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Sample records for quantitative cell dynamic

  1. A quantitative and dynamic model for plant stem cell regulation.

    Directory of Open Access Journals (Sweden)

    Florian Geier

    Full Text Available Plants maintain pools of totipotent stem cells throughout their entire life. These stem cells are embedded within specialized tissues called meristems, which form the growing points of the organism. The shoot apical meristem of the reference plant Arabidopsis thaliana is subdivided into several distinct domains, which execute diverse biological functions, such as tissue organization, cell-proliferation and differentiation. The number of cells required for growth and organ formation changes over the course of a plants life, while the structure of the meristem remains remarkably constant. Thus, regulatory systems must be in place, which allow for an adaptation of cell proliferation within the shoot apical meristem, while maintaining the organization at the tissue level. To advance our understanding of this dynamic tissue behavior, we measured domain sizes as well as cell division rates of the shoot apical meristem under various environmental conditions, which cause adaptations in meristem size. Based on our results we developed a mathematical model to explain the observed changes by a cell pool size dependent regulation of cell proliferation and differentiation, which is able to correctly predict CLV3 and WUS over-expression phenotypes. While the model shows stem cell homeostasis under constant growth conditions, it predicts a variation in stem cell number under changing conditions. Consistent with our experimental data this behavior is correlated with variations in cell proliferation. Therefore, we investigate different signaling mechanisms, which could stabilize stem cell number despite variations in cell proliferation. Our results shed light onto the dynamic constraints of stem cell pool maintenance in the shoot apical meristem of Arabidopsis in different environmental conditions and developmental states.

  2. Dynamic quantitative microscopy and nanoscopy of red blood cells in sickle cell disease

    Science.gov (United States)

    Shaked, Natan T.; Satterwhite, Lisa L.; Telen, Marilyn J.; Truskey, George A.; Wax, Adam

    2012-03-01

    We have applied wide-field digital interferometric techniques to quantitatively image sickle red blood cells (RBCs) [1] in a noncontact label-free manner, and measure the nanometer-scale fluctuations in their thickness as an indication of their stiffness. The technique can simultaneously measure the fluctuations for multiple spatial points on the RBC and thus yields a map describing the stiffness of each RBC in the field of view. Using this map, the local rigidity regions of the RBC are evaluated quantitatively. Since wide-field digital interferometry is a quantitative holographic imaging technique rather than one-point measurement, it can be used to simultaneously evaluate cell transverse morphology plus thickness in addition to its stiffness profile. Using this technique, we examine the morphology and dynamics of RBCs from individuals who suffer from sickle cell disease, and find that the sickle RBCs are significantly stiffer than healthy RBCs. Furthermore, we show that the technique is sensitive enough to distinguish various classes of sickle RBCs, including sickle RBCs with visibly-normal morphology, compared to the stiffer crescent-shaped sickle RBCs.

  3. Measuring dynamic membrane fluctuations in cell membrane using quantitative phase imaging (Conference Presentation)

    Science.gov (United States)

    Lee, SangYun; Kim, Kyoohyun; Park, YongKeun

    2017-02-01

    There is a strong correlation between the dynamic membrane fluctuations and the biomechanical properties of living cells. The dynamic membrane fluctuation consists of submicron displacements, and can be altered by changing the cells' pathophysiological conditions. These results have significant relevance to the understanding of RBC biophysics and pathology, as follows. RBCs must withstand large mechanical deformations during repeated passages through the microvasculature and the fenestrated walls of the splenic sinusoids. This essential ability is diminished with senescence, resulting in physiological destruction of the aging RBCs. Pathological destruction of the red cells, however, occurs in cells affected by a host of diseases such as spherocytosis, malaria, and Sickle cell disease, as RBCs depart from their normal discoid shape and lose their deformability. Therefore, quantifying the RBC deformability insight into a variety of problems regarding the interplay of cell structure, dynamics, and function. Furthermore, the ability to monitor mechanical properties of RBCs is of vital interest in monitoring disease progression or response to treatment as molecular and pharmaceutical approaches for treatment of chronic diseases. Here, we present the measurements of dynamic membrane fluctuations in live cells using quantitative phase imaging techniques. Measuring both the 3-D refractive index maps and the dynamic phase images of live cells are simultaneously measured, from which dynamic membrane fluctuation and deformability of cells are precisely calculated. We also present its applications to various diseases ranging from sickle cell diseases, babesiosis, and to diabetes.

  4. Quantitative imaging with Fucci and mathematics to uncover temporal dynamics of cell cycle progression.

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    Saitou, Takashi; Imamura, Takeshi

    2016-01-01

    Cell cycle progression is strictly coordinated to ensure proper tissue growth, development, and regeneration of multicellular organisms. Spatiotemporal visualization of cell cycle phases directly helps us to obtain a deeper understanding of controlled, multicellular, cell cycle progression. The fluorescent ubiquitination-based cell cycle indicator (Fucci) system allows us to monitor, in living cells, the G1 and the S/G2/M phases of the cell cycle in red and green fluorescent colors, respectively. Since the discovery of Fucci technology, it has found numerous applications in the characterization of the timing of cell cycle phase transitions under diverse conditions and various biological processes. However, due to the complexity of cell cycle dynamics, understanding of specific patterns of cell cycle progression is still far from complete. In order to tackle this issue, quantitative approaches combined with mathematical modeling seem to be essential. Here, we review several studies that attempted to integrate Fucci technology and mathematical models to obtain quantitative information regarding cell cycle regulatory patterns. Focusing on the technological development of utilizing mathematics to retrieve meaningful information from the Fucci producing data, we discuss how the combined methods advance a quantitative understanding of cell cycle regulation.

  5. Dynamics of Natural Killer Cell Receptor Revealed by Quantitative Analysis of Photoswitchable Protein

    Science.gov (United States)

    Pageon, Sophie V.; Aquino, Gerardo; Lagrue, Kathryn; Köhler, Karsten; Endres, Robert G.; Davis, Daniel M.

    2013-11-01

    Natural Killer (NK) cell activation is dynamically regulated by numerous activating and inhibitory surface receptors that accumulate at the immune synapse. Quantitative analysis of receptor dynamics has been limited by methodologies which rely on indirect measurements such as fluorescence recovery after photobleaching. Here, we report a novel approach to study how proteins traffic to and from the immune synapse using NK cell receptors tagged with the photoswitchable fluorescent protein tdEosFP, which can be irreversibly photoswitched from a green to red fluorescent state by ultraviolet light. Thus, following a localized switching event, the movement of the photoswitched molecules can be temporally and spatially resolved by monitoring fluorescence in two regions of interest. By comparing images with mathematical models, we evaluated the diffusion coefficient of the receptor KIR2DL1 (0.23 +- 0.06 micron^2/s) and assessed how synapse formation affects receptor dynamics. Our data conclude that the inhibitory NK cell receptor KIR2DL1 is continually trafficked into the synapse and remains surprisingly stable there. Unexpectedly however, in NK cells forming synapses with multiple target cells simultaneously, KIR2DL1 at one synapse can relocate to another synapse. Thus, our results reveal a previously undetected inter-synaptic exchange of protein.

  6. Quantitative single cell analysis of cell population dynamics during submandibular salivary gland development and differentiation

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    Deirdre A. Nelson

    2013-04-01

    Epithelial organ morphogenesis involves reciprocal interactions between epithelial and mesenchymal cell types to balance progenitor cell retention and expansion with cell differentiation for evolution of tissue architecture. Underlying submandibular salivary gland branching morphogenesis is the regulated proliferation and differentiation of perhaps several progenitor cell populations, which have not been characterized throughout development, and yet are critical for understanding organ development, regeneration, and disease. Here we applied a serial multiplexed fluorescent immunohistochemistry technology to map the progressive refinement of the epithelial and mesenchymal cell populations throughout development from embryonic day 14 through postnatal day 20. Using computational single cell analysis methods, we simultaneously mapped the evolving temporal and spatial location of epithelial cells expressing subsets of differentiation and progenitor markers throughout salivary gland development. We mapped epithelial cell differentiation markers, including aquaporin 5, PSP, SABPA, and mucin 10 (acinar cells; cytokeratin 7 (ductal cells; and smooth muscle α-actin (myoepithelial cells and epithelial progenitor cell markers, cytokeratin 5 and c-kit. We used pairwise correlation and visual mapping of the cells in multiplexed images to quantify the number of single- and double-positive cells expressing these differentiation and progenitor markers at each developmental stage. We identified smooth muscle α-actin as a putative early myoepithelial progenitor marker that is expressed in cytokeratin 5-negative cells. Additionally, our results reveal dynamic expansion and redistributions of c-kit- and K5-positive progenitor cell populations throughout development and in postnatal glands. The data suggest that there are temporally and spatially discreet progenitor populations that contribute to salivary gland development and homeostasis.

  7. Compact, common path quantitative phase microscopic techniques for imaging cell dynamics

    Indian Academy of Sciences (India)

    A Anand; P Vora; S Mahajan; V Trivedi; V Chhaniwal; A Singh; R Leitgeb; B Javidi

    2014-01-01

    Microscopy using visible electromagnetic radiation can be used to investigate living cells in various environments. But bright field microscopy only provides two-dimensional (2D) intensity distribution at a single object plane. One of the ways to retrieve object height/thickness information is to employ quantitative phase microscopic (QPM) techniques. Interferometric QPM techniques are widely used for this. Digital holographic microscopy (DHM) is one of the stateof-the-art methods for quantitative three-dimensional (3D) imaging. Usually it is implemented in two-beam geometry, which is prone to mechanical vibrations. But to study dynamics of objects like red blood cells, one needs temporal stability much better than the fluctuations of the object, which the two-beam geometry fails to deliver. One way to overcome this hurdle is to use selfreferencing techniques, in which a portion of the object beam will act as the reference beam. Here the development of self-referencing QPM techniques is described along with the results.

  8. Quantitative analysis of organelle distribution and dynamics in Physcomitrella patens protonemal cells

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    Furt Fabienne

    2012-05-01

    Full Text Available Abstract Background In the last decade, the moss Physcomitrella patens has emerged as a powerful plant model system, amenable for genetic manipulations not possible in any other plant. This moss is particularly well suited for plant polarized cell growth studies, as in its protonemal phase, expansion is restricted to the tip of its cells. Based on pollen tube and root hair studies, it is well known that tip growth requires active secretion and high polarization of the cellular components. However, such information is still missing in Physcomitrella patens. To gain insight into the mechanisms underlying the participation of organelle organization in tip growth, it is essential to determine the distribution and the dynamics of the organelles in moss cells. Results We used fluorescent protein fusions to visualize and track Golgi dictyosomes, mitochondria, and peroxisomes in live protonemal cells. We also visualized and tracked chloroplasts based on chlorophyll auto-fluorescence. We showed that in protonemata all four organelles are distributed in a gradient from the tip of the apical cell to the base of the sub-apical cell. For example, the density of Golgi dictyosomes is 4.7 and 3.4 times higher at the tip than at the base in caulonemata and chloronemata respectively. While Golgi stacks are concentrated at the extreme tip of the caulonemata, chloroplasts and peroxisomes are totally excluded. Interestingly, caulonemata, which grow faster than chloronemata, also contain significantly more Golgi dictyosomes and fewer chloroplasts than chloronemata. Moreover, the motility analysis revealed that organelles in protonemata move with low persistency and average instantaneous speeds ranging from 29 to 75 nm/s, which are at least three orders of magnitude slower than those of pollen tube or root hair organelles. Conclusions To our knowledge, this study reports the first quantitative analysis of organelles in Physcomitrella patens and will make possible

  9. Quantitative Live Imaging of Human Embryonic Stem Cell Derived Neural Rosettes Reveals Structure-Function Dynamics Coupled to Cortical Development.

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    Omer Ziv

    2015-10-01

    Full Text Available Neural stem cells (NSCs are progenitor cells for brain development, where cellular spatial composition (cytoarchitecture and dynamics are hypothesized to be linked to critical NSC capabilities. However, understanding cytoarchitectural dynamics of this process has been limited by the difficulty to quantitatively image brain development in vivo. Here, we study NSC dynamics within Neural Rosettes--highly organized multicellular structures derived from human pluripotent stem cells. Neural rosettes contain NSCs with strong epithelial polarity and are expected to perform apical-basal interkinetic nuclear migration (INM--a hallmark of cortical radial glial cell development. We developed a quantitative live imaging framework to characterize INM dynamics within rosettes. We first show that the tendency of cells to follow the INM orientation--a phenomenon we referred to as radial organization, is associated with rosette size, presumably via mechanical constraints of the confining structure. Second, early forming rosettes, which are abundant with founder NSCs and correspond to the early proliferative developing cortex, show fast motions and enhanced radial organization. In contrast, later derived rosettes, which are characterized by reduced NSC capacity and elevated numbers of differentiated neurons, and thus correspond to neurogenesis mode in the developing cortex, exhibit slower motions and decreased radial organization. Third, later derived rosettes are characterized by temporal instability in INM measures, in agreement with progressive loss in rosette integrity at later developmental stages. Finally, molecular perturbations of INM by inhibition of actin or non-muscle myosin-II (NMII reduced INM measures. Our framework enables quantification of cytoarchitecture NSC dynamics and may have implications in functional molecular studies, drug screening, and iPS cell-based platforms for disease modeling.

  10. Quantitative Live Imaging of Human Embryonic Stem Cell Derived Neural Rosettes Reveals Structure-Function Dynamics Coupled to Cortical Development.

    Science.gov (United States)

    Ziv, Omer; Zaritsky, Assaf; Yaffe, Yakey; Mutukula, Naresh; Edri, Reuven; Elkabetz, Yechiel

    2015-10-01

    Neural stem cells (NSCs) are progenitor cells for brain development, where cellular spatial composition (cytoarchitecture) and dynamics are hypothesized to be linked to critical NSC capabilities. However, understanding cytoarchitectural dynamics of this process has been limited by the difficulty to quantitatively image brain development in vivo. Here, we study NSC dynamics within Neural Rosettes--highly organized multicellular structures derived from human pluripotent stem cells. Neural rosettes contain NSCs with strong epithelial polarity and are expected to perform apical-basal interkinetic nuclear migration (INM)--a hallmark of cortical radial glial cell development. We developed a quantitative live imaging framework to characterize INM dynamics within rosettes. We first show that the tendency of cells to follow the INM orientation--a phenomenon we referred to as radial organization, is associated with rosette size, presumably via mechanical constraints of the confining structure. Second, early forming rosettes, which are abundant with founder NSCs and correspond to the early proliferative developing cortex, show fast motions and enhanced radial organization. In contrast, later derived rosettes, which are characterized by reduced NSC capacity and elevated numbers of differentiated neurons, and thus correspond to neurogenesis mode in the developing cortex, exhibit slower motions and decreased radial organization. Third, later derived rosettes are characterized by temporal instability in INM measures, in agreement with progressive loss in rosette integrity at later developmental stages. Finally, molecular perturbations of INM by inhibition of actin or non-muscle myosin-II (NMII) reduced INM measures. Our framework enables quantification of cytoarchitecture NSC dynamics and may have implications in functional molecular studies, drug screening, and iPS cell-based platforms for disease modeling.

  11. Heterogeneous Structure of Stem Cells Dynamics: Statistical Models and Quantitative Predictions

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    Bogdan, Paul; Deasy, Bridget M.; Gharaibeh, Burhan; Roehrs, Timo; Marculescu, Radu

    2014-04-01

    Understanding stem cell (SC) population dynamics is essential for developing models that can be used in basic science and medicine, to aid in predicting cells fate. These models can be used as tools e.g. in studying patho-physiological events at the cellular and tissue level, predicting (mal)functions along the developmental course, and personalized regenerative medicine. Using time-lapsed imaging and statistical tools, we show that the dynamics of SC populations involve a heterogeneous structure consisting of multiple sub-population behaviors. Using non-Gaussian statistical approaches, we identify the co-existence of fast and slow dividing subpopulations, and quiescent cells, in stem cells from three species. The mathematical analysis also shows that, instead of developing independently, SCs exhibit a time-dependent fractal behavior as they interact with each other through molecular and tactile signals. These findings suggest that more sophisticated models of SC dynamics should view SC populations as a collective and avoid the simplifying homogeneity assumption by accounting for the presence of more than one dividing sub-population, and their multi-fractal characteristics.

  12. Workshop on quantitative dynamic stratigraphy

    Energy Technology Data Exchange (ETDEWEB)

    Cross, T.A.

    1988-04-01

    This document discusses the development of quantitative simulation models for the investigation of geologic systems. The selection of variables, model verification, evaluation, and future directions in quantitative dynamic stratigraphy (QDS) models are detailed. Interdisciplinary applications, integration, implementation, and transfer of QDS are also discussed. (FI)

  13. Quantitative analysis of mechanisms that govern red blood cell age structure and dynamics during anaemia.

    Directory of Open Access Journals (Sweden)

    Nicholas J Savill

    2009-06-01

    Full Text Available Mathematical modelling has proven an important tool in elucidating and quantifying mechanisms that govern the age structure and population dynamics of red blood cells (RBCs. Here we synthesise ideas from previous experimental data and the mathematical modelling literature with new data in order to test hypotheses and generate new predictions about these mechanisms. The result is a set of competing hypotheses about three intrinsic mechanisms: the feedback from circulating RBC concentration to production rate of immature RBCs (reticulocytes in bone marrow, the release of reticulocytes from bone marrow into the circulation, and their subsequent ageing and clearance. In addition we examine two mechanisms specific to our experimental system: the effect of phenylhydrazine (PHZ and blood sampling on RBC dynamics. We performed a set of experiments to quantify the dynamics of reticulocyte proportion, RBC concentration, and erythropoietin concentration in PHZ-induced anaemic mice. By quantifying experimental error we are able to fit and assess each hypothesis against our data and recover parameter estimates using Markov chain Monte Carlo based Bayesian inference. We find that, under normal conditions, about 3% of reticulocytes are released early from bone marrow and upon maturation all cells are released immediately. In the circulation, RBCs undergo random clearance but have a maximum lifespan of about 50 days. Under anaemic conditions reticulocyte production rate is linearly correlated with the difference between normal and anaemic RBC concentrations, and their release rate is exponentially correlated with the same. PHZ appears to age rather than kill RBCs, and younger RBCs are affected more than older RBCs. Blood sampling caused short aperiodic spikes in the proportion of reticulocytes which appear to have a different developmental pathway than normal reticulocytes. We also provide evidence of large diurnal oscillations in serum erythropoietin levels

  14. Dynamics of single human embryonic stem cells and their pairs: a quantitative analysis

    CERN Document Server

    Wadkin, L E; Neganova, I; Parker, N G; Chichagova, V; Swan, G; Laude, A; Lako, M; Shukurov, A

    2016-01-01

    Numerous biological approaches are available to characterise the mechanisms which govern the formation of human embryonic stem cell (hESC) colonies. To understand how the kinematics of single and pairs of hESCs impact colony formation, we study their mobility characteristics using time-lapse imaging. We perform a detailed statistical analysis of their speed, survival, directionality, distance travelled and diffusivity. We confirm that single and pairs of cells migrate as a diffusive random walk. Moreover, we show that the presence of Cell Tracer significantly reduces hESC mobility. Our results open the path to employ the theoretical framework of the diffusive random walk for the prognostic modelling and optimisation of the growth of hESC colonies. Indeed, we employ this random walk model to estimate the seeding density required to minimise the occurrence of hESC colonies arising from more than one founder cell and the minimal cell number needed for successful colony formation. We believe that our prognostic m...

  15. A high-content image-based method for quantitatively studying context-dependent cell population dynamics.

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    Garvey, Colleen M; Spiller, Erin; Lindsay, Danika; Chiang, Chun-Te; Choi, Nathan C; Agus, David B; Mallick, Parag; Foo, Jasmine; Mumenthaler, Shannon M

    2016-01-01

    Tumor progression results from a complex interplay between cellular heterogeneity, treatment response, microenvironment and heterocellular interactions. Existing approaches to characterize this interplay suffer from an inability to distinguish between multiple cell types, often lack environmental context, and are unable to perform multiplex phenotypic profiling of cell populations. Here we present a high-throughput platform for characterizing, with single-cell resolution, the dynamic phenotypic responses (i.e. morphology changes, proliferation, apoptosis) of heterogeneous cell populations both during standard growth and in response to multiple, co-occurring selective pressures. The speed of this platform enables a thorough investigation of the impacts of diverse selective pressures including genetic alterations, therapeutic interventions, heterocellular components and microenvironmental factors. The platform has been applied to both 2D and 3D culture systems and readily distinguishes between (1) cytotoxic versus cytostatic cellular responses; and (2) changes in morphological features over time and in response to perturbation. These important features can directly influence tumor evolution and clinical outcome. Our image-based approach provides a deeper insight into the cellular dynamics and heterogeneity of tumors (or other complex systems), with reduced reagents and time, offering advantages over traditional biological assays.

  16. Semiautomatic determination of arterial input functions for quantitative dynamic contrast-enhanced magnetic resonance imaging in non-small cell lung cancer patients.

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    Chung, Julius; Kim, Jae-Hun; Lee, Eun Ju; Kim, Yoo Na; Yi, Chin A

    2015-03-01

    The aim of this study was to validate a semiautomatic detection method for the arterial input functions (AIFs) using Kendall coefficient of concordance (KCC) for quantitative analysis of dynamic contrast-enhanced magnetic resonance imaging in non-small cell lung cancer patients. We prospectively enrolled 28 patients (17 men, 11 women; mean age, 62 years) who had biopsy-proven non-small cell lung cancer. All enrolled patients underwent dynamic contrast-enhanced magnetic resonance imaging of the entire thorax. For the quantitative measurement of pharmacokinetic parameters, K and ve, of the lung cancers, AIFs were determined in 2 different ways: a manual method that involved 3 independent thoracic radiologists selecting a region of interest (ROI) within the aortic arch in the 2D coronal plane and a semiautomatic method that used in-house software to establish a KCC score, which provided a measure of similarity to typical AIF pattern. Three independent readers selected voxel clusters with high KCC scores calculated 3-dimensionally across planes in the data set. K and ve were correlated using intraclass correlation coefficients (ICCs), and Bland-Altman plots were used to examine agreement across methods and reproducibility within a method. Arterial input functions were determined using the data from ROI volumes that were significantly larger in the semiautomatic method (mean ± SD, 3360 ± 768 mm) than in the manual method (677 ± 380 mm) (P methods. The reproducibility for K (ICCmanual, 0.813 and ICCsemiautomatic, 0.998; P method than the manual method. We found semiautomated detection using KCC to be a robust method for determining the AIF. This method allows for larger ROIs specified in 3D across planes as opposed to manually selected ROIs restricted to the 2D coronal images seen by the reader and provides increased reproducibility that is comparable with manual specification.

  17. Label-free quantitative cell division monitoring of endothelial cells by digital holographic microscopy

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    Kemper, Björn; Bauwens, Andreas; Vollmer, Angelika; Ketelhut, Steffi; Langehanenberg, Patrik; Müthing, Johannes; Karch, Helge; von Bally, Gert

    2010-05-01

    Digital holographic microscopy (DHM) enables quantitative multifocus phase contrast imaging for nondestructive technical inspection and live cell analysis. Time-lapse investigations on human brain microvascular endothelial cells demonstrate the use of DHM for label-free dynamic quantitative monitoring of cell division of mother cells into daughter cells. Cytokinetic DHM analysis provides future applications in toxicology and cancer research.

  18. Leo Szilard Lectureship Award Talk - Universal Scaling Laws from Cells to Cities; A Physicist's Search for Quantitative, Unified Theories of Biological and Social Structure and Dynamics

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    West, Geoffrey

    2013-04-01

    Many of the most challenging, exciting and profound questions facing science and society, from the origins of life to global sustainability, fall under the banner of ``complex adaptive systems.'' This talk explores how scaling can be used to begin to develop physics-inspired quantitative, predictive, coarse-grained theories for understanding their structure, dynamics and organization based on underlying mathematisable principles. Remarkably, most physiological, organisational and life history phenomena in biology and socio-economic systems scale in a simple and ``universal'' fashion: metabolic rate scales approximately as the 3/4-power of mass over 27 orders of magnitude from complex molecules to the largest organisms. Time-scales (such as lifespans and growth-rates) and sizes (such as genome lengths and RNA densities) scale with exponents which are typically simple multiples of 1/4, suggesting that fundamental constraints underlie much of the generic structure and dynamics of living systems. These scaling laws follow from dynamical and geometrical properties of space-filling, fractal-like, hierarchical branching networks, presumed optimised by natural selection. This leads to a general framework that potentially captures essential features of diverse systems including vasculature, ontogenetic growth, cancer, aging and mortality, sleep, cell size, and DNA nucleotide substitution rates. Cities and companies also scale: wages, profits, patents, crime, disease, pollution, road lengths scale similarly across the globe, reflecting underlying universal social network dynamics which point to general principles of organization transcending their individuality. These have dramatic implications for global sustainability: innovation and wealth creation that fuel social systems, left unchecked, potentially sow the seeds for their inevitable collapse.

  19. Evaluation of phosphopeptide enrichment strategies for quantitative TMT analysis of complex network dynamics in cancer-associated cell signalling

    Directory of Open Access Journals (Sweden)

    Benedetta Lombardi

    2015-03-01

    Full Text Available Defining alterations in signalling pathways in normal and malignant cells is becoming a major field in proteomics. A number of different approaches have been established to isolate, identify and quantify phosphorylated proteins and peptides. In the current report, a comparison between SCX prefractionation versus an antibody based approach, both coupled to TiO2 enrichment and applied to TMT labelled cellular lysates, is described. The antibody strategy was more complete for enriching phosphopeptides and allowed the identification of a large set of proteins known to be phosphorylated (715 protein groups with a minimum number of not previously known phosphorylated proteins (2.

  20. Nonlinear dynamics and quantitative EEG analysis.

    Science.gov (United States)

    Jansen, B H

    1996-01-01

    Quantitative, computerized electroencephalogram (EEG) analysis appears to be based on a phenomenological approach to EEG interpretation, and is primarily rooted in linear systems theory. A fundamentally different approach to computerized EEG analysis, however, is making its way into the laboratories. The basic idea, inspired by recent advances in the area of nonlinear dynamics and chaos theory, is to view an EEG as the output of a deterministic system of relatively simple complexity, but containing nonlinearities. This suggests that studying the geometrical dynamics of EEGs, and the development of neurophysiologically realistic models of EEG generation may produce more successful automated EEG analysis techniques than the classical, stochastic methods. A review of the fundamentals of chaos theory is provided. Evidence supporting the nonlinear dynamics paradigm to EEG interpretation is presented, and the kind of new information that can be extracted from the EEG is discussed. A case is made that a nonlinear dynamic systems viewpoint to EEG generation will profoundly affect the way EEG interpretation is currently done.

  1. Transcription Dynamics in Living Cells.

    Science.gov (United States)

    Lenstra, Tineke L; Rodriguez, Joseph; Chen, Huimin; Larson, Daniel R

    2016-07-01

    The transcription cycle can be roughly divided into three stages: initiation, elongation, and termination. Understanding the molecular events that regulate all these stages requires a dynamic view of the underlying processes. The development of techniques to visualize and quantify transcription in single living cells has been essential in revealing the transcription kinetics. They have revealed that (a) transcription is heterogeneous between cells and (b) transcription can be discontinuous within a cell. In this review, we discuss the progress in our quantitative understanding of transcription dynamics in living cells, focusing on all parts of the transcription cycle. We present the techniques allowing for single-cell transcription measurements, review evidence from different organisms, and discuss how these experiments have broadened our mechanistic understanding of transcription regulation.

  2. Quantitative mass spectrometry of histones H3.2 and H3.3 in Suz12-deficient mouse embryonic stem cells reveals distinct, dynamic post-translational modifications at Lys-27 and Lys-36

    DEFF Research Database (Denmark)

    Jung, Hye Ryung; Pasini, Diego; Helin, Kristian

    2010-01-01

    SUZ12 is a core component of the polycomb repressive complex 2 (PRC2) and is required for the differentiation of mouse embryonic stem cells (ESCs). PRC2 is associated with transcriptional repression via methylation of H3 Lys-27. We applied quantitative mass spectrometry to investigate the effects....... The combined use of ETD and CID MS/MS increased the total number of identified modified peptides. Comparative quantitative analysis of histones from wild type and Suz12-deficient ESCs using stable isotope labeling with amino acids in cell culture and LC-MS/MS revealed a dramatic reduction of H3K27me2 and H3K27...... analysis of the dynamics of coexisting post-translational modifications in proteins....

  3. Workshop on quantitative dynamic stratigraphy. Final conference report

    Energy Technology Data Exchange (ETDEWEB)

    Cross, T.A.

    1988-04-01

    This document discusses the development of quantitative simulation models for the investigation of geologic systems. The selection of variables, model verification, evaluation, and future directions in quantitative dynamic stratigraphy (QDS) models are detailed. Interdisciplinary applications, integration, implementation, and transfer of QDS are also discussed. (FI)

  4. Biological Dynamics Markup Language (BDML): an open format for representing quantitative biological dynamics data.

    Science.gov (United States)

    Kyoda, Koji; Tohsato, Yukako; Ho, Kenneth H L; Onami, Shuichi

    2015-04-01

    Recent progress in live-cell imaging and modeling techniques has resulted in generation of a large amount of quantitative data (from experimental measurements and computer simulations) on spatiotemporal dynamics of biological objects such as molecules, cells and organisms. Although many research groups have independently dedicated their efforts to developing software tools for visualizing and analyzing these data, these tools are often not compatible with each other because of different data formats. We developed an open unified format, Biological Dynamics Markup Language (BDML; current version: 0.2), which provides a basic framework for representing quantitative biological dynamics data for objects ranging from molecules to cells to organisms. BDML is based on Extensible Markup Language (XML). Its advantages are machine and human readability and extensibility. BDML will improve the efficiency of development and evaluation of software tools for data visualization and analysis. A specification and a schema file for BDML are freely available online at http://ssbd.qbic.riken.jp/bdml/. Supplementary data are available at Bioinformatics online. © The Author 2014. Published by Oxford University Press.

  5. Quantitative analysis of ChIP-seq data uncovers dynamic and sustained H3K4me3 and H3K27me3 modulation in cancer cells under hypoxia.

    Science.gov (United States)

    Adriaens, Michiel E; Prickaerts, Peggy; Chan-Seng-Yue, Michelle; van den Beucken, Twan; Dahlmans, Vivian E H; Eijssen, Lars M; Beck, Timothy; Wouters, Bradly G; Voncken, Jan Willem; Evelo, Chris T A

    2016-01-01

    A comprehensive assessment of the epigenetic dynamics in cancer cells is the key to understanding the molecular mechanisms underlying cancer and to improving cancer diagnostics, prognostics and treatment. By combining genome-wide ChIP-seq epigenomics and microarray transcriptomics, we studied the effects of oxygen deprivation and subsequent reoxygenation on histone 3 trimethylation of lysine 4 (H3K4me3) and lysine 27 (H3K27me3) in a breast cancer cell line, serving as a model for abnormal oxygenation in solid tumors. A priori, epigenetic markings and gene expression levels not only are expected to vary greatly between hypoxic and normoxic conditions, but also display a large degree of heterogeneity across the cell population. Where traditionally ChIP-seq data are often treated as dichotomous data, the model and experiment here necessitate a quantitative, data-driven analysis of both datasets. We first identified genomic regions with sustained epigenetic markings, which provided a sample-specific reference enabling quantitative ChIP-seq data analysis. Sustained H3K27me3 marking was located around centromeres and intergenic regions, while sustained H3K4me3 marking is associated with genes involved in RNA binding, translation and protein transport and localization. Dynamic marking with both H3K4me3 and H3K27me3 (hypoxia-induced bivalency) was found in CpG-rich regions at loci encoding factors that control developmental processes, congruent with observations in embryonic stem cells. In silico-identified epigenetically sustained and dynamic genomic regions were confirmed through ChIP-PCR in vitro, and obtained results are corroborated by published data and current insights regarding epigenetic regulation.

  6. Tumor Delineation and Quantitative Assessment of Glucose Metabolic Rate within Histologic Subtypes of Non-Small Cell Lung Cancer by Using Dynamic (18)F Fluorodeoxyglucose PET.

    Science.gov (United States)

    Meijer, Tineke W H; de Geus-Oei, Lioe-Fee; Visser, Eric P; Oyen, Wim J G; Looijen-Salamon, Monika G; Visvikis, Dimitris; Verhagen, Ad F T M; Bussink, Johan; Vriens, Dennis

    2016-11-15

    Purpose To assess whether dynamic fluorine 18 ((18)F) fluorodeoxyglucose (FDG) positron emission tomography (PET) has added value over static (18)F-FDG PET for tumor delineation in non-small cell lung cancer (NSCLC) radiation therapy planning by using pathology volumes as the reference standard and to compare pharmacokinetic rate constants of (18)F-FDG metabolism, including regional variation, between NSCLC histologic subtypes. Materials and Methods The study was approved by the institutional review board. Patients gave written informed consent. In this prospective observational study, 1-hour dynamic (18)F-FDG PET/computed tomographic examinations were performed in 35 patients (36 resectable NSCLCs) between 2009 and 2014. Static and parametric images of glucose metabolic rate were obtained to determine lesion volumes by using three delineation strategies. Pathology volume was calculated from three orthogonal dimensions (n = 32). Whole tumor and regional rate constants and blood volume fraction (VB) were computed by using compartment modeling. Results Pathology volumes were larger than PET volumes (median difference, 8.7-25.2 cm(3); Wilcoxon signed rank test, P segmentation on static (18)F-FDG PET images is in best agreement with pathology volume and could be useful for NSCLC autocontouring. Differences in glycolytic rate and VB between SCC and AC are relevant for research in targeting agents and radiation therapy dose escalation. (©) RSNA, 2016 Online supplemental material is available for this article.

  7. Dynamics of cell orientation

    Science.gov (United States)

    de, Rumi; Zemel, Assaf; Safran, Samuel A.

    2007-09-01

    Many physiological processes depend on the response of biological cells to mechanical forces generated by the contractile activity of the cell or by external stresses. Using a simple theoretical model that includes the forces due to both the mechanosensitivity of cells and the elasticity of the matrix, we predict the dynamics and orientation of cells in both the absence and presence of applied stresses. The model predicts many features observed in measurements of cellular forces and orientation including the increase with time of the cellular forces in the absence of applied stress and the consequent decrease of the force in the presence of quasi-static stresses. We also explain the puzzling observation of parallel alignment of cells for static and quasi-static stresses and of nearly perpendicular alignment for dynamically varying stresses. In addition, we predict the response of the cellular orientation to a sinusoidally varying applied stress as a function of frequency.

  8. Quantitative Map of Proteome Dynamics during Neuronal Differentiation

    Directory of Open Access Journals (Sweden)

    Christian K. Frese

    2017-02-01

    Full Text Available Neuronal differentiation is a multistep process that shapes and re-shapes neurons by progressing through several typical stages, including axon outgrowth, dendritogenesis, and synapse formation. To systematically profile proteome dynamics throughout neuronal differentiation, we took cultured rat hippocampal neurons at different developmental stages and monitored changes in protein abundance using a combination of stable isotope labeling and high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS. Almost one third of all 4,500 proteins quantified underwent a more than 2-fold expression change during neuronal differentiation, indicating extensive remodeling of the neuron proteome. To highlight the strength of our resource, we studied the neural-cell-adhesion molecule 1 (NCAM1 and found that it stimulates dendritic arbor development by promoting actin filament growth at the dendritic growth cone. We anticipate that our quantitative map of neuronal proteome dynamics is a rich resource for further analyses of the many identified proteins in various neurodevelopmental processes.

  9. Controlled surface chemistries and quantitative cell response

    Science.gov (United States)

    Plant, Anne L.

    2002-03-01

    Living cells experience a large number of signaling cues from their extracellular matrix. As a result of these inputs, a variety of intracellular signaling pathways are apparently initiated simultaneously. The vast array of alternative responses that result from the integration of these inputs suggests that it may be reasonable to look for cellular response not as an 'on' or 'off' condition but as a distribution of responses. A difficult challenge is to determine whether variations in responses from individual cells arise from the complexity of intracellular signals or are due to variations in the cell culture environment. By controlling surface chemistry so that every cell 'sees' the same chemical and physical environment, we can begin to assess how the distribution of cell response is affected strictly by changes in the chemistry of the cell culture surface. Using the gene for green fluorescent protein linked to the gene for the promoter of the extracellular matrix protein, tenascin, we can easily probe the end product in a signaling pathway that is purported to be linked to surface protein chemistry and to cell shape. Cell response to well-controlled, well-characterized, and highly reproducible surfaces prepared using soft lithography techniques are compared with more conventional ways of preparing extracellular matrix proteins for cell culture. Using fluorescence microscopy and image analysis of populations of cells on these surfaces, we probe quantitatively the relationship between surface chemistry, cell shape and variations in gene expression endpoint.

  10. Single cell dynamic phenotyping

    OpenAIRE

    Katherin Patsch; Chi-Li Chiu; Mark Engeln; Agus, David B.; Parag Mallick; Shannon M. Mumenthaler; Daniel Ruderman

    2016-01-01

    Live cell imaging has improved our ability to measure phenotypic heterogeneity. However, bottlenecks in imaging and image processing often make it difficult to differentiate interesting biological behavior from technical artifact. Thus there is a need for new methods that improve data quality without sacrificing throughput. Here we present a 3-step workflow to improve dynamic phenotype measurements of heterogeneous cell populations. We provide guidelines for image acquisition, phenotype track...

  11. Quantitative MRI analysis of dynamic enhancement of focal liver lesions

    Directory of Open Access Journals (Sweden)

    S. S. Bagnenko

    2012-01-01

    Full Text Available In our study 45 patients with different focal liver lesions (110 nodules were examined using high field MR-system (1,5 T. During this investigation quantitative MRI analysis of dynamic enhancement of various hepatic lesions and parenchymatous organs of abdomen were performed. It was shown that quantitative evaluation of enhanced MRI improves understanding of vascular transformation processes in pathologic hepatic focuses and in liver itself that is important for differential diagnoses of these diseases.

  12. A novel quantitative explanation for the autonomic modulation of cardiac pacemaker cell automaticity via a dynamic system of sarcolemmal and intracellular proteins.

    Science.gov (United States)

    Maltsev, Victor A; Lakatta, Edward G

    2010-06-01

    Classical numerical models have attributed the regulation of normal cardiac automaticity in sinoatrial node cells (SANCs) largely to G protein-coupled receptor (GPCR) modulation of sarcolemmal ion currents. More recent experimental evidence, however, has indicated that GPCR modulation of SANCs automaticity involves spontaneous, rhythmic, local Ca(2+) releases (LCRs) from the sarcoplasmic reticulum (SR). We explored the GPCR rate modulation of SANCs using a unique and novel numerical model of SANCs in which Ca(2+)-release characteristics are graded by variations in the SR Ca(2+) pumping capability, mimicking the modulation by phospholamban regulated by cAMP-mediated, PKA-activated signaling. The model faithfully predicted the entire range of physiological chronotropic modulation of SANCs by the activation of beta-adrenergic receptors or cholinergic receptors only when experimentally documented changes of sarcolemmal ion channels are combined with a simultaneous increase/decrease in SR Ca(2+) pumping capability. The novel numerical mechanism of GPCR rate modulation is based on numerous complex synergistic interactions between sarcolemmal and intracellular processes via membrane voltage and Ca(2+). Major interactions include changes of diastolic Na(+)/Ca(2+) exchanger current that couple earlier/later diastolic Ca(2+) releases (predicting the experimentally defined LCR period shift) of increased/decreased amplitude (predicting changes in LCR signal mass, i.e., the product of LCR spatial size, amplitude, and number per cycle) to the diastolic depolarization and ultimately to the spontaneous action potential firing rate. Concomitantly, larger/smaller and more/less frequent activation of L-type Ca(2+) current shifts the cellular Ca(2+) balance to support the respective Ca(2+) cycling changes. In conclusion, our model simulations corroborate recent experimental results in rabbit SANCs pointing to a new paradigm for GPCR heart rate modulation by a complex system of

  13. SSBD: a database of quantitative data of spatiotemporal dynamics of biological phenomena.

    Science.gov (United States)

    Tohsato, Yukako; Ho, Kenneth H L; Kyoda, Koji; Onami, Shuichi

    2016-11-15

    Rapid advances in live-cell imaging analysis and mathematical modeling have produced a large amount of quantitative data on spatiotemporal dynamics of biological objects ranging from molecules to organisms. There is now a crucial need to bring these large amounts of quantitative biological dynamics data together centrally in a coherent and systematic manner. This will facilitate the reuse of this data for further analysis. We have developed the Systems Science of Biological Dynamics database (SSBD) to store and share quantitative biological dynamics data. SSBD currently provides 311 sets of quantitative data for single molecules, nuclei and whole organisms in a wide variety of model organisms from Escherichia coli to Mus musculus The data are provided in Biological Dynamics Markup Language format and also through a REST API. In addition, SSBD provides 188 sets of time-lapse microscopy images from which the quantitative data were obtained and software tools for data visualization and analysis. SSBD is accessible at http://ssbd.qbic.riken.jp CONTACT: sonami@riken.jp. © The Author 2016. Published by Oxford University Press.

  14. Quantitative analysis of organelle abundance, morphology and dynamics

    NARCIS (Netherlands)

    van Zutphen, Tim; van der Klei, Ida J.

    2011-01-01

    Recent data indicate that morphological characteristics of cell organelles are important for their function in the cell. These characteristics include not only their shape, number and size, but also their distribution in the cell. Moreover, the dynamics of processes that result in changes in these c

  15. Dynamic Quantitative T1 Mapping in Orthotopic Brain Tumor Xenografts

    Directory of Open Access Journals (Sweden)

    Kelsey Herrmann

    2016-04-01

    Full Text Available Human brain tumors such as glioblastomas are typically detected using conventional, nonquantitative magnetic resonance imaging (MRI techniques, such as T2-weighted and contrast enhanced T1-weighted MRI. In this manuscript, we tested whether dynamic quantitative T1 mapping by MRI can localize orthotopic glioma tumors in an objective manner. Quantitative T1 mapping was performed by MRI over multiple time points using the conventional contrast agent Optimark. We compared signal differences to determine the gadolinium concentration in tissues over time. The T1 parametric maps made it easy to identify the regions of contrast enhancement and thus tumor location. Doubling the typical human dose of contrast agent resulted in a clearer demarcation of these tumors. Therefore, T1 mapping of brain tumors is gadolinium dose dependent and improves detection of tumors by MRI. The use of T1 maps provides a quantitative means to evaluate tumor detection by gadolinium-based contrast agents over time. This dynamic quantitative T1 mapping technique will also enable future quantitative evaluation of various targeted MRI contrast agents.

  16. A quantitative assessment of LCOs for operations using system dynamics

    Energy Technology Data Exchange (ETDEWEB)

    Kyung, Min Kang [Department of Nuclear Engineering, Hanyang University, 17 Haengdang-Dong, Sungdong-Gu, Seoul 133-791 (Korea, Republic of); Jae, Moosung [Department of Nuclear Engineering, Hanyang University, 17 Haengdang-Dong, Sungdong-Gu, Seoul 133-791 (Korea, Republic of)]. E-mail: jae@hanyang.ac.kr

    2005-02-01

    Limiting conditions for operations (LCOs) define the allowed outage times (AOTs) and the actions to be taken if the repair cannot be completed within the AOT. Typically plant shutdown is required. In situations where the risk associated with the action, i.e. the risk of plant shutdown given a failure of the safety system, may be substantial, a strategy is needed to control the plant risk. In this study the changing operation modes are evaluated quantitatively and dynamically using the tool of system dynamics. System dynamics has been developed to analyze the dynamic reliability of a complicated system. System dynamics using the Vensim software have been applied to LCOs assessment for an example system, the auxiliary feed water system of a reference nuclear power plant. Analysis results of both full power operation and shutdown operation have been compared for a measure of core damage frequency. The increase in core damage frequency is used as a measure in this study. A time dependent framework developed in this study has been shown to be very flexible in that it can be applied to assess LCOs quantitatively under any operational context of the Technical Specifications in Final Safety Analysis Report of the reference plant.

  17. Exploring Neural Cell Dynamics with Digital Holographic Microscopy

    KAUST Repository

    Marquet, Pierre

    2013-04-21

    In this talk, I will present how digital holographic microscopy, as a powerful quantitative phase technique, can non-invasively measure cell dynamics and especially resolve local neuronal network activity through simultaneous multiple site optical recording.

  18. Quantitative imaging of heterogeneous dynamics in drying and aging paints.

    Science.gov (United States)

    van der Kooij, Hanne M; Fokkink, Remco; van der Gucht, Jasper; Sprakel, Joris

    2016-09-29

    Drying and aging paint dispersions display a wealth of complex phenomena that make their study fascinating yet challenging. To meet the growing demand for sustainable, high-quality paints, it is essential to unravel the microscopic mechanisms underlying these phenomena. Visualising the governing dynamics is, however, intrinsically difficult because the dynamics are typically heterogeneous and span a wide range of time scales. Moreover, the high turbidity of paints precludes conventional imaging techniques from reaching deep inside the paint. To address these challenges, we apply a scattering technique, Laser Speckle Imaging, as a versatile and quantitative tool to elucidate the internal dynamics, with microscopic resolution and spanning seven decades of time. We present a toolbox of data analysis and image processing methods that allows a tailored investigation of virtually any turbid dispersion, regardless of the geometry and substrate. Using these tools we watch a variety of paints dry and age with unprecedented detail.

  19. Microbial Cell Dynamics Lab (MCDL)

    Data.gov (United States)

    Federal Laboratory Consortium — The Microbial Cell Dynamics Laboratory at PNNL enables scientists to study the molecular details of microbes under relevant environmental conditions. The MCDL seeks...

  20. Lessons Learned from Quantitative Dynamical Modeling in Systems Biology

    Science.gov (United States)

    Bachmann, Julie; Matteson, Andrew; Schelke, Max; Kaschek, Daniel; Hug, Sabine; Kreutz, Clemens; Harms, Brian D.; Theis, Fabian J.; Klingmüller, Ursula; Timmer, Jens

    2013-01-01

    Due to the high complexity of biological data it is difficult to disentangle cellular processes relying only on intuitive interpretation of measurements. A Systems Biology approach that combines quantitative experimental data with dynamic mathematical modeling promises to yield deeper insights into these processes. Nevertheless, with growing complexity and increasing amount of quantitative experimental data, building realistic and reliable mathematical models can become a challenging task: the quality of experimental data has to be assessed objectively, unknown model parameters need to be estimated from the experimental data, and numerical calculations need to be precise and efficient. Here, we discuss, compare and characterize the performance of computational methods throughout the process of quantitative dynamic modeling using two previously established examples, for which quantitative, dose- and time-resolved experimental data are available. In particular, we present an approach that allows to determine the quality of experimental data in an efficient, objective and automated manner. Using this approach data generated by different measurement techniques and even in single replicates can be reliably used for mathematical modeling. For the estimation of unknown model parameters, the performance of different optimization algorithms was compared systematically. Our results show that deterministic derivative-based optimization employing the sensitivity equations in combination with a multi-start strategy based on latin hypercube sampling outperforms the other methods by orders of magnitude in accuracy and speed. Finally, we investigated transformations that yield a more efficient parameterization of the model and therefore lead to a further enhancement in optimization performance. We provide a freely available open source software package that implements the algorithms and examples compared here. PMID:24098642

  1. Lessons learned from quantitative dynamical modeling in systems biology.

    Directory of Open Access Journals (Sweden)

    Andreas Raue

    Full Text Available Due to the high complexity of biological data it is difficult to disentangle cellular processes relying only on intuitive interpretation of measurements. A Systems Biology approach that combines quantitative experimental data with dynamic mathematical modeling promises to yield deeper insights into these processes. Nevertheless, with growing complexity and increasing amount of quantitative experimental data, building realistic and reliable mathematical models can become a challenging task: the quality of experimental data has to be assessed objectively, unknown model parameters need to be estimated from the experimental data, and numerical calculations need to be precise and efficient. Here, we discuss, compare and characterize the performance of computational methods throughout the process of quantitative dynamic modeling using two previously established examples, for which quantitative, dose- and time-resolved experimental data are available. In particular, we present an approach that allows to determine the quality of experimental data in an efficient, objective and automated manner. Using this approach data generated by different measurement techniques and even in single replicates can be reliably used for mathematical modeling. For the estimation of unknown model parameters, the performance of different optimization algorithms was compared systematically. Our results show that deterministic derivative-based optimization employing the sensitivity equations in combination with a multi-start strategy based on latin hypercube sampling outperforms the other methods by orders of magnitude in accuracy and speed. Finally, we investigated transformations that yield a more efficient parameterization of the model and therefore lead to a further enhancement in optimization performance. We provide a freely available open source software package that implements the algorithms and examples compared here.

  2. Quantitative multivariate analysis of dynamic multicellular morphogenic trajectories.

    Science.gov (United States)

    White, Douglas E; Sylvester, Jonathan B; Levario, Thomas J; Lu, Hang; Streelman, J Todd; McDevitt, Todd C; Kemp, Melissa L

    2015-07-01

    Interrogating fundamental cell biology principles that govern tissue morphogenesis is critical to better understanding of developmental biology and engineering novel multicellular systems. Recently, functional micro-tissues derived from pluripotent embryonic stem cell (ESC) aggregates have provided novel platforms for experimental investigation; however elucidating the factors directing emergent spatial phenotypic patterns remains a significant challenge. Computational modelling techniques offer a unique complementary approach to probe mechanisms regulating morphogenic processes and provide a wealth of spatio-temporal data, but quantitative analysis of simulations and comparison to experimental data is extremely difficult. Quantitative descriptions of spatial phenomena across multiple systems and scales would enable unprecedented comparisons of computational simulations with experimental systems, thereby leveraging the inherent power of computational methods to interrogate the mechanisms governing emergent properties of multicellular biology. To address these challenges, we developed a portable pattern recognition pipeline consisting of: the conversion of cellular images into networks, extraction of novel features via network analysis, and generation of morphogenic trajectories. This novel methodology enabled the quantitative description of morphogenic pattern trajectories that could be compared across diverse systems: computational modelling of multicellular structures, differentiation of stem cell aggregates, and gastrulation of cichlid fish. Moreover, this method identified novel spatio-temporal features associated with different stages of embryo gastrulation, and elucidated a complex paracrine mechanism capable of explaining spatiotemporal pattern kinetic differences in ESC aggregates of different sizes.

  3. HIV-Envelope–Dependent Cell-Cell Fusion: Quantitative Studies

    Directory of Open Access Journals (Sweden)

    Leonor Huerta

    2009-01-01

    Full Text Available Interaction in vitro between cells infected with human immunodeficiency virus (HIV and surrounding, uninfected, target cells often leads to cell fusion and the formation of multinucleated cells, called syncytia. The presence in HIV-infected individuals of virus strains able to induce syncytia in cultures of T cells is associated with disease progression and AIDS. Even in the asymptomatic stage of infection, multinucleated cells have been observed in different organs, indicating that fused cells may be generated and remain viable in the tissues of patients. We used lymphocytic cells transfected for the expression of the HIV-envelope (Env glycoproteins to develop a method for the direct quantification of fusion events by flow cytometry (Huerta et al., 2006, J. Virol. Methods 138, 17–23; López-Balderas et al., 2007, Virus Res. 123, 138–146. The method involves the staining of fusion partners with lipophilic probes and the use of fluorescence resonance energy transfer (FRET to distinguish between fused and aggregated cells. We have shown that such a flow-cytometry assay is appropriate for the screening of compounds that have the potential to modulate HIV-Env–mediated cell fusion. Even those syncytia that are small or few in numbers can be detected. Quantitative analysis of the fusion products was performed with this technique; the results indicated that the time of reaction and initial proportion of fusion partners determine the number, relative size, and average cellular composition of syncytia. Heterogeneity of syncytia generated by HIV-Env–mediated cell-cell fusion may result in a variety of possible outcomes that, in turn, may influence the biological properties of the syncytia and surrounding cells, as well as replication of virus. Given the myriad immune abnormalities leading to AIDS, the full understanding of the extent, diverse composition, and role of fused cells in the pathogenesis of, and immune response to, HIV infection is an

  4. HIV-envelope-dependent cell-cell fusion: quantitative studies.

    Science.gov (United States)

    Huerta, Leonor; López-Balderas, Nayali; Rivera-Toledo, Evelyn; Sandoval, Guadalupe; Gómez-Icazbalceta, Guillermo; Villarreal, Carlos; Lamoyi, Edmundo; Larralde, Carlos

    2009-08-11

    Interaction in vitro between cells infected with human immunodeficiency virus (HIV) and surrounding, uninfected, target cells often leads to cell fusion and the formation of multinucleated cells, called syncytia. The presence in HIV-infected individuals of virus strains able to induce syncytia in cultures of T cells is associated with disease progression and AIDS. Even in the asymptomatic stage of infection, multinucleated cells have been observed in different organs, indicating that fused cells may be generated and remain viable in the tissues of patients. We used lymphocytic cells transfected for the expression of the HIV-envelope (Env) glycoproteins to develop a method for the direct quantification of fusion events by flow cytometry (Huerta et al., 2006, J. Virol. Methods 138, 17-23; López-Balderas et al., 2007, Virus Res. 123, 138-146). The method involves the staining of fusion partners with lipophilic probes and the use of fluorescence resonance energy transfer (FRET) to distinguish between fused and aggregated cells. We have shown that such a flow-cytometry assay is appropriate for the screening of compounds that have the potential to modulate HIV-Env-mediated cell fusion. Even those syncytia that are small or few in numbers can be detected. Quantitative analysis of the fusion products was performed with this technique; the results indicated that the time of reaction and initial proportion of fusion partners determine the number, relative size, and average cellular composition of syncytia. Heterogeneity of syncytia generated by HIV-Env-mediated cell-cell fusion may result in a variety of possible outcomes that, in turn, may influence the biological properties of the syncytia and surrounding cells, as well as replication of virus. Given the myriad immune abnormalities leading to AIDS, the full understanding of the extent, diverse composition, and role of fused cells in the pathogenesis of, and immune response to, HIV infection is an important, pending issue.

