Alcanivorax borkumensis biofilms enhance oil degradation by interfacial tubulation.

During the consumption of alkanes, Alcanivorax borkumensis will form a biofilm around an oil droplet, but the role this plays during degradation remains unclear. We identified a shift in biofilm morphology that depends on adaptation to oil consumption: Longer exposure leads to the appearance of dendritic biofilms optimized for oil consumption effected through tubulation of the interface. In situ microfluidic tracking enabled us to correlate tubulation to localized defects in the interfacial cell ordering. We demonstrate control over droplet deformation by using confinement to position defects, inducing dimpling in the droplets. We developed a model that elucidates biofilm morphology, linking tubulation to decreased interfacial tension and increased cell hydrophobicity.

Shaping the zebrafish myotome by intertissue friction and active stress.

Organ formation is an inherently biophysical process, requiring large-scale tissue deformations. Yet, understanding how complex organ shape emerges during development remains a major challenge. During zebrafish embryogenesis, large muscle segments, called myotomes, acquire a characteristic chevron morphology, which is believed to aid swimming. Myotome shape can be altered by perturbing muscle cell differentiation or the interaction between myotomes and surrounding tissues during morphogenesis. To disentangle the mechanisms contributing to shape formation of the myotome, we combine single-cell resolution live imaging with quantitative image analysis and theoretical modeling. We find that, soon after segmentation from the presomitic mesoderm, the future myotome spreads across the underlying tissues. The mechanical coupling between the future myotome and the surrounding tissues appears to spatially vary, effectively resulting in spatially heterogeneous friction. Using a vertex model combined with experimental validation, we show that the interplay of tissue spreading and friction is sufficient to drive the initial phase of chevron shape formation. However, local anisotropic stresses, generated during muscle cell differentiation, are necessary to reach the acute angle of the chevron in wild-type embryos. Finally, tissue plasticity is required for formation and maintenance of the chevron shape, which is mediated by orientated cellular rearrangements. Our work sheds light on how a spatiotemporal sequence of local cellular events can have a nonlocal and irreversible mechanical impact at the tissue scale, leading to robust organ shaping.

Dual energy-band excitation from a low power Rh anode X-ray tube for the simultaneous determination of low Z and high Z elements (Na-U) using total-reflection X-ray fluorescence analysis (TXRF).

This article presents results from an experimental setup for a dual energy-band vacuum spectrometer for total-reflection X-ray fluorescence analysis allowing simultaneous efficient excitation of low, medium, and high Z elements. The spectrometer is equipped with an air-cooled 35 W low power Rh X-ray tube and a 17 mm2 silicon drift detector with a thin 8 µm beryllium window. A Pd/B4C multilayer monochromator is used at the same time as a Bragg reflector for Rh-Kα radiation and as a high-energy cut-off reflector above 5 keV, where the characteristic Rh-L radiation is totally reflected and present in the spectrum of the exciting radiation. This leaves one broad low energy band below 5 keV and one high energy band around the energy of Rh-Kα. As Rh-L radiation would be absorbed on its path through air, a new beam entrance system was designed in order to guide the Rh-L photons into the vacuum chamber for efficient excitation of low Z elements. With this setup, elements down to sodium (Z = 11, E = 1.04 keV) could be detected. First results are presented, and spectra obtained in air as well as in vacuum are compared and discussed. Detection limits in the range of 1000 µg/kg for Na and around 140 µg/kg for Mg were achieved using the NIST SRM 1640 (trace elements in water).

Spontaneous shear flow in confined cellular nematics.

In embryonic development or tumor evolution, cells often migrate collectively within confining tracks defined by their microenvironment 1,2. In some of these situations, the displacements within a cell strand are antiparallel 3, giving rise to shear flows. However, the mechanisms underlying these spontaneous flows remain poorly understood. Here, we show that an ensemble of spindle-shaped cells plated in a well-defined stripe spontaneously develop a shear flow whose characteristics depend on the width of the stripe. On wide stripes, the cells self-organize in a nematic phase with a director at a well-defined angle with the stripe's direction, and develop a shear flow close to the stripe's edges. However, on stripes narrower than a critical width, the cells perfectly align with the stripe's direction and the net flow vanishes. A hydrodynamic active gel theory provides an understanding of these observations and identifies the transition between the non-flowing phase oriented along the stripe and the tilted phase exhibiting shear flow as a Fréedericksz transition driven by the activity of the cells. This physical theory is grounded in the active nature of the cells and based on symmetries and conservation laws, providing a generic mechanism to interpret in vivo antiparallel cell displacements.

