CytoBinning: Immunological insights from multi-dimensional data

New cytometric techniques continue to push the boundaries of multi-parameter quantitative data acquisition at the single-cell level particularly in immunology and medicine. Sophisticated analysis methods for such ever higher dimensional datasets are rapidly emerging, with advanced data representations and dimensional reduction approaches. However, these are not yet standardized and clinical scientists and cell biologists are not yet experienced in their interpretation. More fundamentally their range of statistical validity is not yet fully established. We therefore propose a new method for the automated and unbiased analysis of high-dimensional single cell datasets that is simple and robust, with the goal of reducing this complex information into a familiar 2D scatter plot representation that is of immediate utility to a range of biomedical and clinical settings. Using publicly available flow cytometry and mass cytometry datasets we demonstrate that this method (termed CytoBinning), recapitulates the results of traditional manual cytometric analyses and leads to new and testable hypotheses

Three-dimensional geometry controls division symmetry in stem cell colonies

Proper control of division orientation and symmetry, largely determined by spindle positioning, is essential to development and homeostasis. Spindle positioning has been extensively studied in cells dividing in two-dimensional (2D) environments and in epithelial tissues, where proteins such as NuMA (also known as NUMA1) orient division along the interphase long axis of the cell. However, little is known about how cells control spindle positioning in three-dimensional (3D) environments, such as early mammalian embryos and a variety of adult tissues. Here, we use mouse embryonic stem cells (ESCs), which grow in 3D colonies, as a model to investigate division in 3D. We observe that, at the periphery of 3D colonies, ESCs display high spindle mobility and divide asymmetrically. Our data suggest that enhanced spindle movements are due to unequal distribution of the cell–cell junction protein E-cadherin between future daughter cells. Interestingly, when cells progress towards differentiation, division becomes more symmetric, with more elongated shapes in metaphase and enhanced cortical NuMA recruitment in anaphase. Altogether, this study suggests that in 3D contexts, the geometry of the cell and its contacts with neighbors control division orientation and symmetry

Response of an artificially blown clarinet to different blowing pressure profiles

Using an artificial mouth with an accurate pressure control, the onset of the pressure oscillations inside the mouthpiece of a simplified clarinet is studied experimentally. Two time profiles are used for the blowing pressure: in a first set of experiments the pressure is increased at constant rates, then decreased at the same rate. In a second set of experiments the pressure rises at a constant rate and is then kept constant for an arbitrary period of time. In both cases the experiments are repeated for different increase rates. Numerical simulations using a simplified clarinet model blown with a constantly increasing mouth pressure are compared to the oscillating pressure obtained inside the mouthpiece. Both show that the beginning of the oscillations appears at a higher pressure values than the theoretical static threshold pressure, a manifestation of bifurcation delay. Experiments performed using an interrupted increase in mouth pressure show that the beginning of the oscillation occurs close to the stop in the increase of the pressure. Experimental results also highlight that the speed of the onset transient of the sound is roughly the same, independently of the duration of the increase phase of the blowing pressure.Comment: 14 page

Three-dimensional geometry controls division symmetry in stem cell colonies.

Proper control of division orientation and symmetry, largely determined by spindle positioning, is essential to development and homeostasis. Spindle positioning has been extensively studied in cells dividing in two-dimensional (2D) environments and in epithelial tissues, where proteins such as NuMA (also known as NUMA1) orient division along the interphase long axis of the cell. However, little is known about how cells control spindle positioning in three-dimensional (3D) environments, such as early mammalian embryos and a variety of adult tissues. Here, we use mouse embryonic stem cells (ESCs), which grow in 3D colonies, as a model to investigate division in 3D. We observe that, at the periphery of 3D colonies, ESCs display high spindle mobility and divide asymmetrically. Our data suggest that enhanced spindle movements are due to unequal distribution of the cell-cell junction protein E-cadherin between future daughter cells. Interestingly, when cells progress towards differentiation, division becomes more symmetric, with more elongated shapes in metaphase and enhanced cortical NuMA recruitment in anaphase. Altogether, this study suggests that in 3D contexts, the geometry of the cell and its contacts with neighbors control division orientation and symmetry. This article has an associated First Person interview with the first author of the paper