  5. Manual for Dynamic Triaxial Cell

    DEFF Research Database (Denmark)

    Pedersen, Thomas Schmidt; Ibsen, Lars Bo

    This report is a test report that describes the test setup for a dynamic triaxial cell at the Laboratory for Geotechnique at Aalborg University.......This report is a test report that describes the test setup for a dynamic triaxial cell at the Laboratory for Geotechnique at Aalborg University....

  6. Dynamized Preparations in Cell Culture

    Directory of Open Access Journals (Sweden)

    Ellanzhiyil Surendran Sunila

    2009-01-01

    Full Text Available Although reports on the efficacy of homeopathic medicines in animal models are limited, there are even fewer reports on the in vitro action of these dynamized preparations. We have evaluated the cytotoxic activity of 30C and 200C potencies of ten dynamized medicines against Dalton's Lymphoma Ascites, Ehrlich's Ascites Carcinoma, lung fibroblast (L929 and Chinese Hamster Ovary (CHO cell lines and compared activity with their mother tinctures during short-term and long-term cell culture. The effect of dynamized medicines to induce apoptosis was also evaluated and we studied how dynamized medicines affected genes expressed during apoptosis. Mother tinctures as well as some dynamized medicines showed significant cytotoxicity to cells during short and long-term incubation. Potentiated alcohol control did not produce any cytotoxicity at concentrations studied. The dynamized medicines were found to inhibit CHO cell colony formation and thymidine uptake in L929 cells and those of Thuja, Hydrastis and Carcinosinum were found to induce apoptosis in DLA cells. Moreover, dynamized Carcinosinum was found to induce the expression of p53 while dynamized Thuja produced characteristic laddering pattern in agarose gel electrophoresis of DNA. These results indicate that dynamized medicines possess cytotoxic as well as apoptosis-inducing properties.

  7. Dynamic headspace generation and quantitation of triacetone triperoxide vapor.

    Science.gov (United States)

    Giordano, Braden C; Lubrano, Adam L; Field, Christopher R; Collins, Greg E

    2014-02-28

    Two methods for quantitation of triacetone triperoxide (TATP) vapor using a programmable temperature vaporization (PTV) inlet coupled to a gas chromatography/mass spectrometer (GC/MS) have been demonstrated. The dynamic headspace of bulk TATP was mixed with clean humid air to produce a TATP vapor stream. Sampling via a heated transfer line to a PTV inlet with a Tenax-TA™ filled liner allowed for direct injection of the vapor stream to a GC/MS for vapor quantitation. TATP was extracted from the vapor stream and subsequently desorbed from the PTV liner for splitless injection on the GC column. Calibration curves were prepared using solution standards with a standard split/splitless GC inlet for quantitation of the TATP vapor. Alternatively, vapor was sampled onto a Tenax-TA™ sample tube and placed into a thermal desorption system. In this instance, vapor was desorbed from the tube and subsequently trapped on a liquid nitrogen cooled PTV inlet. Calibration curves for this method were prepared from direct liquid injection of standards onto samples tube with the caveat that a vacuum is applied to the tube during deposition to ensure that the volatile TATP penetrates into the tube. Vapor concentration measurements, as determined by either GC/MS analysis or mass gravimetry of the bulk TATP, were statistically indistinguishable. Different approaches to broaden the TATP vapor dynamic range, including diluent air flow, sample chamber temperature, sample vial orifice size, and sample size are discussed. Vapor concentrations between 50 and 5400ngL(-1) are reported, with stable vapor generation observed for as long as 60 consecutive hours.

  8. Quantitative evaluation of the reticuloendothelial system function with dynamic MRI.

    Directory of Open Access Journals (Sweden)

    Ting Liu

    Full Text Available PURPOSE: To evaluate the reticuloendothelial system (RES function by real-time imaging blood clearance as well as hepatic uptake of superparamagnetic iron oxide nanoparticle (SPIO using dynamic magnetic resonance imaging (MRI with two-compartment pharmacokinetic modeling. MATERIALS AND METHODS: Kinetics of blood clearance and hepatic accumulation were recorded in young adult male 01b74 athymic nude mice by dynamic T2* weighted MRI after the injection of different doses of SPIO nanoparticles (0.5, 3 or 10 mg Fe/kg. Association parameter, Kin, dissociation parameter, Kout, and elimination constant, Ke, derived from dynamic data with two-compartment model, were used to describe active binding to Kupffer cells and extrahepatic clearance. The clodrosome and liposome were utilized to deplete macrophages and block the RES function to evaluate the capability of the kinetic parameters for investigation of macrophage function and density. RESULTS: The two-compartment model provided a good description for all data and showed a low sum squared residual for all mice (0.27±0.03. A lower Kin, a lower Kout and a lower Ke were found after clodrosome treatment, whereas a lower Kin, a higher Kout and a lower Ke were observed after liposome treatment in comparison to saline treatment (P<0.005. CONCLUSION: Dynamic SPIO-enhanced MR imaging with two-compartment modeling can provide information on RES function on both a cell number and receptor function level.

  9. Quantitative Susceptibility Mapping and Dynamic Contrast Enhanced Quantitative Perfusion in Cerebral Cavernous Angiomas

    Science.gov (United States)

    Mikati, Abdul Ghani; Tan, Huan; Shenkar, Robert; Li, Luying; Zhang, Lingjiao; Guo, Xiaodong; Shi, Changbin; Liu, Tian; Wang, Yi; Shah, Akash; Edelman, Robert; Christoforidis, Gregory; Awad, Issam

    2015-01-01

    Background Hyperpermeability and iron deposition are two central pathophysiological phenomena in human cerebral cavernous malformation (CCM) disease. Here we used two novel magnetic resonance imaging (MRI) techniques to establish a relationship between these phenomena. Methods Subjects with CCM disease (4 sporadic and 18 familial) underwent MRI imaging using the Dynamic Contrast Enhanced Quantitative Perfusion (DCEQP) and Quantitative Susceptibility Mapping (QSM) techniques that measure hemodynamic factors of vessel leak and iron deposition respectively, previously demonstrated in CCM disease. Regions of interest encompassing the CCM lesions were analyzed using these techniques Results Susceptibility measured by QSM was positively correlated with permeability of lesions measured using DCEQP (r=0.49, p=<0.0001). The correlation was not affected by factors including familial predisposition, lesion volume, the contrast agent and the use of statin medication. Susceptibility was correlated with lesional blood volume (r=0.4, p=0.0001), but not with lesional blood flow. Conclusion The correlation between QSM and DCEQP suggests that the phenomena of permeability and iron deposition are related in CCM; hence “more leaky lesions” also manifest a more cumulative iron burden. These techniques might be used as biomarkers to monitor the course of this disease and the effect of therapy. PMID:24302484

  10. A quantitative assessment of LCOs using system dynamics

    Energy Technology Data Exchange (ETDEWEB)

    Kang, K. M.; Jey, M. S. [Hanyang Univ., Seoul (Korea, Republic of); Seong, C. K. [Korea Electric Power Research Institute, Taejon (Korea, Republic of)

    2002-10-01

    The research on the technical specifications improvements for too conservative TS has been conducted recently using PSA techniques. In this study a tool of Vensim, which one of System Dynamics software products, is applied to evaluate the Limiting Conditions for Operation (LCOs) quantitatively. A value of core damage frequency in PSA is used as risk measure. The analysis of both full power operation and shutdown operation has been compared for the value of the CDF. The time dependent framework developed in this study has been applied to an example problem accompanied by the operation of Wolsong Nuclear Power plants and it is shown that it is flexible in that it can be applied to any operational context of the technical specification00.

  11. Quantitative-PCR Assessment of Cryptosporidium parvum Cell Culture Infection

    OpenAIRE

    Di Giovanni, George D.; LeChevallier, Mark W.

    2005-01-01

    A quantitative TaqMan PCR method was developed for assessing the Cryptosporidium parvum infection of in vitro cultivated human ileocecal adenocarcinoma (HCT-8) cell cultures. This method, termed cell culture quantitative sequence detection (CC-QSD), has numerous applications, several of which are presented. CC-QSD was used to investigate parasite infection in cell culture over time, the effects of oocyst treatment on infectivity and infectivity assessment of different C. parvum isolates. CC-Q...

  12. Quantitative cell biology: the essential role of theory.

    Science.gov (United States)

    Howard, Jonathon

    2014-11-05

    Quantitative biology is a hot area, as evidenced by the recent establishment of institutes, graduate programs, and conferences with that name. But what is quantitative biology? What should it be? And how can it contribute to solving the big questions in biology? The past decade has seen very rapid development of quantitative experimental techniques, especially at the single-molecule and single-cell levels. In this essay, I argue that quantitative biology is much more than just the quantitation of these experimental results. Instead, it should be the application of the scientific method by which measurement is directed toward testing theories. In this view, quantitative biology is the recognition that theory and models play critical roles in biology, as they do in physics and engineering. By tying together experiment and theory, quantitative biology promises a deeper understanding of underlying mechanisms, when the theory works, or to new discoveries, when it does not.

  13. Quantitative and dynamic analysis of PTEN phosphorylation by NMR.

    Science.gov (United States)

    Cordier, Florence; Chaffotte, Alain; Wolff, Nicolas

    2015-05-01

    The dual lipid and protein phosphatase PTEN is a tumor suppressor controlling key biological processes, such as cell growth, proliferation and neuro-survival. Its activity and intracellular trafficking is finely regulated notably by multi-site phosphorylation of its C-terminal tail. The reversible and highly dynamic character of these regulatory events confers a temporal dimension to the cell for triggering crucial decisions. In this review, we describe how a recently developed time-resolved NMR spectroscopy approach unveils the dynamic establishment of the phosphorylation events of PTEN C-terminal tail controlled by CK2 and GSK3β kinases. Two cascades of reactions have been identified, in vitro and in extracts of human neuroblastoma cells. They are triggered independently on two nearby clusters of sites (S380-S385 and S361-S370) and occur on different timescales. In each cascade, the reactions follow an ordered model with a distributive kinetic mechanism. The vision of these cascades as two delay timers activating distinct or time-delayed regulatory responses gives a temporal dimension on PTEN regulation and is discussed in relation to the known functional roles of each cluster. Copyright © 2014 Elsevier Inc. All rights reserved.

  14. Deep Learning Automates the Quantitative Analysis of Individual Cells in Live-Cell Imaging Experiments.

    Science.gov (United States)

    Van Valen, David A; Kudo, Takamasa; Lane, Keara M; Macklin, Derek N; Quach, Nicolas T; DeFelice, Mialy M; Maayan, Inbal; Tanouchi, Yu; Ashley, Euan A; Covert, Markus W

    2016-11-01

    Live-cell imaging has opened an exciting window into the role cellular heterogeneity plays in dynamic, living systems. A major critical challenge for this class of experiments is the problem of image segmentation, or determining which parts of a microscope image correspond to which individual cells. Current approaches require many hours of manual curation and depend on approaches that are difficult to share between labs. They are also unable to robustly segment the cytoplasms of mammalian cells. Here, we show that deep convolutional neural networks, a supervised machine learning method, can solve this challenge for multiple cell types across the domains of life. We demonstrate that this approach can robustly segment fluorescent images of cell nuclei as well as phase images of the cytoplasms of individual bacterial and mammalian cells from phase contrast images without the need for a fluorescent cytoplasmic marker. These networks also enable the simultaneous segmentation and identification of different mammalian cell types grown in co-culture. A quantitative comparison with prior methods demonstrates that convolutional neural networks have improved accuracy and lead to a significant reduction in curation time. We relay our experience in designing and optimizing deep convolutional neural networks for this task and outline several design rules that we found led to robust performance. We conclude that deep convolutional neural networks are an accurate method that require less curation time, are generalizable to a multiplicity of cell types, from bacteria to mammalian cells, and expand live-cell imaging capabilities to include multi-cell type systems.

  15. Quantitative Aspects of Cyclosis in Plant Cells.

    Science.gov (United States)

    Howells, K. F.; Fell, D. A.

    1979-01-01

    Describes an exercise which is currently used in a course in cell physiology at Oxford Polytechnic in England. This exercise can give students some idea of the molecular events involved in bringing about movement of chloroplasts (and other organelles) in plant cells. (HM)

  16. Elucidating dynamic metabolic physiology through network integration of quantitative time-course metabolomics

    Science.gov (United States)

    Bordbar, Aarash; Yurkovich, James T.; Paglia, Giuseppe; Rolfsson, Ottar; Sigurjónsson, Ólafur E.; Palsson, Bernhard O.

    2017-01-01

    The increasing availability of metabolomics data necessitates novel methods for deeper data analysis and interpretation. We present a flux balance analysis method that allows for the computation of dynamic intracellular metabolic changes at the cellular scale through integration of time-course absolute quantitative metabolomics. This approach, termed “unsteady-state flux balance analysis” (uFBA), is applied to four cellular systems: three dynamic and one steady-state as a negative control. uFBA and FBA predictions are contrasted, and uFBA is found to be more accurate in predicting dynamic metabolic flux states for red blood cells, platelets, and Saccharomyces cerevisiae. Notably, only uFBA predicts that stored red blood cells metabolize TCA intermediates to regenerate important cofactors, such as ATP, NADH, and NADPH. These pathway usage predictions were subsequently validated through 13C isotopic labeling and metabolic flux analysis in stored red blood cells. Utilizing time-course metabolomics data, uFBA provides an accurate method to predict metabolic physiology at the cellular scale for dynamic systems. PMID:28387366

  17. Quantitative stem cell biology: the threat and the glory.

    Science.gov (United States)

    Pollard, Steven M

    2016-11-15

    Major technological innovations over the past decade have transformed our ability to extract quantitative data from biological systems at an unprecedented scale and resolution. These quantitative methods and associated large datasets should lead to an exciting new phase of discovery across many areas of biology. However, there is a clear threat: will we drown in these rivers of data? On 18th July 2016, stem cell biologists gathered in Cambridge for the 5th annual Cambridge Stem Cell Symposium to discuss 'Quantitative stem cell biology: from molecules to models'. This Meeting Review provides a summary of the data presented by each speaker, with a focus on quantitative techniques and the new biological insights that are emerging. © 2016. Published by The Company of Biologists Ltd.

  18. Quantitative high dynamic range beam profiling for fluorescence microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Mitchell, T. J., E-mail: t.j.mitchell@dur.ac.uk; Saunter, C. D.; O’Nions, W.; Girkin, J. M.; Love, G. D. [Centre for Advanced Instrumentation and Biophysical Sciences Institute, Department of Physics, Durham University, Durham DH1 3LE (United Kingdom)

    2014-10-15

    Modern developmental biology relies on optically sectioning fluorescence microscope techniques to produce non-destructive in vivo images of developing specimens at high resolution in three dimensions. As optimal performance of these techniques is reliant on the three-dimensional (3D) intensity profile of the illumination employed, the ability to directly record and analyze these profiles is of great use to the fluorescence microscopist or instrument builder. Though excitation beam profiles can be measured indirectly using a sample of fluorescent beads and recording the emission along the microscope detection path, we demonstrate an alternative approach where a miniature camera sensor is used directly within the illumination beam. Measurements taken using our approach are solely concerned with the illumination optics as the detection optics are not involved. We present a miniature beam profiling device and high dynamic range flux reconstruction algorithm that together are capable of accurately reproducing quantitative 3D flux maps over a large focal volume. Performance of this beam profiling system is verified within an optical test bench and demonstrated for fluorescence microscopy by profiling the low NA illumination beam of a single plane illumination microscope. The generality and success of this approach showcases a widely flexible beam amplitude diagnostic tool for use within the life sciences.

  19. Quantitative photoacoustic imaging of nanoparticles in cells and tissues.

    Science.gov (United States)

    Cook, Jason R; Frey, Wolfgang; Emelianov, Stanislav

    2013-02-26

    Quantitative visualization of nanoparticles in cells and tissues, while preserving the spatial information, is very challenging. A photoacoustic imaging technique to depict the presence and quantity of nanoparticles is presented. This technique is based on the dependence of the photoacoustic signal on both the nanoparticle quantity and the laser fluence. Quantitative photoacoustic imaging is a robust technique that does not require knowledge of the local fluence, but a relative change in the fluence. This eliminates the need for sophisticated methods or models to determine the energy distribution of light in turbid media. Quantitative photoacoustic imaging was first applied to nanoparticle-loaded cells, and quantitation was validated by inductively coupled plasma mass spectrometry. Quantitative photoacoustic imaging was then extended to xenograft tumor tissue sections, and excellent agreement with traditional histopathological analysis was demonstrated. Our results suggest that quantitative photoacoustic imaging may be used in many applications including the determination of the efficiency and effectiveness of molecular targeting strategies for cell studies and animal models, the quantitative assessment of photoacoustic contrast agent biodistribution, and the validation of in vivo photoacoustic imaging.

  20. Maximum entropy reconstructions of dynamic signaling networks from quantitative proteomics data.

    Science.gov (United States)

    Locasale, Jason W; Wolf-Yadlin, Alejandro

    2009-08-26

    Advances in mass spectrometry among other technologies have allowed for quantitative, reproducible, proteome-wide measurements of levels of phosphorylation as signals propagate through complex networks in response to external stimuli under different conditions. However, computational approaches to infer elements of the signaling network strictly from the quantitative aspects of proteomics data are not well established. We considered a method using the principle of maximum entropy to infer a network of interacting phosphotyrosine sites from pairwise correlations in a mass spectrometry data set and derive a phosphorylation-dependent interaction network solely from quantitative proteomics data. We first investigated the applicability of this approach by using a simulation of a model biochemical signaling network whose dynamics are governed by a large set of coupled differential equations. We found that in a simulated signaling system, the method detects interactions with significant accuracy. We then analyzed a growth factor mediated signaling network in a human mammary epithelial cell line that we inferred from mass spectrometry data and observe a biologically interpretable, small-world structure of signaling nodes, as well as a catalog of predictions regarding the interactions among previously uncharacterized phosphotyrosine sites. For example, the calculation places a recently identified tumor suppressor pathway through ARHGEF7 and Scribble, in the context of growth factor signaling. Our findings suggest that maximum entropy derived network models are an important tool for interpreting quantitative proteomics data.

  1. Quantitative Assessment of Molecular Dynamics Sampling for Flexible Systems.

    Science.gov (United States)

    Nemec, Mike; Hoffmann, Daniel

    2017-02-14

    Molecular dynamics (MD) simulation is a natural method for the study of flexible molecules but at the same time is limited by the large size of the conformational space of these molecules. We ask by how much the MD sampling quality for flexible molecules can be improved by two means: the use of diverse sets of trajectories starting from different initial conformations to detect deviations between samples and sampling with enhanced methods such as accelerated MD (aMD) or scaled MD (sMD) that distort the energy landscape in controlled ways. To this end, we test the effects of these approaches on MD simulations of two flexible biomolecules in aqueous solution, Met-Enkephalin (5 amino acids) and HIV-1 gp120 V3 (a cycle of 35 amino acids). We assess the convergence of the sampling quantitatively with known, extensive measures of cluster number Nc and cluster distribution entropy Sc and with two new quantities, conformational overlap Oconf and density overlap Odens, both conveniently ranging from 0 to 1. These new overlap measures quantify self-consistency of sampling in multitrajectory MD experiments, a necessary condition for converged sampling. A comprehensive assessment of sampling quality of MD experiments identifies the combination of diverse trajectory sets and aMD as the most efficient approach among those tested. However, analysis of Odens between conventional and aMD trajectories also reveals that we have not completely corrected aMD sampling for the distorted energy landscape. Moreover, for V3, the courses of Nc and Odens indicate that much higher resources than those generally invested today will probably be needed to achieve convergence. The comparative analysis also shows that conventional MD simulations with insufficient sampling can be easily misinterpreted as being converged.

  2. Elucidating dynamic metabolic physiology through network integration of quantitative time-course metabolomics

    DEFF Research Database (Denmark)

    Bordbar, Aarash; Yurkovich, James T.; Paglia, Giuseppe

    2017-01-01

    The increasing availability of metabolomics data necessitates novel methods for deeper data analysis and interpretation. We present a flux balance analysis method that allows for the computation of dynamic intracellular metabolic changes at the cellular scale through integration of time-course ab......The increasing availability of metabolomics data necessitates novel methods for deeper data analysis and interpretation. We present a flux balance analysis method that allows for the computation of dynamic intracellular metabolic changes at the cellular scale through integration of time......-course absolute quantitative metabolomics. This approach, termed " unsteady-state flux balance analysis" (uFBA), is applied to four cellular systems: three dynamic and one steady-state as a negative control. uFBA and FBA predictions are contrasted, and uFBA is found to be more accurate in predicting dynamic...... isotopic labeling and metabolic flux analysis in stored red blood cells. Utilizing time-course metabolomics data, uFBA provides an accurate method to predict metabolic physiology at the cellular scale for dynamic systems....

  3. Quantitation of glucocorticoid receptor DNA-binding dynamics by single-molecule microscopy and FRAP.

    Directory of Open Access Journals (Sweden)

    Femke L Groeneweg

    Full Text Available Recent advances in live cell imaging have provided a wealth of data on the dynamics of transcription factors. However, a consistent quantitative description of these dynamics, explaining how transcription factors find their target sequences in the vast amount of DNA inside the nucleus, is still lacking. In the present study, we have combined two quantitative imaging methods, single-molecule microscopy and fluorescence recovery after photobleaching, to determine the mobility pattern of the glucocorticoid receptor (GR and the mineralocorticoid receptor (MR, two ligand-activated transcription factors. For dexamethasone-activated GR, both techniques showed that approximately half of the population is freely diffusing, while the remaining population is bound to DNA. Of this DNA-bound population about half the GRs appeared to be bound for short periods of time (∼ 0.7 s and the other half for longer time periods (∼ 2.3 s. A similar pattern of mobility was seen for the MR activated by aldosterone. Inactive receptors (mutant or antagonist-bound receptors show a decreased DNA binding frequency and duration, but also a higher mobility for the diffusing population. Likely, very brief (≤ 1 ms interactions with DNA induced by the agonists underlie this difference in diffusion behavior. Surprisingly, different agonists also induce different mobilities of both receptors, presumably due to differences in ligand-induced conformational changes and receptor complex formation. In summary, our data provide a consistent quantitative model of the dynamics of GR and MR, indicating three types of interactions with DNA, which fit into a model in which frequent low-affinity DNA binding facilitates the search for high-affinity target sequences.

  4. A Quantitative Analysis of Dressing Dynamics for Robotic Dressing Assistance

    Directory of Open Access Journals (Sweden)

    Greg Chance

    2017-05-01

    Full Text Available Assistive robots have a great potential to address issues related to an aging population and an increased demand for caregiving. Successful deployment of robots working in close proximity with people requires consideration of both safety and human–robot interaction (HRI. One of the established activities of daily living where robots could play an assistive role is dressing. Using the correct force profile for robot control will be essential in this application of HRI requiring careful exploration of factors related to the user’s pose and the type of garments involved. In this paper, a Baxter robot was used to dress a jacket onto a mannequin and human participants considering several combinations of user pose and clothing type (base layers, while recording dynamic data from the robot, a load cell, and an IMU. We also report on suitability of these sensors for identifying dressing errors, e.g., fabric snagging. Data were analyzed by comparing the overlap of confidence intervals to determine sensitivity to dressing. We expand the analysis to include classification techniques such as decision tree and support vector machines using k-fold cross-validation. The 6-axis load cell successfully discriminated between clothing types with predictive model accuracies between 72 and 97%. Used independently, the IMU and Baxter sensors were insufficient to discriminate garment types with the IMU showing 40–72% accuracy, but when used in combination this pair of sensors achieved an accuracy similar to the more expensive load cell (98%. When observing dressing errors (snagging, Baxter’s sensors and the IMU data demonstrated poor sensitivity but applying machine learning methods resulted in model with high predicative accuracy and low false negative rates (≤5%. The results show that the load cell could be used independently for this application with good accuracy but a combination of the lower cost sensors could also be used without a significant loss in

  5. Quantitative detection of Vibrio cholera toxin by real-time and dynamic cytotoxicity monitoring.

    Science.gov (United States)

    Jin, Dazhi; Luo, Yun; Zheng, Min; Li, Haijing; Zhang, Jing; Stampfl, Melinda; Xu, Xiao; Ding, Gangqiang; Zhang, Yanjun; Tang, Yi-Wei

    2013-12-01

    We report here the quantitative detection of Vibrio cholerae toxin (CT) in isolates and stool specimens by dynamic monitoring of the full course of CT-mediated cytotoxicity in a real-time cell analysis (RTCA) system. Four cell lines, including Y-1 mouse adrenal tumor cells, Chinese hamster ovary (CHO) cells, small intestine epithelial (FHs74Int) cells, and mouse adrenal gland (PC12-Adh) cells, were evaluated for their suitability for CT-induced cytotoxicity testing. Among them, the Y-1 line was demonstrated to be the most sensitive for CT-mediated cytotoxicity, with limits of detection of 7.0 pg/ml for purified CT and 0.11 ng/ml for spiked CT in pooled negative stool specimens. No CT-mediated cytotoxicity was observed for nontoxigenic V. cholerae, non-V. cholerae species, or non-V. cholerae enterotoxins. The CT-RTCA assay was further validated with 100 stool specimens consecutively collected from patients with diarrhea and 200 V. cholerae isolates recovered from patients and the environment, in comparison to a reference using three detection methods. The CT-RTCA assay had sensitivities and specificities of 97.5% and 100.0%, respectively, for V. cholerae isolates and 90.0% and 97.2% for stool specimens. For stool specimens spiked with CT concentrations ranging from 3.5 pg/ml to 1.8 ng/ml, the inoculation-to-detection time was 1.12 ± 0.38 h, and the values were inversely correlated with CT concentrations (ρ = -1; P = 0.01). The results indicate that the CT-RTCA assay with the Y-1 cell line provides a rapid and sensitive tool for the quantitative detection of CT activities in clinical specimens.

  6. Deep Learning Automates the Quantitative Analysis of Individual Cells in Live-Cell Imaging Experiments.

    Directory of Open Access Journals (Sweden)

    David A Van Valen

    2016-11-01

    Full Text Available Live-cell imaging has opened an exciting window into the role cellular heterogeneity plays in dynamic, living systems. A major critical challenge for this class of experiments is the problem of image segmentation, or determining which parts of a microscope image correspond to which individual cells. Current approaches require many hours of manual curation and depend on approaches that are difficult to share between labs. They are also unable to robustly segment the cytoplasms of mammalian cells. Here, we show that deep convolutional neural networks, a supervised machine learning method, can solve this challenge for multiple cell types across the domains of life. We demonstrate that this approach can robustly segment fluorescent images of cell nuclei as well as phase images of the cytoplasms of individual bacterial and mammalian cells from phase contrast images without the need for a fluorescent cytoplasmic marker. These networks also enable the simultaneous segmentation and identification of different mammalian cell types grown in co-culture. A quantitative comparison with prior methods demonstrates that convolutional neural networks have improved accuracy and lead to a significant reduction in curation time. We relay our experience in designing and optimizing deep convolutional neural networks for this task and outline several design rules that we found led to robust performance. We conclude that deep convolutional neural networks are an accurate method that require less curation time, are generalizable to a multiplicity of cell types, from bacteria to mammalian cells, and expand live-cell imaging capabilities to include multi-cell type systems.

  7. Impact of implementation choices on quantitative predictions of cell-based computational models

    Science.gov (United States)

    Kursawe, Jochen; Baker, Ruth E.; Fletcher, Alexander G.

    2017-09-01

    'Cell-based' models provide a powerful computational tool for studying the mechanisms underlying the growth and dynamics of biological tissues in health and disease. An increasing amount of quantitative data with cellular resolution has paved the way for the quantitative parameterisation and validation of such models. However, the numerical implementation of cell-based models remains challenging, and little work has been done to understand to what extent implementation choices may influence model predictions. Here, we consider the numerical implementation of a popular class of cell-based models called vertex models, which are often used to study epithelial tissues. In two-dimensional vertex models, a tissue is approximated as a tessellation of polygons and the vertices of these polygons move due to mechanical forces originating from the cells. Such models have been used extensively to study the mechanical regulation of tissue topology in the literature. Here, we analyse how the model predictions may be affected by numerical parameters, such as the size of the time step, and non-physical model parameters, such as length thresholds for cell rearrangement. We find that vertex positions and summary statistics are sensitive to several of these implementation parameters. For example, the predicted tissue size decreases with decreasing cell cycle durations, and cell rearrangement may be suppressed by large time steps. These findings are counter-intuitive and illustrate that model predictions need to be thoroughly analysed and implementation details carefully considered when applying cell-based computational models in a quantitative setting.

  8. Quantitative volumetric Raman imaging of three dimensional cell cultures

    Science.gov (United States)

    Kallepitis, Charalambos; Bergholt, Mads S.; Mazo, Manuel M.; Leonardo, Vincent; Skaalure, Stacey C.; Maynard, Stephanie A.; Stevens, Molly M.

    2017-03-01

    The ability to simultaneously image multiple biomolecules in biologically relevant three-dimensional (3D) cell culture environments would contribute greatly to the understanding of complex cellular mechanisms and cell-material interactions. Here, we present a computational framework for label-free quantitative volumetric Raman imaging (qVRI). We apply qVRI to a selection of biological systems: human pluripotent stem cells with their cardiac derivatives, monocytes and monocyte-derived macrophages in conventional cell culture systems and mesenchymal stem cells inside biomimetic hydrogels that supplied a 3D cell culture environment. We demonstrate visualization and quantification of fine details in cell shape, cytoplasm, nucleus, lipid bodies and cytoskeletal structures in 3D with unprecedented biomolecular specificity for vibrational microspectroscopy.

  9. A proteomic approach for quantitation of phosphorylation using stable isotope labeling in cell culture.

    Science.gov (United States)

    Ibarrola, Nieves; Kalume, Dario E; Gronborg, Mads; Iwahori, Akiko; Pandey, Akhilesh

    2003-11-15

    Posttranslational modifications are major mechanisms of regulating protein activity and function in vertebrate cells. It is essential to obtain qualitative information about posttranslational modification patterns of proteins to understand signal transduction mechanisms in greater detail. However, it is equally important to measure the dynamics of posttranslational modifications such as phosphorylation to approach signaling networks from a systems biology perspective. Despite a number of advances, methods to quantitate posttranslational modifications remain difficult to implement due to a number of factors including lack of a generic method, elaborate chemical steps, and requirement for large amounts of sample. We have previously shown that stable isotope-containing amino acids in cell culture (SILAC) can be used to differentially label growing cell populations for quantitation of protein levels. In this report, we extend the use of SILAC as a novel proteomic approach for the relative quantitation of posttranslational modifications such as phosphorylation. We have used SILAC to quantitate the extent of known phosphorylation sites as well as to identify and quantitate novel phosphorylation sites.

  10. Quantitative volumetric Raman imaging of three dimensional cell cultures

    KAUST Repository

    Kallepitis, Charalambos

    2017-03-22

    The ability to simultaneously image multiple biomolecules in biologically relevant three-dimensional (3D) cell culture environments would contribute greatly to the understanding of complex cellular mechanisms and cell–material interactions. Here, we present a computational framework for label-free quantitative volumetric Raman imaging (qVRI). We apply qVRI to a selection of biological systems: human pluripotent stem cells with their cardiac derivatives, monocytes and monocyte-derived macrophages in conventional cell culture systems and mesenchymal stem cells inside biomimetic hydrogels that supplied a 3D cell culture environment. We demonstrate visualization and quantification of fine details in cell shape, cytoplasm, nucleus, lipid bodies and cytoskeletal structures in 3D with unprecedented biomolecular specificity for vibrational microspectroscopy.

  11. Plasmonic Nanoparticles with Quantitatively Controlled Bioconjugation for Photoacoustic Imaging of Live Cancer Cells.

    Science.gov (United States)

    Tian, Chao; Qian, Wei; Shao, Xia; Xie, Zhixing; Cheng, Xu; Liu, Shengchun; Cheng, Qian; Liu, Bing; Wang, Xueding

    2016-12-01

    Detection and imaging of single cancer cells is critical for cancer diagnosis and understanding of cellular dynamics. Photoacoustic imaging (PAI) provides a potential tool for the study of cancer cell dynamics, but faces the challenge that most cancer cells lack sufficient endogenous contrast. Here, a type of colloidal gold nanoparticles (AuNPs) are physically fabricated and are precisely functionalized with quantitative amounts of functional ligands (i.e., polyethyleneglycol (PEG) and (Arginine(R)-Glycine(G)-Aspartic(D))4 (RGD) peptides) to serve as an exogenous contrast agent for PAI of single cells. The functionalized AuNPs, with a fixed number of PEG but different RGD densities, are delivered into human prostate cancer cells. Radioactivity and photoacoustic analyses show that, although cellular uptake efficiency of the AuNPs linearly increases along with RGD density, photoacoustic signal generation efficiency does not and only maximize at a moderate RGD density. The functionalization of the AuNPs is in turn optimized based on the experimental finding, and single cancer cells are imaged using a custom photoacoustic microscopy with high-resolution. The quantitatively functionalized AuNPs together with the high-resolution PAI system provide a unique platform for the detection and imaging of single cancer cells, and may impact not only basic science but also clinical diagnostics on a range of cancers.

  12. Mechanosensing Dynamics of Red blood Cells

    Science.gov (United States)

    Wan, Jiandi

    2015-11-01

    Mechanical stress-induced deformation of human red blood cells (RBCs) plays important physiopathological roles in oxygen delivery, blood rheology, transfusion, and malaria. Recent studies demonstrate that, in response to mechanical deformation, RBCs release adenosine-5'-triphosphate (ATP), suggesting the existence of mechanotransductive pathways in RBCs. Most importantly, the released ATP from RBCs regulates vascular tone and impaired release of ATP from RBCs has been linked to diseases such as type II diabetes and cystic fibrosis. To date, however, the mechanisms of mechanotransductive release of ATP from RBCs remain unclear. Given that RBCs experience shear stresses continuously during the circulation cycle and the released ATP plays a central role in vascular physiopathology, understanding the mechanotransductive release of ATP from RBCs will provide not only fundamental insights to the role of RBCs in vascular homeostasis but also novel therapeutic strategies for red cell dysfunction and vascular disease. This talk describes the main research in my group on integrating microfluidic-based approaches to study the mechanosensing dynamics of RBCs. Specifically, I will introduce a micro?uidic approach that can probe the dynamics of shear-induced ATP release from RBCs with millisecond resolution and provide quantitative understandings of the mechanosensitive ATP release processes in RBCs. Furthermore, I will also describe our recent findings about the roles of the Piezo1 channel, a newly discovered mechanosensitive cation channel in the mechanotransductive ATP release in RBCs. Last, possible functions of RBCs in the regulation of cerebral blood flow will be discussed.

  13. Quantitative analysis of the deformation of polypropylene foam under dynamic loading

    Science.gov (United States)

    Plougonven, Erwan; Bernard, Dominique; Viot, Philippe

    2006-08-01

    A dynamic crash loading experiment is performed on a polypropylene foam. Several interrupted shocks are conducted, in between which microtomographic acquisitions are made, showing the evolution of the sample during its compression. This data can help construct and validate predictive models, although, because this material is multiscale (consitutive grains at the mesoscopic scale are made of microscopic closed cells), image processing is required to extract useful quantitative measures. Such processing is described here, so as to determine a representative volume for each grain of the sample, in order to associate to each grain and to each stage of the compression values such as grain density. This can help build a predictive model at the mesoscopic scale.

  14. Quantitative Map of Proteome Dynamics during Neuronal Differentiation

    NARCIS (Netherlands)

    Frese, Christian K; Mikhaylova, Marina; Stucchi, Riccardo; Gautier, Violette; Liu, Qingyang; Mohammed, Shabaz; Heck, Albert J R; Altelaar, A F Maarten; Hoogenraad, Casper C

    2017-01-01

    Neuronal differentiation is a multistep process that shapes and re-shapes neurons by progressing through several typical stages, including axon outgrowth, dendritogenesis, and synapse formation. To systematically profile proteome dynamics throughout neuronal differentiation, we took cultured rat hip

  15. Protein turnover analysis in Salmonella Typhimurium during infection by dynamic SILAC, Topograph, and quantitative proteomics.

    Science.gov (United States)

    Wang, Zhe; Han, Qiang-Qiang; Zhou, Mao-Tian; Chen, Xi; Guo, Lin

    2016-07-01

    Protein turnover affects protein abundance and phenotypes. Comprehensive investigation of protein turnover dynamics has the potential to provide substantial information about gene expression. Here we report a large-scale protein turnover study in Salmonella Typhimurium during infection by quantitative proteomics. Murine macrophage-like RAW 264.7 cells were infected with SILAC labeled Salmonella. Bacterial cells were extracted after 0, 30, 60, 120, and 240 min. Mass spectrometry analyses yielded information about Salmonella protein turnover dynamics and a software program named Topograph was used for the calculation of protein half lives. The half lives of 311 proteins from intracellular Salmonella were obtained. For bacteria cultured in control medium (DMEM), the half lives for 870 proteins were obtained. The calculated median of protein half lives was 69.13 and 99.30 min for the infection group and the DMEM group, respectively, indicating an elevated protein turnover at the initial stage of infection. Gene ontology analyses revealed that a number of protein functional groups were significantly regulated by infection, including proteins involved in ribosome, periplasmic space, cellular amino acid metabolic process, ion binding, and catalytic activity. The half lives of proteins involved in purine metabolism pathway were found to be significantly shortened during infection.

  16. Making microscopy count: quantitative light microscopy of dynamic processes in living plants.

    Science.gov (United States)

    Fricker, Mark D; Moger, Julian; Littlejohn, George R; Deeks, Michael J

    2016-08-01

    Cell theory has officially reached 350 years of age as the first use of the word 'cell' in a biological context can be traced to a description of plant material by Robert Hooke in his historic publication 'Micrographia: or some physiological definitions of minute bodies'. The 2015 Royal Microscopical Society Botanical Microscopy meeting was a celebration of the streams of investigation initiated by Hooke to understand at the subcellular scale how plant cell function and form arises. Much of the work presented, and Honorary Fellowships awarded, reflected the advanced application of bioimaging informatics to extract quantitative data from micrographs that reveal dynamic molecular processes driving cell growth and physiology. The field has progressed from collecting many pixels in multiple modes to associating these measurements with objects or features that are meaningful biologically. The additional complexity involves object identification that draws on a different type of expertise from computer science and statistics that is often impenetrable to biologists. There are many useful tools and approaches being developed, but we now need more interdisciplinary exchange to use them effectively. In this review we show how this quiet revolution has provided tools available to any personal computer user. We also discuss the oft-neglected issue of quantifying algorithm robustness and the exciting possibilities offered through the integration of physiological information generated by biosensors with object detection and tracking. © 2016 The Authors Journal of Microscopy © 2016 Royal Microscopical Society.

  17. Phosphorylation site dynamics of early T-cell receptor signaling

    DEFF Research Database (Denmark)

    Chylek, Lily A; Akimov, Vyacheslav; Dengjel, Jörn

    2014-01-01

    a systems-level understanding of how these components cooperate to control signaling dynamics, especially during the crucial first seconds of stimulation. Here, we used quantitative proteomics to characterize reshaping of the T-cell phosphoproteome in response to TCR/CD28 co-stimulation, and found...... that diverse dynamic patterns emerge within seconds. We detected phosphorylation dynamics as early as 5 s and observed widespread regulation of key TCR signaling proteins by 30 s. Development of a computational model pointed to the presence of novel regulatory mechanisms controlling phosphorylation of sites...

  18. Redox environment in stem and differentiated cells: A quantitative approach

    Directory of Open Access Journals (Sweden)

    O.G. Lyublinskaya

    2017-08-01

    Full Text Available Stem cells are believed to maintain a specific intracellular redox status through a combination of enhanced removal capacity and limited production of ROS. In the present study, we challenge this assumption by developing a quantitative approach for the analysis of the pro- and antioxidant ability of human embryonic stem cells in comparison with their differentiated descendants, as well as adult stem and non-stem cells. Our measurements showed that embryonic stem cells are characterized by low ROS level, low rate of extracellular hydrogen peroxide removal and low threshold for peroxide-induced cytotoxicity. However, biochemical normalization of these parameters to cell volume/protein leads to matching of normalized values in stem and differentiated cells and shows that tested in the present study cells (human embryonic stem cells and their fibroblast-like progenies, adult mesenchymal stem cells, lymphocytes, HeLa maintain similar intracellular redox status. Based on these observations, we propose to use ROS concentration averaged over the cell volume instead of ROS level as a measure of intracellular redox balance. We show that attempts to use ROS level for comparative analysis of redox status of morphologically different cells could lead to false conclusions. Methods for the assessment of ROS concentration based on flow cytometry analysis with the use of H2DCFDA dye and HyPer, genetically encoded probe for hydrogen peroxide, are discussed.

  19. [Quantitative study on esophageal cytology. I. Quantitative morphologic studies of normal, dysplastic and malignant squamous cells].

    Science.gov (United States)

    Xiang, Y V

    1990-03-01

    On cytosmears of esophageal epithelium of individuals from high-risk area of esophageal cancer squamous epithelial cells, according to standard cytologic diagnostic criteria, can be categorized as normal, hyperplasia, severely dysplastic grade I and grade II, nearly-carcinoma and early carcinoma. Cytosmears from 60 patients, 10 for each category, were studied with a semiautomatic image analysis system. Thirteen morphologic parameters so obtained were further analyzed by computer-based stepwise regression and linear correlation analyses. The results showed that the following 5 parameters could be used to judge the nature of the cells, i.e. a) cytoplasmic area, b) cytoplasmic mean diameter, c) cytoplasmic form factor, d) nuclear form factor and e) N/C ratio. Comparing with cells of the other categories, values of the first 4 parameters for early cancer cells were decreased whereas that of the fifth parameter was significantly increased. From normal to hyperplastic and to dysplastic cells, the nuclear area and mean nuclear diameter were gradually increasing. Therefore, they were the major parameters in judging the degree of hyperplasia and dysplasia. These numerical features of morphologic quantitation conformed with the cytologic diagnostic criteria for cancer, hyperplasia and dysplasia under light microscope. It indicates that visual judgement is relatively accurate and application of the ocular micrometer to measure the cells would make this grading more objective.

  20. Quantitative method of measuring cancer cell urokinase and metastatic potential

    Science.gov (United States)

    Morrison, Dennis R. (Inventor)

    1993-01-01

    The metastatic potential of tumors can be evaluated by the quantitative detection of urokinase and DNA. The cell sample selected for examination is analyzed for the presence of high levels of urokinase and abnormal DNA using analytical flow cytometry and digital image analysis. Other factors such as membrane associated urokinase, increased DNA synthesis rates and certain receptors can be used in the method for detection of potentially invasive tumors.

  1. Macromolecular Dynamics in Red Blood Cells Investigated Using Neutron Spectroscopy

    CERN Document Server

    Stadler, Andreas Maximilian; Demmel, Franz; Artmann, Gerhard; 10.1098/rsif.2010.0306

    2011-01-01

    We present neutron scattering measurements on the dynamics of hemoglobin (Hb) in human red blood cells in vivo. Global and internal Hb dynamics were measured in the ps to ns time- and {\\AA} length-scale using quasielastic neutron backscattering spectroscopy. We observed the cross-over from global Hb short-time to long-time self-diffusion. Both short- and long-time diffusion coefficients agree quantitatively with predicted values from hydrodynamic theory of non-charged hard-sphere suspensions when a bound water fraction of around 0.23g H2O/ g Hb is taken into account. The higher amount of water in the cells facilitates internal protein fluctuations in the ps time-scale when compared to fully hydrated Hb powder. Slower internal dynamics of Hb in red blood cells in the ns time-range were found to be rather similar to results obtained with fully hydrated protein powders, solutions and E. coli cells.

  2. Shape dynamics of growing cell walls

    CERN Document Server

    Banerjee, Shiladitya; Dinner, Aaron R

    2015-01-01

    We introduce a general theoretical framework to study the shape dynamics of actively growing and remodeling surfaces. Using this framework we develop a physical model for growing bacterial cell walls and study the interplay of cell shape with the dynamics of growth and constriction. The model allows us to derive constraints on cell wall mechanical energy based on the observed dynamics of cell shape. We predict that exponential growth in cell size requires a constant amount of cell wall energy to be dissipated per unit volume. We use the model to understand and contrast growth in bacteria with different shapes such as spherical, ellipsoidal, cylindrical and toroidal morphologies. Coupling growth to cell wall constriction, we predict a discontinuous shape transformation, from partial constriction to cell division, as a function of the chemical potential driving cell-wall synthesis. Our model for cell wall energy and shape dynamics relates growth kinetics with cell geometry, and provides a unified framework to d...

  3. A Quantitative Analysis of Photovoltaic Modules Using Halved Cells

    Directory of Open Access Journals (Sweden)

    S. Guo

    2013-01-01

    Full Text Available In a silicon wafer-based photovoltaic (PV module, significant power is lost due to current transport through the ribbons interconnecting neighbour cells. Using halved cells in PV modules is an effective method to reduce the resistive power loss which has already been applied by some major PV manufacturers (Mitsubishi, BP Solar in their commercial available PV modules. As a consequence, quantitative analysis of PV modules using halved cells is needed. In this paper we investigate theoretically and experimentally the difference between modules made with halved and full-size solar cells. Theoretically, we find an improvement in fill factor of 1.8% absolute and output power of 90 mW for the halved cell minimodule. Experimentally, we find an improvement in fill factor of 1.3% absolute and output power of 60 mW for the halved cell module. Also, we investigate theoretically how this effect confers to the case of large-size modules. It is found that the performance increment of halved cell PV modules is even higher for high-efficiency solar cells. After that, the resistive loss of large-size modules with different interconnection schemes is analysed. Finally, factors influencing the performance and cost of industrial halved cell PV modules are discussed.

  4. Lentivirus Live Cell Array for Quantitative Assessment of Gene and Pathway Activation during Myogenic Differentiation of Mesenchymal Stem Cells.

    Directory of Open Access Journals (Sweden)

    Janhavi Moharil

    Full Text Available Stem cell differentiation involves multiple cascades of transcriptional regulation that govern the cell fate. To study the real-time dynamics of this complex process, quantitative and high throughput live cell assays are required. Herein, we developed a lentiviral library of promoters and transcription factor binding sites to quantitatively capture the gene expression dynamics over a period of several days during myogenic differentiation of human mesenchymal stem cells (MSCs harvested from two different anatomic locations, bone marrow and hair follicle. Our results enabled us to monitor the sequential activation of signaling pathways and myogenic gene promoters at various stages of differentiation. In conjunction with chemical inhibitors, the lentiviral array (LVA results also revealed the relative contribution of key signaling pathways that regulate the myogenic differentiation. Our study demonstrates the potential of LVA to monitor the dynamics of gene and pathway activation during MSC differentiation as well as serve as a platform for discovery of novel molecules, genes and pathways that promote or inhibit complex biological processes.

  5. 3D ECG- and respiratory-gated non-contrast-enhanced (CE) perfusion MRI for postoperative lung function prediction in non-small-cell lung cancer patients: A comparison with thin-section quantitative computed tomography, dynamic CE-perfusion MRI, and perfusion scan.

    Science.gov (United States)

    Ohno, Yoshiharu; Seki, Shinichiro; Koyama, Hisanobu; Yoshikawa, Takeshi; Matsumoto, Sumiaki; Takenaka, Daisuke; Kassai, Yoshimori; Yui, Masao; Sugimura, Kazuro

    2015-08-01

    To compare predictive capabilities of non-contrast-enhanced (CE)- and dynamic CE-perfusion MRIs, thin-section multidetector computed tomography (CT) (MDCT), and perfusion scan for postoperative lung function in non-small cell lung cancer (NSCLC) patients. Sixty consecutive pathologically diagnosed NSCLC patients were included and prospectively underwent thin-section MDCT, non-CE-, and dynamic CE-perfusion MRIs and perfusion scan, and had their pre- and postoperative forced expiratory volume in one second (FEV1 ) measured. Postoperative percent FEV1 (po%FEV1 ) was then predicted from the fractional lung volume determined on semiquantitatively assessed non-CE- and dynamic CE-perfusion MRIs, from the functional lung volumes determined on quantitative CT, from the number of segments observed on qualitative CT, and from uptakes detected on perfusion scans within total and resected lungs. Predicted po%FEV1 s were then correlated with actual po%FEV1 s, which were %FEV1 s measured postoperatively. The limits of agreement were also determined. All predicted po%FEV1 s showed significant correlation (0.73 ≤ r ≤ 0.93, P CE-perfusion MRI: 0.3 ± 10.0%, dynamic CE-perfusion MRI: 1.0 ± 10.8%, perfusion scan: 2.2 ± 14.1%, quantitative CT: 1.2 ± 9.0%, qualitative CT: 1.5 ± 10.2%). Non-CE-perfusion MRI may be able to predict postoperative lung function more accurately than qualitatively assessed MDCT and perfusion scan. © 2014 Wiley Periodicals, Inc.

  6. Quantitative Comparison of Abundance Structures of Generalized Communities: From B-Cell Receptor Repertoires to Microbiomes

    Science.gov (United States)

    Saeedghalati, Mohammadkarim; Farahpour, Farnoush; Lange, Anja; Westendorf, Astrid M.; Seifert, Marc; Küppers, Ralf

    2017-01-01

    The community, the assemblage of organisms co-existing in a given space and time, has the potential to become one of the unifying concepts of biology, especially with the advent of high-throughput sequencing experiments that reveal genetic diversity exhaustively. In this spirit we show that a tool from community ecology, the Rank Abundance Distribution (RAD), can be turned by the new MaxRank normalization method into a generic, expressive descriptor for quantitative comparison of communities in many areas of biology. To illustrate the versatility of the method, we analyze RADs from various generalized communities, i.e. assemblages of genetically diverse cells or organisms, including human B cells, gut microbiomes under antibiotic treatment and of different ages and countries of origin, and other human and environmental microbial communities. We show that normalized RADs enable novel quantitative approaches that help to understand structures and dynamics of complex generalized communities. PMID:28114391

  7. Dynamic Quantitative Trait Locus Analysis of Plant Phenomic Data.

    Science.gov (United States)

    Li, Zitong; Sillanpää, Mikko J

    2015-12-01

    Advanced platforms have recently become available for automatic and systematic quantification of plant growth and development. These new techniques can efficiently produce multiple measurements of phenotypes over time, and introduce time as an extra dimension to quantitative trait locus (QTL) studies. Functional mapping utilizes a class of statistical models for identifying QTLs associated with the growth characteristics of interest. A major benefit of functional mapping is that it integrates information over multiple timepoints, and therefore could increase the statistical power for QTL detection. We review the current development of computationally efficient functional mapping methods which provide invaluable tools for analyzing large-scale timecourse data that are readily available in our post-genome era. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. Quantitative validation of PEDFLOW for description of unidirectional pedestrian dynamics

    CERN Document Server

    Zhang, J; Chraibi, M; Loehner, R; Haug, E; Gawenat, B

    2015-01-01

    The results of a systematic quantitative validation of PEDFLOW based on the experimental data from FZJ are presented. Unidirectional flow experiments, totaling 28 different combinations with varying entry, corridor and exit widths, were considered. The condition imposed on PEDFLOW was that all the cases should be run with the same input parameters. The exit times and fundamental diagrams for the measuring region were evaluated and compared. This validation process led to modifications and enhancements of the model underlying PEDFLOW. The preliminary conclusions indicate that the results agree well for densities smaller than 3 m-2 and a good agreement is observed even at high densities for the corridors with bcor = 2.4 m, and bcor = 3.0 m. For densities between 1 and 2 m-2 the specific flow and velocities are underpredicted by PEDFLOW.