Soft inclusion in a confined fluctuating active gel.

We study stochastic dynamics of a point and extended inclusion within a one-dimensional confined active viscoelastic gel. We show that the dynamics of a point inclusion can be described by a Langevin equation with a confining potential and multiplicative noise. Using a systematic adiabatic elimination over the fast variables, we arrive at an overdamped equation with a proper definition of the multiplicative noise. To highlight various features and to appeal to different biological contexts, we treat the inclusion in turn as a rigid extended element, an elastic element, and a viscoelastic (Kelvin-Voigt) element. The dynamics for the shape and position of the extended inclusion can be described by coupled Langevin equations. Deriving exact expressions for the corresponding steady-state probability distributions, we find that the active noise induces an attraction to the edges of the confining domain. In the presence of a competing centering force, we find that the shape of the probability distribution exhibits a sharp transition upon varying the amplitude of the active noise. Our results could help understanding the positioning and deformability of biological inclusions, e.g., organelles in cells, or nucleus and cells within tissues.

Maximal Fluctuations of Confined Actomyosin Gels: Dynamics of the Cell Nucleus.

We investigate the effect of stress fluctuations on the stochastic dynamics of an inclusion embedded in a viscous gel. We show that, in nonequilibrium systems, stress fluctuations give rise to an effective attraction towards the boundaries of the confining domain, which is reminiscent of an active Casimir effect. We apply this generic result to the dynamics of deformations of the cell nucleus, and we demonstrate the appearance of a fluctuation maximum at a critical level of activity, in agreement with recent experiments [E. Makhija, D. S. Jokhun, and G. V. Shivashankar, Proc. Natl. Acad. Sci. U.S.A. 113, E32 (2016)PNASA60027-842410.1073/pnas.1513189113].

Cell-like pressure sensors reveal increase of mechanical stress towards the core of multicellular spheroids under compression.

The surrounding microenvironment limits tumour expansion, imposing a compressive stress on the tumour, but little is known how pressure propagates inside the tumour. Here we present non-destructive cell-like microsensors to locally quantify mechanical stress distribution in three-dimensional tissue. Our sensors are polyacrylamide microbeads of well-defined elasticity, size and surface coating to enable internalization within the cellular environment. By isotropically compressing multicellular spheroids (MCS), which are spherical aggregates of cells mimicking a tumour, we show that the pressure is transmitted in a non-trivial manner inside the MCS, with a pressure rise towards the core. This observed pressure profile is explained by the anisotropic arrangement of cells and our results suggest that such anisotropy alone is sufficient to explain the pressure rise inside MCS composed of a single cell type. Furthermore, such pressure distribution suggests a direct link between increased mechanical stress and previously observed lack of proliferation within the spheroids core.

Synchrotron radiation micro X-ray fluorescence spectroscopy of thin structures in bone samples: comparison of confocal and color X-ray camera setups.

In the quest for finding the ideal synchrotron-radiation-induced imaging method for the investigation of trace element distributions in human bone samples, experiments were performed using both a scanning confocal synchrotron radiation micro X-ray fluorescence (SR-µXRF) (FLUO beamline at ANKA) setup and a full-field color X-ray camera (BAMline at BESSY-II) setup. As zinc is a trace element of special interest in bone, the setups were optimized for its detection. The setups were compared with respect to count rate, required measurement time and spatial resolution. It was demonstrated that the ideal method depends on the element of interest. Although for Ca (a major constituent of the bone with a low energy of 3.69 keV for its Kα XRF line) the color X-ray camera provided a higher resolution in the plane, for Zn (a trace element in bone) only the confocal SR-µXRF setup was able to sufficiently image the distribution.

A novel vacuum spectrometer for total reflection x-ray fluorescence analysis with two exchangeable low power x-ray sources for the analysis of low, medium, and high Z elements in sequence.

