NO2 jet cooled visible excitation spectrum - Vibronic chaos induced by the X2A1-A2B2 interaction

International audienceSignificant improvements have been obtained on measurements of the NO2 jet cooled excitation spectrum in the 16 300-18 502 cm-1 range, previously obtained by Smalley et al. [J. Chem. Phys. 63,4977 ( 1975), Persch et al. [ Ber. Bunsenges. Phys. Chem. 92,312 ( 1988), and Hiraoka et al. [J. Mol. Spectrosc. 126,427 ( 1987). The improvements concern first the rotational analysis, owing to a better resolution ( 150 MHz) and absolute precision ( 500 MHz), and second the completeness and purity of the resulting vibronic sequence, owing to a better sensitivity. As a result, 159 vibronic energy levels have been observed in the 16 500-18 500 cm-1 energy range, where 210 ± 10 are expected. A detailed comparison with previous results is presented. The statistical analysis of the corresponding energy spacings shows that long range correlations up to 50 mean levels spacings are present, confirming the chaotic behavior of this set of vibronic levels. Furthermore, we analyze the observed rovibronic interactions (or rotational perturbations) that are responsible for the very irregular rotational behavior of the visible absorption spectrum of NO2 at room temperature

Multi-confocal fluorescence correlation spectroscopy in living cells using a complementary metal oxide semiconductor-single photon avalanche diode array

International audienceLiving cells are heterogeneous and rapidly changing biological samples. It is thus desirable to measure molecular concentration and dynamics in many locations at the same time. In this note, we present a multi-confocal setup capable of performing simultaneous fluorescence correlation spectroscopy measurements, by focusing the spots with a spatial light modulator and acquiring data with a monolithic 32 × 32 single-photon avalanche photodiode array. A post-processing method is proposed to correct cross-talk effects between neighboring spots. We demonstrate the applicability of our system by simultaneously measuring the diffusion of free enhanced Green Fluorescent Protein (eGFP) molecules at nine different points in living cells

Utilisation de la couleur pour l'appariement et l'indexation d'images.

De nombreux travaux récents ont montré que l'utilisation directe du signal des images en niveaux de gris permet de développer des systèmes puissants d'appariement, d'indexation et de reconnaissance d'images. Le présent document explore l'utilisation d'images en couleur pour ces mêmes objectifs. Les principaux points en sont le choix du modèle de variation de la couleur, l'utilisation d'invariants colorimétriques, et la normalisation des images

Capturing and Quantifying Particle Transcytosis with Microphysiological Intestine-on-Chip Models

Understanding the intestinal transport of particles is critical in several fields ranging from optimizing drug delivery systems to capturing health risks from the increased presence of nano- and micro-sized particles in human environment. While Caco-2 cell monolayers grown on permeable supports are the traditional in vitro model used to probe intestinal absorption of dis-solved molecules, they fail to recapitulate the transcytotic activity of polar-ized enterocytes. Here, an intestine-on-chip model is combined with in silico modeling to demonstrate that the rate of particle transcytosis is ≈350× higher across Caco-2 cell monolayers exposed to fluid shear stress compared to Caco-2 cells in standard “static” configuration. This relates to profound phe-notypical alterations and highly polarized state of cells grown under mechan-ical stimulation and it is shown that transcytosis in the microphysiological model is energy-dependent and involves both clathrin and macropinocytosis mediated endocytic pathways. Finally, it is demonstrated that the increased rate of transcytosis through cells exposed to flow is explained by a higher rate of internal particle transport (i.e., vesicular cellular trafficking and baso-lateral exocytosis), rather than a change in apical uptake (i.e., binding and endocytosis). Taken together, the findings have important implications for addressing research questions concerning intestinal transport of engineered and environmental particles.Ludivine C. Delon, Matthew Faria, Zhengyang Jia, Stuart Johnston, Rachel Gibson, Clive A. Prestidge, and Benjamin Thierr