  9. Quantitative phase imaging for cell culture quality control.

    Science.gov (United States)

    Kastl, Lena; Isbach, Michael; Dirksen, Dieter; Schnekenburger, Jürgen; Kemper, Björn

    2017-05-01

    The potential of quantitative phase imaging (QPI) with digital holographic microscopy (DHM) for quantification of cell culture quality was explored. Label-free QPI of detached single cells in suspension was performed by Michelson interferometer-based self-interference DHM. Two pancreatic tumor cell lines were chosen as cellular model and analyzed for refractive index, volume, and dry mass under varying culture conditions. Firstly, adequate cell numbers for reliable statistics were identified. Then, to characterize the performance and reproducibility of the method, we compared results from independently repeated measurements and quantified the cellular response to osmolality changes of the cell culture medium. Finally, it was demonstrated that the evaluation of QPI images allows the extraction of absolute cell parameters which are related to cell layer confluence states. In summary, the results show that QPI enables label-free imaging cytometry, which provides novel complementary integral biophysical data sets for sophisticated quantification of cell culture quality with minimized sample preparation. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.

  10. Cells and Stripes: A novel quantitative photo-manipulation technique.

    Science.gov (United States)

    Mistrik, Martin; Vesela, Eva; Furst, Tomas; Hanzlikova, Hana; Frydrych, Ivo; Gursky, Jan; Majera, Dusana; Bartek, Jiri

    2016-01-18

    Laser micro-irradiation is a technology widely used in the DNA damage response, checkpoint signaling, chromatin remodeling and related research fields, to assess chromatin modifications and recruitment of diverse DNA damage sensors, mediators and repair proteins to sites of DNA lesions. While this approach has aided numerous discoveries related to cell biology, maintenance of genome integrity, aging and cancer, it has so far been limited by a tedious manual definition of laser-irradiated subcellular regions, with the ensuing restriction to only a small number of cells treated and analyzed in a single experiment. Here, we present an improved and versatile alternative to the micro-irradiation approach: Quantitative analysis of photo-manipulated samples using innovative settings of standard laser-scanning microscopes. Up to 200 cells are simultaneously exposed to a laser beam in a defined pattern of collinear rays. The induced striation pattern is then automatically evaluated by a simple algorithm, which provides a quantitative assessment of various laser-induced phenotypes in live or fixed cells. Overall, this new approach represents a more robust alternative to existing techniques, and provides a versatile tool for a wide range of applications in biomedicine.

  11. High-Content Screening for Quantitative Cell Biology.

    Science.gov (United States)

    Mattiazzi Usaj, Mojca; Styles, Erin B; Verster, Adrian J; Friesen, Helena; Boone, Charles; Andrews, Brenda J

    2016-08-01

    High-content screening (HCS), which combines automated fluorescence microscopy with quantitative image analysis, allows the acquisition of unbiased multiparametric data at the single cell level. This approach has been used to address diverse biological questions and identify a plethora of quantitative phenotypes of varying complexity in numerous different model systems. Here, we describe some recent applications of HCS, ranging from the identification of genes required for specific biological processes to the characterization of genetic interactions. We review the steps involved in the design of useful biological assays and automated image analysis, and describe major challenges associated with each. Additionally, we highlight emerging technologies and future challenges, and discuss how the field of HCS might be enhanced in the future.

  12. plusTipTracker: Quantitative image analysis software for the measurement of microtubule dynamics.

    Science.gov (United States)

    Applegate, Kathryn T; Besson, Sebastien; Matov, Alexandre; Bagonis, Maria H; Jaqaman, Khuloud; Danuser, Gaudenz

    2011-11-01

    Here we introduce plusTipTracker, a Matlab-based open source software package that combines automated tracking, data analysis, and visualization tools for movies of fluorescently-labeled microtubule (MT) plus end binding proteins (+TIPs). Although +TIPs mark only phases of MT growth, the plusTipTracker software allows inference of additional MT dynamics, including phases of pause and shrinkage, by linking collinear, sequential growth tracks. The algorithm underlying the reconstruction of full MT trajectories relies on the spatially and temporally global tracking framework described in Jaqaman et al. (2008). Post-processing of track populations yields a wealth of quantitative phenotypic information about MT network architecture that can be explored using several visualization modalities and bioinformatics tools included in plusTipTracker. Graphical user interfaces enable novice Matlab users to track thousands of MTs in minutes. In this paper, we describe the algorithms used by plusTipTracker and show how the package can be used to study regional differences in the relative proportion of MT subpopulations within a single cell. The strategy of grouping +TIP growth tracks for the analysis of MT dynamics has been introduced before (Matov et al., 2010). The numerical methods and analytical functionality incorporated in plusTipTracker substantially advance this previous work in terms of flexibility and robustness. To illustrate the enhanced performance of the new software we thus compare computer-assembled +TIP-marked trajectories to manually-traced MT trajectories from the same movie used in Matov et al. (2010).

  13. Exploring neural cell dynamics with digital holographic microscopy

    KAUST Repository

    Marquet, Pierre

    2013-07-11

    In this review, we summarize how the new concept of digital optics applied to the field of holographic microscopy has allowed the development of a reliable and flexible digital holographic quantitative phase microscopy (DH-QPM) technique at the nanoscale particularly suitable for cell imaging. Particular emphasis is placed on the original biological ormation provided by the quantitative phase signal. We present the most relevant DH-QPM applications in the field of cell biology, including automated cell counts, recognition, classification, three-dimensional tracking, discrimination between physiological and pathophysiological states, and the study of cell membrane fluctuations at the nanoscale. In the last part, original results show how DH-QPM can address two important issues in the field of neurobiology, namely, multiple-site optical recording of neuronal activity and noninvasive visualization of dendritic spine dynamics resulting from a full digital holographic microscopy tomographic approach. Copyright © 2013 by Annual Reviews.

  14. Quantitative Adaptation Analytics for Assessing Dynamic Systems of Systems.

    Energy Technology Data Exchange (ETDEWEB)

    Gauthier, John H.; Miner, Nadine E.; Wilson, Michael L.; Le, Hai D.; Kao, Gio K; Melander, Darryl J.; Longsine, Dennis Earl [Sandia National Laboratories, Unknown, Unknown; Vander Meer, Robert Charles,

    2015-01-01

    Our society is increasingly reliant on systems and interoperating collections of systems, known as systems of systems (SoS). These SoS are often subject to changing missions (e.g., nation- building, arms-control treaties), threats (e.g., asymmetric warfare, terrorism), natural environments (e.g., climate, weather, natural disasters) and budgets. How well can SoS adapt to these types of dynamic conditions? This report details the results of a three year Laboratory Directed Research and Development (LDRD) project aimed at developing metrics and methodologies for quantifying the adaptability of systems and SoS. Work products include: derivation of a set of adaptability metrics, a method for combining the metrics into a system of systems adaptability index (SoSAI) used to compare adaptability of SoS designs, development of a prototype dynamic SoS (proto-dSoS) simulation environment which provides the ability to investigate the validity of the adaptability metric set, and two test cases that evaluate the usefulness of a subset of the adaptability metrics and SoSAI for distinguishing good from poor adaptability in a SoS. Intellectual property results include three patents pending: A Method For Quantifying Relative System Adaptability, Method for Evaluating System Performance, and A Method for Determining Systems Re-Tasking.

  15. A quantitative model for assessing community dynamics of pleistocene mammals.

    Science.gov (United States)

    Lyons, S Kathleen

    2005-06-01

    Previous studies have suggested that species responded individualistically to the climate change of the last glaciation, expanding and contracting their ranges independently. Consequently, many researchers have concluded that community composition is plastic over time. Here I quantitatively assess changes in community composition over broad timescales and assess the effect of range shifts on community composition. Data on Pleistocene mammal assemblages from the FAUNMAP database were divided into four time periods (preglacial, full glacial, postglacial, and modern). Simulation analyses were designed to determine whether the degree of change in community composition is consistent with independent range shifts, given the distribution of range shifts observed. Results indicate that many of the communities examined in the United States were more similar through time than expected if individual range shifts were completely independent. However, in each time transition examined, there were areas of nonanalogue communities. I conducted sensitivity analyses to explore how the results were affected by the assumptions of the null model. Conclusions about changes in mammalian distributions and community composition are robust with respect to the assumptions of the model. Thus, whether because of biotic interactions or because of common environmental requirements, community structure through time is more complex than previously thought.

  16. [Diagnostic value of quantitative pharmacokinetic parameters and relative quantitative pharmacokinetic parameters in breast lesions with dynamic contrast-enhanced MRI].

    Science.gov (United States)

    Sun, T T; Liu, W H; Zhang, Y Q; Li, L H; Wang, R; Ye, Y Y

    2017-08-01

    Objective: To explore the differential between the value of dynamic contrast-enhanced MRI quantitative pharmacokinetic parameters and relative pharmacokinetic quantitative parameters in breast lesions. Methods: Retrospective analysis of 255 patients(262 breast lesions) who was obtained by clinical palpation , ultrasound or full-field digital mammography , and then all lessions were pathologically confirmed in Zhongda Hospital, Southeast University from May 2012 to May 2016. A 3.0 T MRI scanner was used to obtain the quantitative MR pharmacokinetic parameters: volume transfer constant (K(trans)), exchange rate constant (k(ep))and extravascular extracellular volume fraction (V(e)). And measured the quantitative pharmacokinetic parameters of normal glands tissues which on the same side of the same level of the lesions; and then calculated the value of relative pharmacokinetic parameters: rK(rans)、rk(ep) and rV(e).To explore the diagnostic value of two pharmacokinetic parameters in differential diagnosis of benign and malignant breast lesions using receiver operating curves and model of logistic regression. Results: (1)There were significant differences between benign lesions and malignant lesions in K(trans) and k(ep) (t=15.489, 15.022, respectively, P0.05). The areas under the ROC curve(AUC)of K(trans), k(ep) and V(e) between malignant and benign lesions were 0.933, 0.948 and 0.387, the sensitivity of K(trans), k(ep) and V(e) were 77.1%, 85.0%, 51.0% , and the specificity of K(trans), k(ep) and V(e) were 96.3%, 93.6%, 60.8% for the differential diagnosis of breast lesions if taken the maximum Youden's index as cut-off. (2)There were significant differences between benign lesions and malignant lesions in rK(trans), rk(ep) and rV(e) (t=14.177, 11.726, 2.477, respectively, Pquantitative pharmacokinetic parameters and the prediction probability of relative quantitative pharmacokinetic parameters(Z=0.867, P=0.195). Conclusion: There was no significant difference between

  17. Nanodomain stabilization dynamics in plasma membranes of biological cells

    Science.gov (United States)

    Das, Tamal; Maiti, Tapas K.; Chakraborty, Suman

    2011-02-01

    We discover that a synergistically amplifying role of stabilizing membrane proteins and continuous lipid recycling can explain the physics governing the stability, polydispersity, and dynamics of lipid raft domains in plasma membranes of biological cells. We establish the conjecture using a generalized order parameter based on theoretical formalism, endorsed by detailed scaling arguments and domain mapping. Quantitative agreements with morphological distributions of raft complexes, as obtained from Förster resonance energy transfer based visualization, support the present theoretical conjecture.

  18. Measuring in action research : four ways of integrating quantitative methods in participatory dynamics

    OpenAIRE

    Martí Olivé, Joel

    2015-01-01

    Background of INCASI Project H2020-MSCA-RISE-2015 GA 691004. WP1: Compilation Although action research uses both qualitative and quantitative methods, few contributions have addressed the specific role of the latter in this kind of research. This paper focuses on how quantitative methods can be integrated with participatory dynamics in action research designs. Four types of integration are defined and exemplified. The paper concludes with some reflections on how the integration of quantita...

  19. Qualitative and quantitative features of Rayleigh-Taylor mixing dynamics

    Science.gov (United States)

    Ramaprabhu, Praveen; Karkhanis, Varad; Lawrie, Andrew; Bhowmick, Aklant; Abarzhi, Snezhana; RTI Collaboration

    2015-11-01

    We consider dynamics of Rayleigh-Taylor (RT) flow in a large aspect ratio three-dimensional domain with square symmetry in the plane for fluids with contrasting densities. In order to quantify the interface evolution from a small amplitude single-mode initial perturbation to advanced stage of RT mixing, we apply numerical simulations using the MOBILE code, theoretical analyses, including group theory and momentum model, as well as parameters describing the interplay between acceleration and turbulence. We find: In RT flow, the fluid motion is intense near the interface and is negligible far from the interface. At late times the growth rates of RT bubbles and spikes may increase without a corresponding increase of length-scales in the direction normal to acceleration. The parameters describing the interplay between acceleration and turbulence in RT mixing are shown to scale well with the flow Reynolds number and Froude number.

  20. Quantitative Phosphoproteomic Analysis of T-Cell Receptor Signaling.

    Science.gov (United States)

    Ahsan, Nagib; Salomon, Arthur R

    2017-01-01

    TCR signaling critically depends on protein phosphorylation across many proteins. Localization of each phosphorylation event relative to the T-cell receptor (TCR) and canonical T-cell signaling proteins will provide clues about the structure of TCR signaling networks. Quantitative phosphoproteomic analysis by mass spectrometry provides a wide-scale view of cellular phosphorylation networks. However, analysis of phosphorylation by mass spectrometry is still challenging due to the relative low abundance of phosphorylated proteins relative to all proteins and the extraordinary diversity of phosphorylation sites across the proteome. Highly selective enrichment of phosphorylated peptides is essential to provide the most comprehensive view of the phosphoproteome. Optimization of phosphopeptide enrichment methods coupled with highly sensitive mass spectrometry workflows significantly improves the sequencing depth of the phosphoproteome to over 10,000 unique phosphorylation sites from complex cell lysates. Here we describe a step-by-step method for phosphoproteomic analysis that has achieved widespread success for identification of serine, threonine, and tyrosine phosphorylation. Reproducible quantification of relative phosphopeptide abundance is provided by intensity-based label-free quantitation. An ideal set of mass spectrometry analysis parameters is also provided that optimize the yield of identified sites. We also provide guidelines for the bioinformatic analysis of this type of data to assess the quality of the data and to comply with proteomic data reporting requirements.

  1. Quantitative phosphoproteomics of proteasome inhibition in multiple myeloma cells.

    Directory of Open Access Journals (Sweden)

    Feng Ge

    Full Text Available BACKGROUND: The proteasome inhibitor bortezomib represents an important advance in the treatment of multiple myeloma (MM. Bortezomib inhibits the activity of the 26S proteasome and induces cell death in a variety of tumor cells; however, the mechanism of cytotoxicity is not well understood. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the differential phosphoproteome upon proteasome inhibition by using stable isotope labeling by amino acids in cell culture (SILAC in combination with phosphoprotein enrichment and LC-MS/MS analysis. In total 233 phosphoproteins were identified and 72 phosphoproteins showed a 1.5-fold or greater change upon bortezomib treatment. The phosphoproteins with expression alterations encompass all major protein classes, including a large number of nucleic acid binding proteins. Site-specific phosphopeptide quantitation revealed that Ser38 phosphorylation on stathmin increased upon bortezomib treatment, suggesting new mechanisms associated to bortezomib-induced apoptosis in MM cells. Further studies demonstrated that stathmin phosphorylation profile was modified in response to bortezomib treatment and the regulation of stathmin by phosphorylation at specific Ser/Thr residues participated in the cellular response induced by bortezomib. CONCLUSIONS/SIGNIFICANCE: Our systematic profiling of phosphorylation changes in response to bortezomib treatment not only advanced the global mechanistic understanding of the action of bortezomib on myeloma cells but also identified previously uncharacterized signaling proteins in myeloma cells.

  2. Nanoliter scale microbioreactor array for quantitative cell biology.

    Science.gov (United States)

    Lee, Philip J; Hung, Paul J; Rao, Vivek M; Lee, Luke P

    2006-05-05

    A nanoliter scale microbioreactor array was designed for multiplexed quantitative cell biology. An addressable 8 x 8 array of three nanoliter chambers was demonstrated for observing the serum response of HeLa human cancer cells in 64 parallel cultures. The individual culture unit was designed with a "C" shaped ring that effectively decoupled the central cell growth regions from the outer fluid transport channels. The chamber layout mimics physiological tissue conditions by implementing an outer channel for convective "blood" flow that feeds cells through diffusion into the low shear "interstitial" space. The 2 microm opening at the base of the "C" ring established a differential fluidic resistance up to 3 orders of magnitude greater than the fluid transport channel within a single mold microfluidic device. Three-dimensional (3D) finite element simulation were used to predict fluid transport properties based on chamber dimensions and verified experimentally. The microbioreactor array provided a continuous flow culture environment with a Peclet number (0.02) and shear stress (0.01 Pa) that approximated in vivo tissue conditions without limiting mass transport (10 s nutrient turnover). This microfluidic design overcomes the major problems encountered in multiplexing nanoliter culture environments by enabling uniform cell loading, eliminating shear, and pressure stresses on cultured cells, providing stable control of fluidic addressing, and permitting continuous on-chip optical monitoring.

  3. Qualitative and quantitative analysis of stability and instability dynamics of positive lattice solitons.

    Science.gov (United States)

    Sivan, Y; Fibich, G; Ilan, B; Weinstein, M I

    2008-10-01

    We present a unified approach for qualitative and quantitative analysis of stability and instability dynamics of positive bright solitons in multidimensional focusing nonlinear media with a potential (lattice), which can be periodic, periodic with defects, quasiperiodic, single waveguide, etc. We show that when the soliton is unstable, the type of instability dynamic that develops depends on which of two stability conditions is violated. Specifically, violation of the slope condition leads to a focusing instability, whereas violation of the spectral condition leads to a drift instability. We also present a quantitative approach that allows one to predict the stability and instability strength.

  4. Quantitative flow analysis of swimming dynamics with coherent Lagrangian vortices

    Science.gov (United States)

    Huhn, F.; van Rees, W. M.; Gazzola, M.; Rossinelli, D.; Haller, G.; Koumoutsakos, P.

    2015-08-01

    Undulatory swimmers flex their bodies to displace water, and in turn, the flow feeds back into the dynamics of the swimmer. At moderate Reynolds number, the resulting flow structures are characterized by unsteady separation and alternating vortices in the wake. We use the flow field from simulations of a two-dimensional, incompressible viscous flow of an undulatory, self-propelled swimmer and detect the coherent Lagrangian vortices in the wake to dissect the driving momentum transfer mechanisms. The detected material vortex boundary encloses a Lagrangian control volume that serves to track back the vortex fluid and record its circulation and momentum history. We consider two swimming modes: the C-start escape and steady anguilliform swimming. The backward advection of the coherent Lagrangian vortices elucidates the geometry of the vorticity field and allows for monitoring the gain and decay of circulation and momentum transfer in the flow field. For steady swimming, momentum oscillations of the fish can largely be attributed to the momentum exchange with the vortex fluid. For the C-start, an additionally defined jet fluid region turns out to balance the high momentum change of the fish during the rapid start.

  5. Dynamic Quantitative Sensory Testing to Characterize Central Pain Processing

    Science.gov (United States)

    Mackey, Ian G.; Dixon, Eric A.; Johnson, Kevin; Kong, Jiang-Ti

    2017-01-01

    Central facilitation and modulation of incoming nociceptive signals play an important role in the perception of pain. Disruption in central pain processing is present in many chronic pain conditions and can influence responses to specific therapies. Thus, the ability to precisely describe the state of central pain processing has profound clinical significance in both prognosis and prediction. Because it is not practical to record neuronal firings directly in the human spinal cord, surrogate behavior tests become an important tool to assess the state of central pain processing. Dynamic QST is one such test, and can probe both the ascending facilitation and descending modulation of incoming nociceptive signals via TS and CPM, respectively. Due to the large between-individual variability in the sensitivity to noxious signals, standardized TS and CPM tests may not yield any meaningful data in up to 50% of the population due to floor or ceiling effects. We present methodologies to individualize TS and CPM so we can capture these measures in a broader range of individuals than previously possible. We have used these methods successfully in several studies at the lab, and data from one ongoing study will be presented to demonstrate feasibility and potential applications of the methods. PMID:28287532

  6. Quantitative analysis of the individual dynamics of Psychology theses

    Directory of Open Access Journals (Sweden)

    Robles, Jaime R.

    2009-12-01

    Full Text Available Three cohorts of undergraduate psychology theses (n = 57 performed by last year undergraduate psychology students from Universidad Católica Andrés Bello, were monitored using 5 longitudinal measurements of progression. A Generalized Additive Model, to predict the completion time of the theses, is tested against two completion times: early and delayed. Effect size measures favor a multiple dimension model over a global progress model. The trajectory of the indicators through the 5 measurements allows the differentiation between early and delayed completion. The completion probabilities estimated by the dimensional model allow the identification of differential oscillation levels for the distinct completion times. The initial progression indicators allow the prediction of early completion with a 71% success rate, while the final measurement shows a success rate of 89%. The results support the effectiveness of the supervisory system and the analysis of the progression dynamics of the theses from a task-delay model, focused on the relationship between the amount of task completion and the deadlines

  7. Quantitative flow analysis of swimming dynamics with coherent Lagrangian vortices.

    Science.gov (United States)

    Huhn, F; van Rees, W M; Gazzola, M; Rossinelli, D; Haller, G; Koumoutsakos, P

    2015-08-01

    Undulatory swimmers flex their bodies to displace water, and in turn, the flow feeds back into the dynamics of the swimmer. At moderate Reynolds number, the resulting flow structures are characterized by unsteady separation and alternating vortices in the wake. We use the flow field from simulations of a two-dimensional, incompressible viscous flow of an undulatory, self-propelled swimmer and detect the coherent Lagrangian vortices in the wake to dissect the driving momentum transfer mechanisms. The detected material vortex boundary encloses a Lagrangian control volume that serves to track back the vortex fluid and record its circulation and momentum history. We consider two swimming modes: the C-start escape and steady anguilliform swimming. The backward advection of the coherent Lagrangian vortices elucidates the geometry of the vorticity field and allows for monitoring the gain and decay of circulation and momentum transfer in the flow field. For steady swimming, momentum oscillations of the fish can largely be attributed to the momentum exchange with the vortex fluid. For the C-start, an additionally defined jet fluid region turns out to balance the high momentum change of the fish during the rapid start.

  8. A quantitative analysis of stem cell homeostasis in the Arabidopsis columella root cap.

    Science.gov (United States)

    Hong, Jing Han; Chu, Huangwei; Zhang, Chen; Ghosh, Dipanjana; Gong, Ximing; Xu, Jian

    2015-01-01

    The Lugol's staining method has been widely used to detect changes in the maintenance of stem cell fate in the columella root cap of Arabidopsis roots since the late 1990s. However, various limitations of this method demand for additional or complementary new approaches. For instance, it is unable to reveal the division rate of columella root cap stem cells. Here we report that, by labeling dividing stem cells with 5-ethynyl-2'-deoxyuridine (EdU), the number and distribution of their labeled progeny can be studied so that the division rate of stem cells can be measured quantitatively and in addition, that the progression of stem cell progeny differentiation can be assessed in combination with Lugol's staining. EdU staining takes few hours and visualization of the stain characteristics of columella root cap can be performed easily under confocal microscopes. This simple technology, when used together with Lugol's staining, provides a novel quantitative method to study the dynamics of stem cell behavior that govern homeostasis in the Arabidopsis columella root cap.

  9. A Quantitative Analysis of Stem Cell Homeostasis in the Arabidopsis Columella Root Cap

    Directory of Open Access Journals (Sweden)

    Jing Han eHong

    2015-03-01

    Full Text Available The Lugol’s staining method has been widely used to detect changes in the maintenance of stem cell fate in the columella root cap of Arabidopsis roots since the late ‘90s. However, various limitations of this method demand for additional or complementary new approaches. For instance, it is unable to reveal the division rate of columella root cap stem cells. Here we report that, by labelling dividing stem cells with 5-ethynyl-2´-deoxyuridine (EdU, the number and distribution of their labeled progeny can be studied so that the division rate of stem cells can be measured quantitatively and in addition, that the progression of stem cell progeny differentiation can be assessed in combination with Lugol’s staining. EdU staining takes few hours and visualization of the stain characteristics of columella root cap can be performed easily under confocal microscopes. This simple technology, when used together with Lugol’s staining, provides a novel quantitative method to study the dynamics of stem cell behaviour that govern homeostasis in the Arabidopsis columella root cap.

  10. About supramolecular systems for dynamically probing cells

    NARCIS (Netherlands)

    Brinkmann, J.; Cavatorta, E.; Sankaran, S.; Schmidt, B.; van Weerd, Jasper; Jonkheijm, Pascal

    2014-01-01

    This article reviews the state of the art in the development of strategies for generating supramolecular systems for dynamic cell studies. Dynamic systems are crucial to further our understanding of cell biology and are consequently at the heart of many medical applications. Increasing interest has

  11. Tunable PIE and synchronized gating detections by FastFLIM for quantitative microscopy measurements of fast dynamics of single molecules

    Science.gov (United States)

    Sun, Yuansheng; Coskun, Ulas; Ferreon, Allan Chris; Barbieri, Beniamino; Liao, Shih-Chu Jeff

    2016-03-01

    The crosstalk between two fluorescent species causes problems in fluorescence microscopy imaging, especially for quantitative measurements such as co-localization, Förster resonance energy transfer (FRET), fluorescence cross correlation spectroscopy (FCCS). In laser scanning confocal microscopy, the lasers can be switched on and off by acousto-optic tunable filters (AOTF) in the microsecond scale for alternative line scanning in order to avoid the crosstalk while minimizing the time delay between two lasers on the same pixel location. In contrast, the pulsed interleaved excitation (PIE) technique synchronizes two pulsed lasers of different wavelengths in the nanosecond scale to enable measuring superfast dynamics of two fluorescent species simultaneously and yet quantitatively without the crosstalk contamination. This feature is critical for many cell biology applications, e.g. accurate determination of stoichiometry in FRET measurements for studying protein-protein interactions or cell signal events, detection of weaker bindings in FCCS by eliminating the false cross correlation due to the crosstalk. The PIE has been used with the time correlated single photon counting (TCSPC) electronics. Here, we describe a novel PIE development using the digital frequency domain (DFD) technique -- FastFLIM, which provides tunable PIE setups and synchronized gating detections, tailored and optimized to specific applications. A few PIE setups by FastFLIM and measurement examples are described. Combined with the sensitivity of Alba and Q2 systems, the PIE allowed us to quantitatively measure the fast dynamics of single molecules.

  12. Quantitative comparisons of analogue models of brittle wedge dynamics

    Science.gov (United States)

    Schreurs, Guido

    2010-05-01

    Analogue model experiments are widely used to gain insights into the evolution of geological structures. In this study, we present a direct comparison of experimental results of 14 analogue modelling laboratories using prescribed set-ups. A quantitative analysis of the results will document the variability among models and will allow an appraisal of reproducibility and limits of interpretation. This has direct implications for comparisons between structures in analogue models and natural field examples. All laboratories used the same frictional analogue materials (quartz and corundum sand) and prescribed model-building techniques (sieving and levelling). Although each laboratory used its own experimental apparatus, the same type of self-adhesive foil was used to cover the base and all the walls of the experimental apparatus in order to guarantee identical boundary conditions (i.e. identical shear stresses at the base and walls). Three experimental set-ups using only brittle frictional materials were examined. In each of the three set-ups the model was shortened by a vertical wall, which moved with respect to the fixed base and the three remaining sidewalls. The minimum width of the model (dimension parallel to mobile wall) was also prescribed. In the first experimental set-up, a quartz sand wedge with a surface slope of ˜20° was pushed by a mobile wall. All models conformed to the critical taper theory, maintained a stable surface slope and did not show internal deformation. In the next two experimental set-ups, a horizontal sand pack consisting of alternating quartz sand and corundum sand layers was shortened from one side by the mobile wall. In one of the set-ups a thin rigid sheet covered part of the model base and was attached to the mobile wall (i.e. a basal velocity discontinuity distant from the mobile wall). In the other set-up a basal rigid sheet was absent and the basal velocity discontinuity was located at the mobile wall. In both types of experiments

  13. Quantitative description of electroluminescence images of polymer solar cells

    Energy Technology Data Exchange (ETDEWEB)

    Seeland, Marco; Roesch, Roland; Hoppe, Harald [Institute of Physics, Ilmenau University of Technology, Ilmenau (Germany)

    2011-07-01

    We present a quantitative description of electroluminescence images obtained on organic solar cells, which is based on a device modeling employing a network of interconnected microdiodes. The equivalent circuit network model takes interface and bulk resistances as well as the sheet resistance of the transparent electrode into account. The application of this model allows direct calculation of the lateral current and voltage distribution as well as determination of internal resistances and the sheet resistance of the higher resistive electrode. Furthermore, we have extended the microdiode-model to also describe and predict current voltage characteristics for devices under illumination. Finally the local nature of this description enables important conclusions concerning the geometry dependent performance of thin film solar cells.

  14. Combining PALM and SOFI for quantitative imaging of focal adhesions in living cells

    Science.gov (United States)

    Deschout, Hendrik; Lukes, Tomas; Sharipov, Azat; Feletti, Lely; Lasser, Theo; Radenovic, Aleksandra

    2017-02-01

    Focal adhesions are complicated assemblies of hundreds of proteins that allow cells to sense their extracellular matrix and adhere to it. Although most focal adhesion proteins have been identified, their spatial organization in living cells remains challenging to observe. Photo-activated localization microscopy (PALM) is an interesting technique for this purpose, especially since it allows estimation of molecular parameters such as the number of fluorophores. However, focal adhesions are dynamic entities, requiring a temporal resolution below one minute, which is difficult to achieve with PALM. In order to address this problem, we merged PALM with super-resolution optical fluctuation imaging (SOFI) by applying both techniques to the same data. Since SOFI tolerates an overlap of single molecule images, it can improve the temporal resolution compared to PALM. Moreover, an adaptation called balanced SOFI (bSOFI) allows estimation of molecular parameters, such as the fluorophore density. We therefore performed simulations in order to assess PALM and SOFI for quantitative imaging of dynamic structures. We demonstrated the potential of our PALM-SOFI concept as a quantitative imaging framework by investigating moving focal adhesions in living cells.

  15. Enlightening intracellular complexity of living cells with quantitative phase microscopy

    Science.gov (United States)

    Martinez Torres, C.; Laperrousaz, B.; Berguiga, L.; Boyer Provera, E.; Elezgaray, J.; Nicolini, F. E.; Maguer-Satta, V.; Arneodo, A.; Argoul, F.

    2016-03-01

    The internal distribution of refractive indices (RIs) of a living cell is much more complex than usually admitted in multi-shell models. The reconstruction of RI maps from single phase images has rarely been achieved for several reasons: (i) we still have very little knowledge of the impact of internal macromolecular complexes on the local RI and (ii) phase changes produced by light propagation through the sample are mixed with diffraction effects by internal cell bodies. We propose the implementation a 2D wavelet-based contour chain detection method to distinguish internal boundaries thanks to their greatest optical path difference gradients. These contour chains correspond to the highest image phase contrast and follow the local RI inhomogeneities linked to the intracellular structural intricacy. Their statistics and spatial distribution are morphological indicators for distinguishing cells of different origins and to follow their transformation in pathologic situations. We use this method to compare non adherent blood cells from primary and laboratory culture origins, in healthy and pathological situations (chronic myelogenous leukaemia). In a second part of this presentation, we concentrate on the temporal dynamics of the phase contour chains and we discuss the spectral decomposition of their dynamics in both health and disease.

  16. Dynamic heterogeneity and DNA methylation in embryonic stem cells.

    KAUST Repository

    Singer, Zakary S

    2014-07-01

    Cell populations can be strikingly heterogeneous, composed of multiple cellular states, each exhibiting stochastic noise in its gene expression. A major challenge is to disentangle these two types of variability and to understand the dynamic processes and mechanisms that control them. Embryonic stem cells (ESCs) provide an ideal model system to address this issue because they exhibit heterogeneous and dynamic expression of functionally important regulatory factors. We analyzed gene expression in individual ESCs using single-molecule RNA-FISH and quantitative time-lapse movies. These data discriminated stochastic switching between two coherent (correlated) gene expression states and burst-like transcriptional noise. We further showed that the "2i" signaling pathway inhibitors modulate both types of variation. Finally, we found that DNA methylation plays a key role in maintaining these metastable states. Together, these results show how ESC gene expression states and dynamics arise from a combination of intrinsic noise, coherent cellular states, and epigenetic regulation.

  17. Quantitative phase imaging of cell division in yeast cells and E.coli using digital holographic microscopy

    Science.gov (United States)

    Pandiyan, Vimal Prabhu; John, Renu

    2015-12-01

    Digital holographic microscope (DHM) is an emerging quantitative phase imaging technique with unique imaging scales and resolutions leading to multitude of applications. DHM is promising as a novel investigational and applied tool for cell imaging, studying the morphology and real time dynamics of cells and a number of related applications. The use of numerical propagation and computational digital optics offer unique flexibility to tune the depth of focus, and compensate for image aberrations. In this work, we report imaging the dynamics of cell division in E.coli and yeast cells using a DHM platform. We demonstrate 3-D and depth imaging as well as reconstruction of phase profiles of E.coli and yeast cells using the system. We record a digital hologram of E.coli and yeast cells and reconstruct the image using Fresnel propagation algorithm. We also use aberration compensation algorithms for correcting the aberrations that are introduced by the microscope objective in the object path using linear least square fitting techniques. This work demonstrates the strong potential of a DHM platform in 3-D live cell imaging, fast clinical quantifications and pathological applications.

  18. An improved UPLC-MS/MS platform for quantitative analysis of glycerophosphoinositol in mammalian cells.

    Directory of Open Access Journals (Sweden)

    Laura Grauso

    Full Text Available The glycerophosphoinositols constitute a class of biologically active lipid-derived mediators whose intracellular levels are modulated during physiological and pathological cell processes. Comprehensive assessment of the role of these compounds expands beyond the cellular biology of lipids and includes rapid and unambiguous measurement in cells and tissues. Here we describe a sensitive and simple liquid chromatography-tandem mass spectrometry (LC-MS/MS method for quantitative analysis of the most abundant among these phosphoinositide derivatives in mammalian cells, the glycerophosphoinositol (GroPIns. The method has been developed in mouse Raw 264.7 macrophages with limits of quantitation at 3 ng/ml. Validation on the same cell line showed excellent response in terms of linear dynamic range (from 3 to 3,000 ng/ml, intra-day and inter-day precision (coefficient of variation ≤ 7.10% and accuracy (between 98.1 and 109.0% in the range 10-320 ng/ml. As proof of concept, a simplified analytical platform based on this method and external calibration was also tested on four stimulated and unstimulated cell lines, including Raw 264.7 macrophages, Jurkat T-cells, A375MM melanoma cells and rat basophilic leukemia RBL-2H3 cells. The results indicate a wide variation in GroPIns levels among different cell lines and stimulation conditions, although the measurements were always in line with the literature. No significant matrix effects were observed thus indicating that the here proposed method can be of general use for similar determinations in cells of different origin.

  19. Intergenerational continuity of cell shape dynamics in Caulobacter crescentus

    Science.gov (United States)

    Wright, Charles S.; Banerjee, Shiladitya; Iyer-Biswas, Srividya; Crosson, Sean; Dinner, Aaron R.; Scherer, Norbert F.

    2015-03-01

    We investigate the intergenerational shape dynamics of single Caulobacter crescentus cells using a novel combination of imaging techniques and theoretical modeling. We determine the dynamics of cell pole-to-pole lengths, cross-sectional widths, and medial curvatures from high accuracy measurements of cell contours. Moreover, these shape parameters are determined for over 250 cells across approximately 10000 total generations, which affords high statistical precision. Our data and model show that constriction is initiated early in the cell cycle and that its dynamics are controlled by the time scale of exponential longitudinal growth. Based on our extensive and detailed growth and contour data, we develop a minimal mechanical model that quantitatively accounts for the cell shape dynamics and suggests that the asymmetric location of the division plane reflects the distinct mechanical properties of the stalked and swarmer poles. Furthermore, we find that the asymmetry in the division plane location is inherited from the previous generation. We interpret these results in terms of the current molecular understanding of shape, growth, and division of C. crescentus.

  20. Performance comparison between static and dynamic cardiac CT on perfusion quantitation and patient classification tasks

    Science.gov (United States)

    Bindschadler, Michael; Modgil, Dimple; Branch, Kelley R.; La Riviere, Patrick J.; Alessio, Adam M.

    2015-03-01

    Cardiac CT acquisitions for perfusion assessment can be performed in a dynamic or static mode. In this simulation study, we evaluate the relative classification and quantification performance of these modes for assessing myocardial blood flow (MBF). In the dynamic method, a series of low dose cardiac CT acquisitions yields data on contrast bolus dynamics over time; these data are fit with a model to give a quantitative MBF estimate. In the static method, a single CT acquisition is obtained, and the relative CT numbers in the myocardium are used to infer perfusion states. The static method does not directly yield a quantitative estimate of MBF, but these estimates can be roughly approximated by introducing assumed linear relationships between CT number and MBF, consistent with the ways such images are typically visually interpreted. Data obtained by either method may be used for a variety of clinical tasks, including 1) stratifying patients into differing categories of ischemia and 2) using the quantitative MBF estimate directly to evaluate ischemic disease severity. Through simulations, we evaluate the performance on each of these tasks. The dynamic method has very low bias in MBF estimates, making it particularly suitable for quantitative estimation. At matched radiation dose levels, ROC analysis demonstrated that the static method, with its high bias but generally lower variance, has superior performance in stratifying patients, especially for larger patients.

  1. Dynamics of Traction Stress Field during Cell Division

    Science.gov (United States)

    Tanimoto, Hirokazu; Sano, Masaki

    2012-12-01

    We report a quantitative measurement of traction stress exerted by dividing eukaryotic cells. The stress field was highly dynamic and sequentially changed as follows: (1) strong and localized as two spots, (2) weak and broadly distributed, and (3) strong and localized as four spots. At the final stage of cytokinesis, the dividing cells exerted strong tensile force on the intercellular bridge. The asymmetry of the traction stress and the orientation of the division axis matched throughout the division process, suggesting the possible role of the mechanical force as a “store” of the orientational information.

  2. Quantitation of ranaviruses in cell culture and tissue samples.

    Science.gov (United States)

    Holopainen, Riikka; Honkanen, Jarno; Jensen, Britt Bang; Ariel, Ellen; Tapiovaara, Hannele

    2011-01-01

    A quantitative real-time PCR (qPCR) based on a standard curve was developed for detection and quantitation of ranaviruses. The target gene for the qPCR was viral DNA polymerase (DNApol). All ten ranavirus isolates studied (Epizootic haematopoietic necrosis virus, EHNV; European catfish virus, ECV; European sheatfish virus, ESV; Frog virus 3, FV3; Bohle iridovirus, BIV; Doctor fish virus, DFV; Guppy virus 6, GV6; Pike-perch iridovirus, PPIV; Rana esculenta virus Italy 282/I02, REV282/I02 and Short-finned eel ranavirus, SERV) were detected with the qPCR assay. In addition, two fish cell lines - epithelioma papulosum cyprini (EPC) and bluegill fry (BF-2) - were infected with four of the isolates (EHNV, ECV, FV3 and DFV), and the viral quantity was determined from seven time points during the first three days after infection. The qPCR was also used to determine the viral load in tissue samples from pike (Esox lucius) fry challenged experimentally with EHNV. Copyright © 2010 Elsevier B.V. All rights reserved.

  3. Quantitative transcription dynamic analysis reveals candidate genes and key regulators for ethanol tolerance in Saccharomyces cerevisiae.

    Science.gov (United States)

    Ma, Menggen; Liu, Lewis Z

    2010-06-10

    Derived from our lignocellulosic conversion inhibitor-tolerant yeast, we generated an ethanol-tolerant strain Saccharomyces cerevisiae NRRL Y-50316 by enforced evolutionary adaptation. Using a newly developed robust mRNA reference and a master equation unifying gene expression data analyses, we investigated comparative quantitative transcription dynamics of 175 genes selected from previous studies for an ethanol-tolerant yeast and its closely related parental strain. A highly fitted master equation was established and applied for quantitative gene expression analyses using pathway-based qRT-PCR array assays. The ethanol-tolerant Y-50316 displayed significantly enriched background of mRNA abundance for at least 35 genes without ethanol challenge compared with its parental strain Y-50049. Under the ethanol challenge, the tolerant Y-50316 responded in consistent expressions over time for numerous genes belonging to groups of heat shock proteins, trehalose metabolism, glycolysis, pentose phosphate pathway, fatty acid metabolism, amino acid biosynthesis, pleiotropic drug resistance gene family and transcription factors. The parental strain showed repressed expressions for many genes and was unable to withstand the ethanol stress and establish a viable culture and fermentation. The distinct expression dynamics between the two strains and their close association with cell growth, viability and ethanol fermentation profiles distinguished the tolerance-response from the stress-response in yeast under the ethanol challenge. At least 82 genes were identified as candidate and key genes for ethanol-tolerance and subsequent fermentation under the stress. Among which, 36 genes were newly recognized by the present study. Most of the ethanol-tolerance candidate genes were found to share protein binding motifs of transcription factors Msn4p/Msn2p, Yap1p, Hsf1p and Pdr1p/Pdr3p. Enriched background of transcription abundance and enhanced expressions of ethanol-tolerance genes

  4. Quantitative transcription dynamic analysis reveals candidate genes and key regulators for ethanol tolerance in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Ma Menggen

    2010-06-01

    Full Text Available Abstract Background Derived from our lignocellulosic conversion inhibitor-tolerant yeast, we generated an ethanol-tolerant strain Saccharomyces cerevisiae NRRL Y-50316 by enforced evolutionary adaptation. Using a newly developed robust mRNA reference and a master equation unifying gene expression data analyses, we investigated comparative quantitative transcription dynamics of 175 genes selected from previous studies for an ethanol-tolerant yeast and its closely related parental strain. Results A highly fitted master equation was established and applied for quantitative gene expression analyses using pathway-based qRT-PCR array assays. The ethanol-tolerant Y-50316 displayed significantly enriched background of mRNA abundance for at least 35 genes without ethanol challenge compared with its parental strain Y-50049. Under the ethanol challenge, the tolerant Y-50316 responded in consistent expressions over time for numerous genes belonging to groups of heat shock proteins, trehalose metabolism, glycolysis, pentose phosphate pathway, fatty acid metabolism, amino acid biosynthesis, pleiotropic drug resistance gene family and transcription factors. The parental strain showed repressed expressions for many genes and was unable to withstand the ethanol stress and establish a viable culture and fermentation. The distinct expression dynamics between the two strains and their close association with cell growth, viability and ethanol fermentation profiles distinguished the tolerance-response from the stress-response in yeast under the ethanol challenge. At least 82 genes were identified as candidate and key genes for ethanol-tolerance and subsequent fermentation under the stress. Among which, 36 genes were newly recognized by the present study. Most of the ethanol-tolerance candidate genes were found to share protein binding motifs of transcription factors Msn4p/Msn2p, Yap1p, Hsf1p and Pdr1p/Pdr3p. Conclusion Enriched background of transcription abundance

  5. Pea border cell maturation and release involve complex cell wall structural dynamics

    DEFF Research Database (Denmark)

    Mravec, Jozef; Guo, Xiaoyuan; Hansen, Aleksander Riise

    2017-01-01

    The adhesion of plant cells is vital for support and protection of the plant body and is maintained by a variety of molecular associations between cell wall components. In some specialized cases though, plant cells are programmed to detach and root cap-derived border cells are examples of this....... Border cells (in some species known as border-like cells) provide an expendable barrier between roots and the environment. Their maturation and release is an important but poorly characterized cell separation event. To gain a deeper insight into the complex cellular dynamics underlying this process, we...... undertook a systematic, detailed analysis of pea (Pisum sativum) root tip cell walls. Our study included immuno-carbohydrate microarray profiling, monosaccharide composition determination, Fourier-transformed infrared microspectroscopy (FT-IR), quantitative RT-PCR of cell wall biosynthetic genes, analysis...

  6. Quantitative Analysis of AGV System in FMS Cell Layout

    Directory of Open Access Journals (Sweden)

    B. Ramana

    1997-01-01

    Full Text Available Material handling is a specialised activity for a modern manufacturing concern. Automated guided vehicles (AGVs are invariably used for material handling in flexible manufacturing Systems (FMSs due to their flexibility. The quantitative analysis of an AGV system is useful for determining the material flow rates, operation times, length of delivery, length of empty move of AGV and the number of AGVs required for a typical FMS cell layout. The efficiency of the material handling system, such as AGV can be improved by reducing the length of empty move. The length of empty move of AGV depends upon despatching and scheduling methods. If these methods of AGVs are not properly planned, the length of empty move of AGV is greater than the length of delivery .This results in increase in material handling time which in turn increases the number of AGVs required in FMS cell. This paper presents a method for optimising the length of empty travel of AGV in a typical FMS cell layout.

  7. Quantitative genetic-interaction mapping in mammalian cells

    Science.gov (United States)

    Roguev, Assen; Talbot, Dale; Negri, Gian Luca; Shales, Michael; Cagney, Gerard; Bandyopadhyay, Sourav; Panning, Barbara; Krogan, Nevan J

    2013-01-01

    Mapping genetic interactions (GIs) by simultaneously perturbing pairs of genes is a powerful tool for understanding complex biological phenomena. Here we describe an experimental platform for generating quantitative GI maps in mammalian cells using a combinatorial RNA interference strategy. We performed ~11,000 pairwise knockdowns in mouse fibroblasts, focusing on 130 factors involved in chromatin regulation to create a GI map. Comparison of the GI and protein-protein interaction (PPI) data revealed that pairs of genes exhibiting positive GIs and/or similar genetic profiles were predictive of the corresponding proteins being physically associated. The mammalian GI map identified pathways and complexes but also resolved functionally distinct submodules within larger protein complexes. By integrating GI and PPI data, we created a functional map of chromatin complexes in mouse fibroblasts, revealing that the PAF complex is a central player in the mammalian chromatin landscape. PMID:23407553

  8. Quantitative Determination of Ceramide Molecular Species in Dendritic Cells

    Directory of Open Access Journals (Sweden)

    Samar Al Makdessi

    2016-09-01

    Full Text Available Background/Aims: The activation of acid sphingomyelinase by cellular stress or receptors or the de novo synthesis lead to the formation of ceramide (N-acylsphingosine, which in turn modifies the biophysical properties of cellular membrane and greatly amplifies the intensity of the initial signal. Ceramide, which acts by re-organizing a given signalosome rather than being a second messenger, has many functions in infection biology, cancer, cardiovascular syndromes, and immune regulation. Experimental studies on the infection of human cells with different bacterial agents demonstrated the activation of the acid sphingomyelinase/ceramide system. Moreover, the release of ceramide was found to be a requisite for the uptake of the pathogen. Considering the particular importance of the cellular role of ceramide, it was necessary to develop sensitive and accurate methods for its quantification. Methods: Here, we describe a method quantifying ceramide in dendritic cells and defining the different fatty acids (FA bound to sphingosine. The main steps of the method include extraction of total lipids, separation of the ceramide by thin-layer chromatography, derivatization of ceramide-fatty acids (Cer-FA, and quantitation of these acids in their methyl form by gas chromatography on polar capillary columns. The identification of FA was achieved by means of known standards and confirmed by mass spectrometry. Results: FA ranging between C10 and C24 could be detected and quantified. The concentration of the sum of Cer-FA amounted to 14.88 ± 8.98 nmol/106 cells (n=10. Oleic acid, which accounted for approximately half of Cer-FA (7.73 ± 6.52 nmol/106 cells was the predominant fatty acid followed by palmitic acid (3.47 ± 1.54 nmol/106 cells. Conclusion: This highly sensitive method allows the quantification of different molecular species of ceramides.

  9. A quantitative study of the division cycle of Caulobacter crescentus stalked cells.

    Directory of Open Access Journals (Sweden)

    Shenghua Li

    2008-01-01

    Full Text Available Progression of a cell through the division cycle is tightly controlled at different steps to ensure the integrity of genome replication and partitioning to daughter cells. From published experimental evidence, we propose a molecular mechanism for control of the cell division cycle in Caulobacter crescentus. The mechanism, which is based on the synthesis and degradation of three "master regulator" proteins (CtrA, GcrA, and DnaA, is converted into a quantitative model, in order to study the temporal dynamics of these and other cell cycle proteins. The model accounts for important details of the physiology, biochemistry, and genetics of cell cycle control in stalked C. crescentus cell. It reproduces protein time courses in wild-type cells, mimics correctly the phenotypes of many mutant strains, and predicts the phenotypes of currently uncharacterized mutants. Since many of the proteins involved in regulating the cell cycle of C. crescentus are conserved among many genera of alpha-proteobacteria, the proposed mechanism may be applicable to other species of importance in agriculture and medicine.

  10. Technical Note: Quantitative dynamic contrast-enhanced MRI of a 3-dimensional artificial capillary network.

    Science.gov (United States)

    Gaass, Thomas; Schneider, Moritz Jörg; Dietrich, Olaf; Ingrisch, Michael; Dinkel, Julien

    2017-04-01

    Variability across devices, patients, and time still hinders widespread recognition of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) as quantitative biomarker. The purpose of this work was to introduce and characterize a dedicated microchannel phantom as a model for quantitative DCE-MRI measurements. A perfusable, MR-compatible microchannel network was constructed on the basis of sacrificial melt-spun sugar fibers embedded in a block of epoxy resin. Structural analysis was performed on the basis of light microscopy images before DCE-MRI experiments. During dynamic acquisition the capillary network was perfused with a standard contrast agent injection system. Flow-dependency, as well as inter- and intrascanner reproducibility of the computed DCE parameters were evaluated using a 3.0 T whole-body MRI. Semi-quantitative and quantitative flow-related parameters exhibited the expected proportionality to the set flow rate (mean Pearson correlation coefficient: 0.991, P quantitative parameters. All evaluated parameters were well in the range of known in vivo results for the applied flow rates. The constructed phantom enables reproducible, flow-dependent, contrast-enhanced MR measurements with the potential to facilitate standardization and comparability of DCE-MRI examinations. © 2017 American Association of Physicists in Medicine.

  11. Cell Division, Differentiation and Dynamic Clustering

    CERN Document Server

    Kaneko, K; Kaneko, Kunihiko; Yomo, Tetsuya

    1993-01-01

    A novel mechanism for cell differentiation is proposed, based on the dynamic clustering in a globally coupled chaotic system. A simple model with metabolic reaction, active transport of chemicals from media, and cell division is found to show three successive stages with the growth of the number of cells; coherent growth, dynamic clustering, and fixed cell differentiation. At the last stage, disparity in activities, germ line segregation, somatic cell differentiation, and homeochaotic stability against external perturbation are found. Our results, in consistency with the experiments of the preceding paper, imply that cell differentiation can occur without a spatial pattern. From dynamical systems viewpoint, the new concept of ``open chaos" is proposed, as a novel and general scenario for systems with growing numbers of elements, also seen in economics and sociology.A

  12. 活体荧光成像对裸鼠肝癌细胞系MHCC97-H原位移植模型的动态量化分析%Dynamic and Quantitative Analysis of Orthotopically Transplanted Nude Mouse Model with MHCC97-H Cells using Bioluminescent Imaging Technology

    Institute of Scientific and Technical Information of China (English)

    曹阳; 韩炜; 刘洋; 张勇; 郭欣; 陈勇

    2013-01-01

    目的:利用生物自发光的裸鼠肝癌原位移植模型,以活体荧光成像技术对肝癌的生长和转移情况进行动态、量化分析.方法:将稳定转染了荧光素酶(luciferase)基因的人肝癌细胞株MHCC97-H-LUC细胞,移植至裸鼠肝脏包膜下,每周利用活体荧光成像系统对裸鼠体内移植瘤的生长部位和范围进行成像,测量肿瘤细胞生物发光量,动态观察肝癌细胞在裸鼠体内的肿瘤数量、生长速度和转移情况.结果:建立可稳定表达荧光素酶的人肝癌细胞株MHCC97-H-LUC并用于进行生物自发光的裸鼠原位移植模型;利用活体荧光成像系统对裸鼠体内的移植瘤成像,见发光部位由肝脏向腹腔扩散,发光量随时间呈指数级增长;病理学观察证实肿瘤细胞长.结论:利用活体荧光成像技术的动态量化分析可灵敏、准确地监测裸鼠肝癌原位移植模型中肿瘤细胞的生长及转移情况,为肿瘤发生、生长、转移机制及对抗肿瘤生长和转移的体内研究提供了科学的量化手段.%Objective: To investigate the growth and metastasis of hepatocellular carcinoma (HCC) dynamically and quantitatively in orthotopically transplanted nude mouse model for HCC by the mean of bioluminescent imaging technology. Method: Human hepatocellular carcinoma cell line MHCC97-H was stably transfected with luciferase gene and transplanted under the capsule of nude mice liver. The location and extent of transplanted tumors in nude mice was detected by using bioluminescent imaging system, and total photon flux emitting from tumor cells was measured weekly. The growth and metastasis of transplanted tumor in nude mice was detected dynamically and quantitatively. Results: Hepatocellular carcinoma cell line MHCC97-H-LUC, stably expressing luciferase, was established and it was used to build an orthotopically transplanted nude mouse model for HCC. By bioluminescent imaging, it was found that the lighting area spread

  13. Dynamic quantitative echocardiographic evaluation of mitral regurgitation in the operating department.

    Science.gov (United States)

    Gisbert, Alejandro; Soulière, Vicky; Denault, André Y; Bouchard, Denis; Couture, Pierre; Pellerin, Michel; Carrier, Michel; Levesque, Sylvie; Ducharme, Anique; Basmadjian, Arsène J

    2006-02-01

    Hemodynamic modifications induced by general anesthesia could lead to underestimation of mitral regurgitation (MR) severity in the operating department and potentially serious consequences. The intraoperative severity of MR was prospectively compared with the preoperative baseline evaluation using dynamic quantitative transesophageal echocardiography in 25 patients who were stable with MR 2/4 or greater undergoing coronary bypass, mitral valve operation, or both. Significant changes in the severity of MR using transesophageal echocardiographic criteria occurred after the induction of general anesthesia and with phenylephrine. Quantitative transesophageal echocardiographic evaluation of MR using effective orifice area and vena contracta, and the use of phenylephrine challenge, were useful to avoid underestimating MR severity in the operating department.