The extension of the detectable elemental range with Total Reflection X-ray Fluorescence (TXRF) analysis is a challenging task. In this paper, it is demonstrated how a TXRF spectrometer is modified to analyze elements from carbon to uranium. Based on the existing design of a vacuum TXRF spectrometer with a 12 specimen sample changer, the following components were renewed: the silicon drift detector with 20 mm(2) active area and having a special ultra-thin polymer window allowing the detection of elements from carbon upwards. Two exchangeable X-ray sources guarantee the efficient excitation of both low and high Z elements. These X-ray sources were two light-weighted easily mountable 35 W air-cooled low-power tubes with Cr and Rh anodes, respectively. The air cooled tubes and the Peltier-cooled detector allowed to construct a transportable tabletop spectrometer with compact dimensions, as neither liquid nitrogen cooling for the detector nor a water cooling circuit and a bulky high voltage generator for the X-ray tubes are required. Due to the excellent background conditions as a result of the TXRF geometry, detection limits of 150 ng for C, 12 ng for F, and 3.3 ng for Na have been obtained using Cr excitation in vacuum. For Rh excitation, the detection limits of 90 pg could be achieved for Sr. Taking 10 to 20 µl of sample volume, extrapolated detection limits in the ng/g (ppb) range are resulting in terms of concentration.

Growth, homeostatic regulation and stem cell dynamics in tissues.

The regulation of cell growth in animal tissues is a question of critical importance: most tissues contain different types of cells in interconversion and the fraction of each type has to be controlled in a precise way, by mechanisms that remain unclear. Here, we provide a theoretical framework for the homeostasis of stem-cell-containing epithelial tissues using mechanical equations, which describe the size of the tissue and kinetic equations, which describe the interconversions of the cell populations. We show that several features, such as the evolution of stem cell fractions during intestinal development, the shape of a developing intestinal wall, as well as the increase in the proliferative compartment in cancer initiation, can be studied and understood from generic modelling which does not rely on a particular regulatory mechanism. Finally, inspired by recent experiments, we propose a model where cell division rates are regulated by the mechanical stresses in the epithelial sheet. We show that pressure-controlled growth can, in addition to the previous features, also explain with few parameters the formation of stem cell compartments as well as the morphologies observed when a colonic crypt becomes cancerous. We also discuss optimal strategies of wound healing, in connection with experiments on the cornea.

The actin cortex as an active wetting layer.

Using active gel theory we study theoretically the properties of the cortical actin layer of animal cells. The cortical layer is described as a non-equilibrium wetting film on the cell membrane. The actin density is approximately constant in the layer and jumps to zero at its edge. The layer thickness is determined by the ratio of the polymerization velocity and the depolymerization rate of actin.

Fluctuation-response theorem for the active noisy oscillator of the hair-cell bundle.

The hair bundle of sensory cells in the vertebrate ear provides an example of a noisy oscillator close to a Hopf bifurcation. The analysis of the data from both spontaneous and forced oscillations shows a strong violation of the fluctuation-dissipation theorem, revealing the presence of an underlying active process that keeps the system out of equilibrium. Nevertheless, we show that a generalized fluctuation-dissipation theorem, valid for nonequilibrium steady states, is fulfilled within the limits of our experimental accuracy and computational approximations, when the adequate conjugate degrees of freedom are chosen.

Tissue dynamics with permeation.

Animal tissues are complex assemblies of cells, extracellular matrix (ECM), and permeating interstitial fluid. Whereas key aspects of the multicellular dynamics can be captured by a one-component continuum description, cell division and apoptosis imply material turnover between different components that can lead to additional mechanical conditions on the tissue dynamics. We extend our previous description of tissues in order to account for a cell/ECM phase and the permeating interstitial fluid independently. In line with our earlier work, we consider the cell/ECM phase to behave as an elastic solid in the absence of cell division and apoptosis. In addition, we consider the interstitial fluid as ideal on the relevant length scales, i.e., we ignore viscous stresses in the interstitial fluid. Friction between the fluid and the cell/ECM phase leads to a Darcy-like relation for the interstitial fluid velocity and introduces a new characteristic length scale. We discuss the dynamics of a tissue confined in a chamber with a permeable piston close to the homeostatic state where cell division and apoptosis balance, and we calculate the rescaled effective diffusion coefficient for cells. For different mass densities of the cell/ECM component and the interstitial fluid, a treadmilling steady state due to gravitational forces can be found.

Pharmacokinetics and safety of roledumab, a novel human recombinant monoclonal anti-RhD antibody with an optimized Fc for improved engagement of FCγRIII, in healthy volunteers.