Abscission couples cell division to embryonic stem cell fate

Cell fate transitions are key to development and homeostasis. It is thus essential to understand the cellular mechanisms controlling fate transitions. Cell division has been implicated in fate decisions in many stem cell types, including neuronal and epithelial progenitors. In other stem cells, such as embryonic stem (ES) cells, the role of division remains unclear. Here, we show that exit from naive pluripotency in mouse ES cells generally occurs after a division. We further show that exit timing is strongly correlated between sister cells, which remain connected by cytoplasmic bridges long after division, and that bridge abscission progressively accelerates as cells exit naive pluripotency. Finally, interfering with abscission impairs naive pluripotency exit, and artificially inducing abscission accelerates it. Altogether, our data indicate that a switch in the division machinery leading to faster abscission regulates pluripotency exit. Our study identifies abscission as a key cellular process coupling cell division to fate transitions

Intuitive control of rolling sound synthesis

International audienceThis paper presents a rolling sound synthesis model which can be intuitively controlled. To propose this model, different aspects of the rolling phenomenon are explored : physical modeling, perceptual attributes and signal morphology. A source-filter model for rolling sounds synthesis is presented with associated intuitive controls

Fluorescence imaging through dynamic scattering media with speckle-encoded ultrasound-modulated light correlation

Fluorescence imaging is indispensable to biomedical research, and yet it remains challenging to image through dynamic scattering samples. Techniques that combine ultrasound and light as exemplified by ultrasound-assisted wavefront shaping have enabled fluorescence imaging through scattering media. However, the translation of these techniques into in vivo applications has been hindered by the lack of high-speed solutions to counter the fast speckle decorrelation of dynamic tissue. Here, we report an ultrasound-enabled optical imaging method that instead leverages the dynamic nature to perform imaging. The method utilizes the correlation between the dynamic speckle-encoded fluorescence and ultrasound-modulated light signal that originate from the same location within a sample. We image fluorescent targets with an improved resolution of ≤75 µm (versus a resolution of 1.3 mm with direct optical imaging) within a scattering medium with 17 ms decorrelation time. This new imaging modality paves the way for fluorescence imaging in highly scattering tissue in vivo

The Necrotic Signal Induced by Mycophenolic Acid Overcomes Apoptosis-Resistance in Tumor Cells

The amount of inosine monophosphate dehydrogenase (IMPDH), a pivotal enzyme for the biosynthesis of the guanosine tri-phosphate (GTP), is frequently increased in tumor cells. The anti-viral agent ribavirin and the immunosuppressant mycophenolic acid (MPA) are potent inhibitors of IMPDH. We recently showed that IMPDH inhibition led to a necrotic signal requiring the activation of Cdc42.Herein, we strengthened the essential role played by this small GTPase in the necrotic signal by silencing Cdc42 and by the ectopic expression of a constitutive active mutant of Cdc42. Since resistance to apoptosis is an essential step for the tumorigenesis process, we next examined the effect of the MPA–mediated necrotic signal on different tumor cells demonstrating various mechanisms of resistance to apoptosis (Bcl2-, HSP70-, Lyn-, BCR-ABL–overexpressing cells). All tested cells remained sensitive to MPA–mediated necrotic signal. Furthermore, inhibition of IMPDH activity in Chronic Lymphocytic Leukemia cells was significantly more efficient at eliminating malignant cells than apoptotic inducers.These findings indicate that necrosis and apoptosis are split signals that share few if any common hub of signaling. In addition, the necrotic signaling pathway induced by depletion of the cellular amount of GTP/GDP would be of great interest to eliminate apoptotic-resistant tumor cells

Time-reversed adapted-perturbation (TRAP) optical focusing onto dynamic objects inside scattering media

The ability to steer and focus light inside scattering media has long been sought for a multitude of applications. At present, the only feasible strategy to form optical foci inside scattering media is to guide photons by using either implanted or virtual guide stars, which can be inconvenient and limits the potential applications. Here we report a scheme for focusing light inside scattering media by employing intrinsic dynamics as guide stars. By adaptively time-reversing the perturbed component of the scattered light, we show that it is possible to focus light to the origin of the perturbation. Using this approach, we demonstrate non-invasive dynamic light focusing onto moving targets and imaging of a time-variant object obscured by highly scattering media. Anticipated applications include imaging and photoablation of angiogenic vessels in tumours, as well as other biomedical uses