  14. Universal signal generator for dynamic cell stimulation.

    Science.gov (United States)

    Piehler, Andreas; Ghorashian, Navid; Zhang, Ce; Tay, Savaş

    2017-06-27

    Dynamic cell stimulation is a powerful technique for probing gene networks and for applications in stem cell differentiation, immunomodulation and signaling. We developed a robust and flexible method and associated microfluidic devices to generate a wide-range of precisely formulated dynamic chemical signals to stimulate live cells and measure their dynamic response. This signal generator is capable of digital to analog conversion (DAC) through combinatoric selection of discrete input concentrations, and outperforms existing methods by both achievable resolution, dynamic range and simplicity in design. It requires no calibration, has minimal space requirements and can be easily integrated into microfluidic cell culture devices. The signal generator hardware and software we developed allows to choose the waveform, period and amplitude of chemical input signals and features addition of well-defined chemical noise to study the role of stochasticity in cellular information processing.

  15. Quantitative microscopy and nanoscopy of sickle red blood cells performed by wide field digital interferometry

    Science.gov (United States)

    Shaked, Natan T.; Satterwhite, Lisa L.; Telen, Marilyn J.; Truskey, George A.; Wax, Adam

    2011-03-01

    We have applied wide-field digital interferometry (WFDI) to examine the morphology and dynamics of live red blood cells (RBCs) from individuals who suffer from sickle cell anemia (SCA), a genetic disorder that affects the structure and mechanical properties of RBCs. WFDI is a noncontact, label-free optical microscopy approach that can yield quantitative thickness profiles of RBCs and measurements of their membrane fluctuations at the nanometer scale reflecting their stiffness. We find that RBCs from individuals with SCA are significantly stiffer than those from a healthy control. Moreover, we show that the technique is sensitive enough to distinguish classes of RBCs in SCA, including sickle RBCs with apparently normal morphology, compared to the stiffer crescent-shaped sickle RBCs. We expect that this approach will be useful for diagnosis of SCA and for determining efficacy of therapeutic agents.

  16. Quantitative Magnetic Particle Imaging Monitors the Transplantation, Biodistribution, and Clearance of Stem Cells In Vivo.

    Science.gov (United States)

    Zheng, Bo; von See, Marc P; Yu, Elaine; Gunel, Beliz; Lu, Kuan; Vazin, Tandis; Schaffer, David V; Goodwill, Patrick W; Conolly, Steven M

    2016-01-01

    Stem cell therapies have enormous potential for treating many debilitating diseases, including heart failure, stroke and traumatic brain injury. For maximal efficacy, these therapies require targeted cell delivery to specific tissues followed by successful cell engraftment. However, targeted delivery remains an open challenge. As one example, it is common for intravenous deliveries of mesenchymal stem cells (MSCs) to become entrapped in lung microvasculature instead of the target tissue. Hence, a robust, quantitative imaging method would be essential for developing efficacious cell therapies. Here we show that Magnetic Particle Imaging (MPI), a novel technique that directly images iron-oxide nanoparticle-tagged cells, can longitudinally monitor and quantify MSC administration in vivo. MPI offers near-ideal image contrast, depth penetration, and robustness; these properties make MPI both ultra-sensitive and linearly quantitative. Here, we imaged, for the first time, the dynamic trafficking of intravenous MSC administrations using MPI. Our results indicate that labeled MSC injections are immediately entrapped in lung tissue and then clear to the liver within one day, whereas standard iron oxide particle (Resovist) injections are immediately taken up by liver and spleen. Longitudinal MPI-CT imaging also indicated a clearance half-life of MSC iron oxide labels in the liver at 4.6 days. Finally, our ex vivo MPI biodistribution measurements of iron in liver, spleen, heart, and lungs after injection showed excellent agreement (R(2) = 0.943) with measurements from induction coupled plasma spectrometry. These results demonstrate that MPI offers strong utility for noninvasively imaging and quantifying the systemic distribution of cell therapies and other therapeutic agents.

  17. Dynamic culture improves cell reprogramming efficiency.

    Science.gov (United States)

    Sia, Junren; Sun, Raymond; Chu, Julia; Li, Song

    2016-06-01

    Cell reprogramming to pluripotency is an inefficient process and various approaches have been devised to improve the yield of induced pluripotent stem cells. However, the effect of biophysical factors on cell reprogramming is not well understood. Here we showed that, for the first time, dynamic culture with orbital shaking significantly improved the reprogramming efficiency in adherent cells. Manipulating the viscosity of the culture medium suggested that the improved efficiency is mainly attributed to convective mixing rather than hydrodynamic shear stress. Temporal studies demonstrated that the enhancement of reprogramming efficiency required the dynamic culture in the middle but not early phase. In the early phase, fibroblasts had a high proliferation rate, but as the culture became over-confluent in the middle phase, expression of p57 was upregulated to inhibit cell proliferation and consequently, cell reprogramming. Subjecting the over confluent culture to orbital shaking prevented the upregulation of p57, thus improving reprogramming efficiency. Seeding cells at low densities to avoid over-confluency resulted in a lower efficiency, and optimal reprogramming efficiency was attained at a high seeding density with dynamic culture. Our findings provide insight into the underlying mechanisms of how dynamic culture condition regulate cell reprogramming, and will have broad impact on cell engineering for regenerative medicine and disease modeling.

  18. Epigenetic dynamics across the cell cycle

    DEFF Research Database (Denmark)

    Kheir, Tony Bou; Lund, Anders H.

    2010-01-01

    Progression of the mammalian cell cycle depends on correct timing and co-ordination of a series of events, which are managed by the cellular transcriptional machinery and epigenetic mechanisms governing genome accessibility. Epigenetic chromatin modifications are dynamic across the cell cycle......, and are shown to influence and be influenced by cell-cycle progression. Chromatin modifiers regulate cell-cycle progression locally by controlling the expression of individual genes and globally by controlling chromatin condensation and chromosome segregation. The cell cycle, on the other hand, ensures...... a correct inheritance of epigenetic chromatin modifications to daughter cells. In this chapter, we summarize the current knowledge on the dynamics of epigenetic chromatin modifications during progression of the cell cycle....

  19. Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) Applied to Quantitative Proteomics of Bacillus subtilis

    DEFF Research Database (Denmark)

    Soufi, Boumediene; Kumar, C.; Gnad, F.

    2010-01-01

    We applied stable isotope labeling by amino acids in cell culture (SILAC) to large-scale quantitative proteomics analyses of the model bacterium Bacillus subtilis in two physiological conditions: growth on succinate and growth under phosphate starvation. Using a B. subtilis strain auxotrophic...... of the most comprehensive quantitative proteomics studies in bacteria, covering more than 75% of the B. subtilis genes expressed in the log phase of growth. Furthermore, we detect and quantify dynamics of 35 Ser/Thr/Tyr phosphorylation sites under growth on succinate, and 10 phosphorylation sites under...

  20. Quantitative Verification of a Force-based Model for Pedestrian Dynamics

    CERN Document Server

    Chraibi, Mohcine; Schadschneider, Andreas; Mackens, Wolfgang

    2009-01-01

    This paper introduces a spatially continuous force-based model for simulating pedestrian dynamics. The main intention of this work is the quantitative description of pedestrian movement through bottlenecks and in corridors. Measurements of flow and density at bottlenecks will be presented and compared with empirical data. Furthermore the fundamental diagram for the movement in a corridor is reproduced. The results of the proposed model show a good agreement with empirical data.

  1. Novel quantitative autophagy analysis by organelle flow cytometry after cell sonication.

    Directory of Open Access Journals (Sweden)

    Michael Degtyarev

    Full Text Available Autophagy is a dynamic process of bulk degradation of cellular proteins and organelles in lysosomes. Current methods of autophagy measurement include microscopy-based counting of autophagic vacuoles (AVs in cells. We have developed a novel method to quantitatively analyze individual AVs using flow cytometry. This method, OFACS (organelle flow after cell sonication, takes advantage of efficient cell disruption with a brief sonication, generating cell homogenates with fluorescently labeled AVs that retain their integrity as confirmed with light and electron microscopy analysis. These AVs could be detected directly in the sonicated cell homogenates on a flow cytometer as a distinct population of expected organelle size on a cytometry plot. Treatment of cells with inhibitors of autophagic flux, such as chloroquine or lysosomal protease inhibitors, increased the number of particles in this population under autophagy inducing conditions, while inhibition of autophagy induction with 3-methyladenine or knockdown of ATG proteins prevented this accumulation. This assay can be easily performed in a high-throughput format and opens up previously unexplored avenues for autophagy analysis.

  2. Automated cell tracking tools for quantitative motility studies

    Science.gov (United States)

    Zimmer, Christophe; Zhang, Bo; Blazquez, Samantha; Labruyère, Elisabeth; Frischknecht, Freddy; Ménard, Robert; Guillén, Nancy; Olivo-Marin, Jean-Christophe

    2005-03-01

    Optical microscopy in 2 or 3 dimensions allows extensive observations of the motility and morphology of living cells, in culture or in tissue. This leads to an exploding accumulation of imaging data and shifts the bottleneck from data acquisition to data analysis. Manual image analysis is often either impossible or exceedingly time-consuming and subject to uncontrollable user bias and errors. Computerized methods promise to ensure fast, accurate and reproducible processing, but the basic image analysis functions available in standard commercial software are generally not adapted to the complexity of biological images. For this reason, we develop methods based on active contours, a powerful and flexible technique to segment and track objects, that has become very popular in computer vision research. Here, we describe the main benefits and limitations of active contours for our application, and our efforts to adapt and improve these methods for the analysis of cellular dynamics.

  3. Dynamical Adaptation in Terrorist Cells/Networks

    DEFF Research Database (Denmark)

    Hussain, Dil Muhammad Akbar; Ahmed, Zaki

    2010-01-01

    Typical terrorist cells/networks have dynamical structure as they evolve or adapt to changes which may occur due to capturing or killing of a member of the cell/network. Analytical measures in graph theory like degree centrality, betweenness and closeness centralities are very common and have long...

  4. Sorting cells by their dynamical properties

    Science.gov (United States)

    Henry, Ewan; Holm, Stefan H.; Zhang, Zunmin; Beech, Jason P.; Tegenfeldt, Jonas O.; Fedosov, Dmitry A.; Gompper, Gerhard

    2016-10-01

    Recent advances in cell sorting aim at the development of novel methods that are sensitive to various mechanical properties of cells. Microfluidic technologies have a great potential for cell sorting; however, the design of many micro-devices is based on theories developed for rigid spherical particles with size as a separation parameter. Clearly, most bioparticles are non-spherical and deformable and therefore exhibit a much more intricate behavior in fluid flow than rigid spheres. Here, we demonstrate the use of cells’ mechanical and dynamical properties as biomarkers for separation by employing a combination of mesoscale hydrodynamic simulations and microfluidic experiments. The dynamic behavior of red blood cells (RBCs) within deterministic lateral displacement (DLD) devices is investigated for different device geometries and viscosity contrasts between the intra-cellular fluid and suspending medium. We find that the viscosity contrast and associated cell dynamics clearly determine the RBC trajectory through a DLD device. Simulation results compare well to experiments and provide new insights into the physical mechanisms which govern the sorting of non-spherical and deformable cells in DLD devices. Finally, we discuss the implications of cell dynamics for sorting schemes based on properties other than cell size, such as mechanics and morphology.

  5. Quantitative nucleation and growth kinetics of gold nanoparticles via model-assisted dynamic spectroscopic approach.

    Science.gov (United States)

    Zhou, Yao; Wang, Huixuan; Lin, Wenshuang; Lin, Liqin; Gao, Yixian; Yang, Feng; Du, Mingming; Fang, Weiping; Huang, Jiale; Sun, Daohua; Li, Qingbiao

    2013-10-01

    Lacking of quantitative experimental data and/or kinetic models that could mathematically depict the redox chemistry and the crystallization issue, bottom-to-up formation kinetics of gold nanoparticles (GNPs) remains a challenge. We measured the dynamic regime of GNPs synthesized by l-ascorbic acid (representing a chemical approach) and/or foliar aqueous extract (a biogenic approach) via in situ spectroscopic characterization and established a redox-crystallization model which allows quantitative and separate parameterization of the nucleation and growth processes. The main results were simplified as the following aspects: (I) an efficient approach, i.e., the dynamic in situ spectroscopic characterization assisted with the redox-crystallization model, was established for quantitative analysis of the overall formation kinetics of GNPs in solution; (II) formation of GNPs by the chemical and the biogenic approaches experienced a slow nucleation stage followed by a growth stage which behaved as a mixed-order reaction, and different from the chemical approach, the biogenic method involved heterogeneous nucleation; (III) also, biosynthesis of flaky GNPs was a kinetic-controlled process favored by relatively slow redox chemistry; and (IV) though GNPs formation consists of two aspects, namely the redox chemistry and the crystallization issue, the latter was the rate-determining event that controls the dynamic regime of the whole physicochemical process.

  6. Rapid and quantitative discrimination of tumour cells on tissue slices

    Science.gov (United States)

    Huang, Kai-Wen; Chieh, Jen-Jie; Liao, Shu-Hsien; Wei, Wen-Chun; Hsiao, Pei-Yi; Yang, Hong-Chang; Horng, Herng-Er

    2016-06-01

    After a needle biopsy, immunohistochemistry is generally used to stain tissue slices for clinically confirming tumours. Currently, tissue slices are immersed in a bioprobe-linked fluorescent reagent for several minutes, washed to remove the unbound reagent, and then observed using a fluorescence microscope. However, the observation must be performed by experienced pathologists, and producing a qualitative analysis is time consuming. Therefore, this study proposes a novel scanning superconducting quantum interference device biosusceptometry (SSB) method for avoiding these drawbacks. First, stain reagents were synthesised for the dual modalities of fluorescent and magnetic imaging by combining iron-oxide magnetic nanoparticles and the currently used fluorescent reagent. The reagent for the proposed approach was stained using the same procedure as that for the current fluorescent reagent, and tissue slices were rapidly imaged using the developed SSB for obtaining coregistered optical and magnetic images. Analysing the total intensity of magnetic spots in SSB images enables quantitatively determining the tumour cells of tissue slices. To confirm the magnetic imaging results, a traditional observation methodology entailing the use of a fluorescence microscope was also performed as the gold standard. This study determined high consistency between the fluorescent and magnetic spots in different regions of the tissue slices, demonstrating the feasibility of the proposed approach, which will benefit future clinical pathology.

  7. Sensitivity Analysis of Centralized Dynamic Cell Selection

    DEFF Research Database (Denmark)

    Lopez, Victor Fernandez; Alvarez, Beatriz Soret; Pedersen, Klaus I.;

    2016-01-01

    mechanism and solutions involving cell switching in general. Simulation results show that such solutions can greatly benefit from the use of receivers with interference suppression capabilities and a larger number of antennas, with a maximum data rate gain of 120%. High performance gains are observed...... with two different traffic models, and it is not necessary to be able to connect to a large number of cells in order to reap most of the benefits of the centralized dynamic cell selection....

  8. Subventricular zone cell migration: lessons from quantitative 2-photon microscopy

    Directory of Open Access Journals (Sweden)

    Rachel eJames

    2011-03-01

    Full Text Available Neuroblasts born in the adult subventricular zone (SVZ migrate long distances in the rostral migratory stream (RMS to the olfactory bulbs where they integrate into circuitry as functional interneurons. As very little was known about the dynamic parameters of SVZ neuroblast migration, we used two-photon time-lapse microscopy to analyze migration in acute slices. This involved analyzing 3-dimensional stacks of images over time and uncovered several novel aspects of SVZ migration: chains remain stable, cells can be immotile for extensive periods, morphology does not necessarily correlate with motility, neuroblasts exhibit local exploratory motility, dorsoventral migration occurs throughout the striatal SVZ and neuroblasts turn at distinctive angles. We investigated these novel findings in the SVZ and RMS from the population to the single cell level. In this review we also discuss some technical considerations when setting up a two-photon microscopic imaging system. Throughout the review we identify several unsolved questions about SVZ neuroblast migration that might be addressed with current or emerging techniques.

  9. Evaluation of static and dynamic perfusion cardiac computed tomography for quantitation and classification tasks.

    Science.gov (United States)

    Bindschadler, Michael; Modgil, Dimple; Branch, Kelley R; La Riviere, Patrick J; Alessio, Adam M

    2016-04-01

    Cardiac computed tomography (CT) acquisitions for perfusion assessment can be performed in a dynamic or static mode. Either method may be used for a variety of clinical tasks, including (1) stratifying patients into categories of ischemia and (2) using a quantitative myocardial blood flow (MBF) estimate to evaluate disease severity. In this simulation study, we compare method performance on these classification and quantification tasks for matched radiation dose levels and for different flow states, patient sizes, and injected contrast levels. Under conditions simulated, the dynamic method has low bias in MBF estimates (0 to [Formula: see text]) compared to linearly interpreted static assessment (0.45 to [Formula: see text]), making it more suitable for quantitative estimation. At matched radiation dose levels, receiver operating characteristic analysis demonstrated that the static method, with its high bias but generally lower variance, had superior performance ([Formula: see text]) in stratifying patients, especially for larger patients and lower contrast doses [area under the curve [Formula: see text] to 96 versus 0.86]. We also demonstrate that static assessment with a correctly tuned exponential relationship between the apparent CT number and MBF has superior quantification performance to static assessment with a linear relationship and to dynamic assessment. However, tuning the exponential relationship to the patient and scan characteristics will likely prove challenging. This study demonstrates that the selection and optimization of static or dynamic acquisition modes should depend on the specific clinical task.

  10. Rotorcraft control system design for uncertain vehicle dynamics using quantitative feedback theory

    Science.gov (United States)

    Hess, R. A.

    1994-01-01

    Quantitative Feedback Theory describes a frequency-domain technique for the design of multi-input, multi-output control systems which must meet time or frequency domain performance criteria when specified uncertainty exists in the linear description of the vehicle dynamics. This theory is applied to the design of the longitudinal flight control system for a linear model of the BO-105C rotorcraft. Uncertainty in the vehicle model is due to the variation in the vehicle dynamics over a range of airspeeds from 0-100 kts. For purposes of exposition, the vehicle description contains no rotor or actuator dynamics. The design example indicates the manner in which significant uncertainty exists in the vehicle model. The advantage of using a sequential loop closure technique to reduce the cost of feedback is demonstrated by example.

  11. Quantitative identification of dynamical transitions in a semiconductor laser with optical feedback

    Science.gov (United States)

    Quintero-Quiroz, C.; Tiana-Alsina, J.; Romà, J.; Torrent, M. C.; Masoller, C.

    2016-01-01

    Identifying transitions to complex dynamical regimes is a fundamental open problem with many practical applications. Semi- conductor lasers with optical feedback are excellent testbeds for studying such transitions, as they can generate a rich variety of output signals. Here we apply three analysis tools to quantify various aspects of the dynamical transitions that occur as the laser pump current increases. These tools allow to quantitatively detect the onset of two different regimes, low-frequency fluctuations and coherence collapse, and can be used for identifying the operating conditions that result in specific dynamical properties of the laser output. These tools can also be valuable for analyzing regime transitions in other complex systems. PMID:27857229

  12. Quantitative analysis of quantum dot dynamics and emission spectra in cavity quantum electrodynamics

    DEFF Research Database (Denmark)

    Madsen, Kristian Høeg; Lodahl, Peter

    2013-01-01

    We present detuning-dependent spectral and decay-rate measurements to study the difference between the spectral and dynamical properties of single quantum dots embedded in micropillar and photonic crystal cavities. For the micropillar cavity, the dynamics is well described by the dissipative Jaynes......–Cummings model, whereas systematic deviations are observed for the emission spectra. The discrepancy for the spectra is attributed to the coupling of other exciton lines to the cavity and interference of different propagation paths toward the detector of the fields emitted by the quantum dot. In contrast......, quantitative information about the system can readily be extracted from the dynamical measurements. In the case of photonic crystal cavities, we observe an anti-crossing in the spectra when detuning a single quantum dot through resonance, which is the spectral signature of a strong coupling. However, time...

  13. Quantitative imaging and measurement of cell-substrate surface deformation by digital holography

    Science.gov (United States)

    Yu, Xiao; Cross, Michael; Liu, Changgeng; Clark, David C.; Haynie, Donald T.; Kim, Myung K.

    2012-10-01

    Quantitative phase microscopy by digital holography (DH-QPM) is introduced to study the cell-substrate interactions and migratory behavior of adhesive cells. A non-wrinkling elastic substrate, collagen-coated polyacrylamide (PAA) has been employed and its surface deformation due to cell adhesion and motility has been visualized as certain tangential and vertical displacement and distortion. The surface deformation on substrates of different elasticity and thickness has been quantitatively imaged and the corresponding cellular traction force of motile fibroblasts has been measured from phase profiles by DH-QPM. DH-QPM is able to yield quantitative measures directly and provide efficient and versatile means for quantitatively analyzing cellular motility.

  14. Quantitative variations of CD4 + CD25 + cells in Peking duckwhite ...

    African Journals Online (AJOL)

    Quantitative variations of CD4 + CD25 + cells in Peking duckwhite leghorn chimeras based on bone marrow mesenchymal stem cells. ... PROMOTING ACCESS TO AFRICAN RESEARCH. AFRICAN JOURNALS ONLINE (AJOL) · Journals ...

  15. Formation Dynamics and Quantitative Prediction of Hydrocarbons of the Superpressure System in the Dongying Sag

    Institute of Scientific and Technical Information of China (English)

    SUI Fenggui; HAO Xuefeng; LIU Qing; ZHUO Qingong; ZHANG Shouchun

    2008-01-01

    Based on the theory of formation dynamics of oil/gas pools, the Dongying sag can be divided into three dynamic systems regarding the accumulation of oil and gas: the superpressure closed system,the semi-closed system and the normal pressure open system. Based on the analysis of genesis of superpressure in the superpressure closed system and the rule of hydrocarbon expulsion,it is found that hydrocarbon generation is related to superpressure, which is the main driving factor of hydrocarbon migration. Micro fractures formed by superpressure are the main channels for hydrocarbon migration. There are three dynamic patterns for hydrocarbon expulsion: free water drainage, hydrocarbon accumulation and drainage through micro fissures. In the superpressure closed system, the oil-driving-water process and oil/gas accumulation were completed in lithologic traps by way of such two dynamic patterns as episodic evolution of superpressure systems and episodic pressure release of faults. The oil-bearing capacity of lithologic traps is intimately related to reservoir-forming dynamic force. Quantitative evaluation of dynamic conditions for pool formation can effectively predict the oil-bearing capability of traps.

  16. Gadolinium uptake by brain cancer cells: Quantitative analysis with X-PEEM spectromicroscopy for cancer therapy

    Science.gov (United States)

    De Stasio, Gelsomina; Gilbert, B.; Perfetti, P.; Margaritondo, G.; Mercanti, D.; Ciotti, M. T.; Casalbore, P.; Larocca, L. M.; Rinelli, A.; Pallini, R.

    2000-05-01

    We present the first X-PEEM spectromicroscopy semi-quantitative data, acquired on Gd in glioblastoma cell cultures from human brain cancer. The cells were treated with a Gd compound for the optimization of GdNCT (Gadolinium Neutron Capture Therapy). We analyzed the kinetics of Gd uptake as a function of exposure time, and verified that a quantitative analytical technique gives the same results as our MEPHISTO X-PEEM, demonstrating the feasibility of semi-quantitative spectromicroscopy.

  17. Comparison of blood flow models and acquisitions for quantitative myocardial perfusion estimation from dynamic CT

    Science.gov (United States)

    Bindschadler, Michael; Modgil, Dimple; Branch, Kelley R.; La Riviere, Patrick J.; Alessio, Adam M.

    2014-04-01

    Myocardial blood flow (MBF) can be estimated from dynamic contrast enhanced (DCE) cardiac CT acquisitions, leading to quantitative assessment of regional perfusion. The need for low radiation dose and the lack of consensus on MBF estimation methods motivates this study to refine the selection of acquisition protocols and models for CT-derived MBF. DCE cardiac CT acquisitions were simulated for a range of flow states (MBF = 0.5, 1, 2, 3 ml (min g)-1, cardiac output = 3, 5, 8 L min-1). Patient kinetics were generated by a mathematical model of iodine exchange incorporating numerous physiological features including heterogenenous microvascular flow, permeability and capillary contrast gradients. CT acquisitions were simulated for multiple realizations of realistic x-ray flux levels. CT acquisitions that reduce radiation exposure were implemented by varying both temporal sampling (1, 2, and 3 s sampling intervals) and tube currents (140, 70, and 25 mAs). For all acquisitions, we compared three quantitative MBF estimation methods (two-compartment model, an axially-distributed model, and the adiabatic approximation to the tissue homogeneous model) and a qualitative slope-based method. In total, over 11 000 time attenuation curves were used to evaluate MBF estimation in multiple patient and imaging scenarios. After iodine-based beam hardening correction, the slope method consistently underestimated flow by on average 47.5% and the quantitative models provided estimates with less than 6.5% average bias and increasing variance with increasing dose reductions. The three quantitative models performed equally well, offering estimates with essentially identical root mean squared error (RMSE) for matched acquisitions. MBF estimates using the qualitative slope method were inferior in terms of bias and RMSE compared to the quantitative methods. MBF estimate error was equal at matched dose reductions for all quantitative methods and range of techniques evaluated. This suggests that

  18. Quantitative dynamic nuclear polarization-NMR on blood plasma for assays of drug metabolism.

    Science.gov (United States)

    Lerche, Mathilde H; Meier, Sebastian; Jensen, Pernille R; Hustvedt, Svein-Olaf; Karlsson, Magnus; Duus, Jens Ø; Ardenkjaer-Larsen, Jan H

    2011-01-01

    Analytical platforms for the fast detection, identification and quantification of circulating drugs with a narrow therapeutic range are vital in clinical pharmacology. As a result of low drug concentrations, analytical tools need to provide high sensitivity and specificity. Dynamic nuclear polarization-NMR (DNP-NMR) in the form of the hyperpolarization-dissolution method should afford the sensitivity and spectral resolution for the direct detection and quantification of numerous isotopically labeled circulating drugs and their metabolites in single liquid-state NMR transients. This study explores the capability of quantitative in vitro DNP-NMR to assay drug metabolites in blood plasma. The lower limit of detection for the anti-epileptic drug (13)C-carbamazepine and its pharmacologically active metabolite (13)C-carbamazepine-10,11-epoxide is 0.08 µg/mL in rabbit blood plasma analyzed by single-scan (13)C DNP-NMR. An internal standard is used for the accurate quantification of drug and metabolite. Comparison of quantitative DNP-NMR data with an established analytical method (liquid chromatography-mass spectrometry) yields a Pearson correlation coefficient r of 0.99. Notably, all DNP-NMR determinations were performed without analyte derivatization or sample purification other than plasma protein precipitation. Quantitative DNP-NMR is an emerging methodology which requires little sample preparation and yields quantitative data with high sensitivity for therapeutic drug monitoring.

  19. A New 3-Dimensional Dynamic Quantitative Analysis System of Facial Motion: An Establishment and Reliability Test

    Science.gov (United States)

    Feng, Guodong; Zhao, Yang; Tian, Xu; Gao, Zhiqiang

    2014-01-01

    This study aimed to establish a 3-dimensional dynamic quantitative facial motion analysis system, and then determine its accuracy and test-retest reliability. The system could automatically reconstruct the motion of the observational points. Standardized T-shaped rod and L-shaped rods were used to evaluate the static and dynamic accuracy of the system. Nineteen healthy volunteers were recruited to test the reliability of the system. The average static distance error measurement was 0.19 mm, and the average angular error was 0.29°. The measuring results decreased with the increase of distance between the cameras and objects, 80 cm of which was considered to be optimal. It took only 58 seconds to perform the full facial measurement process. The average intra-class correlation coefficient for distance measurement and angular measurement was 0.973 and 0.794 respectively. The results demonstrated that we successfully established a practical 3-dimensional dynamic quantitative analysis system that is accurate and reliable enough to meet both clinical and research needs. PMID:25390881

  20. Dynamic and Quantitative Method of Analyzing Service Consistency Evolution Based on Extended Hierarchical Finite State Automata

    Directory of Open Access Journals (Sweden)

    Linjun Fan

    2014-01-01

    Full Text Available This paper is concerned with the dynamic evolution analysis and quantitative measurement of primary factors that cause service inconsistency in service-oriented distributed simulation applications (SODSA. Traditional methods are mostly qualitative and empirical, and they do not consider the dynamic disturbances among factors in service’s evolution behaviors such as producing, publishing, calling, and maintenance. Moreover, SODSA are rapidly evolving in terms of large-scale, reusable, compositional, pervasive, and flexible features, which presents difficulties in the usage of traditional analysis methods. To resolve these problems, a novel dynamic evolution model extended hierarchical service-finite state automata (EHS-FSA is constructed based on finite state automata (FSA, which formally depict overall changing processes of service consistency states. And also the service consistency evolution algorithms (SCEAs based on EHS-FSA are developed to quantitatively assess these impact factors. Experimental results show that the bad reusability (17.93% on average is the biggest influential factor, the noncomposition of atomic services (13.12% is the second biggest one, and the service version’s confusion (1.2% is the smallest one. Compared with previous qualitative analysis, SCEAs present good effectiveness and feasibility. This research can guide the engineers of service consistency technologies toward obtaining a higher level of consistency in SODSA.

  1. Quantitative mathematical modeling of PSA dynamics of prostate cancer patients treated with intermittent androgen suppression

    Institute of Scientific and Technical Information of China (English)

    Yoshito Hirata; Koichiro Akakura; Celestia S.Higano; Nicholas Bruchovsky; Kazuyuki Aihara

    2012-01-01

    If a mathematical model is to be used in the diagnosis,treatment,or prognosis of a disease,it must describe the inherent quantitative dynamics of the state.An ideal candidate disease is prostate cancer owing to the fact that it is characterized by an excellent biomarker,prostate-specific antigen (PSA),and also by a predictable response to treatment in the form of androgen suppression therapy.Despite a high initial response rate,the cancer will often relapse to a state of androgen independence which no longer responds to manipulations of the hormonal environment.In this paper,we present relevant background information and a quantitative mathematical model that potentially can be used in the optimal management of patients to cope with biochemical relapse as indicated by a rising PSA.

  2. Quantitation of progressive muscle fatigue during dynamic leg exercise in humans

    DEFF Research Database (Denmark)

    Fulco, C S; Lewis, S F; Frykman, Peter

    1995-01-01

    to quadriceps femoris. The slope of the linear relationship between O2 uptake and work rate was 13.7 ml O2/W (r = 0.93). This slope and the increase of heart rate relative to increasing work intensity agreed with published values for D leg exercise. Test-retest values for O2 uptake were similar (P > 0...... (*P rates). Virtually identical declines in MVC were observed by the end of graded work rate DKE and submaximal constant work rate DKE tests. Quantitation of progressive muscle fatigue during D leg exercise provides a framework to study the effects of a variety......There is virtually no published information on muscle fatigue, defined as a gradual decline in force-generating capacity, during conventional dynamic (D) leg exercise. To quantitate progression of fatigue, we developed 1) a model featuring integration of maximal voluntary static contraction (MVC...

  3. Quantitative analysis of signal transduction in motile and phototactic cells by computerized light stimulation and model based tracking

    Science.gov (United States)

    Streif, Stefan; Staudinger, Wilfried Franz; Oesterhelt, Dieter; Marwan, Wolfgang

    2009-02-01

    To investigate the responses of Halobacterium salinarum to stimulation with light (phototaxis and photokinesis), we designed an experimental setup consisting of optical devices for automatic video image acquisition and computer-controlled light stimulation, and developed algorithms to analyze physiological responses of the cells. Cells are categorized as motile and nonmotile by a classification scheme based on the square displacement of cell positions. Computerized tracking based on a dynamic model of the stochastic cell movement and a Kalman filter-based algorithm allows smoothed estimates of the cell tracks and the detection of physiological responses to complex stimulus patterns. The setup and algorithms were calibrated which allows quantitative measurements and systematic analysis of cellular sensing and response. Overall, the setup is flexible, extensible, and consists mainly of commercially available products. This facilitates modifications of the setup and algorithms for physiological studies of the motility of cells or microorganisms.

  4. Quantitative analysis of signal transduction in motile and phototactic cells by computerized light stimulation and model based tracking.

    Science.gov (United States)

    Streif, Stefan; Staudinger, Wilfried Franz; Oesterhelt, Dieter; Marwan, Wolfgang

    2009-02-01

    To investigate the responses of Halobacterium salinarum to stimulation with light (phototaxis and photokinesis), we designed an experimental setup consisting of optical devices for automatic video image acquisition and computer-controlled light stimulation, and developed algorithms to analyze physiological responses of the cells. Cells are categorized as motile and nonmotile by a classification scheme based on the square displacement of cell positions. Computerized tracking based on a dynamic model of the stochastic cell movement and a Kalman filter-based algorithm allows smoothed estimates of the cell tracks and the detection of physiological responses to complex stimulus patterns. The setup and algorithms were calibrated which allows quantitative measurements and systematic analysis of cellular sensing and response. Overall, the setup is flexible, extensible, and consists mainly of commercially available products. This facilitates modifications of the setup and algorithms for physiological studies of the motility of cells or microorganisms.

  5. Quantitative Contribution of IL2Rγ to the Dynamic Formation of IL2-IL2R Complexes.

    Directory of Open Access Journals (Sweden)

    Luis F Ponce

    Full Text Available Interleukin-2 (IL2 is a growth factor for several immune cells and its function depends on its binding to IL2Rs in the cell membrane. The most accepted model for the assembling of IL2-IL2R complexes in the cell membrane is the Affinity Conversion Model (ACM. This model postulates that IL2R receptor association is sequential and dependent on ligand binding. Most likely free IL2 binds first to IL2Rα, and then this complex binds to IL2Rβ, and finally to IL2Rγ (γc. However, in previous mathematical models representing this process, the binding of γc has not been taken into account. In this work, the quantitative contribution of the number of IL2Rγ chain to the IL2-IL2R apparent binding affinity and signaling is studied. A mathematical model of the affinity conversion process including the γ chain in the dynamic, has been formulated. The model was calibrated by fitting it to experimental data, specifically, Scatchard plots obtained using human cell lines. This paper demonstrates how the model correctly explains available experimental observations. It was estimated, for the first time, the value of the kinetic coefficients of IL2-IL2R complexes interaction in the cell membrane. Moreover, the number of IL2R components in different cell lines was also estimated. It was obtained a variable distribution in the number of IL2R components depending on the cell type and the activation state. Of most significance, the study predicts that not only the number of IL2Rα and IL2Rβ, but also the number of γc determine the capacity of the cell to capture and retain IL2 in signalling complexes. Moreover, it is also showed that different cells might use different pathways to bind IL2 as consequence of its IL2R components distribution in the membrane.

  6. Nonlinear dynamics of cell orientation

    Science.gov (United States)

    Safran, S. A.; de, Rumi

    2009-12-01

    The nonlinear dependence of cellular orientation on an external, time-varying stress field determines the distribution of orientations in the presence of noise and the characteristic time, τc , for the cell to reach its steady-state orientation. The short, local cytoskeletal relaxation time distinguishes between high-frequency (nearly perpendicular) and low-frequency (random or parallel) orientations. However, τc is determined by the much longer, orientational relaxation time. This behavior is related to experiments for which we predict the angle and characteristic time as a function of frequency.

  7. Improved generalized cell mapping for global analysis of dynamical systems

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Three main parts of generalized cell mapping are improved for global analysis. A simple method, which is not based on the theory of digraphs, is presented to locate complete self-cycling sets that corre- spond to attractors and unstable invariant sets involving saddle, unstable periodic orbit and chaotic saddle. Refinement for complete self-cycling sets is developed to locate attractors and unstable in- variant sets with high degree of accuracy, which can start with a coarse cell structure. A nonuniformly interior-and-boundary sampling technique is used to make the refinement robust. For homeomorphic dissipative dynamical systems, a controlled boundary sampling technique is presented to make gen- eralized cell mapping method with refinement extremely accurate to obtain invariant sets. Recursive laws of group absorption probability and expected absorption time are introduced into generalized cell mapping, and then an optimal order for quantitative analysis of transient cells is established, which leads to the minimal computational work. The improved method is applied to four examples to show its effectiveness in global analysis of dynamical systems.

  8. Improved generalized cell mapping for global analysis of dynamical systems

    Institute of Scientific and Technical Information of China (English)

    ZOU HaiLin; XU JianXue

    2009-01-01

    Three main parts of generalized cell mapping are improved for global analysis. A simple method, whichis not based on the theory of digraphs, is presented to locate complete self-cycling sets that corre-spond to attractors and unstable invariant sets involving saddle, unstable periodic orbit and chaotic saddle. Refinement for complete self-cycling sets is developed to locate attractors and unstable in-variant sets with high degree of accuracy, which can start with a coarse cell structure. A nonuniformly interior-and-boundary sampling technique is used to make the refinement robust. For homeomorphic dissipative dynamical systems, a controlled boundary sampling technique is presented to make gen-eralized cell mapping method with refinement extremely accurate to obtain invariant sets. Recursive laws of group absorption probability and expected absorption time are introduced into generalized cell mapping, and then an optimal order for quantitative analysis of transient cells is established, which leads to the minimal computational work. The improved method is applied to four examples to show its effectiveness in global analysis of dynamical systems.

  9. Modeling and Quantitative Analysis of GNSS/INS Deep Integration Tracking Loops in High Dynamics

    Directory of Open Access Journals (Sweden)

    Yalong Ban

    2017-09-01

    Full Text Available To meet the requirements of global navigation satellite systems (GNSS precision applications in high dynamics, this paper describes a study on the carrier phase tracking technology of the GNSS/inertial navigation system (INS deep integration system. The error propagation models of INS-aided carrier tracking loops are modeled in detail in high dynamics. Additionally, quantitative analysis of carrier phase tracking errors caused by INS error sources is carried out under the uniform high dynamic linear acceleration motion of 100 g. Results show that the major INS error sources, affecting the carrier phase tracking accuracy in high dynamics, include initial attitude errors, accelerometer scale factors, gyro noise and gyro g-sensitivity errors. The initial attitude errors are usually combined with the receiver acceleration to impact the tracking loop performance, which can easily cause the failure of carrier phase tracking. The main INS error factors vary with the vehicle motion direction and the relative position of the receiver and the satellites. The analysis results also indicate that the low-cost micro-electro mechanical system (MEMS inertial measurement units (IMU has the ability to maintain GNSS carrier phase tracking in high dynamics.

  10. A novel benzene quantitative analysis method using miniaturized metal ionization gas sensor and non-linear bistable dynamic system.

    Science.gov (United States)

    Tang, Xuxiang; Liu, Fuqi

    2015-01-01

    In this paper, a novel benzene quantitative analysis method utilizing miniaturized metal ionization gas sensor and non-linear bistable dynamic system was investigated. Al plate anodic gas-ionization sensor was installed for electrical current-voltage data measurement. Measurement data was analyzed by non-linear bistable dynamics system. Results demonstrated that this method realized benzene concentration quantitative determination. This method is promising in laboratory safety management in benzene leak detection.

  11. Quantitation of recombinant protein in whole cells and cell extracts via solid-state NMR spectroscopy.

    Science.gov (United States)

    Vogel, Erica P; Weliky, David P

    2013-06-25

    Recombinant proteins (RPs) are commonly expressed in bacteria followed by solubilization and chromatography. Purified RP yield can be diminished by losses at any step with very different changes in methods that can improve the yield. Time and labor can therefore be saved by first identifying the specific reason for the low yield. This study describes a new solid-state nuclear magnetic resonance approach to RP quantitation in whole cells or cell extracts without solubilization or purification. The method is straightforward and inexpensive and requires only ∼50 mL culture and a low-field spectrometer.

  12. Application of quantitative second-harmonic generation microscopy to dynamic conditions.

    Science.gov (United States)

    Kabir, Mohammad M; Inavalli, V V G Krishna; Lau, Tung-Yuen; Toussaint, Kimani C

    2013-01-01

    We present a quantitative second-harmonic generation (SHG) imaging technique that quantifies the 2D spatial organization of collagen fiber samples under dynamic conditions, as an image is acquired. The technique is demonstrated for both a well-aligned tendon sample and a randomly aligned, sparsely distributed collagen scaffold sample. For a fixed signal-to-noise ratio, we confirm the applicability of this method for various window sizes (pixel areas) as well as with using a gridded overlay map that allows for correlations of fiber orientations within a given image. This work has direct impact to in vivo biological studies by incorporating simultaneous SHG image acquisition and analysis.

  13. Dynamics of actin waves on patterned substrates: a quantitative analysis of circular dorsal ruffles.

    Directory of Open Access Journals (Sweden)

    Erik Bernitt

    Full Text Available Circular Dorsal Ruffles (CDRs have been known for decades, but the mechanism that organizes these actin waves remains unclear. In this article we systematically analyze the dynamics of CDRs on fibroblasts with respect to characteristics of current models of actin waves. We studied CDRs on heterogeneously shaped cells and on cells that we forced into disk-like morphology. We show that CDRs exhibit phenomena such as periodic cycles of formation, spiral patterns, and mutual wave annihilations that are in accord with an active medium description of CDRs. On cells of controlled morphologies, CDRs exhibit extremely regular patterns of repeated wave formation and propagation, whereas on random-shaped cells the dynamics seem to be dominated by the limited availability of a reactive species. We show that theoretical models of reaction-diffusion type incorporating conserved species capture partially the behavior we observe in our data.

  14. High-throughput quantitative analysis with cell growth kinetic curves for low copy number mutant cells.

    Science.gov (United States)

    Xing, James Z; Gabos, Stephan; Huang, Biao; Pan, Tianhong; Huang, Min; Chen, Jie

    2012-10-01

    The mutation rate in cells induced by environmental genotoxic hazards is very low and difficult to detect using traditional cell counting assays. The established genetic toxicity tests currently recognized by regulatory authorities, such as conventional Ames and hypoxanthine guanine phosphoribosyl-transferase (HPRT) assays, are not well suited for higher-throughput screening as they require large amounts of test compounds and are very time consuming. In this study, we developed a novel cell-based assay for quantitative analysis of low numbers of cell copies with HPRT mutation induced by an environmental mutagen. The HPRT gene mutant cells induced by the mutagen were selected by 6-thioguanine (6-TG) and the cell's kinetic growth curve monitored by a real-time cell electronic sensor (RT-CES) system. When a threshold is set at a certain cell index (CI) level, samples with different initial mutant cell copies take different amounts of time in order for their growth (or CI accumulation) to cross this threshold. The more cells that are initially seeded in the test well, the faster the cell accumulation and therefore the shorter the time required to cross this threshold. Therefore, the culture time period required to cross the threshold of each sample corresponds to the original number of cells in the sample. A mutant cell growth time threshold (MT) value of each sample can be calculated to predict the number of original mutant cells. For mutagenesis determination, the RT-CES assay displayed an equal sensitivity (p > 0.05) and coefficients of variation values with good correlation to conventional HPRT mutagenic assays. Most importantly, the RT-CES mutation assay has a higher throughput than conventional cellular assays.

  15. Quantitative dynamic contrast-enhanced MR imaging analysis of complex adnexal masses: a preliminary study.

    Science.gov (United States)

    Thomassin-Naggara, Isabelle; Balvay, Daniel; Aubert, Emilie; Daraï, Emile; Rouzier, Roman; Cuenod, Charles A; Bazot, Marc

    2012-04-01

    To evaluate the ability of quantitative dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) to differentiate malignant from benign adnexal tumours. Fifty-six women with 38 malignant and 18 benign tumours underwent MR imaging before surgery for complex adnexal masses. Microvascular parameters were extracted from high temporal resolution DCE-MRI series, using a pharmacokinetic model in the solid tissue of adnexal tumours. These parameters were tissue blood flow (F(T)), blood volume fraction (Vb), permeability-surface area product (PS), interstitial volume fraction (Ve), lag time (Dt) and area under the enhancing curve (rAUC). Area under the receiver operating curve (AUROC) was calculated as a descriptive tool to assess the overall discrimination of parameters. Malignant tumours displayed higher F(T), Vb, rAUC and lower Ve than benign tumours (P < 0.0001, P = 0.0006, P = 0.04 and P = 0.0002, respectively). F(T) was the most relevant factor for discriminating malignant from benign tumours (AUROC = 0.86). Primary ovarian invasive tumours displayed higher F(T) and shorter Dt than borderline tumours. Malignant adnexal tumours with associated peritoneal carcinomatosis at surgery displayed a shorter Dt than those without peritoneal carcinomatosis at surgery (P = 0.01). Quantitative DCE-MRI is a feasible and accurate technique to differentiate malignant from benign adnexal tumours and could potentially help oncologists with management decisions. Quantitative DCE MR imaging allows accurate differentiation between malignant and benign tumours. Quantitative DCE MRI may help predict peritoneal carcinomatosis associated with ovarian tumors. Quantitative DCE MRI helps distinguish between invasive and borderline primary ovarian tumours.

  16. Human T Cell Memory: A Dynamic View

    Directory of Open Access Journals (Sweden)

    Derek C. Macallan

    2017-02-01

    Full Text Available Long-term T cell-mediated protection depends upon the formation of a pool of memory cells to protect against future pathogen challenge. In this review we argue that looking at T cell memory from a dynamic viewpoint can help in understanding how memory populations are maintained following pathogen exposure or vaccination. For example, a dynamic view resolves the apparent paradox between the relatively short lifespans of individual memory cells and very long-lived immunological memory by focussing on the persistence of clonal populations, rather than individual cells. Clonal survival is achieved by balancing proliferation, death and differentiation rates within and between identifiable phenotypic pools; such pools correspond broadly to sequential stages in the linear differentiation pathway. Each pool has its own characteristic kinetics, but only when considered as a population; single cells exhibit considerable heterogeneity. In humans, we tend to concentrate on circulating cells, but memory T cells in non-lymphoid tissues and bone marrow are increasingly recognised as critical for immune defence; their kinetics, however, remain largely unexplored. Considering vaccination from this viewpoint shifts the focus from the size of the primary response to the survival of the clone and enables identification of critical system pinch-points and opportunities to improve vaccine efficacy.

  17. Collective dynamics of cell migration and cell rearrangements

    Science.gov (United States)

    Kabla, Alexandre

    Understanding multicellular processes such as embryo development or cancer metastasis requires to decipher the contributions of local cell autonomous behaviours and long range interactions with the tissue environment. A key question in this context concerns the emergence of large scale coordination in cell behaviours, a requirement for collective cell migration or convergent extension. I will present a few examples where physical and mechanical aspects play a significant role in driving tissue scale dynamics. Geometrical confinement is one of the key external factors influencing large scale coordination during collective migration. Using a combination of in vitro experiments and numerical simulations, we show that the velocity correlation length, measured in unconfined conditions, provides a convenient length scale to predict the dynamic response under confinement. The same length scale can also be used to quantify the influence range of directional cues within the cell population. Heterogeneity within motile cell populations is frequently associated with an increase in their invasive capability and appears to play an important role during cancer metastasis. Using in silico experiments, we studied the way cell invasion is influenced by both the degree of cell coordination and the amount of variability in the motile force of the invading cells. Results suggest that mechanical heterogeneity dramatically enhances the invasion rate through an emerging cooperative process between the stronger and weaker cells, accounting for a number of observed invasion phenotypes. Effective convergent extension requires on a consistent orientation of cell intercalation at the tissue scale, most often in relation with planar cell polarity mechanisms to define the primary axes of deformation. Using a novel modelling approach for cells mechanical interactions, we studied the dynamics of substrate free motile cell populations. Ongoing work shows in particular that nematic order emerges

  18. Separation of Cells using a Fluidic MEMS Device and a Quantitative Analysis of Cell Movement

    Science.gov (United States)

    Isoda, Takaaki; Ishida, Yasuaki

    Fluidic micro electro mechanical system (fluidic MEMS) devices, composed of a micro pump, mixer, valve, reactor, sensor and an electric circuit on a chip, have been widely applied in biotechnology and medical analyses. This study describes the design and fabrication of a fluidic MEMS device that can separate living leukocyte cells from a single droplet of blood (blood sample was flowed into the gap (40μm) between the two substrates driven by the difference in surface tension of the two materials. Leukocyte cells were left adhered to the lower hydrophobic surface, whereas red corpuscles flowed toward the exit of the fluidic MEMS device. The separation rate of the red corpuscles has been achieved to 91 ± 9% in a unit area of 0.1 mm2. Further, the change in an area of a living leukocyte cell separated in the chip, was quantitatively analyzed. This study proposes a method for separating and measuring living cells in a fluidic MEMS device.

  19. Quantitative imaging of glutathione in live cells using a reversible reaction-based ratiometric fluorescent probe

    Science.gov (United States)

    Glutathione (GSH) plays an important role in maintaining redox homeostasis inside cells. Currently, there are no methods available to quantitatively assess the GSH concentration in live cells. Live cell fluorescence imaging revolutionized the understanding of cell biology and has become an indispens...

  20. Measuring cell surface area and deformability of individual human red blood cells over blood storage using quantitative phase imaging

    Science.gov (United States)

    Park, Hyunjoo; Lee, Sangyun; Ji, Misuk; Kim, Kyoohyun; Son, Yonghak; Jang, Seongsoo; Park, Yongkeun

    2016-10-01

    The functionality and viability of stored human red blood cells (RBCs) is an important clinical issue in transfusions. To systematically investigate changes in stored whole blood, the hematological properties of individual RBCs were quantified in blood samples stored for various periods with and without a preservation solution called citrate phosphate dextrose adenine-1 (CPDA-1). With 3-D quantitative phase imaging techniques, the optical measurements for 3-D refractive index (RI) distributions and membrane fluctuations were done at the individual cell level. From the optical measurements, the morphological (volume, surface area and sphericity), biochemical (hemoglobin content and concentration), and mechanical parameters (dynamic membrane fluctuation) were simultaneously quantified to investigate the functionalities and progressive alterations of stored RBCs. Our results show that stored RBCs without CPDA-1 had a dramatic morphological transformation from discocytes to spherocytes within two weeks which was accompanied by significant decreases in cell deformability and cell surface area, and increases in sphericity. However, the stored RBCs with CPDA-1 maintained their morphology and deformability for up to 6 weeks.

  1. Dynamics of B cells in germinal centres.

    Science.gov (United States)

    De Silva, Nilushi S; Klein, Ulf

    2015-03-01

    Humoral immunity depends on the germinal centre (GC) reaction during which somatically mutated high-affinity memory B cells and plasma cells are generated. Recent studies have uncovered crucial cues that are required for the formation and the maintenance of GCs and for the selection of high-affinity antibody mutants. In addition, it is now clear that these events are promoted by the dynamic movements of cells within and between GCs. These findings have resolved the complexities of the GC reaction in greater detail than ever before. This Review focuses on these recent advances and discusses their implications for the establishment of humoral immunity.

  2. Quantitative Assessment of Heart Rate Dynamics during Meditation: An ECG Based Study with Multi-Fractality and Visibility Graph.

    Science.gov (United States)

    Bhaduri, Anirban; Ghosh, Dipak

    2016-01-01

    The cardiac dynamics during meditation is explored quantitatively with two chaos-based non-linear techniques viz. multi-fractal detrended fluctuation analysis and visibility network analysis techniques. The data used are the instantaneous heart rate (in beats/minute) of subjects performing Kundalini Yoga and Chi meditation from PhysioNet. The results show consistent differences between the quantitative parameters obtained by both the analysis techniques. This indicates an interesting phenomenon of change in the complexity of the cardiac dynamics during meditation supported with quantitative parameters. The results also produce a preliminary evidence that these techniques can be used as a measure of physiological impact on subjects performing meditation.