BACKGROUND AND OBJECTIVES: A human recombinant monoclonal anti-RhD IgG may be useful to prevent RhD allo-immunization. Roledumab is such an antibody with a glycosylation pattern optimized for biological activity. The objective of the study was to assess the safety and pharmacokinetics of roledumab in healthy RhD-negative volunteers. MATERIALS AND METHODS: A total of 46 subjects received doses of 30-3000 µg i.v. of roledumab or placebo using a double-blind escalating single-dose design; 12 of these subjects also received 300 µg i.m. of roledumab. Subjects were followed for 6 months after administration. Serum roledumab concentrations were determined using flow cytometry. RESULTS: Fourteen treatment-emergent adverse events related to treatment were reported in nine subjects, with no apparent difference in their frequency or nature after placebo or roledumab administration. No anti-roledumab antibodies were detected. AUC(last) increased from 4·4 ng/ml.day at 30 µg i.v. to 2257 ng/ml.day at 3000 g i.v. The t(½) ranged from 18 to 22 days, and the absolute bioavailability after i.m. administration was between 73% and 80%. CONCLUSION: Roledumab is safe and well tolerated in healthy RhD-negative volunteers and shows a pharmacokinetic profile similar to that of polyclonal anti-RhD immunoglobulin.

Bidirectional motion of motor assemblies and the weak-noise escape problem.

We present a detailed calculation that enables us to estimate the reversal time of a molecular motor assembly that displays bidirectional motion in the limit of weak noise. We derive a Fokker-Planck equation by taking a large volume expansion of a master equation, and we consider a simple choice of transition rates that enables us to reduce the number of variables to 2. We use the Wentzell-Freidlin theory to define an effective nonequilibrium potential and analytically estimate the reversal time. We also present the results of stochastic simulations that match very well our simulation results.

Instabilities of monolayered epithelia: shape and structure of villi and crypts.

We study theoretically the shapes of a dividing epithelial monolayer of cells lying on top of an elastic stroma. The negative tension created by cell division provokes a buckling instability at a finite wave vector leading to the formation of periodic arrays of villi and crypts. The instability is similar to the buckling of a metallic plate under compression. We use the results to rationalize the various structures of the intestinal lining observed in vivo. Taking into account the coupling between cell division and local curvature, we obtain different patterns of villi and crypts, which could explain the different morphologies of the small intestine and the colon.

Dynamical behavior of molecular motor assemblies in the rigid and crossbridge models.

We present a detailed analysis of the dynamical instabilities appearing in two kinetic theories for the collective behavior of molecular motors: the rigid two-state model and the two-state crossbridge (or power-stroke) model with continuous binding sites. We calculate force-velocity relations, discuss their stability, plot a diagram that summarizes the oscillation regimes, identify the location of the Hopf bifurcation with a memory effect, discuss the oscillation frequency and make a link with single-molecule experiments. We show that the instabilities present in these models naturally translate into non-linearities in force-displacement relations, and at linear order give forces that are similar to the delayed stretch activation observed in oscillating muscles. We also find that instabilities can appear for both apparent load-decelerated and load-accelerated detachment rates in a 3-state crossbridge model.

Motion reversal of molecular motor assemblies due to weak noise.

Bidirectional motion is an example of collective behavior of molecular motors. It occurs at finite noise level in a nonequilibrium system. We consider this problem as a first exit problem. We identify the noise strength by doing an expansion of a master equation and apply the Wentzell-Freidlin theory to define an effective nonequilibrium potential and provide analytical estimates of the reversal time. Our results match very well with the results of stochastic simulations.

Analysis of CD16+CD56dim NK cells from CLL patients: evidence supporting a therapeutic strategy with optimized anti-CD20 monoclonal antibodies.

Although anti-CD20 monoclonal antibodies (mAbs) show promise for the treatment of chronic lymphocytic leukemia (CLL), the success of the anti-CD20 mAb rituximab in CLL treatment has been limited. Novel anti-CD20 mAbs with more potent cytotoxic activity have recently been engineered, but so far most have only been tested in vitro with natural killer (NK) cells from healthy donors. Because it is still unclear whether these optimized cytotoxic mAbs will improve NK-cell killing of tumor cells in CLL patients, we characterized the relevant phenotypic and functional features of NK cells from CLL patients in detail. Expression of inhibitory and activating NK-cell receptors and of Fc gamma receptor IIIA (FcγRIIIA) is well preserved in CD16(+)CD56(dim) cytotoxic NK cells from these patients, independently of disease progression. These cells are fully functional following cytokine stimulation. In addition, the FcγRIIIA-optimized LFB-R603 anti-CD20 mAb mediates 100 times greater antibody-dependent cell-mediated cytotoxicity by NK cells from CLL patients and healthy donors than rituximab. Enhanced degranulation against autologous B-CLL cells is observed at lower concentrations of LFB-R603 than rituximab, regardless of CLL prognostic factors. These findings strongly justify further clinical development of anti-CD20 mAbs optimized for FcγR engagement in CLL patients.