Vascular Smooth Muscle Cell Stiffness and Adhesion to Collagen I Modified by Vasoactive Agonists

In vascular smooth muscle cells (VSMCs) integrin-mediated adhesion to extracellular matrix (ECM) proteins play important roles in sustaining vascular tone and resistance. The main goal of this study was to determine whether VSMCs adhesion to type I collagen (COL-I) was altered in parallel with the changes in the VSMCs contractile state induced by vasoconstrictors and vasodilators. VSMCs were isolated from rat cremaster skeletal muscle arterioles and maintained in primary culture without passage. Cell adhesion and cell E-modulus were assessed using atomic force microscopy (AFM) by repetitive nano-indentation of the AFM probe on the cell surface at 0.1 Hz sampling frequency and 3200 nm Z-piezo travelling distance (approach and retraction). AFM probes were tipped with a 5 μm diameter microbead functionalized with COL-I (1mg\ml). Results showed that the vasoconstrictor angiotensin II (ANG-II; 10−6 ) significantly increased (p<0.05) VSMC E-modulus and adhesion probability to COL-I by approximately 35% and 33%, respectively. In contrast, the vasodilator adenosine (ADO; 10−4 ) significantly decreased (p<0.05) VSMC E-modulus and adhesion probability by approximately −33% and −17%, respectively. Similarly, the NO donor (PANOate, 10−6 M), a potent vasodilator, also significantly decreased (p<0.05) the VSMC E-modulus and COL-I adhesion probability by −38% and −35%, respectively. These observations support the hypothesis that integrin-mediated VSMC adhesion to the ECM protein COL-I is dynamically regulated in parallel with VSMC contractile activation. These data suggest that the signal transduction pathways modulating VSMC contractile activation and relaxation, in addition to ECM adhesion, interact during regulation of contractile state

Pathogenic Mouse Hepatitis Virus or Poly(I:C) Induce IL-33 in Hepatocytes in Murine Models of Hepatitis.

International audienceThe IL-33/ST2 axis is known to be involved in liver pathologies. Although, the IL-33 levels increased in sera of viral hepatitis patients in human, the cellular sources of IL-33 in viral hepatitis remained obscure. Therefore, we aimed to investigate the expression of IL-33 in murine fulminant hepatitis induced by a Toll like receptor (TLR3) viral mimetic, poly(I:C) or by pathogenic mouse hepatitis virus (L2-MHV3). The administration of poly(I:C) plus D-galactosamine (D-GalN) in mice led to acute liver injury associated with the induction of IL-33 expression in liver sinusoidal endothelial cells (LSEC) and vascular endothelial cells (VEC), while the administration of poly(I:C) alone led to hepatocyte specific IL-33 expression in addition to vascular IL-33 expression. The hepatocyte-specific IL-33 expression was down-regulated in NK-depleted poly(I:C) treated mice suggesting a partial regulation of IL-33 by NK cells. The CD1d KO (NKT deficient) mice showed hepatoprotection against poly(I:C)-induced hepatitis in association with increased number of IL-33 expressing hepatocytes in CD1d KO mice than WT controls. These results suggest that hepatocyte-specific IL-33 expression in poly(I:C) induced liver injury was partially dependent of NK cells and with limited role of NKT cells. In parallel, the L2-MHV3 infection in mice induced fulminant hepatitis associated with up-regulated IL-33 expression as well as pro-inflammatory cytokine microenvironment in liver. The LSEC and VEC expressed inducible expression of IL-33 following L2-MHV3 infection but the hepatocyte-specific IL-33 expression was only evident between 24 to 32h of post infection. In conclusion, the alarmin cytokine IL-33 was over-expressed during fulminant hepatitis in mice with LSEC, VEC and hepatocytes as potential sources of IL-33

Bayesian Model Selection Applied to the Analysis of Fluorescence Correlation Spectroscopy Data of Fluorescent Proteins in Vitro and in Vivo