  3. 定量动态增强MRI对肾透明细胞癌及乏脂肪肾血管平滑肌脂肪瘤的鉴别诊断价值%The value of quantitative dynamic contrast enhanced MRI in differential diagnosis of renal clear cell carcinoma and renal angiomyolipomas with minimal fat

    Institute of Scientific and Technical Information of China (English)

    苗燕平; 高阳; 曹鹏

    2015-01-01

    目的:探讨定量动态增强磁共振成像(dynamic contrast-enhanced MRI, DCE-MRI)对肾透明细胞癌、乏脂肪肾血管平滑肌脂肪瘤的鉴别能力。材料与方法收集38例经手术病理证实的肾脏肿瘤患者,其中肾透明细胞癌26例,乏脂肪肾血管平滑肌脂肪瘤12例,术前均行常规MRI、DCE-MRI扫描,选取感兴趣区测量肿瘤的动态增强定量参数Ktrans(容量转移常数)、Kep(速率常数)、Ve(血管外细胞外间隙容积比),并对得到的数据进行分析。结果经DCE-MRI检查的26例肾透明细胞癌患者中病灶区在动态增强扫描的早期强化,定量参数Ktrans、Kep、Ve均值分别为(0.625±0.313) min-1、(1.764±1.105) min-1、(-0.341±0.207);12例乏脂肪肾血管平滑肌脂肪瘤的定量参数K trans、K ep、Ve均值分别为(0.061±0.023) min-1、(0.916±0.313) min-1、(-0.146±0.074)。Ktrans在肾透明细胞癌与乏脂肪肾血管平滑肌脂肪瘤间的差异有统计学意义(t=4.063,P<0.05), Kep、Ve在肾透明细胞癌与乏脂肪肾血管平滑肌脂肪瘤间的差异无统计学意义(t值分别为2.153、0.5,P>0.05)。结论定量DCE-MRI技术对肾透明细胞癌与乏脂肪肾血管平滑肌脂肪瘤有良好的鉴别诊断作用。%AbstractObjective:To explore the differential diagnosis value of quantitative dynamic contrast-enhanced MRI(DCE-MRI) of renal clear cell carcinoma and renal angiomyolipomas with minimal fat. Materials and Methods:Twenty-six cases with renal clear cell carcinoma, twelve cases with renal angiomyolipomas with minimal fat confirmed by operation, underwent the examination of MRI conventional scanning, and DCE-MRI. ROI were drawn to record the quantitative parameters average values of Ktrans, Kep, Ve. Results were statistically treated with SPSS 13.0.Results:Twenty-six cases with renal clear cell carcinoma showed obvious enhancement in the early phase of DCE-MRI. The Ktrans, Kep, Ve average value of renal cell

  4. A label-free proteome analysis strategy for identifying quantitative changes in erythrocyte membranes induced by red cell disorders.

    Science.gov (United States)

    Pesciotta, Esther N; Sriswasdi, Sira; Tang, Hsin-Yao; Mason, Philip J; Bessler, Monica; Speicher, David W

    2012-12-05

    Red blood cells have been extensively studied but many questions regarding membrane properties and pathophysiology remain unanswered. Proteome analysis of red cell membranes is complicated by a very wide dynamic range of protein concentrations as well as the presence of proteins that are very large, very hydrophobic, or heterogeneously glycosylated. This study investigated the removal of other blood cell types, red cell membrane extraction, differing degrees of fractionation using 1-D SDS gels, and label-free quantitative methods to determine optimized conditions for proteomic comparisons of clinical blood samples. The results showed that fractionation of red cell membranes on 1-D SDS gels was more efficient than low-ionic-strength extractions followed by 1-D gel fractionation. When gel lanes were sliced into 30 uniform slices, a good depth of analysis that included the identification of most well-characterized, low-abundance red cell membrane proteins including those present at 500 to 10,000 copies per cell was obtained. Furthermore, the size separation enabled detection of changes due to proteolysis or in vivo protein crosslinking. A combination of Rosetta Elucidator quantitation and subsequent statistical analysis enabled the robust detection of protein differences that could be used to address unresolved questions in red cell disorders. This article is part of a Special Issue entitled: Integrated omics.

  5. Quantitative Analysis of the Nanopore Translocation Dynamics of Simple Structured Polynucleotides

    Science.gov (United States)

    Schink, Severin; Renner, Stephan; Alim, Karen; Arnaut, Vera; Simmel, Friedrich C.; Gerland, Ulrich

    2012-01-01

    Nanopore translocation experiments are increasingly applied to probe the secondary structures of RNA and DNA molecules. Here, we report two vital steps toward establishing nanopore translocation as a tool for the systematic and quantitative analysis of polynucleotide folding: 1), Using α-hemolysin pores and a diverse set of different DNA hairpins, we demonstrate that backward nanopore force spectroscopy is particularly well suited for quantitative analysis. In contrast to forward translocation from the vestibule side of the pore, backward translocation times do not appear to be significantly affected by pore-DNA interactions. 2), We develop and verify experimentally a versatile mesoscopic theoretical framework for the quantitative analysis of translocation experiments with structured polynucleotides. The underlying model is based on sequence-dependent free energy landscapes constructed using the known thermodynamic parameters for polynucleotide basepairing. This approach limits the adjustable parameters to a small set of sequence-independent parameters. After parameter calibration, the theoretical model predicts the translocation dynamics of new sequences. These predictions can be leveraged to generate a baseline expectation even for more complicated structures where the assumptions underlying the one-dimensional free energy landscape may no longer be satisfied. Taken together, backward translocation through α-hemolysin pores combined with mesoscopic theoretical modeling is a promising approach for label-free single-molecule analysis of DNA and RNA folding. PMID:22225801

  6. Quantitative imaging of single mRNA splice variants in living cells

    Science.gov (United States)

    Lee, Kyuwan; Cui, Yi; Lee, Luke P.; Irudayaraj, Joseph

    2014-06-01

    Alternative messenger RNA (mRNA) splicing is a fundamental process of gene regulation, and errors in RNA splicing are known to be associated with a variety of different diseases. However, there is currently a lack of quantitative technologies for monitoring mRNA splice variants in cells. Here, we show that a combination of plasmonic dimer probes and hyperspectral imaging can be used to detect and quantify mRNA splice variants in living cells. The probes are made from gold nanoparticles functionalized with oligonucleotides and can hybridize to specific mRNA sequences, forming nanoparticle dimers that exhibit distinct spectral shifts due to plasmonic coupling. With this approach, we show that the spatial and temporal distribution of three selected splice variants of the breast cancer susceptibility gene, BRCA1, can be monitored at single-copy resolution by measuring the hybridization dynamics of the nanoplasmonic dimers. Our study provides insights into RNA and its transport in living cells, which could improve our understanding of cellular protein complexes, pharmacogenomics, genetic diagnosis and gene therapies.

  7. Image registration and analysis for quantitative myocardial perfusion: application to dynamic circular cardiac CT

    Energy Technology Data Exchange (ETDEWEB)

    Isola, A A [Philips Research Laboratories, X-ray Imaging Systems Department, Weisshausstrasse 2, D-52066 Aachen (Germany); Schmitt, H; Van Stevendaal, U; Grass, M [Philips Research Laboratories, Sector Digital Imaging, Roentgenstrasse 24-26, D-22335 Hamburg (Germany); Begemann, P G [Department of Radiology, University Hospital Hamburg-Eppendorf, Martinistrasse 52, D-20246 Hamburg (Germany); Coulon, P [Philips Healthcare France, 33 rue de Verdun, F-92150 Suresnes Cedex (France); Boussel, L, E-mail: Alfonso.Isola@Philips.com [Department of Radiology, Louis Pradel Hospital, CREATIS, UMR CNRS 5515, INSERM U630, Lyon (France)

    2011-09-21

    Large area detector computed tomography systems with fast rotating gantries enable volumetric dynamic cardiac perfusion studies. Prospectively, ECG-triggered acquisitions limit the data acquisition to a predefined cardiac phase and thereby reduce x-ray dose and limit motion artefacts. Even in the case of highly accurate prospective triggering and stable heart rate, spatial misalignment of the cardiac volumes acquired and reconstructed per cardiac cycle may occur due to small motion pattern variations from cycle to cycle. These misalignments reduce the accuracy of the quantitative analysis of myocardial perfusion parameters on a per voxel basis. An image-based solution to this problem is elastic 3D image registration of dynamic volume sequences with variable contrast, as it is introduced in this contribution. After circular cone-beam CT reconstruction of cardiac volumes covering large areas of the myocardial tissue, the complete series is aligned with respect to a chosen reference volume. The results of the registration process and the perfusion analysis with and without registration are evaluated quantitatively in this paper. The spatial alignment leads to improved quantification of myocardial perfusion for three different pig data sets.

  8. Measuring the Nonuniform Evaporation Dynamics of Sprayed Sessile Microdroplets with Quantitative Phase Imaging.

    Science.gov (United States)

    Edwards, Chris; Arbabi, Amir; Bhaduri, Basanta; Wang, Xiaozhen; Ganti, Raman; Yunker, Peter J; Yodh, Arjun G; Popescu, Gabriel; Goddard, Lynford L

    2015-10-13

    We demonstrate real-time quantitative phase imaging as a new optical approach for measuring the evaporation dynamics of sessile microdroplets. Quantitative phase images of various droplets were captured during evaporation. The images enabled us to generate time-resolved three-dimensional topographic profiles of droplet shape with nanometer accuracy and, without any assumptions about droplet geometry, to directly measure important physical parameters that characterize surface wetting processes. Specifically, the time-dependent variation of the droplet height, volume, contact radius, contact angle distribution along the droplet's perimeter, and mass flux density for two different surface preparations are reported. The studies clearly demonstrate three phases of evaporation reported previously: pinned, depinned, and drying modes; the studies also reveal instances of partial pinning. Finally, the apparatus is employed to investigate the cooperative evaporation of the sprayed droplets. We observe and explain the neighbor-induced reduction in evaporation rate, that is, as compared to predictions for isolated droplets. In the future, the new experimental methods should stimulate the exploration of colloidal particle dynamics on the gas-liquid-solid interface.

  9. Dynamic and quantitative assessment of blood coagulation using optical coherence elastography

    Science.gov (United States)

    Xu, Xiangqun; Zhu, Jiang; Chen, Zhongping

    2016-04-01

    Reliable clot diagnostic systems are needed for directing treatment in a broad spectrum of cardiovascular diseases and coagulopathy. Here, we report on non-contact measurement of elastic modulus for dynamic and quantitative assessment of whole blood coagulation using acoustic radiation force orthogonal excitation optical coherence elastography (ARFOE-OCE). In this system, acoustic radiation force (ARF) is produced by a remote ultrasonic transducer, and a shear wave induced by ARF excitation is detected by the optical coherence tomography (OCT) system. During porcine whole blood coagulation, changes in the elastic property of the clots increase the shear modulus of the sample, altering the propagating velocity of the shear wave. Consequently, dynamic blood coagulation status can be measured quantitatively by relating the velocity of the shear wave with clinically relevant coagulation metrics, including reaction time, clot formation kinetics and maximum shear modulus. The results show that the ARFOE-OCE is sensitive to the clot formation kinetics and can differentiate the elastic properties of the recalcified porcine whole blood, blood added with kaolin as an activator, and blood spiked with fibrinogen.

  10. Image registration and analysis for quantitative myocardial perfusion: application to dynamic circular cardiac CT

    Science.gov (United States)

    Isola, A. A.; Schmitt, H.; van Stevendaal, U.; Begemann, P. G.; Coulon, P.; Boussel, L.; Grass, M.

    2011-09-01

    Large area detector computed tomography systems with fast rotating gantries enable volumetric dynamic cardiac perfusion studies. Prospectively, ECG-triggered acquisitions limit the data acquisition to a predefined cardiac phase and thereby reduce x-ray dose and limit motion artefacts. Even in the case of highly accurate prospective triggering and stable heart rate, spatial misalignment of the cardiac volumes acquired and reconstructed per cardiac cycle may occur due to small motion pattern variations from cycle to cycle. These misalignments reduce the accuracy of the quantitative analysis of myocardial perfusion parameters on a per voxel basis. An image-based solution to this problem is elastic 3D image registration of dynamic volume sequences with variable contrast, as it is introduced in this contribution. After circular cone-beam CT reconstruction of cardiac volumes covering large areas of the myocardial tissue, the complete series is aligned with respect to a chosen reference volume. The results of the registration process and the perfusion analysis with and without registration are evaluated quantitatively in this paper. The spatial alignment leads to improved quantification of myocardial perfusion for three different pig data sets.

  11. Prospects and challenges of quantitative phase imaging in tumor cell biology

    Science.gov (United States)

    Kemper, Björn; Götte, Martin; Greve, Burkhard; Ketelhut, Steffi

    2016-03-01

    Quantitative phase imaging (QPI) techniques provide high resolution label-free quantitative live cell imaging. Here, prospects and challenges of QPI in tumor cell biology are presented, using the example of digital holographic microscopy (DHM). It is shown that the evaluation of quantitative DHM phase images allows the retrieval of different parameter sets for quantification of cellular motion changes in migration and motility assays that are caused by genetic modifications. Furthermore, we demonstrate simultaneously label-free imaging of cell growth and morphology properties.

  12. Cell wall staining with Trypan Blue enables quantitative analysis of morphological changes in yeast cells

    Directory of Open Access Journals (Sweden)

    Johannes eLiesche

    2015-02-01

    Full Text Available Yeast cells are protected by a cell wall that plays an important role in the exchange of substances with the environment. The cell wall structure is dynamic and can adapt to different physiological states or environmental conditions. For the investigation of morphological changes, selective staining with fluorescent dyes is a valuable tool. Furthermore, cell wall staining is used to facilitate sub-cellular localization experiments with fluorescently-labeled proteins and the detection of yeast cells in non-fungal host tissues. Here, we report staining of Saccharomyces cerevisiae cell wall with Trypan Blue, which emits strong red fluorescence upon binding to chitin and yeast glucan; thereby, it facilitates cell wall analysis by confocal and super-resolution microscopy. The staining pattern of Trypan Blue was similar to that of the widely used UV-excitable, blue fluorescent cell wall stain Calcofluor White. Trypan Blue staining facilitated quantification of cell size and cell wall volume when utilizing the optical sectioning capacity of a confocal microscope. This enabled the quantification of morphological changes during growth under anaerobic conditions and in the presence of chemicals, demonstrating the potential of this approach for morphological investigations or screening assays.

  13. Dynamic synchrony of local cell assembly.

    Science.gov (United States)

    Sakurai, Yoshio; Takahashi, Susumu

    2008-01-01

    In the present paper, we focus on the problem of the dynamic size of a cell assembly and discuss how we can detect synchronized firing of a local cell assembly consisting of closely neighboring neurons in the working brain. A local cell assembly is difficult to detect because of the problem of spike overlapping of neighboring neurons, which cannot be overcome by ordinary spike-sorting techniques. We introduce a unique technique of spike-sorting that combines independent component analysis (ICA) and an ordinary sorting method to separate individual neighboring neurons and analyze their firing synchrony in behaving animals. One of our experiments employing this method showed that some closely neighboring neurons in the monkey prefrontal cortex have dynamic and sharp synchrony of firing reflecting local cell assemblies during working-memory processes. Another experiment showed that our other method (ICSort) of novel spike-sorting by ICA using special electrodes (dodecatrodes) can distinguish firing signals from the soma and those from the dendrites of individual neurons in behaving rats and suggests that the somatic and dendritic signals have different roles in information processing. This indicates that functional connectivity among neurons may be more dynamic and complex and spikes from the soma and dendrites of individual neurons should be considered in the investigation of the activity of local cell assemblies. We finally propose that detailed and real features of a local cell assembly consisting of closely neighboring neurons should be examined further and detection of local cell assemblies could be applied to the development of neuronal prosthetic devices, that is, brain-machine interfaces (BMIs).

  14. Quantitative analysis of HIV-1 protease inhibitors in cell lysates using MALDI-FTICR mass spectrometry.

    NARCIS (Netherlands)

    Kampen, JJ van; Burgers, P.C.; Groot, R. de; Osterhaus, A.D.; Reedijk, M.L.; Verschuren, E.J.; Gruters, R.A.; Luider, T.M.

    2008-01-01

    In this report we explore the use of MALDI-FTICR mass spectrometry for the quantitative analysis of five HIV-1 protease inhibitors in cell lysates. 2,5-Dihydroxybenzoic acid (DHB) was used as the matrix. From a quantitative perspective, DHB is usually a poor matrix due to its poor shot-to-shot and p

  15. Quantitative Investigation of Solitary Pulmonary Nodules with Dynamic Contrast-Enhanced Functional CT

    Institute of Scientific and Technical Information of China (English)

    MinmingZhang; HuaZhou; YuZou

    2004-01-01

    OBJECTIVE To evaluate solitary pulmonary nodules (SPNs) using quantitative-dynamic contrast-enhancedfunctional-computed tomography(CT); and to illustrate its clinical efficacy in differential diagnosis of benign and malignant pulmonary nodules.METHODS Eighty patients with non-calcified SPNs (diameter, 5-30mm) were studied with dynamic contrast-enhanced CT. Patterns of the time-density curves (TDC) were assessed. The precontrast density, peak height in density (PH: the maximum value of the TDC) and S/A ratio (the ratio of the PH of SPN to aorta) were recorded. Precontrast density and enhancement patterns of SPNs were also recorded. Perfusion of the SPNs was calculated.RESULTS Malignant, benign and inflammatory nodules showed quite different patterns in the TDC. The PH and S/A ratios of the malignant and inflammatory nodules were significantly higher than that of the benig nnodules (P<0.001; P<0.001), while no statistical difference of either the PH or S/A ratio was found between the malignant and inflammatory nodules. Precontrast density of the inflammatory nodules was lower than that of the malignant nodules (P <0.05). Both the malignant and inflammatory nodules showed significantly higher perfusions than that of the benign nodules (P<0.01; P<0.01). However, the difference between the peffusion of the malignant nodules and inflammatory nodules was not significant.CONCLUSION Dynamic contrast-enhanced functional CT can provide quantitative information regarding blood flow patterns of SPNs and proved to be an alternate non-invasive option in the evaluation and management of solitary pulmonary nodules.

  16. A quantitative study of exocytosis of titanium dioxide nanoparticles from neural stem cells

    Science.gov (United States)

    Wang, Yanli; Wu, Qiuxia; Sui, Keke; Chen, Xin-Xin; Fang, Jie; Hu, Xuefeng; Wu, Minghong; Liu, Yuanfang

    2013-05-01

    Nanoparticles (NPs) have been widely studied and applied in biomedicine and other fields. It is important to know the basic process of interaction between NPs and cells in terms of cellular endocytosis and exocytosis. However, little attention has been paid to the cellular exocytosis of NPs. Herein, using a multi-step cellular subculture method, we ascertain quantitatively the endocytosis and exocytosis of widely used TiO2 NPs using the neural stem cells (NSC) as a cellular model and ICP-AES as an analytic measure. Irrespective of the type and dose of TiO2 NPs, approximately 30% of the total TiO2 NPs entered NSCs after 48 h incubation. In the first 24 h after removing TiO2NPs, from the culture medium, about 35.0%, 34.6% and 41.7% of NP1 (50 nm), NP2 (30 nm) and NTs (nanotubes, 100 nm × 4-6 nm) were released (exocytosed) from cells, respectively. The release decreased over time, and became negligible at 72 h. Exocytosis did not happen during cell division. In addition, our results suggested that both endocytosis and exocytosis of TiO2NPs were energy-dependent processes, and NPs uptake by cells was influenced by serum proteins. Furthermore, we achieved primary dynamic confocal imaging of the exocytosis, allowing tracking of TiO2 NPs from NSCs. These findings may benefit studies on nanotoxicology and nanomedicine of TiO2 NPs.Nanoparticles (NPs) have been widely studied and applied in biomedicine and other fields. It is important to know the basic process of interaction between NPs and cells in terms of cellular endocytosis and exocytosis. However, little attention has been paid to the cellular exocytosis of NPs. Herein, using a multi-step cellular subculture method, we ascertain quantitatively the endocytosis and exocytosis of widely used TiO2 NPs using the neural stem cells (NSC) as a cellular model and ICP-AES as an analytic measure. Irrespective of the type and dose of TiO2 NPs, approximately 30% of the total TiO2 NPs entered NSCs after 48 h incubation. In the

  17. Sequential processing of quantitative phase images for the study of cell behaviour in real-time digital holographic microscopy.

    Science.gov (United States)

    Zikmund, T; Kvasnica, L; Týč, M; Křížová, A; Colláková, J; Chmelík, R

    2014-11-01

    Transmitted light holographic microscopy is particularly used for quantitative phase imaging of transparent microscopic objects such as living cells. The study of the cell is based on extraction of the dynamic data on cell behaviour from the time-lapse sequence of the phase images. However, the phase images are affected by the phase aberrations that make the analysis particularly difficult. This is because the phase deformation is prone to change during long-term experiments. Here, we present a novel algorithm for sequential processing of living cells phase images in a time-lapse sequence. The algorithm compensates for the deformation of a phase image using weighted least-squares surface fitting. Moreover, it identifies and segments the individual cells in the phase image. All these procedures are performed automatically and applied immediately after obtaining every single phase image. This property of the algorithm is important for real-time cell quantitative phase imaging and instantaneous control of the course of the experiment by playback of the recorded sequence up to actual time. Such operator's intervention is a forerunner of process automation derived from image analysis. The efficiency of the propounded algorithm is demonstrated on images of rat fibrosarcoma cells using an off-axis holographic microscope.

  18. Segmentation of vascular structures and hematopoietic cells in 3D microscopy images and quantitative analysis

    Science.gov (United States)

    Mu, Jian; Yang, Lin; Kamocka, Malgorzata M.; Zollman, Amy L.; Carlesso, Nadia; Chen, Danny Z.

    2015-03-01

    In this paper, we present image processing methods for quantitative study of how the bone marrow microenvironment changes (characterized by altered vascular structure and hematopoietic cell distribution) caused by diseases or various factors. We develop algorithms that automatically segment vascular structures and hematopoietic cells in 3-D microscopy images, perform quantitative analysis of the properties of the segmented vascular structures and cells, and examine how such properties change. In processing images, we apply local thresholding to segment vessels, and add post-processing steps to deal with imaging artifacts. We propose an improved watershed algorithm that relies on both intensity and shape information and can separate multiple overlapping cells better than common watershed methods. We then quantitatively compute various features of the vascular structures and hematopoietic cells, such as the branches and sizes of vessels and the distribution of cells. In analyzing vascular properties, we provide algorithms for pruning fake vessel segments and branches based on vessel skeletons. Our algorithms can segment vascular structures and hematopoietic cells with good quality. We use our methods to quantitatively examine the changes in the bone marrow microenvironment caused by the deletion of Notch pathway. Our quantitative analysis reveals property changes in samples with deleted Notch pathway. Our tool is useful for biologists to quantitatively measure changes in the bone marrow microenvironment, for developing possible therapeutic strategies to help the bone marrow microenvironment recovery.

  19. A simple optical fiber device for quantitative fluorescence microscopy of single living cells

    NARCIS (Netherlands)

    Graft, van Marja; Oosterhuis, Bernard; Werf, van der Kees O.; Grooth, de Bart G.; Greve, Jan

    1993-01-01

    simple and relatively inexpensive system is described for obtaining quantitative fluorescence measurements on single living cells loaded with a fluorescent probe to study cell physiological processes. The light emitted from the fluorescent cells is captured by and transported through an optical fibe

  20. Consensus strategy to quantitate malignant cells in myeloma patients is validated in a multicenter study

    NARCIS (Netherlands)

    Willems, P; Verhagen, O; Segeren, C; Veenhuizen, P; Guikema, J; Wiemer, E; Groothuis, L; Buitenweg-de Jong, T; Kok, H; Bloem, A; Bos, N; Vellenga, E; Mensink, E; Sonneveld, P; van der Schoot, HLE; Raymakers, R

    2000-01-01

    Recently the Belgium-Dutch Hematology-Oncology group initiated a multicenter study to evaluate whether myeloma patients treated with intensive chemotherapy benefit from additional peripheral stem cell transplantation. To determine treatment response accurately, we decided to quantitate malignant cel

  1. Rotorcraft flight control design using quantitative feedback theory and dynamic crossfeeds

    Science.gov (United States)

    Cheng, Rendy P.

    1995-01-01

    A multi-input, multi-output controls design with robust crossfeeds is presented for a rotorcraft in near-hovering flight using quantitative feedback theory (QFT). Decoupling criteria are developed for dynamic crossfeed design and implementation. Frequency dependent performance metrics focusing on piloted flight are developed and tested on 23 flight configurations. The metrics show that the resulting design is superior to alternative control system designs using conventional fixed-gain crossfeeds and to feedback-only designs which rely on high gains to suppress undesired off-axis responses. The use of dynamic, robust crossfeeds prior to the QFT design reduces the magnitude of required feedback gain and results in performance that meets current handling qualities specifications relative to the decoupling of off-axis responses. The combined effect of the QFT feedback design following the implementation of low-order, dynamic crossfeed compensator successfully decouples ten of twelve off-axis channels. For the other two channels it was not possible to find a single, low-order crossfeed that was effective.

  2. Modeling cell dynamics under mobile phone radiation.

    Science.gov (United States)

    Minelli, Tullio Antonio; Balduzzo, Maurizio; Milone, Francesco Ferro; Nofrate, Valentina

    2007-04-01

    Perturbations by pulse-modulated microwave radiation from GSM mobile phones on neuron cell membrane gating and calcium oscillations have been suggested as a possible mechanism underlying activation of brain states and electroencephalographic epiphenomena. As the employ of UMTS phones seems to reveal other symptoms, a unified phenomenological framework is needed. In order to explain possible effects of mobile phone radiation on cell oscillations, GSM and UMTS low-frequency envelopes have been detected, recorded and used as input in cell models. Dynamical systems endowed with contiguous regular and chaotic regimes suitable to produce stochastic resonance can both account for the perturbation of the neuro-electrical activity and even for the low intensity of the signal perceived by high sensitive subjects. Neuron models of this kind can be employed as a reductionist hint for the mentioned phenomenology. The Hindmarsh-Rose model exhibits frequency enhancement and regularization phenomena induced by weak GSM and UMTS. More realistic simulations of cell membrane gating and calcium oscillations have been performed with the help of an adaptation of the Chay-Keizer dynamical system. This scheme can explain the suspected subjective sensitivity to mobile phone signals under the thermal threshold, in terms of cell calcium regularity mechanisms. Concerning the two kinds of emission, the stronger occupation of the ELF band of last generation UMTS phones is compensated by lower power emitted.

  3. Metabolic remodeling of the human red blood cell membrane measured by quantitative phase microscopy

    Science.gov (United States)

    Park, YongKeun; Best, Catherine; Auth, Thorsten; Gov, Nir S.; Safran, Samuel; Popescu, Gabriel

    2011-02-01

    We have quantitatively and systemically measured the morphologies and dynamics of fluctuations in human RBC membranes using a full-field laser interferometry technique that accurately measures dynamic membrane fluctuations. We present conclusive evidence that the presence of adenosine 5'-triphosphate (ATP) facilitates nonequilibrium dynamic fluctuations in the RBC membrane and that these fluctuations are highly correlated with specific regions in the biconcave shape of RBCs. Spatial analysis reveals that these nonequilibrium membrane fluctuations are enhanced at the scale of the spectrin mesh size. Our results indicate the presence of dynamic remodeling in the RBC membrane cortex powered by ATP, which results in nonequilibrium membrane fluctuations.

  4. Hydrophobic ionic liquids for quantitative bacterial cell lysis with subsequent DNA quantification.

    Science.gov (United States)

    Fuchs-Telka, Sabine; Fister, Susanne; Mester, Patrick-Julian; Wagner, Martin; Rossmanith, Peter

    2017-02-01

    DNA is one of the most frequently analyzed molecules in the life sciences. In this article we describe a simple and fast protocol for quantitative DNA isolation from bacteria based on hydrophobic ionic liquid supported cell lysis at elevated temperatures (120-150 °C) for subsequent PCR-based analysis. From a set of five hydrophobic ionic liquids, 1-butyl-1-methylpyrrolidinium bis(trifluoromethylsulfonyl)imide was identified as the most suitable for quantitative cell lysis and DNA extraction because of limited quantitative PCR inhibition by the aqueous eluate as well as no detectable DNA uptake. The newly developed method was able to efficiently lyse Gram-negative bacterial cells, whereas Gram-positive cells were protected by their thick cell wall. The performance of the final protocol resulted in quantitative DNA extraction efficiencies for Gram-negative bacteria similar to those obtained with a commercial kit, whereas the number of handling steps, and especially the time required, was dramatically reduced. Graphical Abstract After careful evaluation of five hydrophobic ionic liquids, 1-butyl-1-methylpyrrolidinium bis(trifluoromethylsulfonyl)imide ([BMPyr (+) ][Ntf 2(-) ]) was identified as the most suitable ionic liquid for quantitative cell lysis and DNA extraction. When used for Gram-negative bacteria, the protocol presented is simple and very fast and achieves DNA extraction efficiencies similar to those obtained with a commercial kit. ddH 2 O double-distilled water, qPCR quantitative PCR.

  5. Discrete dynamic modeling of T cell survival signaling networks

    Science.gov (United States)

    Zhang, Ranran

    2009-03-01

    Biochemistry-based frameworks are often not applicable for the modeling of heterogeneous regulatory systems that are sparsely documented in terms of quantitative information. As an alternative, qualitative models assuming a small set of discrete states are gaining acceptance. This talk will present a discrete dynamic model of the signaling network responsible for the survival and long-term competence of cytotoxic T cells in the blood cancer T-LGL leukemia. We integrated the signaling pathways involved in normal T cell activation and the known deregulations of survival signaling in leukemic T-LGL, and formulated the regulation of each network element as a Boolean (logic) rule. Our model suggests that the persistence of two signals is sufficient to reproduce all known deregulations in leukemic T-LGL. It also indicates the nodes whose inactivity is necessary and sufficient for the reversal of the T-LGL state. We have experimentally validated several model predictions, including: (i) Inhibiting PDGF signaling induces apoptosis in leukemic T-LGL. (ii) Sphingosine kinase 1 and NFκB are essential for the long-term survival of T cells in T-LGL leukemia. (iii) T box expressed in T cells (T-bet) is constitutively activated in the T-LGL state. The model has identified potential therapeutic targets for T-LGL leukemia and can be used for generating long-term competent CTL necessary for tumor and cancer vaccine development. The success of this model, and of other discrete dynamic models, suggests that the organization of signaling networks has an determining role in their dynamics. Reference: R. Zhang, M. V. Shah, J. Yang, S. B. Nyland, X. Liu, J. K. Yun, R. Albert, T. P. Loughran, Jr., Network Model of Survival Signaling in LGL Leukemia, PNAS 105, 16308-16313 (2008).

  6. A census of cells in time: quantitative genetics meets developmental biology.

    Science.gov (United States)

    Chitwood, Daniel H; Sinha, Neelima R

    2013-02-01

    Quantitative genetics has become a popular method for determining the genetic basis of natural variation. Combined with genomic methods, it provides a tool for discerning the genetic basis of gene expression. So-called genetical genomics approaches yield a wealth of genomic information, but by necessity, because of cost and time, fail to resolve the differences between organs, tissues, and/or cell types. Similarly, quantitative approaches in development that might potentially address these issues are seldom applied to quantitative genetics. We discuss recent advances in cell type-specific isolation methods, the quantitative analysis of phenotype, and developmental modeling that are compatible with quantitative genetics and, with time, promise to bridge the gap between these two powerful disciplines yielding unprecedented biological insight.

  7. Addressing metabolic heterogeneity in clear cell renal cell carcinoma with quantitative Dixon MRI

    Science.gov (United States)

    Zhang, Yue; Udayakumar, Durga; Cai, Ling; Hu, Zeping; Kapur, Payal; Kho, Eun-Young; Pavía-Jiménez, Andrea; Fulkerson, Michael; de Leon, Alberto Diaz; Yuan, Qing; Dimitrov, Ivan E.; Ye, Jin; Mitsche, Matthew A.; Kim, Hyeonwoo; McDonald, Jeffrey G.; Madhuranthakam, Ananth J.; Dwivedi, Durgesh K.; Lenkinski, Robert E.; Cadeddu, Jeffrey A.; Margulis, Vitaly; Brugarolas, James; DeBerardinis, Ralph J.

    2017-01-01

    BACKGROUND. Dysregulated lipid and glucose metabolism in clear cell renal cell carcinoma (ccRCC) has been implicated in disease progression, and whole tumor tissue–based assessment of these changes is challenged by the tumor heterogeneity. We studied a noninvasive quantitative MRI method that predicts metabolic alterations in the whole tumor. METHODS. We applied Dixon-based MRI for in vivo quantification of lipid accumulation (fat fraction [FF]) in targeted regions of interest of 45 primary ccRCCs and correlated these MRI measures to mass spectrometry–based lipidomics and metabolomics of anatomically colocalized tissue samples isolated from the same tumor after surgery. RESULTS. In vivo tumor FF showed statistically significant (P quantitative Dixon-based MRI as a biomarker of reprogrammed lipid metabolism in ccRCC, which may serve as a predictor of tumor aggressiveness before surgical intervention. FUNDING. NIH R01CA154475 (YZ, MF, PK, IP), NIH P50CA196516 (IP, JB, RJD, JAC, PK), Welch Foundation I-1832 (JY), and NIH P01HL020948 (JGM). PMID:28768909

  8. DYNAMIC MATHEMATICS SOFTWARE: A QUANTITATIVE ANALYSIS IN THE CONTEXT OF THE PREPARATION OF MATH TEACHER

    Directory of Open Access Journals (Sweden)

    Olena V. Semenikhina

    2015-09-01

    Full Text Available The results of the pedagogical experiment to clarify the number of dynamic mathematics software, which modern math teacher should know, are described.Quantitative results of the questionnaire survey of math teachers concerning the use of DMS in the learning process are given. Thus, the preferences of the teachers are software Gran and GeoGebra, the preferences of the students are software GeoGebra and MathKit. It is noted that math teachers in determining the number of DMS, which they should know and be able to use in the future, notice 3-5 DMS and students, future math teachers, notice 5-7 DMS. Statistical processing of the results was made based on the sign test. It allowed to justify the conclusion, that the study of 5 DMS is needed, at the significance level of 0.05.

  9. Quantitative calculation of reaction performance in sonochemical reactor by bubble dynamics

    Science.gov (United States)

    Xu, Zheng; Yasuda, Keiji; Liu, Xiao-Jun

    2015-10-01

    In order to design a sonochemical reactor with high reaction efficiency, it is important to clarify the size and intensity of the sonochemical reaction field. In this study, the reaction field in a sonochemical reactor is estimated from the distribution of pressure above the threshold for cavitation. The quantitation of hydroxide radical in a sonochemical reactor is obtained from the calculation of bubble dynamics and reaction equations. The distribution of the reaction field of the numerical simulation is consistent with that of the sonochemical luminescence. The sound absorption coefficient of liquid in the sonochemical reactor is much larger than that attributed to classical contributions which are heat conduction and shear viscosity. Under the dual irradiation, the reaction field becomes extensive and intensive because the acoustic pressure amplitude is intensified by the interference of two ultrasonic waves. Project supported by the National Natural Science Foundation of China (Grant Nos. 11404245, 11204129, and 11211140039).

  10. Quantitative dynamic nuclear polarization‐NMR on blood plasma for assays of drug metabolism

    DEFF Research Database (Denmark)

    Lerche, Mathilde Hauge; Meier, Sebastian; Jensen, Pernille Rose

    2011-01-01

    Analytical platforms for the fast detection, identification and quantification of circulating drugs with a narrow therapeutic range are vital in clinical pharmacology. As a result of low drug concentrations, analytical tools need to provide high sensitivity and specificity. Dynamic nuclear...... polarization‐NMR (DNP‐NMR) in the form of the hyperpolarization–dissolution method should afford the sensitivity and spectral resolution for the direct detection and quantification of numerous isotopically labeled circulating drugs and their metabolites in single liquid‐state NMR transients. This study......‐scan 13C DNP‐NMR. An internal standard is used for the accurate quantification of drug and metabolite. Comparison of quantitative DNP‐NMR data with an established analytical method (liquid chromatography‐mass spectrometry) yields a Pearson correlation coefficient r of 0.99. Notably, all DNP...

  11. Digital PCR dynamic range is approaching that of real-time quantitative PCR.

    Science.gov (United States)

    Jones, Gerwyn M; Busby, Eloise; Garson, Jeremy A; Grant, Paul R; Nastouli, Eleni; Devonshire, Alison S; Whale, Alexandra S

    2016-12-01

    Digital PCR (dPCR) has been reported to be more precise and sensitive than real-time quantitative PCR (qPCR) in a variety of models and applications. However, in the majority of commercially available dPCR platforms, the dynamic range is dependent on the number of partitions analysed and so is typically limited to four orders of magnitude; reduced compared with the typical seven orders achievable by qPCR. Using two different biological models (HIV DNA analysis and KRAS genotyping), we have demonstrated that the RainDrop Digital PCR System (RainDance Technologies) is capable of performing accurate and precise quantification over six orders of magnitude thereby approaching that achievable by qPCR.

  12. Quantitative calculation of reaction performance in sonochemical reactor by bubble dynamics

    Institute of Scientific and Technical Information of China (English)

    徐峥; 安田启司; 刘晓峻

    2015-01-01

    In order to design a sonochemical reactor with high reaction efficiency, it is important to clarify the size and intensity of the sonochemical reaction field. In this study, the reaction field in a sonochemical reactor is estimated from the distribution of pressure above the threshold for cavitation. The quantitation of hydroxide radical in a sonochemical reactor is obtained from the calculation of bubble dynamics and reaction equations. The distribution of the reaction field of the numerical simulation is consistent with that of the sonochemical luminescence. The sound absorption coefficient of liquid in the sonochemical reactor is much larger than that attributed to classical contributions which are heat conduction and shear viscosity. Under the dual irradiation, the reaction field becomes extensive and intensive because the acoustic pressure amplitude is intensified by the interference of two ultrasonic waves.

  13. On quantitative analysis of interband recombination dynamics: Theory and application to bulk ZnO

    Energy Technology Data Exchange (ETDEWEB)

    Lettieri, S. [Institute for Superconductors, Oxides and Innovative Materials, National Research Council (CNR-SPIN), U.O.S. Napoli, Via Cintia, I-80126 Napoli (Italy); Capello, V.; Santamaria, L. [Physics Department, University of Naples “Federico II,” Via Cintia I-80126 Napoli (Italy); Maddalena, P. [Institute for Superconductors, Oxides and Innovative Materials, National Research Council (CNR-SPIN), U.O.S. Napoli, Via Cintia, I-80126 Napoli (Italy); Physics Department, University of Naples “Federico II,” Via Cintia I-80126 Napoli (Italy)

    2013-12-09

    The issue of the quantitative analysis of time-resolved photoluminescence experiments is addressed by developing and describing two approaches for determination of unimolecular lifetime, bimolecular recombination coefficient, and equilibrium free-carrier concentration, based on a quite general second-order expression of the electron-hole recombination rate. Application to the case of band-edge emission of ZnO single crystals is reported, evidencing the signature of sub-nanosecond second-order recombination dynamics for optical transitions close to the interband excitation edge. The resulting findings are in good agreement with the model prediction and further confirm the presence, formerly evidenced in literature by non-optical methods, of near-surface conductive layers in ZnO crystals with sheet charge densities of about 3–5×10{sup 13} cm{sup −2}.

  14. Quantitative assessment of cytosolic Salmonella in epithelial cells.

    Directory of Open Access Journals (Sweden)

    Leigh A Knodler

    Full Text Available Within mammalian cells, Salmonella enterica serovar Typhimurium (S. Typhimurium inhabits a membrane-bound vacuole known as the Salmonella-containing vacuole (SCV. We have recently shown that wild type S. Typhimurium also colonizes the cytosol of epithelial cells. Here we sought to quantify the contribution of cytosolic Salmonella to the total population over a time course of infection in different epithelial cell lines and under conditions of altered vacuolar escape. We found that the lysosomotropic agent, chloroquine, acts on vacuolar, but not cytosolic, Salmonella. After chloroquine treatment, vacuolar bacteria are not transcriptionally active or replicative and appear degraded. Using a chloroquine resistance assay, in addition to digitonin permeabilization, we found that S. Typhimurium lyses its nascent vacuole in numerous epithelial cell lines, albeit with different frequencies, and hyper-replication in the cytosol is also widespread. At later times post-infection, cytosolic bacteria account for half of the total population in some epithelial cell lines, namely HeLa and Caco-2 C2Bbe1. Both techniques accurately measured increased vacuole lysis in epithelial cells upon treatment with wortmannin. By chloroquine resistance assay, we also determined that Salmonella pathogenicity island-1 (SPI-1, but not SPI-2, the virulence plasmid nor the flagellar apparatus, was required for vacuolar escape and cytosolic replication in epithelial cells. Together, digitonin permeabilization and the chloroquine resistance assay will be useful, complementary tools for deciphering the mechanisms of SCV lysis and Salmonella replication in the epithelial cell cytosol.

  15. (19)F MRI for quantitative in vivo cell tracking.

    NARCIS (Netherlands)

    Srinivas, M.; Heerschap, A.; Ahrens, E.T.; Figdor, C.G.; Vries, I.J.M. de

    2010-01-01

    Cellular therapy, including stem cell transplants and dendritic cell vaccines, is typically monitored for dosage optimization, accurate delivery, and localization using noninvasive imaging, of which magnetic resonance imaging (MRI) is a key modality. (19)F MRI retains the advantages of MRI as an ima

  16. Improved Protein Arrays for Quantitative Systems Analysis of the Dynamics of Signaling Pathway Interactions

    Energy Technology Data Exchange (ETDEWEB)

    YANG, CHIN-RANG [NHLBI, NIH

    2013-12-11

    Astronauts and workers in nuclear plants who repeatedly exposed to low doses of ionizing radiation (IR, <10 cGy) are likely to incur specific changes in signal transduction and gene expression in various tissues of their body. Remarkable advances in high throughput genomics and proteomics technologies enable researchers to broaden their focus from examining single gene/protein kinetics to better understanding global gene/protein expression profiling and biological pathway analyses, namely Systems Biology. An ultimate goal of systems biology is to develop dynamic mathematical models of interacting biological systems capable of simulating living systems in a computer. This Glue Grant is to complement Dr. Boothman’s existing DOE grant (No. DE-FG02-06ER64186) entitled “The IGF1/IGF-1R-MAPK-Secretory Clusterin (sCLU) Pathway: Mediator of a Low Dose IR-Inducible Bystander Effect” to develop sensitive and quantitative proteomic technology that suitable for low dose radiobiology researches. An improved version of quantitative protein array platform utilizing linear Quantum dot signaling for systematically measuring protein levels and phosphorylation states for systems biology modeling is presented. The signals are amplified by a confocal laser Quantum dot scanner resulting in ~1000-fold more sensitivity than traditional Western blots and show the good linearity that is impossible for the signals of HRP-amplification. Therefore this improved protein array technology is suitable to detect weak responses of low dose radiation. Software is developed to facilitate the quantitative readout of signaling network activities. Kinetics of EGFRvIII mutant signaling was analyzed to quantify cross-talks between EGFR and other signaling pathways.

  17. Two-photon microscopy for non-invasive, quantitative monitoring of stem cell differentiation.

    Directory of Open Access Journals (Sweden)

    William L Rice

    Full Text Available BACKGROUND: The engineering of functional tissues is a complex multi-stage process, the success of which depends on the careful control of culture conditions and ultimately tissue maturation. To enable the efficient optimization of tissue development protocols, techniques suitable for monitoring the effects of added stimuli and induced tissue changes are needed. METHODOLOGY/PRINCIPAL FINDINGS: Here, we present the quantitative use of two-photon excited fluorescence (TPEF and second harmonic generation (SHG as a noninvasive means to monitor the differentiation of human mesenchymal stem cells (hMSCs using entirely endogenous sources of contrast. We demonstrate that the individual fluorescence contribution from the intrinsic cellular fluorophores NAD(PH, flavoproteins and lipofuscin can be extracted from TPEF images and monitored dynamically from the same cell population over time. Using the redox ratio, calculated from the contributions of NAD(PH and flavoproteins, we identify distinct patterns in the evolution of the metabolic activity of hMSCs maintained in either propagation, osteogenic or adipogenic differentiation media. The differentiation of these cells is mirrored by changes in cell morphology apparent in high resolution TPEF images and by the detection of collagen production via SHG imaging. Finally, we find dramatic increases in lipofuscin levels in hMSCs maintained at 20% oxygen vs. those in 5% oxygen, establishing the use of this chromophore as a potential biomarker for oxidative stress. CONCLUSIONS/SIGNIFICANCE: In this study we demonstrate that it is possible to monitor the metabolic activity, morphology, ECM production and oxidative stress of hMSCs in a non-invasive manner. This is accomplished using generally available multiphoton microscopy equipment and simple data analysis techniques, such that the method can widely adopted by laboratories with a diversity of comparable equipment. This method therefore represents a powerful tool

  18. Identifying hazard parameter to develop quantitative and dynamic hazard map of an active volcano in Indonesia

    Science.gov (United States)

    Suminar, Wulan; Saepuloh, Asep; Meilano, Irwan

    2016-05-01

    Analysis of hazard assessment to active volcanoes is crucial for risk management. The hazard map of volcano provides information to decision makers and communities before, during, and after volcanic crisis. The rapid and accurate hazard assessment, especially to an active volcano is necessary to be developed for better mitigation on the time of volcanic crises in Indonesia. In this paper, we identified the hazard parameters to develop quantitative and dynamic hazard map of an active volcano. The Guntur volcano in Garut Region, West Java, Indonesia was selected as study area due population are resided adjacent to active volcanoes. The development of infrastructures, especially related to tourism at the eastern flank from the Summit, are growing rapidly. The remote sensing and field investigation approaches were used to obtain hazard parameters spatially. We developed a quantitative and dynamic algorithm to map spatially hazard potential of volcano based on index overlay technique. There were identified five volcano hazard parameters based on Landsat 8 and ASTER imageries: volcanic products including pyroclastic fallout, pyroclastic flows, lava and lahar, slope topography, surface brightness temperature, and vegetation density. Following this proposed technique, the hazard parameters were extracted, indexed, and calculated to produce spatial hazard values at and around Guntur Volcano. Based on this method, the hazard potential of low vegetation density is higher than high vegetation density. Furthermore, the slope topography, surface brightness temperature, and fragmental volcanic product such as pyroclastics influenced to the spatial hazard value significantly. Further study to this proposed approach will be aimed for effective and efficient analyses of volcano risk assessment.

  19. Quantitative dynamic contrast-enhanced MR imaging analysis of complex adnexal masses: a preliminary study

    Energy Technology Data Exchange (ETDEWEB)

    Thomassin-Naggara, Isabelle [Hopital Tenon, Assistance Publique-Hopitaux de Paris, Department of Radiology, Paris (France); Laboratoire de recherche en imagerie - UMR 970 INSERM - Universite Rene Descartes, Paris (France); Service de Radiologie, Hopital Tenon, Paris (France); Balvay, Daniel [Laboratoire de recherche en imagerie - UMR 970 INSERM - Universite Rene Descartes, Paris (France); Aubert, Emilie; Bazot, Marc [Hopital Tenon, Assistance Publique-Hopitaux de Paris, Department of Radiology, Paris (France); Darai, Emile; Rouzier, Roman [Hopital Tenon, Assistance Publique-Hopitaux de Paris, Department of Gynaecology-Obstetrics, Paris (France); Cuenod, Charles A. [Laboratoire de recherche en imagerie - UMR 970 INSERM - Universite Rene Descartes, Paris (France); Hopital Europeen Georges Pompidou (HEGP), Department of Radiology, Paris (France)

    2012-04-15

    To evaluate the ability of quantitative dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) to differentiate malignant from benign adnexal tumours. Fifty-six women with 38 malignant and 18 benign tumours underwent MR imaging before surgery for complex adnexal masses. Microvascular parameters were extracted from high temporal resolution DCE-MRI series, using a pharmacokinetic model in the solid tissue of adnexal tumours. These parameters were tissue blood flow (F{sub T}), blood volume fraction (Vb), permeability-surface area product (PS), interstitial volume fraction (Ve), lag time (Dt) and area under the enhancing curve (rAUC). Area under the receiver operating curve (AUROC) was calculated as a descriptive tool to assess the overall discrimination of parameters. Malignant tumours displayed higher F{sub T}, Vb, rAUC and lower Ve than benign tumours (P < 0.0001, P = 0.0006, P = 0.04 and P = 0.0002, respectively). F{sub T} was the most relevant factor for discriminating malignant from benign tumours (AUROC = 0.86). Primary ovarian invasive tumours displayed higher F{sub T} and shorter Dt than borderline tumours. Malignant adnexal tumours with associated peritoneal carcinomatosis at surgery displayed a shorter Dt than those without peritoneal carcinomatosis at surgery (P = 0.01). Quantitative DCE-MRI is a feasible and accurate technique to differentiate malignant from benign adnexal tumours and could potentially help oncologists with management decisions. (orig.)

  20. Quantitative and dynamic measurements of biological fresh samples with X-ray phase contrast tomography

    Energy Technology Data Exchange (ETDEWEB)

    Hoshino, Masato, E-mail: hoshino@spring8.or.jp; Uesugi, Kentaro [Japan Synchrotron Radiation Research Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5198 (Japan); Tsukube, Takuro [Japanese Red Cross Kobe Hospital, 1-3-1 Wakinohamakaigandori, Chuo-ku, Kobe, Hyogo 651-0073 (Japan); Yagi, Naoto [Japan Synchrotron Radiation Research Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5198 (Japan)

    2014-10-08

    Quantitative measurements of biological fresh samples based on three-dimensional densitometry using X-ray phase contrast tomography are presented. X-ray phase contrast tomography using a Talbot grating interferometer was applied to biological fresh samples which were not fixed by any fixatives. To achieve a high-throughput measurement for the fresh samples the X-ray phase contrast tomography measurement procedure was improved. The three-dimensional structure of a fresh mouse fetus was clearly depicted as a mass density map using X-ray phase contrast tomography. The mouse fetus measured in the fresh state was then fixed by formalin and measured in the fixed state. The influence of the formalin fixation on soft tissue was quantitatively evaluated by comparing the fresh and fixed samples. X-ray phase contrast tomography was also applied to the dynamic measurement of a biological fresh sample. Morphological changes of a ring-shaped fresh pig aorta were measured tomographically under different degrees of stretching.

  1. Semi-quantitative assessment of pulmonary perfusion in children using dynamic contrast-enhanced MRI

    Science.gov (United States)

    Fetita, Catalin; Thong, William E.; Ou, Phalla

    2013-03-01

    This paper addresses the study of semi-quantitative assessment of pulmonary perfusion acquired from dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) in a study population mainly composed of children with pulmonary malformations. The automatic analysis approach proposed is based on the indicator-dilution theory introduced in 1954. First, a robust method is developed to segment the pulmonary artery and the lungs from anatomical MRI data, exploiting 2D and 3D mathematical morphology operators. Second, the time-dependent contrast signal of the lung regions is deconvolved by the arterial input function for the assessment of the local hemodynamic system parameters, ie. mean transit time, pulmonary blood volume and pulmonary blood flow. The discrete deconvolution method implements here a truncated singular value decomposition (tSVD) method. Parametric images for the entire lungs are generated as additional elements for diagnosis and quantitative follow-up. The preliminary results attest the feasibility of perfusion quantification in pulmonary DCE-MRI and open an interesting alternative to scintigraphy for this type of evaluation, to be considered at least as a preliminary decision in the diagnostic due to the large availability of the technique and to the non-invasive aspects.

  2. Evaluation of acetazolamine response in patients with cerebellar ataxia using dynamic quantitative F-18-FDG PET

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Y. K.; Lee, D. S.; Lee, J. S.; Kim, M. H.; Lee, K. M.; Yeo, J. S.; Chung, J. K.; Lee, M. C. [College of Medicine, Seoul National Univ., Seoul (Korea, Republic of)

    2001-07-01

    Cerebellar Ataxia (CA) usually shows dramatic response to acetazolamide treatment. But few cases of acetazolamide unresponse CA were reported recently. Using dynamic FDG PET, we tried to evaluate the metabolic abnormality and its drug response in CA. Quantitative F-18-FDG PET was performed prior and after treatment of acetazolamide (250 mg qid for 10 days) in two patient suspected episodic cerebellar ataxia. Using Model-based clustering method, the regional cerebral glucose metabolic rate (rCMRglu) was calculated. Two patients showed different treatment response to acetazolamide. In one patient who showed markedly reduced frequency of the ataxic attack after treatment. FDG PET showed that mean cerebellar glucose metabolism was increased after treatment ({delta}rCMRglu:9%). However, in the other who showed poor response to acetazolamide, FDG PET showed the more decrease metabolism in cerebellar metabolism after treatment ({delta}rCMRglu:-17%). The change of the cerebellar glucose metabolism on FDG PET reflected the symptomatic improvement after acetazolamide in these two CA patients. We could expected that FDG PET might be a very useful tool to quantitatively predict the treatment response in CA and other neurologic disorder.

  3. A quantitative SMRT cell sequencing method for ribosomal amplicons.

    Science.gov (United States)

    Jones, Bethan M; Kustka, Adam B

    2017-04-01

    Advances in sequencing technologies continue to provide unprecedented opportunities to characterize microbial communities. For example, the Pacific Biosciences Single Molecule Real-Time (SMRT) platform has emerged as a unique approach harnessing DNA polymerase activity to sequence template molecules, enabling long reads at low costs. With the aim to simultaneously classify and enumerate in situ microbial populations, we developed a quantitative SMRT (qSMRT) approach that involves the addition of exogenous standards to quantify ribosomal amplicons derived from environmental samples. The V7-9 regions of 18S SSU rDNA were targeted and quantified from protistan community samples collected in the Ross Sea during the Austral summer of 2011. We used three standards of different length and optimized conditions to obtain accurate quantitative retrieval across the range of expected amplicon sizes, a necessary criterion for analyzing taxonomically diverse 18S rDNA molecules from natural environments. The ability to concurrently identify and quantify microorganisms in their natural environment makes qSMRT a powerful, rapid and cost-effective approach for defining ecosystem diversity and function.

