effect correlated with the compaction of chromatin. We conclude that alteration in chromatin density directly influences the mobility of protein assemblies within the nucleus.

Nuclear body movement is determined by chromatin accessibility and dynamics.

Promyelocytic leukemia (PML) and Cajal bodies are mobile subnuclear organelles, which are involved in activities like RNA processing, transcriptional regulation, and antiviral defense. A key parameter in understanding their biological functions is their mobility. The diffusion properties of PML and Cajal bodies were compared with a biochemically inactive body formed by aggregates of murine Mx1 by using single-particle tracking methods. The artificial Mx1-yellow fluorescent protein body showed a very similar mobility compared with PML and Cajal bodies. The data are described quantitatively by a mechanism of nuclear body movement consisting of two components: diffusion of the body within a chromatin corral and its translocation resulting from chromatin diffusion. This finding suggests that the body mobility reflects the dynamics and accessibility of the chromatin environment, which might target bodies to specific nuclear subcompartments where they exert their biological function.

Trichostatin A-induced histone acetylation causes decondensation of interphase chromatin.

The effect of trichostatin A (TSA)-induced histone acetylation on the interphase chromatin structure was visualized in vivo with a HeLa cell line stably expressing histone H2A, which was fused to enhanced yellow fluorescent protein. The globally increased histone acetylation caused a reversible decondensation of dense chromatin regions and led to a more homogeneous distribution. These structural changes were quantified by image correlation spectroscopy and by spatially resolved scaling analysis. The image analysis revealed that a chromatin reorganization on a length scale from 200 nm to >1 microm was induced consistent with the opening of condensed chromatin domains containing several Mb of DNA. The observed conformation changes could be assigned to the folding of chromatin during G1 phase by characterizing the effect of TSA on cell cycle progression and developing a protocol that allowed the identification of G1 phase cells on microscope coverslips. An analysis by flow cytometry showed that the addition of TSA led to a significant arrest of cells in S phase and induced apoptosis. The concentration dependence of both processes was studied.