Fluorescence correlation spectroscopy (FCS) is a powerful technique to investigate molecular dynamics with single molecule sensitivity. In particular, in the life sciences it has found widespread application using fluorescent proteins as molecularly specific labels. However, FCS data analysis and interpretation using fluorescent proteins remains challenging due to typically low signal-to-noise ratio of FCS data and correlated noise in autocorrelated data sets. As a result, naive fitting procedures that ignore these important issues typically provide similarly good fits for multiple competing models without clear distinction of which model is preferred given the signal-to-noise ratio present in the data. Recently, we introduced a Bayesian model selection procedure to overcome this issue with FCS data analysis. The method accounts for the highly correlated noise that is present in FCS data sets and additionally penalizes model complexity to prevent over interpretation of FCS data. Here, we apply this procedure to evaluate FCS data from fluorescent proteins assayed in vitro and in vivo. Consistent with previous work, we demonstrate that model selection is strongly dependent on the signal-to-noise ratio of the measurement, namely, excitation intensity and measurement time, and is sensitive to saturation artifacts. Under fixed, low intensity excitation conditions, physical transport models can unambiguously be identified. However, at excitation intensities that are considered moderate in many studies, unwanted artifacts are introduced that result in nonphysical models to be preferred. We also determined the appropriate fitting models of a GFP tagged secreted signaling protein, Wnt3, in live zebrafish embryos, which is necessary for the investigation of Wnt3 expression and secretion in development. Bayes model selection therefore provides a robust procedure to determine appropriate transport and photophysical models for fluorescent proteins when appropriate models are provided, to help detect and eliminate experimental artifacts in solution, cells, and in living organisms.National Science Foundation (U.S.). Physics of Living Systems ProgramNational Institute of Mental Health (U.S.) (Award U01MH106011

Spinal muscular atrophy diagnosis and carrier screening from genome sequencing data.

PURPOSE: Spinal muscular atrophy (SMA), caused by loss of the SMN1 gene, is a leading cause of early childhood death. Due to the near identical sequences of SMN1 and SMN2, analysis of this region is challenging. Population-wide SMA screening to quantify the SMN1 copy number (CN) is recommended by the American College of Medical Genetics and Genomics. METHODS: We developed a method that accurately identifies the CN of SMN1 and SMN2 using genome sequencing (GS) data by analyzing read depth and eight informative reference genome differences between SMN1/2. RESULTS: We characterized SMN1/2 in 12,747 genomes, identified 1568 samples with SMN1 gains or losses and 6615 samples with SMN2 gains or losses, and calculated a pan-ethnic carrier frequency of 2%, consistent with previous studies. Additionally, 99.8% of our SMN1 and 99.7% of SMN2 CN calls agreed with orthogonal methods, with a recall of 100% for SMA and 97.8% for carriers, and a precision of 100% for both SMA and carriers. CONCLUSION: This SMN copy-number caller can be used to identify both carrier and affected status of SMA, enabling SMA testing to be offered as a comprehensive test in neonatal care and an accurate carrier screening tool in GS sequencing projects

Barcoding T Cell Calcium Response Diversity with Methods for Automated and Accurate Analysis of Cell Signals (MAAACS)

International audienceWe introduce a series of experimental procedures enabling sensitive calcium monitoring in T cell populations by confocal video-microscopy. Tracking and post-acquisition analysis was performed using Methods for Automated and Accurate Analysis of Cell Signals (MAAACS), a fully customized program that associates a high throughput tracking algorithm, an intuitive reconnection routine and a statistical platform to provide, at a glance, the calcium barcode of a population of individual T-cells. Combined with a sensitive calcium probe, this method allowed us to unravel the heterogeneity in shape and intensity of the calcium response in T cell populations and especially in naive T cells, which display intracellular calcium oscillations upon stimulation by antigen presenting cells