  4. Characterizing motility dynamics in human RPE cells

    Science.gov (United States)

    Liu, Zhuolin; Kurokawa, Kazuhiro; Zhang, Furu; Miller, Donald T.

    2017-02-01

    Retinal pigment epithelium (RPE) cells are vital to health of the outer retina, however, are often compromised in ageing and ocular diseases that lead to blindness. Early manifestation of RPE disruption occurs at the cellular level, but while in vivo biomarkers at this scale hold considerable promise, RPE cells have proven extremely challenging to image in the living human eye. Recently we addressed this problem by using organelle motility as a novel contrast agent to enhance the RPE cell in conjunction with 3D resolution of adaptive optics-optical coherence tomography (AO-OCT) to section the RPE layer. In this study, we expand on the central novelty of our method - organelle motility - by characterizing the dynamics of the motility in individual RPE cells, important because of its direct link to RPE physiology. To do this, AO-OCT videos of the same retinal patch were acquired at approximately 1 min intervals or less, time stamped, and registered in 3D with sub-cellular accuracy. Motility was quantified by an exponential decay time constant, the time for motility to decorrelate the speckle field across an RPE cell. In two normal subjects, we found the decay time constant to be just 3 seconds, thus indicating rapid motility in normal RPE cells.

  5. Decoding brain cancer dynamics: a quantitative histogram-based approach using temporal MRI

    Science.gov (United States)

    Zhou, Mu; Hall, Lawrence O.; Goldgof, Dmitry B.; Russo, Robin; Gillies, Robert J.; Gatenby, Robert A.

    2015-03-01

    Brain tumor heterogeneity remains a challenge for probing brain cancer evolutionary dynamics. In light of evolution, it is a priority to inspect the cancer system from a time-domain perspective since it explicitly tracks the dynamics of cancer variations. In this paper, we study the problem of exploring brain tumor heterogeneity from temporal clinical magnetic resonance imaging (MRI) data. Our goal is to discover evidence-based knowledge from such temporal imaging data, where multiple clinical MRI scans from Glioblastoma multiforme (GBM) patients are generated during therapy. In particular, we propose a quantitative histogram-based approach that builds a prediction model to measure the difference in histograms obtained from pre- and post-treatment. The study could significantly assist radiologists by providing a metric to identify distinctive patterns within each tumor, which is crucial for the goal of providing patient-specific treatments. We examine the proposed approach for a practical application - clinical survival group prediction. Experimental results show that our approach achieved 90.91% accuracy.

  6. The long-term reliability of static and dynamic Quantitative Sensory Testing in healthy individuals.

    Science.gov (United States)

    Marcuzzi, Anna; Wrigley, Paul J; Dean, Catherine M; Adams, Roger; Hush, Julia M

    2017-03-21

    Quantitative sensory tests (QST) have been increasingly used to investigate alterations in somatosensory function in a wide range of painful conditions. The interpretation of these findings is based on the assumption that the measures are stable and reproducible. To date, reliability of QST has been investigated for short test-retest intervals. The aim of this study was to investigate the long-term reliability of a multimodal QST assessment in healthy people, with testing conducted on three occasions over 4-months. Forty-two healthy people were enrolled in the study. Static and dynamic tests were performed, including cold and heat pain threshold (CPT, HPT), mechanical wind up (WUR), pressure pain threshold (PPT), two-point discrimination (TPD) and conditioned pain modulation (CPM). Systematic bias, relative reliability and agreement were analysed using repeated measure ANOVA, intraclass correlation coefficients (ICCs3,1) and standard error of the measurement (SEM), respectively. Static QST (CPT, HPT, PPT and TPD) showed good to excellent reliability (ICCs: 0.68 to 0.90). Dynamic QST (WUR and CPM) showed poor to good reliability (ICCs: 0.35 to 0.61). A significant linear decrease over time was observed for mechanical QST at the back (PPT and TPD) and for CPM (p<0.01). Static QST were stable over a period of 4 months; however, a small systematic decrease over time has been observed for mechanical QST. Dynamic QST showed considerable variability over time; in particular, CPM using PPT as the test stimulus did not show adequate reliability, suggesting that this test paradigm may be less useful for monitoring individuals over time.

  7. High temperature and high pressure gas cell for quantitative spectroscopic measurements

    DEFF Research Database (Denmark)

    Christiansen, Caspar; Stolberg-Rohr, Thomine; Fateev, Alexander

    2016-01-01

    A high temperature and high pressure gas cell (HTPGC) has been manufactured for quantitative spectroscopic measurements in the pressure range 1-200 bar and temperature range 300-1300 K. In the present work the cell was employed at up to 100 bar and 1000 K, and measured absorption coefficients...

  8. Quantitation of ranaviruses in cell culture and tissue samples

    DEFF Research Database (Denmark)

    Holopainen, Riikka; Honkanen, Jarno; Jensen, Britt Bang

    2011-01-01

    ; European sheatfish virus, ESV; Frog virus 3, FV3; Bohle iridovirus, BIV; Doctor fish virus, DFV; Guppy virus 6, GV6; Pike-perch iridovirus, PPIV; Rana esculenta virus Italy 282/I02, REV282/I02 and Short-finned eel ranavirus, SERV) were detected with the qPCR assay. In addition, two fish cell lines...

  9. Swimming motility plays a key role in the stochastic dynamics of cell clumping

    Science.gov (United States)

    Qi, Xianghong; Nellas, Ricky B.; Byrn, Matthew W.; Russell, Matthew H.; Bible, Amber N.; Alexandre, Gladys; Shen, Tongye

    2013-04-01

    Dynamic cell-to-cell interactions are a prerequisite to many biological processes, including development and biofilm formation. Flagellum induced motility has been shown to modulate the initial cell-cell or cell-surface interaction and to contribute to the emergence of macroscopic patterns. While the role of swimming motility in surface colonization has been analyzed in some detail, a quantitative physical analysis of transient interactions between motile cells is lacking. We examined the Brownian dynamics of swimming cells in a crowded environment using a model of motorized adhesive tandem particles. Focusing on the motility and geometry of an exemplary motile bacterium Azospirillum brasilense, which is capable of transient cell-cell association (clumping), we constructed a physical model with proper parameters for the computer simulation of the clumping dynamics. By modulating mechanical interaction (‘stickiness’) between cells and swimming speed, we investigated how equilibrium and active features affect the clumping dynamics. We found that the modulation of active motion is required for the initial aggregation of cells to occur at a realistic time scale. Slowing down the rotation of flagellar motors (and thus swimming speeds) is correlated to the degree of clumping, which is consistent with the experimental results obtained for A. brasilense.

  10. Quantitative phosphoproteomic analysis of prion-infected neuronal cells

    Directory of Open Access Journals (Sweden)

    Löwer Johannes

    2010-09-01

    Full Text Available Abstract Prion diseases or transmissible spongiform encephalopathies (TSEs are fatal diseases associated with the conversion of the cellular prion protein (PrPC to the abnormal prion protein (PrPSc. Since the molecular mechanisms in pathogenesis are widely unclear, we analyzed the global phospho-proteome and detected a differential pattern of tyrosine- and threonine phosphorylated proteins in PrPSc-replicating and pentosan polysulfate (PPS-rescued N2a cells in two-dimensional gel electrophoresis. To quantify phosphorylated proteins, we performed a SILAC (stable isotope labeling by amino acids in cell culture analysis and identified 105 proteins, which showed a regulated phosphorylation upon PrPSc infection. Among those proteins, we validated the dephosphorylation of stathmin and Cdc2 and the induced phosphorylation of cofilin in PrPSc-infected N2a cells in Western blot analyses. Our analysis showed for the first time a differentially regulated phospho-proteome in PrPSc infection, which could contribute to the establishment of novel protein markers and to the development of novel therapeutic intervention strategies in targeting prion-associated disease.

  11. Quantitative assessment of early experimental diabetes in rats using dynamic contrast-enhanced computed tomography

    Energy Technology Data Exchange (ETDEWEB)

    Murase, Kenya [Department of Medical Physics and Engineering, Division of Medical Technology and Science, Faculty of Health Science, Graduate School of Medicine, Osaka University, 1-7 Yamadaoka, Suita, Osaka 565-0871 (Japan)], E-mail: murase@sahs.med.osaka-u.ac.jp; Kitamura, Akihiro; Tachibana, Atsushi; Kusakabe, Yoshinori; Matsuura, Risa; Miyazaki, Shohei [Department of Medical Physics and Engineering, Division of Medical Technology and Science, Faculty of Health Science, Graduate School of Medicine, Osaka University, 1-7 Yamadaoka, Suita, Osaka 565-0871 (Japan)

    2010-04-15

    Purpose: To quantitatively assess the time course of changes of the renal volume and function in the early phase of streptozotocin (STZ)-induced diabetes in rats using dynamic contrast-enhanced computed tomography (DCE-CT). Methods: The DCE-CT studies were performed in 24 male Sprague-Dawley rats (n = 6 for control and n = 18 for STZ-treated group) on days 0, 4, 7, 11, and 14 using a multi-detector row CT. The rats of an STZ-treated group were given intraperitoneally 65 mg/kg body weight of STZ on day 0, and were divided into two groups based on the blood glucose concentration on day 4 being less than 300 mg/dL [STZ-treated group (L), n = 8] or greater than 300 mg/dL [STZ-treated group (G), n = 10]. The contrast clearance per unit renal volume (K{sub 1}) was estimated from the DCE-CT data using the Patlak model. The renal volume (V{sub CT}) was calculated by manually delineating the kidney on the contrast-enhanced CT image. The contrast clearance of the entire kidney (K) was obtained by K{sub 1} x V{sub CT}. Results: V{sub CT} in the STZ-treated group was significantly enlarged on day 4 compared to that on day 0 and continued until day 14. Although there were no significant changes in the time course of K{sub 1} in all groups, K in the STZ-treated groups (L) and (G) significantly increased on days 7 and 4, respectively, and continued until day 14, suggesting that hyperfiltration occurs in parallel with renal volume enlargement. Conclusion: The present method appears useful for quantitatively evaluating the time course of STZ-induced diabetes in rats, because it allows repeated and simultaneous evaluation of renal morphology and function.

  12. Label-free quantitative proteomics of CD133-positive liver cancer stem cells

    Directory of Open Access Journals (Sweden)

    Tsai Sheng-Ta

    2012-11-01

    Full Text Available Abstract Background CD133-positive liver cancer stem cells, which are characterized by their resistance to conventional chemotherapy and their tumor initiation ability at limited dilutions, have been recognized as a critical target in liver cancer therapeutics. In the current work, we developed a label-free quantitative method to investigate the proteome of CD133-positive liver cancer stem cells for the purpose of identifying unique biomarkers that can be utilized for targeting liver cancer stem cells. Label-free quantitation was performed in combination with ID-based Elution time Alignment by Linear regression Quantitation (IDEAL-Q and MaxQuant. Results Initially, IDEAL-Q analysis revealed that 151 proteins were differentially expressed in the CD133-positive hepatoma cells when compared with CD133-negative cells. We then analyzed these 151 differentially expressed proteins by MaxQuant software and identified 10 significantly up-regulated proteins. The results were further validated by RT-PCR, western blot, flow cytometry or immunofluorescent staining which revealed that prominin-1, annexin A1, annexin A3, transgelin, creatine kinase B, vimentin, and EpCAM were indeed highly expressed in the CD133-positive hepatoma cells. Conclusions These findings confirmed that mass spectrometry-based label-free quantitative proteomics can be used to gain insights into liver cancer stem cells.

  13. Hierarchical and dynamic seascapes: A quantitative framework for scaling pelagic biogeochemistry and ecology

    Science.gov (United States)

    Kavanaugh, Maria T.; Hales, Burke; Saraceno, Martin; Spitz, Yvette H.; White, Angelicque E.; Letelier, Ricardo M.

    2014-01-01

    monitoring and comparing oceanographic biophysical dynamics and an objective, quantitative basis by which to scale data from local experiments and observations to regional and global biogeochemical cycles.

  14. Quantitative characterization of single cells by use of immunocytochemistry combined with multiplex LA-ICP-MS.

    Science.gov (United States)

    Mueller, Larissa; Herrmann, Antje J; Techritz, Sandra; Panne, Ulrich; Jakubowski, Norbert

    2017-05-01

    Actual research demonstrates that LA-ICP-MS is capable of being used as an imaging tool with cellular resolution. The aim of this investigation was the method development for LA-ICP-MS to extend the versatility to quantitative and multiplexing imaging of single eukaryotic cells. For visualization of individual cells selected, lanthanide-labeled antibodies were optimized for immuno-imaging of single cells with LA-ICP-MS. The molar content of the artificial introduced labels per cell was quantified using self-made nitrocellulose-coated slides for matrix-matched calibration and calculated amounts were in the range of 3.1 to 17.8 atmol per cell. Furthermore, the quantification strategy allows a conversion of 2D intensity profiles based on counts per second (cps) to quantitative 2D profiles representing the molar amount of the artificial introduced elemental probes per pixel for each individual cell. Graphical abstract ᅟ.

  15. Comparison of DNA fragmentation and color thresholding for objective quantitation of apoptotic cells

    Science.gov (United States)

    Plymale, D. R.; Ng Tang, D. S.; Fermin, C. D.; Lewis, D. E.; Martin, D. S.; Garry, R. F.

    1995-01-01

    Apoptosis is a process of cell death characterized by distinctive morphological changes and fragmentation of cellular DNA. Using video imaging and color thresholding techniques, we objectively quantitated the number of cultured CD4+ T-lymphoblastoid cells (HUT78 cells, RH9 subclone) displaying morphological signs of apoptosis before and after exposure to gamma-irradiation. The numbers of apoptotic cells measured by objective video imaging techniques were compared to numbers of apoptotic cells measured in the same samples by sensitive apoptotic assays that quantitate DNA fragmentation. DNA fragmentation assays gave consistently higher values compared with the video imaging assays that measured morphological changes associated with apoptosis. These results suggest that substantial DNA fragmentation can precede or occur in the absence of the morphological changes which are associated with apoptosis in gamma-irradiated RH9 cells.

  16. Quantitative Functional Imaging Using Dynamic Positron Computed Tomography and Rapid Parameter Estimation Techniques

    Science.gov (United States)

    Koeppe, Robert Allen

    Positron computed tomography (PCT) is a diagnostic imaging technique that provides both three dimensional imaging capability and quantitative measurements of local tissue radioactivity concentrations in vivo. This allows the development of non-invasive methods that employ the principles of tracer kinetics for determining physiological properties such as mass specific blood flow, tissue pH, and rates of substrate transport or utilization. A physiologically based, two-compartment tracer kinetic model was derived to mathematically describe the exchange of a radioindicator between blood and tissue. The model was adapted for use with dynamic sequences of data acquired with a positron tomograph. Rapid estimation techniques were implemented to produce functional images of the model parameters by analyzing each individual pixel sequence of the image data. A detailed analysis of the performance characteristics of three different parameter estimation schemes was performed. The analysis included examination of errors caused by statistical uncertainties in the measured data, errors in the timing of the data, and errors caused by violation of various assumptions of the tracer kinetic model. Two specific radioindicators were investigated. ('18)F -fluoromethane, an inert freely diffusible gas, was used for local quantitative determinations of both cerebral blood flow and tissue:blood partition coefficient. A method was developed that did not require direct sampling of arterial blood for the absolute scaling of flow values. The arterial input concentration time course was obtained by assuming that the alveolar or end-tidal expired breath radioactivity concentration is proportional to the arterial blood concentration. The scale of the input function was obtained from a series of venous blood concentration measurements. The method of absolute scaling using venous samples was validated in four studies, performed on normal volunteers, in which directly measured arterial concentrations

  17. Use of MRI in Differentiation of Papillary Renal Cell Carcinoma Subtypes: Qualitative and Quantitative Analysis.

    Science.gov (United States)

    Doshi, Ankur M; Ream, Justin M; Kierans, Andrea S; Bilbily, Matthew; Rusinek, Henry; Huang, William C; Chandarana, Hersh

    2016-03-01

    The purpose of this study was to determine whether qualitative and quantitative MRI feature analysis is useful for differentiating type 1 from type 2 papillary renal cell carcinoma (PRCC). This retrospective study included 21 type 1 and 17 type 2 PRCCs evaluated with preoperative MRI. Two radiologists independently evaluated various qualitative features, including signal intensity, heterogeneity, and margin. For the quantitative analysis, a radiology fellow and a medical student independently drew 3D volumes of interest over the entire tumor on T2-weighted HASTE images, apparent diffusion coefficient parametric maps, and nephrographic phase contrast-enhanced MR images to derive first-order texture metrics. Qualitative and quantitative features were compared between the groups. For both readers, qualitative features with greater frequency in type 2 PRCC included heterogeneous enhancement, indistinct margin, and T2 heterogeneity (all, p Quantitative analysis revealed that apparent diffusion coefficient, HASTE, and contrast-enhanced entropy were greater in type 2 PRCC (p quantitative and qualitative model had an AUC of 0.859. Qualitative features within the model had interreader concordance of 84-95%, and the quantitative data had intraclass coefficients of 0.873-0.961. Qualitative and quantitative features can help discriminate between type 1 and type 2 PRCC. Quantitative analysis may capture useful information that complements the qualitative appearance while benefiting from high interobserver agreement.

  18. Sperm Cell Dynamics in Shallow Chambers

    Science.gov (United States)

    Condat, Carlos; Marconi, Veronica; Guidobaldi, Alejandro; Giojalas, Laura; Silhanek, Alejandro; Jeyaram, Yogesh; Moshchalkov, Victor

    2015-03-01

    Self-propelled microorganisms are attracted to surfaces. This makes their dynamic behavior in restricted geometries very different from that observed in the bulk. Here we analyze the motion of spermatozoids confined to shallow chambers, investigating the nature of the cell trajectories and their accumulation near the side boundaries. Observed cell trajectories are composed of a succession of quasi-circular and quasi-linear segments. This suggests that the cells follow a path of intermittent trappings near the top and down surfaces separated by stretches of quasi-free motion near the center of the gap. Use of microstructured petal-shaped edges limits accumulation near the borders and contributes to increase the concentration in the chamber interior. System stabilization occurs over times of the order of minutes, which agrees well with a theoretical estimate that assumes that the cell mean-square displacement is largely due to the quasi-linear segments. Pure quasi-circular trajectories would require several hours to stabilize. Our estimates also indicate that stabilization proceeds 2.5 times faster in the rosette geometries than in the smooth-edged chambers, which is another practical reason to prefer the former.

  19. Optical characterization of red blood cells from individuals with sickle cell trait and disease in Tanzania using quantitative phase imaging

    Science.gov (United States)

    Jung, Jaehwang; Matemba, Lucas E.; Lee, Kyeoreh; Kazyoba, Paul E.; Yoon, Jonghee; Massaga, Julius J.; Kim, Kyoohyun; Kim, Dong-Jin; Park, Yongkeun

    2016-08-01

    Sickle cell disease (SCD) is common across Sub-Saharan Africa. However, the investigation of SCD in this area has been significantly limited mainly due to the lack of research facilities and skilled personnel. Here, we present optical measurements of individual red blood cells from healthy individuals and individuals with SCD and sickle cell trait in Tanzania using the quantitative phase imaging technique. By employing a quantitative phase imaging unit, an existing microscope in a clinic is transformed into a powerful quantitative phase microscope providing measurements on the morphological, biochemical, and biomechanical properties of individual cells. The present approach will open up new opportunities for cost-effective investigation and diagnosis of several diseases in low resource environments.

  20. Optical characterization of red blood cells from individuals with sickle cell trait and disease in Tanzania using quantitative phase imaging

    CERN Document Server

    Jung, JaeHwang; Lee, KyeoReh; Kazyoba, Paul E; Yoon, Jonghee; Massaga, Julius J; Kim, Kyoohyun; Kim, Dong-Jin; Park, YongKeun

    2016-01-01

    Sickle cell disease (SCD) is common across Sub-Saharan Africa. However, the investigation of SCD in this area has been significantly limited mainly due to the lack of research facilities and skilled personnel. Here, we present optical measurements of individual red blood cells (RBCs) from healthy individuals and individuals with SCD and sickle cell trait in Tanzania using the quantitative phase imaging technique. By employing a quantitative phase imaging unit (QPIU), an existing microscope in a clinic is transformed into a powerful quantitative phase microscope providing measurements on the morphological, biochemical, and biomechanical properties of individual cells. The present approach will open up new opportunities for cost-effective investigation and diagnosis of several diseases in low resource environments.

  1. Quantitative assessment of Heart Rate Dynamics during meditation: An ECG based study with Multi-fractality and visibility graph

    Directory of Open Access Journals (Sweden)

    Anirban eBhaduri

    2016-02-01

    Full Text Available Abstract: Abstract: The cardiac dynamics during meditation is explored quantitatively with two chaos-based non-linear techniques viz. multi-fractal detrended fluctuation analysis and visibility network analysis techniques. The data used are the instantaneous heart rate (in beats/minute of subjects performing Kundalini Yoga and Chi meditation from PhysioNet. The results show consistent differences between the quantitative parameters obtained by both the analysis techniques. This indicates an interesting phenomenon of change in the complexity of the cardiac dynamics during meditation supported with quantitative parameters.The results also produce a preliminary evidence that these techniques can be used as a measure of physiological impact on subjects performing meditation.

  2. The bias associated with amplicon sequencing does not affect the quantitative assessment of bacterial community dynamics.

    Directory of Open Access Journals (Sweden)

    Federico M Ibarbalz

    Full Text Available The performance of two sets of primers targeting variable regions of the 16S rRNA gene V1-V3 and V4 was compared in their ability to describe changes of bacterial diversity and temporal turnover in full-scale activated sludge. Duplicate sets of high-throughput amplicon sequencing data of the two 16S rRNA regions shared a collection of core taxa that were observed across a series of twelve monthly samples, although the relative abundance of each taxon was substantially different between regions. A case in point was the changes in the relative abundance of filamentous bacteria Thiothrix, which caused a large effect on diversity indices, but only in the V1-V3 data set. Yet the relative abundance of Thiothrix in the amplicon sequencing data from both regions correlated with the estimation of its abundance determined using fluorescence in situ hybridization. In nonmetric multidimensional analysis samples were distributed along the first ordination axis according to the sequenced region rather than according to sample identities. The dynamics of microbial communities indicated that V1-V3 and the V4 regions of the 16S rRNA gene yielded comparable patterns of: 1 the changes occurring within the communities along fixed time intervals, 2 the slow turnover of activated sludge communities and 3 the rate of species replacement calculated from the taxa-time relationships. The temperature was the only operational variable that showed significant correlation with the composition of bacterial communities over time for the sets of data obtained with both pairs of primers. In conclusion, we show that despite the bias introduced by amplicon sequencing, the variable regions V1-V3 and V4 can be confidently used for the quantitative assessment of bacterial community dynamics, and provide a proper qualitative account of general taxa in the community, especially when the data are obtained over a convenient time window rather than at a single time point.

  3. Quantitative absorption cytometry for measuring red blood cell hemoglobin mass and volume.

    Science.gov (United States)

    Schonbrun, Ethan; Malka, Roy; Di Caprio, Giuseppe; Schaak, Diane; Higgins, John M

    2014-04-01

    We present an optical system, called the quantitative absorption cytometer (QAC), to measure the volume and hemoglobin mass of red blood cells flowing through a microfluidic channel. In contrast to clinical hematology analyzers, where cells are sphered in order for both volume and hemoglobin to be measured accurately, the QAC measures cells in their normal physiological shape. Human red blood cells are suspended in a refractive index-matching absorbing buffer, driven through a microfluidic channel, and imaged using a transmission light microscope onto a color camera. A red and a blue LED illuminate cells and images at each color are used to independently retrieve cell volume and hemoglobin mass. This system shows good agreement with red blood cell indices retrieved by a clinical hematology analyzer and in fact measures a smaller coefficient of variation of hemoglobin concentration. In addition to cell indices, the QAC returns height and mass maps of each measured cell. These quantitative images are valuable for analyzing the detailed morphology of individual cells as well as statistical outliers found in the data. We also measured red blood cells in hypertonic and hypotonic buffers to quantify the correlation between volume and hemoglobin mass under osmotic stress. Because this method is invariant to cell shape, even extremely nonspherical cells in hypertonic buffers can be measured accurately.

  4. Quantitative comparison between microfluidic and microtiter plate formats for cell-based assays.

    Science.gov (United States)

    Yin, Huabing; Pattrick, Nicola; Zhang, Xunli; Klauke, Norbert; Cordingley, Hayley C; Haswell, Steven J; Cooper, Jonathan M

    2008-01-01

    In this paper, we compare a quantitative cell-based assay measuring the intracellular Ca2+ response to the agonist uridine 5'-triphosphate in Chinese hamster ovary cells, in both microfluidic and microtiter formats. The study demonstrates that, under appropriate hydrodynamic conditions, there is an excellent agreement between traditional well-plate assays and those obtained on-chip for both suspended immobilized cells and cultured adherent cells. We also demonstrate that the on-chip assay, using adherent cells, provides the possibility of faster screening protocols with the potential for resolving subcellular information about local Ca2+ flux.

  5. Micro/nano-computed tomography technology for quantitative dynamic, multi-scale imaging of morphogenesis.

    Science.gov (United States)

    Gregg, Chelsea L; Recknagel, Andrew K; Butcher, Jonathan T

    2015-01-01

    Tissue morphogenesis and embryonic development are dynamic events challenging to quantify, especially considering the intricate events that happen simultaneously in different locations and time. Micro- and more recently nano-computed tomography (micro/nanoCT) has been used for the past 15 years to characterize large 3D fields of tortuous geometries at high spatial resolution. We and others have advanced micro/nanoCT imaging strategies for quantifying tissue- and organ-level fate changes throughout morphogenesis. Exogenous soft tissue contrast media enables visualization of vascular lumens and tissues via extravasation. Furthermore, the emergence of antigen-specific tissue contrast enables direct quantitative visualization of protein and mRNA expression. Micro-CT X-ray doses appear to be non-embryotoxic, enabling longitudinal imaging studies in live embryos. In this chapter we present established soft tissue contrast protocols for obtaining high-quality micro/nanoCT images and the image processing techniques useful for quantifying anatomical and physiological information from the data sets.

  6. Quantitative Agent Based Model of Opinion Dynamics: Polish Elections of 2015.

    Directory of Open Access Journals (Sweden)

    Pawel Sobkowicz

    Full Text Available We present results of an abstract, agent based model of opinion dynamics simulations based on the emotion/information/opinion (E/I/O approach, applied to a strongly polarized society, corresponding to the Polish political scene between 2005 and 2015. Under certain conditions the model leads to metastable coexistence of two subcommunities of comparable size (supporting the corresponding opinions-which corresponds to the bipartisan split found in Poland. Spurred by the recent breakdown of this political duopoly, which occurred in 2015, we present a model extension that describes both the long term coexistence of the two opposing opinions and a rapid, transitory change due to the appearance of a third party alternative. We provide quantitative comparison of the model with the results of polls and elections in Poland, testing the assumptions related to the modeled processes and the parameters used in the simulations. It is shown, that when the propaganda messages of the two incumbent parties differ in emotional tone, the political status quo may be unstable. The asymmetry of the emotions within the support bases of the two parties allows one of them to be 'invaded' by a newcomer third party very quickly, while the second remains immune to such invasion.

  7. Ambiguity, logic, simplicity, and dynamics: Wittgensteinian evaluative criteria in peer review of quantitative research on categorization.

    Science.gov (United States)

    Shimp, Charles P

    2004-06-30

    Research on categorization has changed over time, and some of these changes resemble how Wittgenstein's views changed from his Tractatus Logico-Philosophicus to his Philosophical Investigations. Wittgenstein initially focused on unambiguous, abstract, parsimonious, logical propositions and rules, and on independent, static, "atomic facts." This approach subsequently influenced the development of logical positivism and thereby may have indirectly influenced method and theory in research on categorization: much animal research on categorization has focused on learning simple, static, logical rules unambiguously interrelating small numbers of independent features. He later rejected logical simplicity and rigor and focused instead on Gestalt ideas about figure-ground reversals and context, the ambiguity of family resemblance, and the function of details of everyday language. Contemporary contextualism has been influenced by this latter position, some features of which appear in contemporary empirical research on categorization. These developmental changes are illustrated by research on avian local and global levels of visual perceptual analysis, categorization of rectangles and moving objects, and artificial grammar learning. Implications are described for peer review of quantitative theory in which ambiguity, logical rigor, simplicity, or dynamics are designed to play important roles.

  8. Improved Dynamic Analysis method for quantitative PIXE and SXRF element imaging of complex materials

    Energy Technology Data Exchange (ETDEWEB)

    Ryan, C.G., E-mail: chris.ryan@csiro.au; Laird, J.S.; Fisher, L.A.; Kirkham, R.; Moorhead, G.F.

    2015-11-15

    The Dynamic Analysis (DA) method in the GeoPIXE software provides a rapid tool to project quantitative element images from PIXE and SXRF imaging event data both for off-line analysis and in real-time embedded in a data acquisition system. Initially, it assumes uniform sample composition, background shape and constant model X-ray relative intensities. A number of image correction methods can be applied in GeoPIXE to correct images to account for chemical concentration gradients, differential absorption effects, and to correct images for pileup effects. A new method, applied in a second pass, uses an end-member phase decomposition obtained from the first pass, and DA matrices determined for each end-member, to re-process the event data with each pixel treated as an admixture of end-member terms. This paper describes the new method and demonstrates through examples and Monte-Carlo simulations how it better tracks spatially complex composition and background shape while still benefitting from the speed of DA.

  9. Quantitative dynamics on stimulating regeneration and sowing seedlings of Larix gmelinii in Daxing'an Mountains

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    To understand the quantitative dynamics and death reason of stimulating regeneration seedlings is significant for stimulating the natural regeneration of Larix gmelinii and implement of conservation project of natural forest. This paper summarized location observations and directly-seeding simulation experiments of six permanent sample plots that were set up after the seed bumper harvest year of Larix gmelinii in 1989. The study showed that stimulating natural regeneration seedlings had a large mortality in the first three years, especially in the first year of seedling emergence. After three years seedlings died less and stepped into the stable regeneration stage. A large number of seedlings died of sunscald as the primary death reason. For those areas of good site conditions and rich soil, damping-off would cause seedlings to death in large quantities. The task of stimulating regeneration is mainly to get rid of the litter (forest floor) on burned areas. By means of promoting measures, emergence rate of sown seeds would be several times and dozens of times higher than that of seed shedding on the condition of retention of forest floor. Promoting the regeneration need to select the suitable site against great slope and low-lying lands; at the same time, be careful of the avoidance of frost heaving by depression water.

  10. Quantitative model of cell cycle arrest and cellular senescence in primary human fibroblasts.

    Directory of Open Access Journals (Sweden)

    Sascha Schäuble

    Full Text Available Primary human fibroblasts in tissue culture undergo a limited number of cell divisions before entering a non-replicative "senescent" state. At early population doublings (PD, fibroblasts are proliferation-competent displaying exponential growth. During further cell passaging, an increasing number of cells become cell cycle arrested and finally senescent. This transition from proliferating to senescent cells is driven by a number of endogenous and exogenous stress factors. Here, we have developed a new quantitative model for the stepwise transition from proliferating human fibroblasts (P via reversibly cell cycle arrested (C to irreversibly arrested senescent cells (S. In this model, the transition from P to C and to S is driven by a stress function γ and a cellular stress response function F which describes the time-delayed cellular response to experimentally induced irradiation stress. The application of this model based on senescence marker quantification at the single-cell level allowed to discriminate between the cellular states P, C, and S and delivers the transition rates between the P, C and S states for different human fibroblast cell types. Model-derived quantification unexpectedly revealed significant differences in the stress response of different fibroblast cell lines. Evaluating marker specificity, we found that SA-β-Gal is a good quantitative marker for cellular senescence in WI-38 and BJ cells, however much less so in MRC-5 cells. Furthermore we found that WI-38 cells are more sensitive to stress than BJ and MRC-5 cells. Thus, the explicit separation of stress induction from the cellular stress response, and the differentiation between three cellular states P, C and S allows for the first time to quantitatively assess the response of primary human fibroblasts towards endogenous and exogenous stress during cellular ageing.

  11. Dynamic cell culture system (7-IML-1)

    Science.gov (United States)

    Cogoli, Augusto

    1992-01-01

    This experiment is one of the Biorack experiments being flown on the International Microgravity Laboratory 1 (MIL-1) mission as part of an investigation studying cell proliferation and performance in space. One of the objectives of this investigation is to assess the potential benefits of bioprocessing in space with the ultimate goal of developing a bioreactor for continuous cell cultures in space. This experiment will test the operation of an automated culture chamber that was designed for use in a Bioreactor in space. The device to be tested is called the Dynamic Cell Culture System (DCCS). It is a simple device in which media are renewed or chemicals are injected automatically, by means of osmotic pumps. This experiment uses four Type I/O experiment containers. One DCCS unit, which contains a culture chamber with renewal of medium and a second chamber without a medium supply fits in each container. Two DCCS units are maintained under zero gravity conditions during the on-orbit period. The other two units are maintained under 1 gh conditions in a 1 g centrifuge. The schedule for incubator transfer is given.

  12. Application of magnetic carriers to two examples of quantitative cell analysis

    Science.gov (United States)

    Zhou, Chen; Qian, Zhixi; Choi, Young Suk; David, Allan E.; Todd, Paul; Hanley, Thomas R.

    2017-04-01

    The use of magnetophoretic mobility as a surrogate for fluorescence intensity in quantitative cell analysis was investigated. The objectives of quantitative fluorescence flow cytometry include establishing a level of labeling for the setting of parameters in fluorescence activated cell sorters (FACS) and the determination of levels of uptake of fluorescently labeled substrates by living cells. Likewise, the objectives of quantitative magnetic cytometry include establishing a level of labeling for the setting of parameters in flowing magnetic cell sorters and the determination of levels of uptake of magnetically labeled substrates by living cells. The magnetic counterpart to fluorescence intensity is magnetophoretic mobility, defined as the velocity imparted to a suspended cell per unit of magnetic ponderomotive force. A commercial velocimeter available for making this measurement was used to demonstrate both applications. Cultured Gallus lymphoma cells were immunolabeled with commercial magnetic beads and shown to have adequate magnetophoretic mobility to be separated by a novel flowing magnetic separator. Phagocytosis of starch nanoparticles having magnetic cores by cultured Chinese hamster ovary cells, a CHO line, was quantified on the basis of magnetophoretic mobility.

  13. Application of Organosilane Monolayer Template to Quantitative Evaluation of Cancer Cell Adhesive Ability

    Science.gov (United States)

    Tanii, Takashi; Sasaki, Kosuke; Ichisawa, Kota; Demura, Takanori; Beppu, Yuichi; Vu, Hoan Anh; Thanh Chi, Hoan; Yamamoto, Hideaki; Sato, Yuko

    2011-06-01

    The adhesive ability of two human pancreatic cancer cell lines was evaluated using organosilane monolayer templates (OMTs). Using the OMT, the spreading area of adhered cells can be limited, and this enables us to focus on the initial attachment process of adhesion. Moreover, it becomes possible to arrange the cells in an array and to quantitatively evaluate the number of attached cells. The adhesive ability of the cancer cells cultured on the OMT was controlled by adding (-)-epigallocatechin-3-gallate (EGCG), which blocks a receptor that mediates cell adhesion and is overexpressed in cancer cells. Measurement of the relative ability of the cancer cells to attach to the OMT revealed that the ability for attachment decreased with increasing EGCG concentration. The results agreed well with the western blot analysis, indicating that the OMT can potentially be employed to evaluate the adhesive ability of various cancer cells.

  14. Dynamic measurements of flowing cells labeled by gold nanoparticles using full-field photothermal interferometric imaging

    Science.gov (United States)

    Turko, Nir A.; Roitshtain, Darina; Blum, Omry; Kemper, Björn; Shaked, Natan T.

    2017-06-01

    We present highly dynamic photothermal interferometric phase microscopy for quantitative, selective contrast imaging of live cells during flow. Gold nanoparticles can be biofunctionalized to bind to specific cells, and stimulated for local temperature increase due to plasmon resonance, causing a rapid change of the optical phase. These phase changes can be recorded by interferometric phase microscopy and analyzed to form an image of the binding sites of the nanoparticles in the cells, gaining molecular specificity. Since the nanoparticle excitation frequency might overlap with the sample dynamics frequencies, photothermal phase imaging was performed on stationary or slowly dynamic samples. Furthermore, the computational analysis of the photothermal signals is time consuming. This makes photothermal imaging unsuitable for applications requiring dynamic imaging or real-time analysis, such as analyzing and sorting cells during fast flow. To overcome these drawbacks, we utilized an external interferometric module and developed new algorithms, based on discrete Fourier transform variants, enabling fast analysis of photothermal signals in highly dynamic live cells. Due to the self-interference module, the cells are imaged with and without excitation in video-rate, effectively increasing signal-to-noise ratio. Our approach holds potential for using photothermal cell imaging and depletion in flow cytometry.

  15. Phenol-formaldehyde resins: A quantitative NMR study of molecular structure and molecular dynamics

    Science.gov (United States)

    Ottenbourgs, Benjamin Tony

    Phenol-formaldehyde (PF) resins have been the subject of this work. 13C liquid-state and solid-state NMR has been used to investigate the molecular structure of mainly novolak and partially of resole resins. 1H wideline in combination with 13C solid-state NMR relaxometry has been applied to study the curing and the molecular dynamics of phenolic resins. It was the intention to provide an insight in the relationship between resin composition, resin structure and subsequent resin properties (by means of the molecular dynamics). An improved 13C liquid-state NMR quantification technique of novolaks in THF-CDCl3 solutions is demonstrated. Full quantitative 13C liquid-state spectra of phenol-formaldehyde resins with high signal- to-noise ratio were obtained by using chromium acetylacetonate under optimized spectral conditions within a few hours spectrometer time. Attached proton test (APT) spectra enabled proper peak assignments in the region with significant overlap. For several novolaks, prepared under different catalytic conditions, the degree of polymerization, degree of branching, number average molecular weight, isomeric distribution, and the number of unreacted ortho and para phenol ring positions was determined with a reduced margin of error, by analyzing and integrating the 13C spectra. The power of 13C solid-state NMR in the analysis of cured PF resins is shown. Particular importance was ascribed to the question of the quantifiability of the experiments when it was desired to measure the degree of conversion by means of a 13C CP/MAS contact time study. The network structure present, and thus also the mechanical properties, is critically dependent upon the final degree of conversion obtained after curing. The degree of conversion, which depended on the cure conditions (cure temperature, cure pressure and cure time), was limited by vitrification as was demonstrated by DSC experiments. Changes in the spin-lattice relaxation time T 1H were observed, providing

  16. Why Do CD8 + T Cells Become Indifferent To Tumors: A Dynamic Modeling Approach

    Directory of Open Access Journals (Sweden)

    Colin eCampbell

    2011-07-01

    Full Text Available CD8+ T cells have the potential to influence the outcome of cancer pathogenesis, including complete tumor eradication or selection of malignant tumor escape variants. The Simian virus 40 large T-antigen oncoprotein promotes tumor formation in T-antigen transgenic mice and also provides multiple target determinants (sites for responding CD8+ T cells in C57BL/6 (H-2b mice. To understand the in vivo quantitative dynamics of CD8+ T cells after encountering T-antigen, we constructed a dynamic model from in vivo-generated data to simulate the interactions between T-antigen expressing cells and CD8+ T cells in distinct scenarios including immunization of wild type C57BL/6 mice and of T-antigen transgenic mice that develop various tumors. In these scenarios the model successfully reproduces the dynamics of both the T-antigen-expressing cells and antigen specific CD8+ T cell responses. The model predicts that the tolerance of the site-specific T cells is dependent on their apoptosis rates and that the net growth of CD8+ T cells is altered in transgenic mice. We experimentally validate both predictions. Our results indicate that site-specific CD8+ T cells have tissue-specific apoptosis rates affecting their tolerance to the tumor antigen. Moreover, the model highlights differences in apoptosis rates that contribute to compromised CD8+ T cell responses and tumor progression, knowledge of which is essential for development of cancer immunotherapy.

  17. Quantitative Study of Cell Invasion Process under Extracellular Stimulation of Cytokine in a Microfluidic Device

    Science.gov (United States)

    Lei, Kin Fong; Tseng, Hsueh-Peng; Lee, Chia-Yi; Tsang, Ngan-Ming

    2016-05-01

    Cell invasion is the first step of cancer metastasis that is the primary cause of death for cancer patients and defined as cell movement through extracellular matrix (ECM). Investigation of the correlation between cell invasive and extracellular stimulation is critical for the inhabitation of metastatic dissemination. Conventional cell invasion assay is based on Boyden chamber assay, which has a number of limitations. In this work, a microfluidic device incorporating with impedance measurement technique was developed for quantitative investigation of cell invasion process. The device consisted of 2 reservoirs connecting with a microchannel filled with hydrogel. Malignant cells invaded along the microchannel and impedance measurement was concurrently conducted by measuring across electrodes located at the bottom of the microchannel. Therefore, cell invasion process could be monitored in real-time and non-invasive manner. Also, cell invasion rate was then calculated to study the correlation between cell invasion and extracellular stimulation, i.e., IL-6 cytokine. Results showed that cell invasion rate was directly proportional to the IL-6 concentration. The microfluidic device provides a reliable and convenient platform for cell-based assays to facilitate more quantitative assessments in cancer research.

  18. Quantitative image analysis in the assessment of diffuse large B-cell lymphoma.

    Science.gov (United States)

    Chabot-Richards, Devon S; Martin, David R; Myers, Orrin B; Czuchlewski, David R; Hunt, Kristin E

    2011-12-01

    Proliferation rates in diffuse large B-cell lymphoma have been associated with conflicting outcomes in the literature, more often with high proliferation associated with poor prognosis. In most studies, the proliferation rate was estimated by a pathologist using an immunohistochemical stain for the monoclonal antibody Ki-67. We hypothesized that a quantitative image analysis algorithm would give a more accurate estimate of the proliferation rate, leading to better associations with survival. In all, 84 cases of diffuse large B-cell lymphoma were selected according to the World Health Organization criteria. Ki-67 percentage positivity estimated by the pathologist was recorded from the original report. The same slides were then scanned using an Aperio ImageScope, and Ki-67 percentage positivity was calculated using a computer-based quantitative immunohistochemistry nuclear algorithm. In addition, chart review was performed and survival time was recorded. The Ki-67 percentage estimated by the pathologist from the original report versus quantitative image analysis was significantly correlated (Pquantitative image analysis (P=0.021). There was less agreement at lower Ki-67 percentages. Comparison of Ki-67 percentage positivity versus survival did not show significant association either with pathologist estimate or quantitative image analysis. However, although not significant, there was a trend of worse survival at higher proliferation rates detected by the pathologist but not by quantitative image analysis. Interestingly, our data suggest that the Ki-67 percentage positivity as assessed by the pathologist may be more closely associated with survival outcome than that identified by quantitative image analysis. This may indicate that pathologists are better at selecting appropriate areas of the slide. More cases are needed to assess whether this finding would be statistically significant. Due to the good correlation between pathologist estimate and quantitative image

  19. Beta cell dynamics: beta cell replenishment, beta cell compensation and diabetes.

    Science.gov (United States)

    Cerf, Marlon E

    2013-10-01

    Type 2 diabetes, characterized by persistent hyperglycemia, arises mostly from beta cell dysfunction and insulin resistance and remains a highly complex metabolic disease due to various stages in its pathogenesis. Glucose homeostasis is primarily regulated by insulin secretion from the beta cells in response to prevailing glycemia. Beta cell populations are dynamic as they respond to fluctuating insulin demand. Beta cell replenishment and death primarily regulate beta cell populations. Beta cells, pancreatic cells, and extra-pancreatic cells represent the three tiers for replenishing beta cells. In rodents, beta cell self-replenishment appears to be the dominant source for new beta cells supported by pancreatic cells (non-beta islet cells, acinar cells, and duct cells) and extra-pancreatic cells (liver, neural, and stem/progenitor cells). In humans, beta cell neogenesis from non-beta cells appears to be the dominant source of beta cell replenishment as limited beta cell self-replenishment occurs particularly in adulthood. Metabolic states of increased insulin demand trigger increased insulin synthesis and secretion from beta cells. Beta cells, therefore, adapt to support their physiology. Maintaining physiological beta cell populations is a strategy for targeting metabolic states of persistently increased insulin demand as in diabetes.

  20. Classifying the expansion kinetics and critical surface dynamics of growing cell populations

    CERN Document Server

    Block, M; Drasdo, D

    2006-01-01

    Based on a cellular automaton model the growth kinetics and the critical surface dynamics of cell monolayers is systematically studied by variation of the cell migration activity, the size of the proliferation zone and the cell cycle time distribution over wide ranges. The model design avoids lattice artifacts and ensures high performance. The monolayer expansion velocity derived from our simulations can be interpreted as a generalization of the velocity relationship for a traveling front in the Fisher-Kolmogorov-Petrovskii-Piskounov (FKPP) equation that is frequently used to model tumor growth phenomena by continuum models. The critical surface dynamics corresponds to the Kardar-Parisi-Zhang (KPZ) universality class for all parameters and model variations studied. While the velocity agrees quantitatively with experimental observations by Bru et al, the critical surface dynamics is in contrast to their interpretation as generic molecular-beam-epitaxy-like growth.

  1. Quantitative determination of optical trapping strength and viscoelastic moduli inside living cells

    DEFF Research Database (Denmark)

    Mas, Josep; Richardson, Andrew Callum; Reihani, S. Nader S.

    2013-01-01

    With the success of in vitro single-molecule force measurements obtained in recent years, the next step is to perform quantitative force measurements inside a living cell. Optical traps have proven excellent tools for manipulation, also in vivo, where they can be essentially non-invasive under...... correct wavelength and exposure conditions. It is a pre-requisite for in vivo quantitative force measurements that a precise and reliable force calibration of the tweezers is performed. There are well-established calibration protocols in purely viscous environments; however, as the cellular cytoplasm...... as the force constant describing the optical trap, thus paving the way for quantitative force measurements inside the living cell. Here, we determine both the spring constant of the optical trap and the elastic contribution from the cytoplasm, influencing the motion of naturally occurring tracer particles...

  2. Dynamic imaging of cell-free and cell-associated viral capture in mature dendritic cells.

    Science.gov (United States)

    Izquierdo-Useros, Nuria; Esteban, Olga; Rodriguez-Plata, Maria T; Erkizia, Itziar; Prado, Julia G; Blanco, Julià; García-Parajo, Maria F; Martinez-Picado, Javier

    2011-12-01

    Dendritic cells (DCs) capture human immunodeficiency virus (HIV) through a non-fusogenic mechanism that enables viral transmission to CD4(+) T cells, contributing to in vivo viral dissemination. Although previous studies have provided important clues to cell-free viral capture by mature DCs (mDCs), dynamic and kinetic insight on this process is still missing. Here, we used three-dimensional video microscopy and single-particle tracking approaches to dynamically dissect both cell-free and cell-associated viral capture by living mDCs. We show that cell-free virus capture by mDCs operates through three sequential phases: virus binding through specific determinants expressed in the viral particle, polarized or directional movements toward concrete regions of the cell membrane and virus accumulation in a sac-like structure where trapped viral particles display a hindered diffusive behavior. Moreover, real-time imaging of cell-associated viral transfer to mDCs showed a similar dynamics to that exhibited by cell-free virus endocytosis leading to viral accumulation in compartments. However, cell-associated HIV type 1 transfer to mDCs was the most effective pathway, boosted throughout enhanced cellular contacts with infected CD4(+) T cells. Our results suggest that in lymphoid tissues, mDC viral uptake could occur either by encountering cell-free or cell-associated virus produced by infected cells generating the perfect scenario to promote HIV pathogenesis and impact disease progression.

  3. Qualitative and quantitative analysis of adenovirus type 5 vector-induced memory CD8 T cells

    DEFF Research Database (Denmark)

    Steffensen, Maria Abildgaard; Holst, Peter Johannes; Steengaard, Sanne Skovvang

    2013-01-01

    adenoviral boosting and, importantly, the generated secondary memory cells cannot be qualitatively differentiated from those induced by primary infection with replicating virus. Quantitatively, DNA priming prior to Ad-vaccination will lead to even higher numbers of memory cells. In this case, the vaccination...... infection with lymphocytic choriomeningitis virus. We found that localized immunization with intermediate doses of Ad vector induce a moderate number of functional CD8 T cells, which qualitatively match those found in LCMV-infected mice. Numbers of these cells may be efficiently increased by additional...

  4. Mitochondrial dynamics and the cell cycle

    Directory of Open Access Journals (Sweden)

    Penny M.A. Kianian

    2014-05-01

    Full Text Available Nuclear-mitochondrial (NM communication impacts many aspects of plant development including vigor, sterility and viability. Dynamic changes in mitochondrial number, shape, size, and cellular location takes place during the cell cycle possibly impacting the process itself and leading to distribution of this organelle into daughter cells. The genes that underlie these changes are beginning to be identified in model plants such as Arabidopsis. In animals disruption of the drp1 gene, a homolog to the plant drp3A and drp3B, delays mitochondrial division. This mutation results in increased aneuploidy due to chromosome mis-segregation. It remains to be discovered if a similar outcome is observed in plants. Alloplasmic lines provide an opportunity to understand the communication between the cytoplasmic organelles and the nucleus. Examples of studies in these lines, especially from the extensive collection in wheat, point to the role of mitochondria in chromosome movement, pollen fertility and other aspects of development. Genes involved in NM interaction also are believed to play a critical role in evolution of species and interspecific cross incompatibilities.

  5. Quantitative Phase Imaging Techniques for the Study of Cell Pathophysiology: From Principles to Applications

    Directory of Open Access Journals (Sweden)

    Hyunjoo Park

    2013-03-01

    Full Text Available A cellular-level study of the pathophysiology is crucial for understanding the mechanisms behind human diseases. Recent advances in quantitative phase imaging (QPI techniques show promises for the cellular-level understanding of the pathophysiology of diseases. To provide important insight on how the QPI techniques potentially improve the study of cell pathophysiology, here we present the principles of QPI and highlight some of the recent applications of QPI ranging from cell homeostasis to infectious diseases and cancer.

  6. Quantitative imaging of collective cell migration during Drosophila gastrulation: multiphoton microscopy and computational analysis

    OpenAIRE

    Supatto, Willy; McMahon, Amy; Fraser, Scott E.; Stathopoulos, Angelike

    2009-01-01

    This protocol describes imaging and computational tools to collect and analyze live imaging data of embryonic cell migration. Our five-step protocol requires a few weeks to move through embryo preparation and four-dimensional (4D) live imaging using multiphoton microscopy, to 3D cell tracking using image processing, registration of tracking data and their quantitative analysis using computational tools. It uses commercially available equipment and requires expertise in microscopy and progr...

  7. Quantitative interpretation of molecular dynamics simulations for X-ray photoelectron spectroscopy of aqueous solutions

    Science.gov (United States)

    Olivieri, Giorgia; Parry, Krista M.; Powell, Cedric J.; Tobias, Douglas J.; Brown, Matthew A.

    2016-04-01

    Over the past decade, energy-dependent ambient pressure X-ray photoelectron spectroscopy (XPS) has emerged as a powerful analytical probe of the ion spatial distributions at the vapor (vacuum)-aqueous electrolyte interface. These experiments are often paired with complementary molecular dynamics (MD) simulations in an attempt to provide a complete description of the liquid interface. There is, however, no systematic protocol that permits a straightforward comparison of the two sets of results. XPS is an integrated technique that averages signals from multiple layers in a solution even at the lowest photoelectron kinetic energies routinely employed, whereas MD simulations provide a microscopic layer-by-layer description of the solution composition near the interface. Here, we use the National Institute of Standards and Technology database for the Simulation of Electron Spectra for Surface Analysis (SESSA) to quantitatively interpret atom-density profiles from MD simulations for XPS signal intensities using sodium and potassium iodide solutions as examples. We show that electron inelastic mean free paths calculated from a semi-empirical formula depend strongly on solution composition, varying by up to 30% between pure water and concentrated NaI. The XPS signal thus arises from different information depths in different solutions for a fixed photoelectron kinetic energy. XPS signal intensities are calculated using SESSA as a function of photoelectron kinetic energy (probe depth) and compared with a widely employed ad hoc method. SESSA simulations illustrate the importance of accounting for elastic-scattering events at low photoelectron kinetic energies (hoc method systematically underestimates the preferential enhancement of anions over cations. Finally, some technical aspects of applying SESSA to liquid interfaces are discussed.

  8. Quantitative proteomics reveals dynamic responses of Synechocystis sp. PCC 6803 to next-generation biofuel butanol.

    Science.gov (United States)

    Tian, Xiaoxu; Chen, Lei; Wang, Jiangxin; Qiao, Jianjun; Zhang, Weiwen

    2013-01-14

    Butanol is a promising biofuel, and recent metabolic engineering efforts have demonstrated the use of photosynthetic cyanobacterial hosts for its production. However, cyanobacteria have very low tolerance to butanol, limiting the economic viability of butanol production from these renewable producing systems. The existing knowledge of molecular mechanism involved in butanol tolerance in cyanobacteria is very limited. To build a foundation necessary to engineer robust butanol-producing cyanobacterial hosts, in this study, the responses of Synechocystis PCC 6803 to butanol were investigated using a quantitative proteomics approach with iTRAQ - LC-MS/MS technologies. The resulting high-quality dataset consisted of 25,347 peptides corresponding to 1452 unique proteins, a coverage of approximately 40% of the predicted proteins in Synechocystis. Comparative quantification of protein abundances led to the identification of 303 differentially regulated proteins by butanol. Annotation and GO term enrichment analysis showed that multiple biological processes were regulated, suggesting that Synechocystis probably employed multiple and synergistic resistance mechanisms in dealing with butanol stress. Notably, the analysis revealed the induction of heat-shock protein and transporters, along with modification of cell membrane and envelope were the major protection mechanisms against butanol. A conceptual cellular model of Synechocystis PCC 6803 responses to butanol stress was constructed to illustrate the putative molecular mechanisms employed to defend against butanol stress. Copyright © 2012 Elsevier B.V. All rights reserved.

  9. Quantitative Analysis of Delta-like 1 Membrane Dynamics Elucidates the Role of Contact Geometry on Notch Signaling

    Directory of Open Access Journals (Sweden)

    Itzhak Khait

    2016-01-01

    Full Text Available Notch signaling is ubiquitously used to coordinate differentiation between adjacent cells across metazoans. Whereas Notch pathway components have been studied extensively, the effect of membrane distribution and dynamics of Notch receptors and ligands remains poorly understood. It is also unclear how cellular morphology affects these distributions and, ultimately, the signaling between cells. Here, we combine live-cell imaging and mathematical modeling to address these questions. We use a FRAP-TIRF assay to measure the diffusion and endocytosis rates of Delta-like 1 (Dll1 in mammalian cells. We find large cell-to-cell variability in the diffusion coefficients of Dll1 measured in single cells within the same population. Using a simple reaction-diffusion model, we show how membrane dynamics and cell morphology affect cell-cell signaling. We find that differences in the diffusion coefficients, as observed experimentally, can dramatically affect signaling between cells. Together, these results elucidate how membrane dynamics and cellular geometry can affect cell-cell signaling.

  10. Quantitative Analysis of Differential Proteome Expression in Bladder Cancer vs. Normal Bladder Cells Using SILAC Method.

    Directory of Open Access Journals (Sweden)

    Ganglong Yang

    Full Text Available The best way to increase patient survival rate is to identify patients who are likely to progress to muscle-invasive or metastatic disease upfront and treat them more aggressively. The human cell lines HCV29 (normal bladder epithelia, KK47 (low grade nonmuscle invasive bladder cancer, NMIBC, and YTS1 (metastatic bladder cancer have been widely used in studies of molecular mechanisms and cell signaling during bladder cancer (BC progression. However, little attention has been paid to global quantitative proteome analysis of these three cell lines. We labeled HCV29, KK47, and YTS1 cells by the SILAC method using three stable isotopes each of arginine and lysine. Labeled proteins were analyzed by 2D ultrahigh-resolution liquid chromatography LTQ Orbitrap mass spectrometry. Among 3721 unique identified and annotated proteins in KK47 and YTS1 cells, 36 were significantly upregulated and 74 were significantly downregulated with >95% confidence. Differential expression of these proteins was confirmed by western blotting, quantitative RT-PCR, and cell staining with specific antibodies. Gene ontology (GO term and pathway analysis indicated that the differentially regulated proteins were involved in DNA replication and molecular transport, cell growth and proliferation, cellular movement, immune cell trafficking, and cell death and survival. These proteins and the advanced proteome techniques described here will be useful for further elucidation of molecular mechanisms in BC and other types of cancer.

  11. Quantitative evaluation of Leydig cells in testicular biopsies of men with varicocele.

    Science.gov (United States)

    Francavilla, S; Bruno, B; Martini, M; Moscardelli, S; Properzi, G; Francavilla, F; Santiemma, V; Fabbrini, A

    1986-01-01

    A quantitative analysis of Leydig cells was performed in 23 testicular biopsies of men with left varicocele and sperm count ranging from zero to 95,000 sperm/mm3. The oligozoospermic patients had more Leydig cells and higher FSH and LH serum levels than the patient group with more than 10,000 sperm/mm3. The Leydig cell density appeared tightly correlated (p less than 0.01) with the serum level of LH. In oligozoospermic subjects, an altered Leydig cell function could trigger an increased LH secretion; this seems likely to be responsible for the stimulation of interstitial cells resulting in an exaggerated recruitment of mature Leydig cells from their precursors. The comparative analysis of left and right testes failed to show differences in Leydig cell density and spermatogenesis in normozoospermic and oligozoospermic patients. This suggests that the two testes are equally involved by a possible, although unknown, detrimental effect of left side varicocele.

  12. Quantitative imaging of epithelial cell scattering identifies specific inhibitors of cell motility and cell-cell dissociation

    NARCIS (Netherlands)

    Loerke, D.; le Duc, Q.; Blonk, I.; Kerstens, A.; Spanjaard, E.; Machacek, M.; Danuser, G.; de Rooij, J.

    2012-01-01

    The scattering of cultured epithelial cells in response to hepatocyte growth factor (HGF) is a model system that recapitulates key features of metastatic cell behavior in vitro, including disruption of cell-cell adhesions and induction of cell migration. We have developed image analysis tools that

  13. QSSPN: dynamic simulation of molecular interaction networks describing gene regulation, signalling and whole-cell metabolism in human cells.

    Science.gov (United States)

    Fisher, Ciarán P; Plant, Nicholas J; Moore, J Bernadette; Kierzek, Andrzej M

    2013-12-15

    Dynamic simulation of genome-scale molecular interaction networks will enable the mechanistic prediction of genotype-phenotype relationships. Despite advances in quantitative biology, full parameterization of whole-cell models is not yet possible. Simulation methods capable of using available qualitative data are required to develop dynamic whole-cell models through an iterative process of modelling and experimental validation. We formulate quasi-steady state Petri nets (QSSPN), a novel method integrating Petri nets and constraint-based analysis to predict the feasibility of qualitative dynamic behaviours in qualitative models of gene regulation, signalling and whole-cell metabolism. We present the first dynamic simulations including regulatory mechanisms and a genome-scale metabolic network in human cell, using bile acid homeostasis in human hepatocytes as a case study. QSSPN simulations reproduce experimentally determined qualitative dynamic behaviours and permit mechanistic analysis of genotype-phenotype relationships. The model and simulation software implemented in C++ are available in supplementary material and at http://sysbio3.fhms.surrey.ac.uk/qsspn/.

  14. Quantitative analysis of the secretion of the MCP family of chemokines by muscle cells

    DEFF Research Database (Denmark)

    Henningsen, Jeanette; Pedersen, Bente Klarlund; Kratchmarova, Irina

    2011-01-01

    by Amino acids in Cell culture (SILAC) method for quantitative analysis resulted in the identification and generation of quantitative profiles of 59 growth factors and cytokines, including 9 classical chemokines. The members of the CC chemokine family of proteins such as monocyte chemotactic proteins 1, 2......, and 3 (MCP-1/CCL2, MCP-2/CCL8, and MCP-3/CCL7) showed a distinct pattern of secretion during differentiation. Further analysis using combinatorial RNA and protein approaches demonstrated that the MCPs are regulated via both post-transcriptional and post-translational mechanisms. Analyses...

  15. Fluid dynamics and noise in bacterial cell-cell and cell-surface scattering

    CERN Document Server

    Drescher, Knut; Cisneros, Luis H; Ganguly, Sujoy; Goldstein, Raymond E; 10.1073/pnas.1019079108

    2011-01-01

    Bacterial processes ranging from gene expression to motility and biofilm formation are constantly challenged by internal and external noise. While the importance of stochastic fluctuations has been appreciated for chemotaxis, it is currently believed that deterministic long-range fluid dynamical effects govern cell-cell and cell-surface scattering - the elementary events that lead to swarming and collective swimming in active suspensions and to the formation of biofilms. Here, we report the first direct measurements of the bacterial flow field generated by individual swimming Escherichia coli both far from and near to a solid surface. These experiments allowed us to examine the relative importance of fluid dynamics and rotational diffusion for bacteria. For cell-cell interactions it is shown that thermal and intrinsic stochasticity drown the effects of long-range fluid dynamics, implying that physical interactions between bacteria are determined by steric collisions and near-field lubrication forces. This dom...

  16. Magnetic fingerprints of rolling cells for quantitative flow cytometry in whole blood

    Science.gov (United States)

    Reisbeck, Mathias; Helou, Michael Johannes; Richter, Lukas; Kappes, Barbara; Friedrich, Oliver; Hayden, Oliver

    2016-09-01

    Over the past 50 years, flow cytometry has had a profound impact on preclinical and clinical applications requiring single cell function information for counting, sub-typing and quantification of epitope expression. At the same time, the workflow complexity and high costs of such optical systems still limit flow cytometry applications to specialized laboratories. Here, we present a quantitative magnetic flow cytometer that incorporates in situ magnetophoretic cell focusing for highly accurate and reproducible rolling of the cellular targets over giant magnetoresistance sensing elements. Time-of-flight analysis is used to unveil quantitative single cell information contained in its magnetic fingerprint. Furthermore, we used erythrocytes as a biological model to validate our methodology with respect to precise analysis of the hydrodynamic cell diameter, quantification of binding capacity of immunomagnetic labels, and discrimination of cell morphology. The extracted time-of-flight information should enable point-of-care quantitative flow cytometry in whole blood for clinical applications, such as immunology and primary hemostasis.

  17. Quantitative Magnetic Separation of Particles and Cells Using Gradient Magnetic Ratcheting.

    Science.gov (United States)

    Murray, Coleman; Pao, Edward; Tseng, Peter; Aftab, Shayan; Kulkarni, Rajan; Rettig, Matthew; Di Carlo, Dino

    2016-04-13

    Extraction of rare target cells from biosamples is enabling for life science research. Traditional rare cell separation techniques, such as magnetic activated cell sorting, are robust but perform coarse, qualitative separations based on surface antigen expression. A quantitative magnetic separation technology is reported using high-force magnetic ratcheting over arrays of magnetically soft micropillars with gradient spacing, and the system is used to separate and concentrate magnetic beads based on iron oxide content (IOC) and cells based on surface expression. The system consists of a microchip of permalloy micropillar arrays with increasing lateral pitch and a mechatronic device to generate a cycling magnetic field. Particles with higher IOC separate and equilibrate along the miropillar array at larger pitches. A semi-analytical model is developed that predicts behavior for particles and cells. Using the system, LNCaP cells are separated based on the bound quantity of 1 μm anti-epithelial cell adhesion molecule (EpCAM) particles as a metric for expression. The ratcheting cytometry system is able to resolve a ±13 bound particle differential, successfully distinguishing LNCaP from PC3 populations based on EpCAM expression, correlating with flow cytometry analysis. As a proof-of-concept, EpCAM-labeled cells from patient blood are isolated with 74% purity, demonstrating potential toward a quantitative magnetic separation instrument.

  18. Analysis of the Trypanosoma brucei cell cycle by quantitative DAPI imaging.

    Science.gov (United States)

    Siegel, T Nicolai; Hekstra, Doeke R; Cross, George A M

    2008-08-01

    Trypanosoma brucei has two DNA compartments: the nucleus and the kinetoplast. DNA replication of these two compartments only partially coincides. Woodward and Gull [Woodward R, Gull K. Timing of nuclear and kinetoplast DNA replication and early morphological events in the cell cycle of Trypanosoma brucei. J Cell Sci 1990;95:49-57] comprehensively studied the relative timing of the replication and segregation of nuclear DNA (nDNA) and kinetoplast DNA (kDNA). Others have since assumed the consistency of morphological indicators of cell-cycle stage among strains and conditions. We report the use of quantitative DAPI imaging to determine the cell-cycle stage of individual procyclic cells. Using this approach, we found that kinetoplast elongation occurs mainly during nuclear S phase and not during G2, as previously assumed. We confirmed this finding by sorting cells by DNA content, followed by fluorescence microscopy. In addition, simultaneous quantitative imaging at two wavelengths can be used to determine the abundance of cell-cycle-regulated proteins during the cell cycle. We demonstrate this technique by co-staining for the non-acetylated state of lysine 4 of histone H4 (H4K4), which is enriched during nuclear S phase.

  19. Quantitative characterization of mitosis-blocked tetraploid cells using high content analysis.

    Science.gov (United States)

    Grove, Linnette E; Ghosh, Richik N

    2006-08-01

    A range of cellular evidence supporting a G1 tetraploidy checkpoint was obtained from different assay methods including flow cytometry, immunoblotting, and microscopy. Cancer research would benefit if these cellular properties could instead be measured by a single, quantitative, automated assay method, such as high content analysis (HCA). Thus, nocodazole-treated cells were fluorescently labeled for different cell cycle-associated properties, including DNA content, retinoblastoma (Rb) and histone H3 phosphorylation, p53 and p21(WAF1) expression, nuclear and cell sizes, and cell morphology, and automatically imaged, analyzed, and correlated using HCA. HCA verified that nocodazole-induced mitosis block resulted in tetraploid cells. Rb and histone H3 were maximally hyperphosphorylated by 24 h of nocodazole treatment, accompanied by cell and nuclear size decreases and cellular rounding. Cells remained tetraploid and mononucleated with longer treatments, but other targets reverted to G1 levels, including Rb and histone H3 dephosphorylation accompanied by cellular respreading. This was accompanied by increased p53 and p21(WAF1) expression levels. The range of effects accompanying nocodazole-induced block of mitosis and the resulting tetraploid cells' reversal to a pseudo-G1 state can be quantitatively measured by HCA in an automated manner, recommending this assay method for the large-scale biology challenges of modern cancer drug discovery.

  20. Quantitative and qualitative in vitro analysis of the stem cell potential of hematopoietic cells purified from murine skeletal muscle

    Institute of Scientific and Technical Information of China (English)

    Celine Haond; Fran(c)oise Farace; Martine Guillier; Yann Lécluse; Frederic Mazurier; William Vainchenker; Ali G Turhan

    2007-01-01

    The murine skeletal muscle contains hematopoietic stem cells, but this potential has so far not been studied quantitatively or qualitatively in vitro. To quantity the hematopoietic stem cell potential, we have used highly purified SP/CD45+ cells in long-term culture initiating cell (LTC-IC) assays. The SP/CD45+ cell population purified from murine muscle was found to have significant stem cell activity with an LTC-IC frequency of 1/640. Single-cell-sorted SP/CD45+ cells from muscle exhibited robust proliferative activity in vitro at day 16 (380-fold amplification), especially after culture with OP-9 layers that also support embryonic stem cells. Amplified cell populations originating from single cells exhibited multilineage differentiation ability with evidence of myeloid, lymphoid and NK cell markers. Thus, our results demonstrate that hematopoietic stem cells that can be quantified by LTC-IC assays exist in the murine skeletal muscle and show also for the first time, at the single-cell level, that these cells exhibit multilineage differentiation ability and major proliferative potential.

  1. Quantitative analysis of rat adipose tissue cell recovery, and non-fat cell volume, in primary cell cultures

    Directory of Open Access Journals (Sweden)

    Floriana Rotondo

    2016-11-01

    Full Text Available Background White adipose tissue (WAT is a complex, diffuse, multifunctional organ which contains adipocytes, and a large proportion of fat, but also other cell types, active in defense, regeneration and signalling functions. Studies with adipocytes often require their isolation from WAT by breaking up the matrix of collagen fibres; however, it is unclear to what extent adipocyte number in primary cultures correlates with their number in intact WAT, since recovery and viability are often unknown. Experimental Design Epididymal WAT of four young adult rats was used to isolate adipocytes with collagenase. Careful recording of lipid content of tissue, and all fraction volumes and weights, allowed us to trace the amount of initial WAT fat remaining in the cell preparation. Functionality was estimated by incubation with glucose and measurement of glucose uptake and lactate, glycerol and NEFA excretion rates up to 48 h. Non-adipocyte cells were also recovered and their sizes (and those of adipocytes were measured. The presence of non-nucleated cells (erythrocytes was also estimated. Results Cell numbers and sizes were correlated from all fractions to intact WAT. Tracing the lipid content, the recovery of adipocytes in the final, metabolically active, preparation was in the range of 70–75%. Cells showed even higher metabolic activity in the second than in the first day of incubation. Adipocytes were 7%, erythrocytes 66% and other stromal (nucleated cells 27% of total WAT cells. However, their overall volumes were 90%, 0.05%, and 0.2% of WAT. Non-fat volume of adipocytes was 1.3% of WAT. Conclusions The methodology presented here allows for a direct quantitative reference to the original tissue of studies using isolated cells. We have also found that the “live cell mass” of adipose tissue is very small: about 13 µL/g for adipocytes and 2 µL/g stromal, plus about 1 µL/g blood (the rats were killed by exsanguination. These data translate (with

  2. Parsing ERK Activation Reveals Quantitatively Equivalent Contributions From Epidermal Growth Factor Receptor and HER2 In Human Mammary Epithelial Cells

    Energy Technology Data Exchange (ETDEWEB)

    Hendriks, Bart S.; Orr, Galya; Wells, Alan H.; Wiley, H. S.; Lauffenburger, Douglas A.

    2005-02-18

    HER2, a member of the EGFR tyrosine kinase family, functions as an accessory EGFR signaling component and alters EGFR trafficking by heterodimerization. HER2 overexpression leads to aberrant cell behavior including enhanced proliferation and motility. Here we apply a combination of computational modeling and quantitative experimental studies of the dynamic interactions between EGFR and HER2, and their downstream activation of extracellular signal-related kinase (ERK) to understand this complex signaling system. Using cells expressing different levels of HER2 relative to the EGFR, we can separate relative contributions of EGFR and HER2 to signaling amplitude and duration. Based on our model calculations, we demonstrate that, in contrast with previous suggestions in the literature, the intrinsic capabilities of EGFR and HER2 to activated ERK are quantitatively equivalent . We find that HER2-mediated effects on EGFR dimerization and trafficking are sufficient to explain the detected HER2-mediated amplification of EGF-induced ERK signaling. Our model suggests that transient amplification of ERK activity by HER2 arises predominantly from the 2-to-1 stoichiometry of receptor kinase to bound ligand in EGFR/HER2 heterodimers compared to the 1-to-1 stoichiometry of the EGFR homodimer, but alterations in receptor trafficking, with resultant EGFR sparing, cause the sustained HER2-mediated enhancement of ERK signaling.

  3. Discharging dynamics in an electrolytic cell

    Science.gov (United States)

    Feicht, Sarah E.; Frankel, Alexandra E.; Khair, Aditya S.

    2016-07-01

    We analyze the dynamics of a discharging electrolytic cell comprised of a binary symmetric electrolyte between two planar, parallel blocking electrodes. When a voltage is initially applied, ions in the electrolyte migrate towards the electrodes, forming electrical double layers. After the system reaches steady state and the external current decays to zero, the applied voltage is switched off and the cell discharges, with the ions eventually returning to a uniform spatial concentration. At voltages on the order of the thermal voltage VT=kBT /q ≃25 mV, where kB is Boltzmann's constant, T is temperature, and q is the charge of a proton, experiments on surfactant-doped nonpolar fluids observe that the temporal evolution of the external current during charging and discharging is not symmetric [V. Novotny and M. A. Hopper, J. Electrochem. Soc. 126, 925 (1979), 10.1149/1.2129195; P. Kornilovitch and Y. Jeon, J. Appl. Phys. 109, 064509 (2011), 10.1063/1.3554445]. In fact, at sufficiently large voltages (several VT), the current during discharging is no longer monotonic: it displays a "reverse peak" before decaying in magnitude to zero. We analyze the dynamics of discharging by solving the Poisson-Nernst-Planck equations governing ion transport via asymptotic and numerical techniques in three regimes. First, in the "linear regime" when the applied voltage V is formally much less than VT, the charging and discharging currents are antisymmetric in time; however, the potential and charge density profiles during charging and discharging are asymmetric. The current evolution is on the R C timescale of the cell, λDL /D , where L is the width of the cell, D is the diffusivity of ions, and λD is the Debye length. Second, in the (experimentally relevant) thin-double-layer limit ɛ =λD/L ≪1 , there is a "weakly nonlinear" regime defined by VT≲V ≲VTln(1 /ɛ ) , where the bulk salt concentration is uniform; thus the R C timescale of the evolution of the current magnitude

  4. Low-frequency quantitative ultrasound imaging of cell death in vivo.

    Science.gov (United States)

    Sadeghi-Naini, Ali; Papanicolau, Naum; Falou, Omar; Tadayyon, Hadi; Lee, Justin; Zubovits, Judit; Sadeghian, Alireza; Karshafian, Raffi; Al-Mahrouki, Azza; Giles, Anoja; Kolios, Michael C; Czarnota, Gregory J

    2013-08-01

    Currently, no clinical imaging modality is used routinely to assess tumor response to cancer therapies within hours to days of the delivery of treatment. Here, the authors demonstrate the efficacy of ultrasound at a clinically relevant frequency to quantitatively detect changes in tumors in response to cancer therapies using preclinical mouse models. Conventional low-frequency and corresponding high-frequency ultrasound (ranging from 4 to 28 MHz) were used along with quantitative spectroscopic and signal envelope statistical analyses on data obtained from xenograft tumors treated with chemotherapy, x-ray radiation, as well as a novel vascular targeting microbubble therapy. Ultrasound-based spectroscopic biomarkers indicated significant changes in cell-death associated parameters in responsive tumors. Specifically changes in the midband fit, spectral slope, and 0-MHz intercept biomarkers were investigated for different types of treatment and demonstrated cell-death related changes. The midband fit and 0-MHz intercept biomarker derived from low-frequency data demonstrated increases ranging approximately from 0 to 6 dBr and 0 to 8 dBr, respectively, depending on treatments administrated. These data paralleled results observed for high-frequency ultrasound data. Statistical analysis of ultrasound signal envelope was performed as an alternative method to obtain histogram-based biomarkers and provided confirmatory results. Histological analysis of tumor specimens indicated up to 61% cell death present in the tumors depending on treatments administered, consistent with quantitative ultrasound findings indicating cell death. Ultrasound-based spectroscopic biomarkers demonstrated a good correlation with histological morphological findings indicative of cell death (r2=0.71, 0.82; p<0.001). In summary, the results provide preclinical evidence, for the first time, that quantitative ultrasound used at a clinically relevant frequency, in addition to high-frequency ultrasound

  5. Low-frequency quantitative ultrasound imaging of cell death in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Sadeghi-Naini, Ali; Falou, Omar; Czarnota, Gregory J. [Imaging Research – Physical Science, Sunnybrook Research Institute, Sunnybrook Health Sciences Centre, Toronto, Ontario M4N 3M5 (Canada); Department of Radiation Oncology, Odette Cancer Centre, Sunnybrook Health Sciences Centre, Toronto, Ontario M4N 3M5 (Canada); Department of Medical Biophysics, Faculty of Medicine, University of Toronto, Toronto, Ontario M4N 3M5 (Canada); Department of Radiation Oncology, Faculty of Medicine, University of Toronto, Toronto, Ontario M4N 3M5 (Canada); Papanicolau, Naum; Tadayyon, Hadi [Imaging Research – Physical Science, Sunnybrook Research Institute, Sunnybrook Health Sciences Centre, Toronto, Ontario M4N 3M5, Canada and Department of Medical Biophysics, Faculty of Medicine, University of Toronto, Toronto, Ontario M4N 3M5 (Canada); Lee, Justin [Department of Radiation Oncology, Odette Cancer Centre, Sunnybrook Health Sciences Centre, Toronto, Ontario M4N 3M5, Canada and Department of Radiation Oncology, Faculty of Medicine, University of Toronto, Toronto, Ontario M4N 3M5 (Canada); Zubovits, Judit [Department of Pathology, Sunnybrook Health Sciences Centre, Toronto, Ontario M4N 3M5 (Canada); Sadeghian, Alireza [Department of Computer Science, Ryerson University, Toronto, Ontario M5B 2K3 (Canada); Karshafian, Raffi [Department of Physics, Ryerson University, Toronto, Ontario M5B 2K3 (Canada); Al-Mahrouki, Azza; Giles, Anoja [Imaging Research – Physical Science, Sunnybrook Research Institute, Sunnybrook Health Sciences Centre, Toronto, Ontario M4N 3M5, Canada and Department of Radiation Oncology, Odette Cancer Centre, Sunnybrook Health Sciences Centre, Toronto, Ontario M4N 3M5 (Canada); Kolios, Michael C. [Department of Medical Biophysics, Faculty of Medicine, University of Toronto, Toronto, Ontario M4N 3M5, Canada and Department of Physics, Ryerson University, Toronto, Ontario M5B 2K3 (Canada)

    2013-08-15

    Purpose: Currently, no clinical imaging modality is used routinely to assess tumor response to cancer therapies within hours to days of the delivery of treatment. Here, the authors demonstrate the efficacy of ultrasound at a clinically relevant frequency to quantitatively detect changes in tumors in response to cancer therapies using preclinical mouse models.Methods: Conventional low-frequency and corresponding high-frequency ultrasound (ranging from 4 to 28 MHz) were used along with quantitative spectroscopic and signal envelope statistical analyses on data obtained from xenograft tumors treated with chemotherapy, x-ray radiation, as well as a novel vascular targeting microbubble therapy.Results: Ultrasound-based spectroscopic biomarkers indicated significant changes in cell-death associated parameters in responsive tumors. Specifically changes in the midband fit, spectral slope, and 0-MHz intercept biomarkers were investigated for different types of treatment and demonstrated cell-death related changes. The midband fit and 0-MHz intercept biomarker derived from low-frequency data demonstrated increases ranging approximately from 0 to 6 dBr and 0 to 8 dBr, respectively, depending on treatments administrated. These data paralleled results observed for high-frequency ultrasound data. Statistical analysis of ultrasound signal envelope was performed as an alternative method to obtain histogram-based biomarkers and provided confirmatory results. Histological analysis of tumor specimens indicated up to 61% cell death present in the tumors depending on treatments administered, consistent with quantitative ultrasound findings indicating cell death. Ultrasound-based spectroscopic biomarkers demonstrated a good correlation with histological morphological findings indicative of cell death (r{sup 2}= 0.71, 0.82; p < 0.001).Conclusions: In summary, the results provide preclinical evidence, for the first time, that quantitative ultrasound used at a clinically relevant frequency

  6. Quantitative mammalian cell mutagenesis and mutagen screening: study with CHO cells

    Energy Technology Data Exchange (ETDEWEB)

    Hsie, A.W.; O' Neill, J.P.; San Sebastian, J.R.; Brimer, P.A.

    1979-01-01

    The CHO/HGPRT system has been developed and defined for quantifying mutation induced by various physical and chemical agents at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in Chinese hamster ovary (CHO) cells. In all direct-acting chemical mutagens studied, mutation induction increases linearly as a function of the concentration, with no apparent threshold. Some chemicals induce mutation at non-cytotoxic concentrations. The mutagenicity of ethyl methanesulfonate has been quantified as a function of exposure concentration x treatment time. The sensitive and quantitative nature of the system enables studies of the structure-activity (mutagenicity) relationships of various classes of chemicals, including alkylating agents, heterocyclic nitrogen mustards, and platinum compounds. When rat liver S/sub 9/-mediated metabolic activation is present, procarcinogens such as benzo(a)pyrene, 2-acetylaminofluorene, and dimethylnitrosamine are mutagenic, whereas their noncarcinogenic structural analogues pyrene, fluorene, and dimethylamine are not. The system has been shown to be useful in determining the interactive effects between physical and chemical agents, and in screening for mutagenicity of fractionated organic mixtures and industrial chemicals in both liquid and gaseous state. For the system to be used successfully in routine screening, further studies should be directed toward the development of a metabolic activation system suitable for a broad spectrum of chemicals, a sensitive and reliable statistical method, and an experimental design to determine compounds with low mutagenicity. The system has been expanded for determination of mutagen-induced chromosome aberration, sister-chromatid exchange, and micronucleus formation in addition to gene mutation and cytotoxicity; it can also be used to study inhibition of DNA synthesis. (ERB)

  7. Quantitative morphological analysis of interatrial muscle cells in the ferret heart.

    Science.gov (United States)

    Marino, T A; Biberstein, D; Cook, P N; Cook, L; Dwyer, S J

    1981-09-01

    Cells located in the interatrial septum of the ferret heart were examined and mean cell volume, surface area, length, width, as well as cell length/width and surface area/volume ratios were obtained. The muscle cells were from two different regions. One region was the area of the middle internodal tract while the other was from the area where the anterior and middle internodal tracts intermingled. Based on the data obtained, at least two different subpopulations of interatrial muscle cells could be defined. The larger cells had a mean cell length of 109.7 micrometer, a mean cell width of 13.1 micrometer, a length/width ratio of 8.61, a mean cell surface area of 5,057.6 micrometer2, a mean cell volume of 5960.8 micrometer3, and a surface area/volume ratio of 0.87. The smaller cells had a mean cell length of 58.0 micrometer, a mean cell width of 12.2 micrometer, a length/width ratio of 4.85, a mean cell surface area of 2494.1 micrometer2, a mean cell volume of 2553.6 micrometer3, and a surface area/volume ratio of 1.00. The large cell population had cells that were myofibril rich and also others that were myofibril poor. These quantitative data indicate that the regions of internodal pathways are not composed of a single specialized cell type, but rather are composed of at least two, if not more, cell types that intermingle with each other.

  8. Incineration of organic solar cells: Efficient end of life management by quantitative silver recovery

    DEFF Research Database (Denmark)

    Søndergaard, Roar R.; Zimmermann, Yannick Serge; Espinosa Martinez, Nieves

    2016-01-01

    Recovery of silver from the electrodes of roll-to-roll processed organic solar cells after incineration has been performed quantitatively by extraction with nitric acid. This procedure is more than 10 times faster than previous reports and the amount of acid needed for the extraction is reduced...... by a factor of 100-150. LCA studies show that the resulting environmental impacts from silver extraction of incinerated ashes are more favourable on almost all standard factors compared to extraction from shredded organic solar cells. The so lessened environmental impacts by efficient recovery fully justify...... the use of Ag as an electrode in scaled production of organic solar cells....

  9. Non-invasive, label-free cell counting and quantitative analysis of adherent cells using digital holography.

    Science.gov (United States)

    Mölder, A; Sebesta, M; Gustafsson, M; Gisselson, L; Wingren, A Gjörloff; Alm, K

    2008-11-01

    Manual cell counting is time consuming and requires a high degree of skill on behalf of the person performing the count. Here we use a technique that utilizes digital holography, allowing label-free and completely non-invasive cell counting directly in cell culture vessels with adherent viable cells. The images produced can provide both quantitative and qualitative phase information from a single hologram. The recently constructed microscope Holomonitor (Phase Holographic Imaging AB, Lund, Sweden) combines the commonly used phase contrast microscope with digital holography, the latter giving us the possibility of achieving quantitative information on cellular shape, area, confluence and optical thickness. This project aimed at determining the accuracy and repeatability of cell counting measurements using digital holography compared to the conventional manual cell counting method using a haemocytometer. The collected data were also used to determine cell size and cellular optical thickness. The results show that digital holography can be used for non-invasive automatic cell counting as precisely as conventional manual cell counting.

  10. Quantitative index imaging of coculture cells by scanning focused refractive index microscopy

    Science.gov (United States)

    Sun, Teng-Qian; Ye, Qing; Hu, Fen; Liu, Shi-ke; Wang, Xiao-Wan; Wang, Jin; Deng, Zhi-Chao; Mei, Jian-Chun; Zhou, Wen-Yuan; Zhang, Chun-Ping; Wang, Xin-Yu; Pan, Lei-Ting; Tian, Jian-Guo

    2016-08-01

    We report the quantitative refractive index (RI) imaging of cocultured cells in their living environment by scanning focused refractive index microscopy (SFRIM). Mouse microglial cells and synovial cells are cocultured on the top surface of a trapezoid prism. The RI imaging of living cells is obtained in a reflection-type method. The RI information is deduced with the simple derivative total internal reflection method, where a complex retrieval algorithm or reconstruction process is unnecessary. The outline of each cell is determined according to the RI value compared with that of the immersion liquid. The cocultured cells can be discriminated in the RI image. The measurement is nondestructive and label-free. The experimental results prove that SFRIM is a promising tool in the field of biological optics.

  11. Germ cell dynamics in the testis of the postnatal common marmoset monkey (Callithrix jacchus).

    Science.gov (United States)

    Albert, S; Ehmcke, J; Wistuba, J; Eildermann, K; Behr, R; Schlatt, S; Gromoll, J

    2010-11-01

    The seminiferous epithelium in the nonhuman primate Callithrix jacchus is similarly organized to man. This monkey has therefore been used as a preclinical model for spermatogenesis and testicular stem cell physiology. However, little is known about the developmental dynamics of germ cells in the postnatal primate testis. In this study, we analyzed testes of newborn, 8-week-old, and adult marmosets employing immunohistochemistry using pluripotent stem cell and germ cell markers DDX4 (VASA), POU5F1 (OCT3/4), and TFAP2C (AP-2γ). Stereological and morphometric techniques were applied for quantitative analysis of germ cell populations and testicular histological changes. Quantitative RT-PCR (qRT-PCR) of testicular mRNA was applied using 16 marker genes establishing the corresponding profiles during postnatal testicular development. Testis size increased during the first 8 weeks of life with the main driver being longitudinal outgrowth of seminiferous cords. The number of DDX4-positive cells per testis doubled between birth and 8 weeks of age whereas TFAP2C- and POU5F1-positive cells remained unchanged. This increase in DDX4-expressing cells indicates dynamic growth of the differentiated A-spermatogonial population. The presence of cells expressing POU5F1 and TFAP2C after 8 weeks reveals the persistence of less differentiated germ cells. The mRNA and protein profiles determined by qRT-PCR and western blot in newborn, 8-week-old, and adult marmosets corroborated the immunohistochemical findings. In conclusion, we demonstrated the presence of distinct spermatogonial subpopulations in the primate testis exhibiting different dynamics during early testicular development. Our study demonstrates the suitability of the marmoset testis as a model for human testicular development.

  12. Single-cell NF-kappaB dynamics reveal digital activation and analogue information processing.

    Science.gov (United States)

    Tay, Savaş; Hughey, Jacob J; Lee, Timothy K; Lipniacki, Tomasz; Quake, Stephen R; Covert, Markus W

    2010-07-08

    Cells operate in dynamic environments using extraordinary communication capabilities that emerge from the interactions of genetic circuitry. The mammalian immune response is a striking example of the coordination of different cell types. Cell-to-cell communication is primarily mediated by signalling molecules that form spatiotemporal concentration gradients, requiring cells to respond to a wide range of signal intensities. Here we use high-throughput microfluidic cell culture and fluorescence microscopy, quantitative gene expression analysis and mathematical modelling to investigate how single mammalian cells respond to different concentrations of the signalling molecule tumour-necrosis factor (TNF)-alpha, and relay information to the gene expression programs by means of the transcription factor nuclear factor (NF)-kappaB. We measured NF-kappaB activity in thousands of live cells under TNF-alpha doses covering four orders of magnitude. We find, in contrast to population-level studies with bulk assays, that the activation is heterogeneous and is a digital process at the single-cell level with fewer cells responding at lower doses. Cells also encode a subtle set of analogue parameters to modulate the outcome; these parameters include NF-kappaB peak intensity, response time and number of oscillations. We developed a stochastic mathematical model that reproduces both the digital and analogue dynamics as well as most gene expression profiles at all measured conditions, constituting a broadly applicable model for TNF-alpha-induced NF-kappaB signalling in various types of cells. These results highlight the value of high-throughput quantitative measurements with single-cell resolution in understanding how biological systems operate.

  13. [Seasonal dynamics of quantitative and morphological traits of poplar fine roots and their differences between successive rotation plantations].

    Science.gov (United States)

    Wang, Yan-ping; Xu, Tan; Zhu, Wan-rui; Wang, Qi-tong; Liu, Meng-ling; Wang, Hua-tian; Li, Chuan-rong; Dong, Yu-feng

    2016-02-01

    Based on the fine root samples of the first and second generations of poplar (Populus x euramericana ' Neva'), this study examined the response of quantitative and morphological traits of fine roots of different orders and the difference between generations. The results showed that, the quantitative traits of fine roots, such as root length, root surface area and root biomass, presented obvious seasonal variation, and the fine root traits had obvious difference among root orders. The quantitative traits of lower-order fine roots showed significant seasonal difference, and the fine root biomass increased in the growing season and then decreased significantly. The specific root length (SRL) of higher-order roots also showed significant change with season, while the root length density (RLD) and root tissue density (RTD) changed a little. The successive rotation resulted in the significant increase of root length, root biomass, SRL and RLD of 1-2 orders in the growing season. The quantitative traits of first order root significantly positively correlated with soil temperature and moisture, and significantly negatively correlated with the soil organic matter and soil available nitrogen content. However, the quantitative traits of second order root only showed significant correlation with soil nutrient content. The seasonal dynamics of poplar fine roots and the difference between successive rotation plantations implied carbon investment change of poplar to roots. Soil nutrient deficiency induced more carbon investment into roots, and this carbon allocation pattern might affect the aboveground productivity of poplar plantation.

  14. Dynamic behaviour of human neuroepithelial cells in the developing forebrain

    Science.gov (United States)

    Subramanian, Lakshmi; Bershteyn, Marina; Paredes, Mercedes F.; Kriegstein, Arnold R.

    2017-01-01

    To understand how diverse progenitor cells contribute to human neocortex development, we examined forebrain progenitor behaviour using timelapse imaging. Here we find that cell cycle dynamics of human neuroepithelial (NE) cells differ from radial glial (RG) cells in both primary tissue and in stem cell-derived organoids. NE cells undergoing proliferative, symmetric divisions retract their basal processes, and both daughter cells regrow a new process following cytokinesis. The mitotic retraction of the basal process is recapitulated by NE cells in cerebral organoids generated from human-induced pluripotent stem cells. In contrast, RG cells undergoing vertical cleavage retain their basal fibres throughout mitosis, both in primary tissue and in older organoids. Our findings highlight developmentally regulated changes in mitotic behaviour that may relate to the role of RG cells to provide a stable scaffold for neuronal migration, and suggest that the transition in mitotic dynamics can be studied in organoid models. PMID:28139695

  15. Dynamic changes in mouse hematopoietic stem cell numbers during aging

    NARCIS (Netherlands)

    de Haan, G; Van Zant, G

    1999-01-01

    To address the fundamental question of whether or not stem cell populations age, we performed quantitative measurements of the cycling status and frequency of hematopoietic stem cells in long-lived C57BL/6 (B6) and short-lived DBA/2 (DBA) mice at different developmental and aging stages. The frequen

  16. Phosphorylation dynamics during early differentiation of human embryonic stem cells

    NARCIS (Netherlands)

    van Hoof, D.; Munoz, J.; Braam, S.R.; Pinkse, M.W.H.; Linding, R.; Heck, A.J.R.; Mummery, C.L.; Krijgsveld, J.

    2009-01-01

    Pluripotent stem cells self-renew indefinitely and possess characteristic protein-protein networks that remodel during differentiation. How this occurs is poorly understood. Using quantitative mass spectrometry, we analyzed the (phospho)proteome of human embryonic stem cells (hESCs) during

  17. Phosphorylation dynamics during early differentiation of human embryonic stem cells

    NARCIS (Netherlands)

    van Hoof, D.; Munoz, J.; Braam, S.R.; Pinkse, M.W.H.; Linding, R.; Heck, A.J.R.; Mummery, C.L.; Krijgsveld, J.

    2009-01-01

    Pluripotent stem cells self-renew indefinitely and possess characteristic protein-protein networks that remodel during differentiation. How this occurs is poorly understood. Using quantitative mass spectrometry, we analyzed the (phospho)proteome of human embryonic stem cells (hESCs) during different

  18. Phosphorylation dynamics during early differentiation of human embryonic stem cells

    DEFF Research Database (Denmark)

    Van Hoof, Dennis; Muñoz, Javier; Braam, Stefan R

    2009-01-01

    Pluripotent stem cells self-renew indefinitely and possess characteristic protein-protein networks that remodel during differentiation. How this occurs is poorly understood. Using quantitative mass spectrometry, we analyzed the (phospho)proteome of human embryonic stem cells (hESCs) during...

  19. Quantitative neuroanatomy of all Purkinje cells with light sheet microscopy and high-throughput image analysis

    Directory of Open Access Journals (Sweden)

    Ludovico eSilvestri

    2015-05-01

    Full Text Available Characterizing the cytoarchitecture of mammalian central nervous system on a brain-wide scale is becoming a compelling need in neuroscience. For example, realistic modeling of brain activity requires the definition of quantitative features of large neuronal populations in the whole brain. Quantitative anatomical maps will also be crucial to classify the cytoarchtitectonic abnormalities associated with neuronal pathologies in a high reproducible and reliable manner. In this paper, we apply recent advances in optical microscopy and image analysis to characterize the spatial distribution of Purkinje cells across the whole cerebellum. Light sheet microscopy was used to image with micron-scale resolution a fixed and cleared cerebellum of an L7-GFP transgenic mouse, in which all Purkinje cells are fluorescently labeled. A fast and scalable algorithm for fully automated cell identification was applied on the image to extract the position of all the fluorescent Purkinje cells. This vectorized representation of the cell population allows a thorough characterization of the complex three-dimensional distribution of the neurons, highlighting the presence of gaps inside the lamellar organization of Purkinje cells, whose density is believed to play a significant role in autism spectrum disorders. Furthermore, clustering analysis of the localized somata permits dividing the whole cerebellum in groups of Purkinje cells with high spatial correlation, suggesting new possibilities of anatomical partition. The quantitative approach presented here can be extended to study the distribution of different types of cell in many brain regions and across the whole encephalon, providing a robust base for building realistic computational models of the brain, and for unbiased morphological tissue screening in presence of pathologies and/or drug treatments.

  20. Quantitative comparison of a human cancer cell surface proteome between interphase and mitosis.

    Science.gov (United States)

    Özlü, Nurhan; Qureshi, Mohammad H; Toyoda, Yusuke; Renard, Bernhard Y; Mollaoglu, Gürkan; Özkan, Nazlı E; Bulbul, Selda; Poser, Ina; Timm, Wiebke; Hyman, Anthony A; Mitchison, Timothy J; Steen, Judith A

    2015-01-13

    The cell surface is the cellular compartment responsible for communication with the environment. The interior of mammalian cells undergoes dramatic reorganization when cells enter mitosis. These changes are triggered by activation of the CDK1 kinase and have been studied extensively. In contrast, very little is known of the cell surface changes during cell division. We undertook a quantitative proteomic comparison of cell surface-exposed proteins in human cancer cells that were tightly synchronized in mitosis or interphase. Six hundred and twenty-eight surface and surface-associated proteins in HeLa cells were identified; of these, 27 were significantly enriched at the cell surface in mitosis and 37 in interphase. Using imaging techniques, we confirmed the mitosis-selective cell surface localization of protocadherin PCDH7, a member of a family with anti-adhesive roles in embryos. We show that PCDH7 is required for development of full mitotic rounding pressure at the onset of mitosis. Our analysis provided basic information on how cell cycle progression affects the cell surface. It also provides potential pharmacodynamic biomarkers for anti-mitotic cancer chemotherapy.

  1. Quantitative analysis of changes in actin microfilament contribution to cell plate development in plant cytokinesis

    Directory of Open Access Journals (Sweden)

    Sano Toshio

    2008-07-01

    Full Text Available Abstract Background Plant cells divide by the formation of new cross walls, known as cell plates, from the center to periphery of each dividing cell. Formation of the cell plate occurs in the phragmoplast, a complex structure composed of membranes, microtubules (MTs and actin microfilaments (MFs. Disruption of phragmoplast MTs was previously found to completely inhibit cell plate formation and expansion, indicative of their crucial role in the transport of cell plate membranes and materials. In contrast, disruption of MFs only delays cell plate expansion but does not completely inhibit cell plate formation. Despite such findings, the significance and molecular mechanisms of MTs and MFs remain largely unknown. Results Time-sequential changes in MF-distribution were monitored by live imaging of tobacco BY-2 cells stably expressing the GFP-actin binding domain 2 (GFP-ABD2 fusion protein, which vitally co-stained with the endocytic tracer, FM4-64, that labels the cell plate. During cytokinesis, MFs accumulated near the newly-separated daughter nuclei towards the emerging cell plate, and subsequently approached the expanding cell plate edges. Treatment with an actin polymerization inhibitor caused a decrease in the cell plate expansion rate, which was quantified using time-lapse imaging and regression analysis. Our results demonstrated time-sequential changes in the contribution of MFs to cell plate expansion; MF-disruption caused about a 10% decrease in the cell plate expansion rate at the early phase of cytokinesis, but about 25% at the late phase. MF-disruption also caused malformation of the emerging cell plate at the early phase, indicative of MF involvement in early cell plate formation and expansion. The dynamic movement of endosomes around the cell plate was also inhibited by treatment with an actin polymerization inhibitor and a myosin ATPase inhibitor, respectively. Furthermore, time-lapse imaging of the endoplasmic reticulum (ER revealed

  2. A parallel and quantitative cell migration assay using a novel multi-well-based device.

    Science.gov (United States)

    Quan, Qianghua; Zhang, Shuwen; Wang, Xudong; Ouyang, Qi; Wang, Yugang; Yang, Gen; Luo, Chunxiong

    2016-12-01

    Cell migration assays for different chemical environments are important for both scientists and clinicians searching for new therapeutics. In this study, we developed a multi-well-based microfluidic chip that has multiple units for different conditions. In each unit, cells can be patterned and then released to observe their migration. Automatic image analysis and model-based data processing were developed to describe the integrated cell migration assay precisely and quickly. As a demonstration, the migration behaviors of two types of cells in eight chemical conditions were studied. The results showed that supplementation with transforming growth factor-β(TGF-β) significantly promoted the migration of MCF-7 and MCF-10 A cells compared to several growth factors, such as Epidermal Growth Factor(EGF) and basic fibroblast growth factor(bFGF), as well as a control sample. Cells can migrate particularly fast with two or more mixed supplementary factors, such as TGF-β + bFGF + EGF, which indicated a synergy effect. Thus, this chip could be used to quantitatively observe cancer cell migration and demonstrated great potential for use in quantitative migration studies and chemical screening.

  3. Quantitative 3D imaging of whole, unstained cells by using X-ray diffraction microscopy.

    Science.gov (United States)

    Jiang, Huaidong; Song, Changyong; Chen, Chien-Chun; Xu, Rui; Raines, Kevin S; Fahimian, Benjamin P; Lu, Chien-Hung; Lee, Ting-Kuo; Nakashima, Akio; Urano, Jun; Ishikawa, Tetsuya; Tamanoi, Fuyuhiko; Miao, Jianwei

    2010-06-22

    Microscopy has greatly advanced our understanding of biology. Although significant progress has recently been made in optical microscopy to break the diffraction-limit barrier, reliance of such techniques on fluorescent labeling technologies prohibits quantitative 3D imaging of the entire contents of cells. Cryoelectron microscopy can image pleomorphic structures at a resolution of 3-5 nm, but is only applicable to thin or sectioned specimens. Here, we report quantitative 3D imaging of a whole, unstained cell at a resolution of 50-60 nm by X-ray diffraction microscopy. We identified the 3D morphology and structure of cellular organelles including cell wall, vacuole, endoplasmic reticulum, mitochondria, granules, nucleus, and nucleolus inside a yeast spore cell. Furthermore, we observed a 3D structure protruding from the reconstructed yeast spore, suggesting the spore germination process. Using cryogenic technologies, a 3D resolution of 5-10 nm should be achievable by X-ray diffraction microscopy. This work hence paves a way for quantitative 3D imaging of a wide range of biological specimens at nanometer-scale resolutions that are too thick for electron microscopy.

  4. A genetic strategy for the dynamic and graded control of cell mechanics, motility, and matrix remodeling.

    Science.gov (United States)

    MacKay, Joanna L; Keung, Albert J; Kumar, Sanjay

    2012-02-08

    Cellular mechanical properties have emerged as central regulators of many critical cell behaviors, including proliferation, motility, and differentiation. Although investigators have developed numerous techniques to influence these properties indirectly by engineering the extracellular matrix (ECM), relatively few tools are available to directly engineer the cells themselves. Here we present a genetic strategy for obtaining graded, dynamic control over cellular mechanical properties by regulating the expression of mutant mechanotransductive proteins from a single copy of a gene placed under a repressible promoter. With the use of constitutively active mutants of RhoA GTPase and myosin light chain kinase, we show that varying the expression level of either protein produces graded changes in stress fiber assembly, traction force generation, cellular stiffness, and migration speed. Using this approach, we demonstrate that soft ECMs render cells maximally sensitive to changes in RhoA activity, and that by modulating the ability of cells to engage and contract soft ECMs, we can dynamically control cell spreading, migration, and matrix remodeling. Thus, in addition to providing quantitative relationships between mechanotransductive signaling, cellular mechanical properties, and dynamic cell behaviors, this strategy enables us to control the physical interactions between cells and the ECM and thereby dictate how cells respond to matrix properties.

  5. Optical coherence tomography of cell dynamics in three-dimensional engineered tissues

    Science.gov (United States)

    Boppart, Stephen A.; Tan, Wei; Ko, Han-Jo; Vinegoni, Claudio

    2005-08-01

    Cell-based engineered tissue models have been increasingly useful in the field of tissue engineering, in in vitro drug screening systems, and in complex cell biology studies. While techniques for engineering tissue models have advanced, there have been few imaging technique capable of assessing the complex 3-D cell behaviors in real-time and at the depths that comprise thick tissues. Understanding cell behavior requires advanced imaging tools to progress from characterizing 2-D cell cultures to complex, highly-scattering, thick 3-D tissue constructs. In this study, we demonstrate that it is possible to use OCT to non-destructively evaluate dynamic cell behavior and function in a quantitative fashion in four dimensions (3-D space plus time). Dynamic processes including cell migration, proliferation, apoptosis, necrosis, and mechanical restructuring are observed during engineering tissue development. With high penetration depth and increased spatial and temporal resolution in 3-D space, OCT will be a useful tool for improving our understanding of cell dynamics in situ and in real-time, for elucidating the complex biological interactions, and for directing our designs toward functional and biomimetic engineered tissues.

  6. Dynamics of Vibrio cholerae abundance in Austrian saline lakes, assessed with quantitative solid-phase cytometry.

    Science.gov (United States)

    Schauer, Sonja; Jakwerth, Stefan; Bliem, Rupert; Baudart, Julia; Lebaron, Philippe; Huhulescu, Steliana; Kundi, Michael; Herzig, Alois; Farnleitner, Andreas H; Sommer, Regina; Kirschner, Alexander

    2015-11-01

    In order to elucidate the main predictors of Vibrio cholerae dynamics and to estimate the risk of Vibrio cholera-related diseases, a recently developed direct detection approach based on fluorescence in situ hybridization and solid-phase cytometry (CARD-FISH/SPC) was applied in comparison to cultivation for water samples from the lake Neusiedler See, Austria and three shallow alkaline lakes over a period of 20 months. Vibrio cholerae attached to crustacean zooplankton was quantified via FISH and epifluorescence microscopy. Concentrations obtained by CARD-FISH/SPC were significantly higher than those obtained by culture in 2011, but were mostly of similar magnitude in 2012. Maximum cell numbers were 1.26 × 10(6) V. cholerae per L in Neusiedler See and 7.59 × 10(7) V. cholerae per L in the shallow alkaline lakes. Only on a few occasions during summer was the crustacean zooplankton the preferred habitat for V. cholerae. In winter, V. cholerae was not culturable but could be quantified at all sites with CARD-FISH/SPC. Beside temperature, suspended solids, zooplankton and ammonium were the main predictors of V. cholerae abundance in Neusiedler See, while in the shallow alkaline lakes it was organic carbon, conductivity and phosphorus. Based on the obtained concentrations a first estimation of the health risk for visitors of the lake could be performed. © 2015 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.

  7. Hologram quantitative structure activity relationship, docking, and molecular dynamics studies of inhibitors for CXCR4.

    Science.gov (United States)

    Zhang, Chongqian; Du, Chunmiao; Feng, Zhiwei; Zhu, Jingyu; Li, Youyong

    2015-02-01

    CXCR4 plays a crucial role as a co-receptor with CCR5 for HIV-1 anchoring to mammalian cell membrane and is implicated in cancer metastasis and inflammation. In the current work, we study the relationship of structure and activity of AMD11070 derivatives and other inhibitors of CXCR4 using HQSAR, docking and molecular dynamics (MD) simulations. We obtain an HQSAR model (q(2) = 0.779), and the HQSAR result illustrates that AMD11070 shows a high antiretroviral activity. As HQSAR only provides 2D information, we perform docking and MD to study the interaction of It1t, AMD3100, and AMD3465 with CXCR4. Our results illustrate that the binding are affected by two crucial residues Asp97 and Glu288. The butyl amine moiety of AMD11070 contributes to its high antiretroviral activity. Without a butyl amine moiety, (2,7a-Dihydro-1H-benzoimidazol-2-ylmethyl)-methyl-(5,6,7,8-tetrahydro-quinolin-8-yl)-amine (compound 5a) shows low antiretroviral activity. Our results provide structural details about the interactions between the inhibitors and CXCR4, which are useful for rational drug design of CXCR4.

  8. Optomechanical properties of cancer cells revealed by light-induced deformation and quantitative phase microscopy

    Science.gov (United States)

    Kastl, Lena; Budde, Björn; Isbach, Michael; Rommel, Christina; Kemper, Björn; Schnekenburger, Jürgen

    2015-05-01

    There is a growing interest in cell biology and clinical diagnostics in label-free, optical techniques as the interaction with the sample is minimized and substances like dyes or fixatives do not affect the investigated cells. Such techniques include digital holographic microscopy (DHM) and the optical stretching by fiber optical two beam traps. DHM enables quantitative phase contrast imaging and thereby the determination of the cellular refractive index, dry mass and the volume, whereas optical cell stretching reveals the deformability of cells. Since optical stretching strongly depends on the optical properties and the shape of the investigated material we combined the usage of fiber optical stretching and DHM for the characterization of pancreatic tumor cells. The risk of tumors is their potential to metastasize, spread through the bloodstream and build distal tumors/metastases. The grade of dedifferentiation in which the cells lose their cell type specific properties is a measure for this metastatic potential. The less differentiated the cells are, the higher is their risk to metastasize. Our results demonstrate that pancreatic tumor cells, which are from the same tumor but vary in their grade of differentiation, show significant differences in their deformability. The retrieved data show that differentiated cells have a higher stiffness than less differentiated cells of the same tumor. Even cells that differ only in the expression of a single tumor suppressor gene which is responsible for cell-cell adhesions can be distinguished by their mechanical properties. Additionally, results from DHM measurements yield that the refractive index shows only few variations, indicating that it does not significantly influence optical cell stretching. The obtained results show a promising new approach for the phenotyping of different cell types, especially in tumor cell characterization and cancer diagnostics.

  9. Quantitative analysis of regional airways obstruction using dynamic hyperpolarized 3He MRI-preliminary results in children with cystic fibrosis.

    Science.gov (United States)

    Koumellis, Panos; van Beek, Edwin J R; Woodhouse, Neil; Fichele, Stan; Swift, Andrew J; Paley, Martyn N J; Hill, Catherine; Taylor, Chris J; Wild, Jim M

    2005-09-01

    To investigate regional airways obstruction in patients with cystic fibrosis (CF) with quantitative analysis of dynamic hyperpolarized (HP) (3)He MRI. Dynamic radial projection MRI of HP (3)He gas was used to study respiratory dynamics in a group of eight children with CF. Signal kinetics in a total of seven regions of interest (ROIs; three in each lung, and one in the trachea) were compared with the results of spirometric pulmonary function tests (PFTs). The tracheal signal intensity was used as a form of "input function" to normalize for input flow effects. A pattern of low flow rate in the upper lobes was observed. When the flow measurements from the peripheral ROIs were averaged to obtain an index of flow in the peripheral lung, a good correlation was found (P = 3.74 x 10(-5)) with the forced expired volume in one second (FEV1). These results suggest that a quantitative measurement of localized airways obstruction in the early stages of CF may be obtained from dynamic (3)He MRI by using the slope of the signal rise as a measure of air flow into the peripheral lung. This study also demonstrates that children can cooperate well with the (3)He MRI technique. (c) 2005 Wiley-Liss, Inc.

  10. Quantitative ultrasound evaluation of tumor cell death response in locally advanced breast cancer patients receiving chemotherapy.

    Science.gov (United States)

    Sadeghi-Naini, Ali; Papanicolau, Naum; Falou, Omar; Zubovits, Judit; Dent, Rebecca; Verma, Sunil; Trudeau, Maureen; Boileau, Jean Francois; Spayne, Jacqueline; Iradji, Sara; Sofroni, Ervis; Lee, Justin; Lemon-Wong, Sharon; Yaffe, Martin; Kolios, Michael C; Czarnota, Gregory J

    2013-04-15

    Quantitative ultrasound techniques have been recently shown to be capable of detecting cell death through studies conducted on in vitro and in vivo models. This study investigates for the first time the potential of early detection of tumor cell death in response to clinical cancer therapy administration in patients using quantitative ultrasound spectroscopic methods. Patients (n = 24) with locally advanced breast cancer received neoadjuvant chemotherapy treatments. Ultrasound data were collected before treatment onset and at 4 times during treatment (weeks 1, 4, and 8, and preoperatively). Quantitative ultrasound parameters were evaluated for clinically responsive and nonresponding patients. Results indicated that quantitative ultrasound parameters showed significant changes for patients who responded to treatment, and no similar alteration was observed in treatment-refractory patients. Such differences between clinically and pathologically determined responding and nonresponding patients were statistically significant (P < 0.05) after 4 weeks of chemotherapy. Responding patients showed changes in parameters related to cell death with, on average, an increase in mid-band fit and 0-MHz intercept of 9.1 ± 1.2 dBr and 8.9 ± 1.9 dBr, respectively, whereas spectral slope was invariant. Linear discriminant analysis revealed a sensitivity of 100% and a specificity of 83.3% for distinguishing nonresponding patients by the fourth week into a course of chemotherapy lasting several months. This study reports for the first time that quantitative ultrasound spectroscopic methods can be applied clinically to evaluate cancer treatment responses noninvasively. The results form a basis for monitoring chemotherapy effects and facilitating the personalization of cancer treatment.

  11. Reliability of static and dynamic quantitative sensory testing in patients with painful chronic pancreatitis.

    NARCIS (Netherlands)

    Olesen, S.S.; Goor, H. van; Bouwense, S.A.W.; Wilder-Smith, O.H.G.; Drewes, A.M.

    2012-01-01

    BACKGROUND AND OBJECTIVES: Quantitative sensory testing (QST) has proven to be an important instrument to characterize mechanisms underlying somatic and neuropathic pain disorders. However, its reliability has not previously been established in patients with visceral pain. We investigated the

  12. A combined static-dynamic single-dose imaging protocol to compare quantitative dynamic SPECT with static conventional SPECT.

    Science.gov (United States)

    Sciammarella, Maria; Shrestha, Uttam M; Seo, Youngho; Gullberg, Grant T; Botvinick, Elias H

    2017-08-03

    SPECT myocardial perfusion imaging (MPI) is a clinical mainstay that is typically performed with static imaging protocols and visually or semi-quantitatively assessed for perfusion defects based upon the relative intensity of myocardial regions. Dynamic cardiac SPECT presents a new imaging technique based on time-varying information of radiotracer distribution, which permits the evaluation of regional myocardial blood flow (MBF) and coronary flow reserve (CFR). In this work, a preliminary feasibility study was conducted in a small patient sample designed to implement a unique combined static-dynamic single-dose one-day visit imaging protocol to compare quantitative dynamic SPECT with static conventional SPECT for improving the diagnosis of coronary artery disease (CAD). Fifteen patients (11 males, four females, mean age 71 ± 9 years) were enrolled for a combined dynamic and static SPECT (Infinia Hawkeye 4, GE Healthcare) imaging protocol with a single dose of (99m)Tc-tetrofosmin administered at rest and a single dose administered at stress in a one-day visit. Out of 15 patients, eleven had selective coronary angiography (SCA), 8 within 6 months and the rest within 24 months of SPECT imaging, without intervening symptoms or interventions. The extent and severity of perfusion defects in each myocardial region was graded visually. Dynamically acquired data were also used to estimate the MBF and CFR. Both visually graded images and estimated CFR were tested against SCA as a reference to evaluate the validity of the methods. Overall, conventional static SPECT was normal in ten patients and abnormal in five patients, dynamic SPECT was normal in 12 patients and abnormal in three patients, and CFR from dynamic SPECT was normal in nine patients and abnormal in six patients. Among those 11 patients with SCA, conventional SPECT was normal in 5, 3 with documented CAD on SCA with an overall accuracy of 64%, sensitivity of 40% and specificity of 83%. Dynamic SPECT image

  13. Expanding the linear dynamic range for quantitative liquid chromatography-high resolution mass spectrometry utilizing natural isotopologue signals

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Hanghui, E-mail: Hanghui.Liu@senomyx.com [Senomyx Inc. 4767 Nexus Centre Dr., San Diego, CA 92121 (United States); Lam, Lily; Yan, Lin; Chi, Bert [Senomyx Inc. 4767 Nexus Centre Dr., San Diego, CA 92121 (United States); Dasgupta, Purnendu K., E-mail: Dasgupta@uta.edu [Department of Chemistry and Biochemistry, The University of Texas at Arlington, Arlington, TX 76019-0065 (United States)

    2014-11-19

    Highlights: • Less abundant isotopologue ions were utilized to decrease detector saturation. • A 25–50 fold increase in the upper limit of dynamic range was demonstrated. • Linear dynamic range was expanded without compromising mass resolution. - Abstract: The linear dynamic range (LDR) for quantitative liquid chromatography–mass spectrometry can be extended until ionization saturation is reached by using a number of target isotopologue ions in addition to the normally used target ion that provides the highest sensitivity. Less abundant isotopologue ions extend the LDR: the lower ion abundance decreases the probability of ion detector saturation. Effectively the sensitivity decreases and the upper limit of the LDR increases. We show in this paper that the technique is particularly powerful with a high resolution time of flight mass spectrometer because the data for all ions are automatically acquired, and we demonstrated this for four small organic molecules; the upper limits of LDRs increased by 25–50 times.

  14. Quantitative glycomics monitoring of induced pluripotent- and embryonic stem cells during neuronal differentiation

    Directory of Open Access Journals (Sweden)

    Michiyo Terashima

    2014-11-01

    Full Text Available Alterations in the structure of cell surface glycoforms occurring during the stages of stem cell differentiation remain unclear. We describe a rapid glycoblotting-based cellular glycomics method for quantitatively evaluating changes in glycoform expression and structure during neuronal differentiation of murine induced pluripotent stem cells (iPSCs and embryonic stem cells (ESCs. Our results show that changes in the expression of cellular N-glycans are comparable during the differentiation of iPSCs and ESCs. The expression of bisect-type N-glycans was significantly up-regulated in neurons that differentiated from both iPSCs and ESCs. From a glycobiological standpoint, iPSCs are an alternative neural cell source in addition to ESCs.

  15. Quantitative imaging of collective cell migration during Drosophila gastrulation: multiphoton microscopy and computational analysis.

    Science.gov (United States)

    Supatto, Willy; McMahon, Amy; Fraser, Scott E; Stathopoulos, Angelike

    2009-01-01

    This protocol describes imaging and computational tools to collect and analyze live imaging data of embryonic cell migration. Our five-step protocol requires a few weeks to move through embryo preparation and four-dimensional (4D) live imaging using multi-photon microscopy, to 3D cell tracking using image processing, registration of tracking data and their quantitative analysis using computational tools. It uses commercially available equipment and requires expertise in microscopy and programming that is appropriate for a biology laboratory. Custom-made scripts are provided, as well as sample datasets to permit readers without experimental data to carry out the analysis. The protocol has offered new insights into the genetic control of cell migration during Drosophila gastrulation. With simple modifications, this systematic analysis could be applied to any developing system to define cell positions in accordance with the body plan, to decompose complex 3D movements and to quantify the collective nature of cell migration.

  16. Fluorescent nanodiamonds enable quantitative tracking of human mesenchymal stem cells in miniature pigs

    Science.gov (United States)

    Su, Long-Jyun; Wu, Meng-Shiue; Hui, Yuen Yung; Chang, Be-Ming; Pan, Lei; Hsu, Pei-Chen; Chen, Yit-Tsong; Ho, Hong-Nerng; Huang, Yen-Hua; Ling, Thai-Yen; Hsu, Hsao-Hsun; Chang, Huan-Cheng

    2017-03-01

    Cell therapy is a promising strategy for the treatment of human diseases. While the first use of cells for therapeutic purposes can be traced to the 19th century, there has been a lack of general and reliable methods to study the biodistribution and associated pharmacokinetics of transplanted cells in various animal models for preclinical evaluation. Here, we present a new platform using albumin-conjugated fluorescent nanodiamonds (FNDs) as biocompatible and photostable labels for quantitative tracking of human placenta choriodecidual membrane-derived mesenchymal stem cells (pcMSCs) in miniature pigs by magnetic modulation. With this background-free detection technique and time-gated fluorescence imaging, we have been able to precisely determine the numbers as well as positions of the transplanted FND-labeled pcMSCs in organs and tissues of the miniature pigs after intravenous administration. The method is applicable to single-cell imaging and quantitative tracking of human stem/progenitor cells in rodents and other animal models as well.

  17. Fluorescent nanodiamonds enable quantitative tracking of human mesenchymal stem cells in miniature pigs

    Science.gov (United States)

    Su, Long-Jyun; Wu, Meng-Shiue; Hui, Yuen Yung; Chang, Be-Ming; Pan, Lei; Hsu, Pei-Chen; Chen, Yit-Tsong; Ho, Hong-Nerng; Huang, Yen-Hua; Ling, Thai-Yen; Hsu, Hsao-Hsun; Chang, Huan-Cheng

    2017-01-01

    Cell therapy is a promising strategy for the treatment of human diseases. While the first use of cells for therapeutic purposes can be traced to the 19th century, there has been a lack of general and reliable methods to study the biodistribution and associated pharmacokinetics of transplanted cells in various animal models for preclinical evaluation. Here, we present a new platform using albumin-conjugated fluorescent nanodiamonds (FNDs) as biocompatible and photostable labels for quantitative tracking of human placenta choriodecidual membrane-derived mesenchymal stem cells (pcMSCs) in miniature pigs by magnetic modulation. With this background-free detection technique and time-gated fluorescence imaging, we have been able to precisely determine the numbers as well as positions of the transplanted FND-labeled pcMSCs in organs and tissues of the miniature pigs after intravenous administration. The method is applicable to single-cell imaging and quantitative tracking of human stem/progenitor cells in rodents and other animal models as well. PMID:28358111

  18. Quantitative photothermal phase imaging of red blood cells using digital holographic photothermal microscope.

    Science.gov (United States)

    Vasudevan, Srivathsan; Chen, George C K; Lin, Zhiping; Ng, Beng Koon

    2015-05-10

    Photothermal microscopy (PTM), a noninvasive pump-probe high-resolution microscopy, has been applied as a bioimaging tool in many biomedical studies. PTM utilizes a conventional phase contrast microscope to obtain highly resolved photothermal images. However, phase information cannot be extracted from these photothermal images, as they are not quantitative. Moreover, the problem of halos inherent in conventional phase contrast microscopy needs to be tackled. Hence, a digital holographic photothermal microscopy technique is proposed as a solution to obtain quantitative phase images. The proposed technique is demonstrated by extracting phase values of red blood cells from their photothermal images. These phase values can potentially be used to determine the temperature distribution of the photothermal images, which is an important study in live cell monitoring applications.

  19. Flow cytometry analysis of cell population dynamics and cell cycle during HIV-1 envelope-mediated formation of syncytia in vitro.

    Science.gov (United States)

    Torres-Castro, Israel; Cortés-Rubio, César N; Sandoval, Guadalupe; Lamoyi, Edmundo; Larralde, Carlos; Huerta, Leonor

    2014-01-01

    Cell fusion occurs in physiological and pathological conditions and plays a role in regulation of cell fate. The analysis of cell population dynamics and cell cycle in cell-cell fusion experiments is necessary to determine changes in the quantitative equilibrium of cell populations and to identify potential bystander effects. Here, using cocultures of Jurkat HIV-1 envelope expressing cells and CD4(+) cells as a model system and flow cytometry for the analysis, the number, viability, and cell cycle status of the populations participating in fusion were determined. In 3-day cocultures, a sustained reduction of the number of CD4(+) cells was observed while they showed high viability and normal cell cycle progression; fusion, but not inhibition of proliferation or death, accounted for their decrease. In contrast, the number of Env(+) cells decreased in cocultures due to fusion, death, and an inherent arrest at G1. Most of syncytia formed in the first 6 h of coculture showed DNA synthesis activity, indicating that the efficient recruitment of proliferating cells contributed to amplify the removal of CD4(+) cells by syncytia formation. Late in cocultures, approximately 50% of syncytia were viable and a subpopulation still underwent DNA synthesis, even when the recruitment of additional cells was prevented by the addition of the fusion inhibitor T-20, indicating that a population of syncytia may progress into the cell cycle. These results show that the quantitative analysis of cellular outcomes of cell-cell fusion can be performed by flow cytometry.

  20. Nonequilibrium population dynamics of phenotype conversion of cancer cells.

    Directory of Open Access Journals (Sweden)

    Joseph Xu Zhou

    Full Text Available Tumorigenesis is a dynamic biological process that involves distinct cancer cell subpopulations proliferating at different rates and interconverting between them. In this paper we proposed a mathematical framework of population dynamics that considers both distinctive growth rates and intercellular transitions between cancer cell populations. Our mathematical framework showed that both growth and transition influence the ratio of cancer cell subpopulations but the latter is more significant. We derived the condition that different cancer cell types can maintain distinctive subpopulations and we also explain why there always exists a stable fixed ratio after cell sorting based on putative surface markers. The cell fraction ratio can be shifted by changing either the growth rates of the subpopulations (Darwinism selection or by environment-instructed transitions (Lamarckism induction. This insight can help us to understand the dynamics of the heterogeneity of cancer cells and lead us to new strategies to overcome cancer drug resistance.

  1. Quantitative Probing of Cu(2+) Ions Naturally Present in Single Living Cells.

    Science.gov (United States)

    Lee, Junho; Lee, Hwa-Rim; Pyo, Jaeyeon; Jung, Youngseob; Seo, Ji-Young; Ryu, Hye Guk; Kim, Kyong-Tai; Je, Jung Ho

    2016-06-01

    Quantitative probing of Cu(2+) ions naturally present in single living cells is realized by developing a quantum-dot-embedded nanowire-waveguide probe. The intracellular Cu(2+) ion concentration is quantified by direct monitoring of photoluminescence quenching during the insertion of the nanowire in a living neuron. The measured intracellular Cu(2+) ion concentration is 3.34 ± 1.04 × 10(-6) m (mean ± s.e.m.) in single hippocampal neurons.

  2. Quantitative assays for new families of esterified oxylipins generated by immune cells

    OpenAIRE

    2010-01-01

    Phospholipid-esterified oxylipins are newly described families of bioactive lipids generated by lipoxygenases in immune cells. Until now, assays for their quantitation were not well developed or widely available. Here, we describe a mass spectrometric protocol that enables accurate measurement of several, in particular hydro(pero)xyeicosatetraenoic acids (H(p)ETEs), hydroxyoctadecadienoic acids (HODEs), hydroxydocosahexaenoic acids (HDOHEs) and keto-eicosatetraenoic acids (KETEs), attached to...

  3. A quantitative evaluation of cell migration by the phagokinetic track motility assay.

    Science.gov (United States)

    Nogalski, Maciej T; Chan, Gary C T; Stevenson, Emily V; Collins-McMillen, Donna K; Yurochko, Andrew D

    2012-12-04

    Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular organisms towards a source of nutrients or away from unsuitable conditions, as well as in multicellular organisms for tissue development, immune surveillance and wound healing, just to mention a few roles(1,2,3). Deregulation of this process can lead to serious neurological, cardiovascular and immunological diseases, as well as exacerbated tumor formation and spread(4,5). Molecularly, actin polymerization and receptor recycling have been shown to play important roles in creating cellular extensions (lamellipodia), that drive the forward movement of the cell(6,7,8). However, many biological questions about cell migration remain unanswered. The central role for cellular motility in human health and disease underlines the importance of understanding the specific mechanisms involved in this process and makes accurate methods for evaluating cell motility particularly important. Microscopes are usually used to visualize the movement of cells. However, cells move rather slowly, making the quantitative measurement of cell migration a resource-consuming process requiring expensive cameras and software to create quantitative time-lapsed movies of motile cells. Therefore, the ability to perform a quantitative measurement of cell migration that is cost-effective, non-laborious, and that utilizes common laboratory equipment is a great need for many researchers. The phagokinetic track motility assay utilizes the ability of a moving cell to clear gold particles from its path to create a measurable track on a colloidal gold-coated glass coverslip(9,10). With the use of freely available software, multiple tracks can be evaluated for each treatment to accomplish statistical requirements. The assay can be utilized to assess motility of many cell types, such as cancer cells(11,12), fibroblasts(9), neutrophils(13), skeletal muscle cells(14

  4. High resolution quantitative phase imaging of live cells with constrained optimization approach

    Science.gov (United States)

    Pandiyan, Vimal Prabhu; Khare, Kedar; John, Renu

    2016-03-01

    Quantitative phase imaging (QPI) aims at studying weakly scattering and absorbing biological specimens with subwavelength accuracy without any external staining mechanisms. Use of a reference beam at an angle is one of the necessary criteria for recording of high resolution holograms in most of the interferometric methods used for quantitative phase imaging. The spatial separation of the dc and twin images is decided by the reference beam angle and Fourier-filtered reconstructed image will have a very poor resolution if hologram is recorded below a minimum reference angle condition. However, it is always inconvenient to have a large reference beam angle while performing high resolution microscopy of live cells and biological specimens with nanometric features. In this paper, we treat reconstruction of digital holographic microscopy images as a constrained optimization problem with smoothness constraint in order to recover only complex object field in hologram plane even with overlapping dc and twin image terms. We solve this optimization problem by gradient descent approach iteratively and the smoothness constraint is implemented by spatial averaging with appropriate size. This approach will give excellent high resolution image recovery compared to Fourier filtering while keeping a very small reference angle. We demonstrate this approach on digital holographic microscopy of live cells by recovering the quantitative phase of live cells from a hologram recorded with nearly zero reference angle.

  5. A polarised population of dynamic microtubules mediates homeostatic length control in animal cells.

    Directory of Open Access Journals (Sweden)

    Remigio Picone

    Full Text Available Because physical form and function are intimately linked, mechanisms that maintain cell shape and size within strict limits are likely to be important for a wide variety of biological processes. However, while intrinsic controls have been found to contribute to the relatively well-defined shape of bacteria and yeast cells, the extent to which individual cells from a multicellular animal control their plastic form remains unclear. Here, using micropatterned lines to limit cell extension to one dimension, we show that cells spread to a characteristic steady-state length that is independent of cell size, pattern width, and cortical actin. Instead, homeostatic length control on lines depends on a population of dynamic microtubules that lead during cell extension, and that are aligned along the long cell axis as the result of interactions of microtubule plus ends with the lateral cell cortex. Similarly, during the development of the zebrafish neural tube, elongated neuroepithelial cells maintain a relatively well-defined length that is independent of cell size but dependent upon oriented microtubules. A simple, quantitative model of cellular extension driven by microtubules recapitulates cell elongation on lines, the steady-state distribution of microtubules, and cell length homeostasis, and predicts the effects of microtubule inhibitors on cell length. Together this experimental and theoretical analysis suggests that microtubule dynamics impose unexpected limits on cell geometry that enable cells to regulate their length. Since cells are the building blocks and architects of tissue morphogenesis, such intrinsically defined limits may be important for development and homeostasis in multicellular organisms.

  6. Decipher the dynamic coordination between enzymatic activity and structural modulation at focal adhesions in living cells

    Science.gov (United States)

    Lu, Shaoying; Seong, Jihye; Wang, Yi; Chang, Shiou-Chi; Eichorst, John Paul; Ouyang, Mingxing; Li, Julie Y.-S.; Chien, Shu; Wang, Yingxiao

    2014-07-01

    Focal adhesions (FAs) are dynamic subcellular structures crucial for cell adhesion, migration and differentiation. It remains an enigma how enzymatic activities in these local complexes regulate their structural remodeling in live cells. Utilizing biosensors based on fluorescence resonance energy transfer (FRET), we developed a correlative FRET imaging microscopy (CFIM) approach to quantitatively analyze the subcellular coordination between the enzymatic Src activation and the structural FA disassembly. CFIM reveals that the Src kinase activity only within the microdomain of lipid rafts at the plasma membrane is coupled with FA dynamics. FA disassembly at cell periphery was linearly dependent on this raft-localized Src activity, although cells displayed heterogeneous levels of response to stimulation. Within lipid rafts, the time delay between Src activation and FA disassembly was 1.2 min in cells seeded on low fibronectin concentration ([FN]) and 4.3 min in cells on high [FN]. CFIM further showed that the level of Src-FA coupling, as well as the time delay, was regulated by cell-matrix interactions, as a tight enzyme-structure coupling occurred in FA populations mediated by integrin αvβ3, but not in those by integrin α5β1. Therefore, different FA subpopulations have distinctive regulation mechanisms between their local kinase activity and structural FA dynamics.

  7. Towards quantitative molecular mapping of cells by Raman microscopy: using AFM for decoupling molecular concentration and cell topography.

    Science.gov (United States)

    Boitor, Radu; Sinjab, Faris; Strohbuecker, Stephanie; Sottile, Virginie; Notingher, Ioan

    2016-06-23

    Raman micro-spectroscopy (RMS) is a non-invasive technique for imaging live cells in vitro. However, obtaining quantitative molecular information from Raman spectra is difficult because the intensity of a Raman band is proportional to the number of molecules in the sampled volume, which depends on the local molecular concentration and the thickness of the cell. In order to understand these effects, we combined RMS with atomic force microscopy (AFM), a technique that can measure accurately the thickness profile of the cells. Solution-based calibration models for RNA and albumin were developed to create quantitative maps of RNA and proteins in individual fixed cells. The maps were built by applying the solution-based calibration models, based on partial least squares fitting (PLS), on raster-scan Raman maps, after accounting for the local cell height obtained from the AFM. We found that concentrations of RNA in the cytoplasm of mouse neuroprogenitor stem cells (NSCs) were as high as 25 ± 6 mg ml(-1), while proteins were distributed more uniformly and reached concentrations as high as ∼50 ± 12 mg ml(-1). The combined AFM-Raman datasets from fixed cells were also used to investigate potential improvements for normalization of Raman spectral maps. For all Raman maps of fixed cells (n = 10), we found a linear relationship between the scores corresponding to the first component (PC1) and the cell height profile obtained by AFM. We used PC1 scores to reconstruct the relative height profiles of independent cells (n = 10), and obtained correlation coefficients with AFM maps higher than 0.99. Using this normalization method, qualitative maps of RNA and protein were used to obtain concentrations for live NSCs. While this study demonstrates the potential of using AFM and RMS for measuring concentration maps for individual NSCs in vitro, further studies are required to establish the robustness of the normalization method based on principal component analysis when comparing

  8. Desmosome dynamics in migrating epithelial cells requires the actin cytoskeleton

    Science.gov (United States)

    Roberts, Brett J.; Pashaj, Anjeza; Johnson, Keith R.; Wahl, James K.

    2011-01-01

    Re-modeling of epithelial tissues requires that the cells in the tissue rearrange their adhesive contacts in order to allow cells to migrate relative to neighboring cells. Desmosomes are prominent adhesive structures found in a variety of epithelial tissues that are believed to inhibit cell migration and invasion. Mechanisms regulating desmosome assembly and stability in migrating cells are largely unknown. In this study we established a cell culture model to examine the fate of desmosomal components during scratch wound migration. Desmosomes are rapidly assembled between epithelial cells at the lateral edges of migrating cells and structures are transported in a retrograde fashion while the structures become larger and mature. Desmosome assembly and dynamics in this system are dependent on the actin cytoskeleton prior to being associated with the keratin intermediate filament cytoskeleton. These studies extend our understanding of desmosome assembly and provide a system to examine desmosome assembly and dynamics during epithelial cell migration. PMID:21945137

  9. Dual mode diffraction phase microscopy for quantitative functional assessment of biological cells

    Science.gov (United States)

    Talaikova, N. A.; Popov, A. P.; Kalyanov, A. L.; Ryabukho, V. P.; Meglinski, I. V.

    2017-10-01

    A diffraction phase microscopy approach with a combined use of transmission and reflection imaging modes has been developed and applied for non-invasive quantitative assessment of the refractive index of red blood cells (RBCs). We present the theoretical background of signal formation for both imaging modes, accompanied by the results of experimental studies. We demonstrate that simultaneous use of the two modes has great potential for accurate assessment of the refractive index of biological cells, and we perform a reconstruction of spatial distribution of the refractive index of RBC in 3D.

  10. SuperSILAC Quantitative Proteome Profiling of Murine Middle Ear Epithelial Cell Remodeling with NTHi.

    Directory of Open Access Journals (Sweden)

    Stéphanie Val

    Full Text Available Chronic Otitis Media with effusion (COME develops after sustained inflammation and is characterized by secretory middle ear epithelial metaplasia and effusion, most frequently mucoid. Non-typeable Haemophilus influenzae (NTHi, the most common acute Otitis Media (OM pathogen, is postulated to promote middle ear epithelial remodeling in the progression of OM from acute to chronic. The goals of this study were to examine histopathological and quantitative proteomic epithelial effects of NTHi challenge in a murine middle ear epithelial cell line.NTHi lysates were generated and used to stimulate murine epithelial cells (mMEEC cultured at air-liquid interface over 48 hours- 1 week. Conditional quantitative Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC of cell lysates was performed to interrogate the global protein production in the cells, using the SuperSILAC technique. Histology of the epithelium over time was done to measure bacterial dependent remodeling.Mass spectrometry analysis identified 2,565 proteins across samples, of which 74 exhibited differential enrichment or depletion in cell lysates (+/-2.0 fold-change; p value<0.05. The key molecular functions regulated by NTHi lysates exposure were related to cell proliferation, death, migration, adhesion and inflammation. Finally, chronic exposure induced significant epithelial thickening of cells grown at air liquid interface.NTHi lysates drive pathways responsible of cell remodeling in murine middle ear epithelium which likely contributes to observed epithelial hyperplasia in vitro. Further elucidation of these mediators will be critical in understanding the progression of OM from acute to chronic at the molecular level.

  11. Highly sensitive quantitative imaging for monitoring single cancer cell growth kinetics and drug response.

    Directory of Open Access Journals (Sweden)

    Mustafa Mir

    Full Text Available The detection and treatment of cancer has advanced significantly in the past several decades, with important improvements in our understanding of the fundamental molecular and genetic basis of the disease. Despite these advancements, drug-screening methodologies have remained essentially unchanged since the introduction of the in vitro human cell line screen in 1990. Although the existing methods provide information on the overall effects of compounds on cell viability, they are restricted by bulk measurements, large sample sizes, and lack capability to measure proliferation kinetics at the individual cell level. To truly understand the nature of cancer cell proliferation and to develop personalized adjuvant therapies, there is a need for new methodologies that provide quantitative information to monitor the effect of drugs on cell growth as well as morphological and phenotypic changes at the single cell level. Here we show that a quantitative phase imaging modality known as spatial light interference microscopy (SLIM addresses these needs and provides additional advantages over existing proliferation assays. We demonstrate these capabilities through measurements on the effects of the hormone estradiol and the antiestrogen ICI182,780 (Faslodex on the growth of MCF-7 breast cancer cells. Along with providing information on changes in the overall growth, SLIM provides additional biologically relevant information. For example, we find that exposure to estradiol results in rapidly growing cells with lower dry mass than the control population. Subsequently blocking the estrogen receptor with ICI results in slower growing cells, with lower dry masses than the control. This ability to measure changes in growth kinetics in response to environmental conditions provides new insight on growth regulation mechanisms. Our results establish the capabilities of SLIM as an advanced drug screening technology that provides information on changes in proliferation

  12. Multispectral fingerprinting for improved in vivo cell dynamics analysis

    Directory of Open Access Journals (Sweden)

    Cooper Cameron HJ

    2010-09-01

    Full Text Available Abstract Background Tracing cell dynamics in the embryo becomes tremendously difficult when cell trajectories cross in space and time and tissue density obscure individual cell borders. Here, we used the chick neural crest (NC as a model to test multicolor cell labeling and multispectral confocal imaging strategies to overcome these roadblocks. Results We found that multicolor nuclear cell labeling and multispectral imaging led to improved resolution of in vivo NC cell identification by providing a unique spectral identity for each cell. NC cell spectral identity allowed for more accurate cell tracking and was consistent during short term time-lapse imaging sessions. Computer model simulations predicted significantly better object counting for increasing cell densities in 3-color compared to 1-color nuclear cell labeling. To better resolve cell contacts, we show that a combination of 2-color membrane and 1-color nuclear cell labeling dramatically improved the semi-automated analysis of NC cell interactions, yet preserved the ability to track cell movements. We also found channel versus lambda scanning of multicolor labeled embryos significantly reduced the time and effort of image acquisition and analysis of large 3D volume data sets. Conclusions Our results reveal that multicolor cell labeling and multispectral imaging provide a cellular fingerprint that may uniquely determine a cell's position within the embryo. Together, these methods offer a spectral toolbox to resolve in vivo cell dynamics in unprecedented detail.

  13. Modeling of microfluidic microbial fuel cells using quantitative bacterial transport parameters

    Science.gov (United States)

    Mardanpour, Mohammad Mahdi; Yaghmaei, Soheila; Kalantar, Mohammad

    2017-02-01

    The objective of present study is to analyze the dynamic modeling of bioelectrochemical processes and improvement of the performance of previous models using quantitative data of bacterial transport parameters. The main deficiency of previous MFC models concerning spatial distribution of biocatalysts is an assumption of initial distribution of attached/suspended bacteria on electrode or in anolyte bulk which is the foundation for biofilm formation. In order to modify this imperfection, the quantification of chemotactic motility to understand the mechanisms of the suspended microorganisms' distribution in anolyte and/or their attachment to anode surface to extend the biofilm is implemented numerically. The spatial and temporal distributions of the bacteria, as well as the dynamic behavior of the anolyte and biofilm are simulated. The performance of the microfluidic MFC as a chemotaxis assay is assessed by analyzing the bacteria activity, substrate variation, bioelectricity production rate and the influences of external resistance on the biofilm and anolyte's features.

  14. Quantitative analysis of cell-type specific gene expression in the green alga Volvox carteri

    Directory of Open Access Journals (Sweden)

    Hallmann Armin

    2006-12-01

    Full Text Available Abstract Background The multicellular alga Volvox carteri possesses only two cell types: mortal, motile somatic cells and potentially immortal, immotile reproductive cells. It is therefore an attractive model system for studying how cell-autonomous cytodifferentiation is programmed within a genome. Moreover, there are ongoing genome projects both in Volvox carteri and in the closely related unicellular alga Chlamydomonas reinhardtii. However, gene sequencing is only the beginning. To identify cell-type specific expression and to determine relative expression rates, we evaluate the potential of real-time RT-PCR for quantifying gene transcript levels. Results Here we analyze a diversified pool of 39 target genes by real-time RT-PCR for each cell type. This gene pool contains previously known genes with unknown localization of cellular expression, 28 novel genes which are described in this study for the first time, and a few known, cell-type specific genes as a control. The respective gene products are, for instance, part of photosynthesis, cellular regulation, stress response, or transport processes. We provide expression data for all these genes. Conclusion The results show that quantitative real-time RT-PCR is a favorable approach to analyze cell-type specific gene expression in Volvox, which can be extended to a much larger number of genes or to developmental or metabolic mutants. Our expression data also provide a basis for a detailed analysis of individual, previously unknown, cell-type specifically expressed genes.

  15. Quantitative proteomics of Spodoptera frugiperda cells during growth and baculovirus infection.

    Science.gov (United States)

    Carinhas, Nuno; Robitaille, Aaron Mark; Moes, Suzette; Carrondo, Manuel José Teixeira; Jenoe, Paul; Oliveira, Rui; Alves, Paula Marques

    2011-01-01

    Baculovirus infection of Spodoptera frugiperda cells is a system of choice to produce a range of recombinant proteins, vaccines and, potentially, gene therapy vectors. While baculovirus genomes are well characterized, the genome of S. frugiperda is not sequenced and the virus-host molecular interplay is sparsely known. Herein, we describe the application of stable isotope labeling by amino acids in cell culture (SILAC) to obtain the first comparative proteome quantitation of S. frugiperda cells during growth and early baculovirus infection. The proteome coverage was maximized by compiling a search database with protein annotations from insect species. Of interest were differentially proteins related to energy metabolism, endoplasmic reticulum and oxidative stress, yet not investigated in the scope of baculovirus infection. Further, the reduced expression of key viral-encoded proteins early in the infection cycle is suggested to be related with decreased viral replication at high cell density culture. These findings have implications for virological research and improvement of baculovirus-based bioprocesses.

  16. Deciphering DNA replication dynamics in eukaryotic cell populations in relation with their averaged chromatin conformations

    DEFF Research Database (Denmark)

    Goldar, A.; Arneodo, A.; Audit, B.

    2016-01-01

    We propose a non-local model of DNA replication that takes into account the observed uncertainty on the position and time of replication initiation in eukaryote cell populations. By picturing replication initiation as a two-state system and considering all possible transition configurations......, and by taking into account the chromatin's fractal dimension, we derive an analytical expression for the rate of replication initiation. This model predicts with no free parameter the temporal profiles of initiation rate, replication fork density and fraction of replicated DNA, in quantitative agreement...... with corresponding experimental data from both S. cerevisiae and human cells and provides a quantitative estimate of initiation site redundancy. This study shows that, to a large extent, the program that regulates the dynamics of eukaryotic DNA replication is a collective phenomenon that emerges from the stochastic...

  17. Deciphering DNA replication dynamics in eukaryotic cell populations in relation with their averaged chromatin conformations

    Science.gov (United States)

    Goldar, A.; Arneodo, A.; Audit, B.; Argoul, F.; Rappailles, A.; Guilbaud, G.; Petryk, N.; Kahli, M.; Hyrien, O.

    2016-03-01

    We propose a non-local model of DNA replication that takes into account the observed uncertainty on the position and time of replication initiation in eukaryote cell populations. By picturing replication initiation as a two-state system and considering all possible transition configurations, and by taking into account the chromatin’s fractal dimension, we derive an analytical expression for the rate of replication initiation. This model predicts with no free parameter the temporal profiles of initiation rate, replication fork density and fraction of replicated DNA, in quantitative agreement with corresponding experimental data from both S. cerevisiae and human cells and provides a quantitative estimate of initiation site redundancy. This study shows that, to a large extent, the program that regulates the dynamics of eukaryotic DNA replication is a collective phenomenon that emerges from the stochastic nature of replication origins initiation.

  18. High-resolution dynamic imaging and quantitative analysis of lung cancer xenografts in nude mice using clinical PET/CT

    Science.gov (United States)

    Wang, Ying Yi; Wang, Kai; Xu, Zuo Yu; Song, Yan; Wang, Chu Nan; Zhang, Chong Qing; Sun, Xi Lin; Shen, Bao Zhong

    2017-01-01

    Considering the general application of dedicated small-animal positron emission tomography/computed tomography is limited, an acceptable alternative in many situations might be clinical PET/CT. To estimate the feasibility of using clinical PET/CT with [F-18]-fluoro-2-deoxy-D-glucose for high-resolution dynamic imaging and quantitative analysis of cancer xenografts in nude mice. Dynamic clinical PET/CT scans were performed on xenografts for 60 min after injection with [F-18]-fluoro-2-deoxy-D-glucose. Scans were reconstructed with or without SharpIR method in two phases. And mice were sacrificed to extracting major organs and tumors, using ex vivo γ-counting as a reference. Strikingly, we observed that the image quality and the correlation between the all quantitive data from clinical PET/CT and the ex vivo counting was better with the SharpIR reconstructions than without. Our data demonstrate that clinical PET/CT scanner with SharpIR reconstruction is a valuable tool for imaging small animals in preclinical cancer research, offering dynamic imaging parameters, good image quality and accurate data quatification. PMID:28881772

  19. Development and dynamics of cell polarity at a glance.

    Science.gov (United States)

    Campanale, Joseph P; Sun, Thomas Y; Montell, Denise J

    2017-04-01

    Cells exhibit morphological and molecular asymmetries that are broadly categorized as cell polarity. The cell polarity established in early embryos prefigures the macroscopic anatomical asymmetries characteristic of adult animals. For example, eggs and early embryos have polarized distributions of RNAs and proteins that generate global anterior/posterior and dorsal/ventral axes. The molecular programs that polarize embryos are subsequently reused in multiple contexts. Epithelial cells require apical/basal polarity to establish their barrier function. Migrating cells polarize in the direction of movement, creating distinct leading and trailing structures. Asymmetrically dividing stem cells partition different molecules between themselves and their daughter cells. Cell polarity can develop de novo, be maintained through rounds of cell division and be dynamically remodeled. In this Cell Science at a Glance review and poster, we describe molecular asymmetries that underlie cell polarity in several cellular contexts. We highlight multiple developmental systems that first establish cell/developmental polarity, and then maintain it. Our poster showcases repeated use of the Par, Scribble and Crumbs polarity complexes, which drive the development of cell polarity in many cell types and organisms. We then briefly discuss the diverse and dynamic changes in cell polarity that occur during cell migration, asymmetric cell division and in planar polarized tissues.

  20. Automated Detection of P. falciparum Using Machine Learning Algorithms with Quantitative Phase Images of Unstained Cells

    Science.gov (United States)

    Park, Han Sang; Rinehart, Matthew T.; Walzer, Katelyn A.; Chi, Jen-Tsan Ashley; Wax, Adam

    2016-01-01

    Malaria detection through microscopic examination of stained blood smears is a diagnostic challenge that heavily relies on the expertise of trained microscopists. This paper presents an automated analysis method for detection and staging of red blood cells infected by the malaria parasite Plasmodium falciparum at trophozoite or schizont stage. Unlike previous efforts in this area, this study uses quantitative phase images of unstained cells. Erythrocytes are automatically segmented using thresholds of optical phase and refocused to enable quantitative comparison of phase images. Refocused images are analyzed to extract 23 morphological descriptors based on the phase information. While all individual descriptors are highly statistically different between infected and uninfected cells, each descriptor does not enable separation of populations at a level satisfactory for clinical utility. To improve the diagnostic capacity, we applied various machine learning techniques, including linear discriminant classification (LDC), logistic regression (LR), and k-nearest neighbor classification (NNC), to formulate algorithms that combine all of the calculated physical parameters to distinguish cells more effectively. Results show that LDC provides the highest accuracy of up to 99.7% in detecting schizont stage infected cells compared to uninfected RBCs. NNC showed slightly better accuracy (99.5%) than either LDC (99.0%) or LR (99.1%) for discriminating late trophozoites from uninfected RBCs. However, for early trophozoites, LDC produced the best accuracy of 98%. Discrimination of infection stage was less accurate, producing high specificity (99.8%) but only 45.0%-66.8% sensitivity with early trophozoites most often mistaken for late trophozoite or schizont stage and late trophozoite and schizont stage most often confused for each other. Overall, this methodology points to a significant clinical potential of using quantitative phase imaging to detect and stage malaria infection

  1. A Quantitative Comparison of Antibodies to Programmed Cell Death 1 Ligand 1.

    Science.gov (United States)

    Gaule, Patricia; Smithy, James W; Toki, Maria; Rehman, Jamaal; Patell-Socha, Farah; Cougot, Delphine; Collin, Philippe; Morrill, Paul; Neumeister, Veronique; Rimm, David L

    2016-08-18

    Assessment of PD-L1 (programmed cell death 1 ligand 1) expression by immunohistochemical analysis has been used as a predictive diagnostic test to identify responders and guide treatment in trials of the PD-1 (programmed cell death 1) axis inhibitors. The definition of PD-L1 positive lacks standardization, and prediction of response by immunohistochemical analysis is additionally limited by the subjective nature of this technique. To examine whether PD-L1 antibody reagents are interchangeable by quantitatively comparing the expression of the PD-L1 protein. In this immunohistochemistry standardization study, 30 randomly selected cases of lung cancer resected from January 1, 2008, through December 31, 2009, were obtained from Yale Pathology Archives with a range of expression of PD-L1. To test for protein measurement, rather than clinical utility, a PD-L1 index tissue microarray, including cell line and tissue controls, was used. The results were then validated on a commercially available, genetically defined PD-L1 engineered cell line array with a range of controlled protein-expressing cell lines using 6 monoclonal antibodies (SP142, E1L3N, 9A11, SP263, 22c3, and 28-8). Protein levels were measured by quantitative immunofluorescence and quantitative chromogenic assessment. Data analysis was performed from September 2015 through May 2016. Concordance between 4 antibodies revealed regression for tumor tissue cores (R2 = 0.42-0.91) and cell line cores (R2 = 0.83-0.97) by quantitative immunofluorescence in the PD-L1 index tissue microarray. All 6 antibodies had high levels of concordance (R2 = 0.76-0.99) when using chromogenic staining in isogenic cell lines. Because the antibodies are highly concordant, these results suggest that assays based on the use of these antibodies could yield concordant results. They further suggest that previously described differences in PD-L1 expression in tissue are independent of the antibody used and likely attributable to

  2. Selection of reference genes for quantitative PCR studies in purified B cells from B cell chronic lymphocytic leukaemia patients

    OpenAIRE

    Valceckiene, Vilma; Kontenyte, Rima; Jakubauskas, Arturas; Griskevicius, Laimonas

    2010-01-01

    Abstract Clinical heterogeneity of B-cell chronic lymphocytic leukaemia (B-CLL) makes it necessary to identify potent prognostic indicators to predict individual clinical course and select risk-adapted therapy. During the last years numerous gene expression models have been suggested as prognostic factors of B-CLL. Today quantitative polymerase chain reaction (qPCR) is a preferred method for rapid quantification of gene expression and validation of microarray data. Reliability of q...

  3. Quantitative trait gene Slit2 positively regulates murine hematopoietic stem cell numbers.

    Science.gov (United States)

    Waterstrat, Amanda; Rector, Kyle; Geiger, Hartmut; Liang, Ying

    2016-08-09

    Hematopoietic stem cells (HSC) demonstrate natural variation in number and function. The genetic factors responsible for the variations (or quantitative traits) are largely unknown. We previously identified a gene whose differential expression underlies the natural variation of HSC numbers in C57BL/6 (B6) and DBA/2 (D2) mice. We now report the finding of another gene, Slit2, on chromosome 5 that also accounts for variation in HSC number. In reciprocal chromosome 5 congenic mice, introgressed D2 alleles increased HSC numbers, whereas B6 alleles had the opposite effect. Using gene array and quantitative polymerase chain reaction, we identified Slit2 as a quantitative trait gene whose expression was positively correlated with the number of HSCs. Ectopic expression of Slit2 not only increased the number of the long-term colony forming HSCs, but also enhanced their repopulation capacity upon transplantation. Therefore, Slit2 is a novel quantitative trait gene and a positive regulator of the number and function of murine HSCs. This finding suggests that Slit2 may be a potential therapeutic target for the effective in vitro and in vivo expansion of HSCs without compromising normal hematopoiesis.

  4. Quantitative analysis of somatic mitochondrial DNA mutations by single-cell single-molecule PCR.

    Science.gov (United States)

    Kraytsberg, Yevgenya; Bodyak, Natalya; Myerow, Susan; Nicholas, Alexander; Ebralidze, Konstantin; Khrapko, Konstantin

    2009-01-01

    Mitochondrial genome integrity is an important issue in somatic mitochondrial genetics. Development of quantitative methods is indispensable to somatic mitochondrial genetics as quantitative studies are required to characterize heteroplasmy and mutation processes, as well as their effects on phenotypic developments. Quantitative studies include the identification and measurement of the load of pathogenic and non-pathogenic clonal mutations, screening mitochondrial genomes for mutations in order to determine the mutation spectra and characterize an ongoing mutation process. Single-molecule PCR (smPCR) has been shown to be an effective method that can be applied to all areas of quantitative studies. It has distinct advantages over conventional vector-based cloning techniques avoiding the well-known PCR-related artifacts such as the introduction of artificial mutations, preferential allelic amplifications, and "jumping" PCR. smPCR is a straightforward and robust method, which can be effectively used for molecule-by-molecule mutational analysis, even when mitochondrial whole genome (mtWG) analysis is involved. This chapter describes the key features of the smPCR method and provides three examples of its applications in single-cell analysis: di-plex smPCR for deletion quantification, smPCR cloning for clonal point mutation quantification, and smPCR cloning for whole genome sequencing (mtWGS).

  5. A dynamic regression analysis tool for quantitative assessment of bacterial growth written in Python.

    Science.gov (United States)

    Hoeflinger, Jennifer L; Hoeflinger, Daniel E; Miller, Michael J

    2017-01-01

    Herein, an open-source method to generate quantitative bacterial growth data from high-throughput microplate assays is described. The bacterial lag time, maximum specific growth rate, doubling time and delta OD are reported. Our method was validated by carbohydrate utilization of lactobacilli, and visual inspection revealed 94% of regressions were deemed excellent.

  6. Protein dynamics in individual human cells: experiment and theory.

    Directory of Open Access Journals (Sweden)

    Ariel Aharon Cohen

    Full Text Available A current challenge in biology is to understand the dynamics of protein circuits in living human cells. Can one define and test equations for the dynamics and variability of a protein over time? Here, we address this experimentally and theoretically, by means of accurate time-resolved measurements of endogenously tagged proteins in individual human cells. As a model system, we choose three stable proteins displaying cell-cycle-dependant dynamics. We find that protein accumulation with time per cell is quadratic for proteins with long mRNA life times and approximately linear for a protein with short mRNA lifetime. Both behaviors correspond to a classical model of transcription and translation. A stochastic model, in which genes slowly switch between ON and OFF states, captures measured cell-cell variability. The data suggests, in accordance with the model, that switching to the gene ON state is exponentially distributed and that the cell-cell distribution of protein levels can be approximated by a Gamma distribution throughout the cell cycle. These results suggest that relatively simple models may describe protein dynamics in individual human cells.

  7. Dynamics of embryonic stem cell differentiation inferred from single-cell transcriptomics show a series of transitions through discrete cell states

    Science.gov (United States)

    Jang, Sumin; Choubey, Sandeep; Furchtgott, Leon; Zou, Ling-Nan; Doyle, Adele; Menon, Vilas; Loew, Ethan B; Krostag, Anne-Rachel; Martinez, Refugio A; Madisen, Linda; Levi, Boaz P; Ramanathan, Sharad

    2017-01-01

    The complexity of gene regulatory networks that lead multipotent cells to acquire different cell fates makes a quantitative understanding of differentiation challenging. Using a statistical framework to analyze single-cell transcriptomics data, we infer the gene expression dynamics of early mouse embryonic stem (mES) cell differentiation, uncovering discrete transitions across nine cell states. We validate the predicted transitions across discrete states using flow cytometry. Moreover, using live-cell microscopy, we show that individual cells undergo abrupt transitions from a naïve to primed pluripotent state. Using the inferred discrete cell states to build a probabilistic model for the underlying gene regulatory network, we further predict and experimentally verify that these states have unique response to perturbations, thus defining them functionally. Our study provides a framework to infer the dynamics of differentiation from single cell transcriptomics data and to build predictive models of the gene regulatory networks that drive the sequence of cell fate decisions during development. DOI: http://dx.doi.org/10.7554/eLife.20487.001 PMID:28296635

  8. Quantitative proteomic analysis of cell cycle of the dinoflagellate Prorocentrum donghaiense (Dinophyceae.

    Directory of Open Access Journals (Sweden)

    Da-Zhi Wang

    Full Text Available Dinoflagellates are the major causative agents of harmful algal blooms in the coastal zone, which has resulted in adverse effects on the marine ecosystem and public health, and has become a global concern. Knowledge of cell cycle regulation in proliferating cells is essential for understanding bloom dynamics, and so this study compared the protein profiles of Prorocentrum donghaiense at different cell cycle phases and identified differentially expressed proteins using 2-D fluorescence difference gel electrophoresis combined with MALDI-TOF-TOF mass spectrometry. The results showed that the synchronized cells of P. donghaiense completed a cell cycle within 24 hours and cell division was phased with the diurnal cycle. Comparison of the protein profiles at four cell cycle phases (G1, S, early and late G2/M showed that 53 protein spots altered significantly in abundance. Among them, 41 were identified to be involved in a variety of biological processes, e.g. cell cycle and division, RNA metabolism, protein and amino acid metabolism, energy and carbon metabolism, oxidation-reduction processes, and ABC transport. The periodic expression of these proteins was critical to maintain the proper order and function of the cell cycle. This study, to our knowledge, for the first time revealed the major biological processes occurring at different cell cycle phases which provided new insights into the mechanisms regulating the cell cycle and growth of dinoflagellates.

  9. Quantitative shape analysis of chemoresistant colon cancer cells: correlation between morphotype and phenotype.

    Science.gov (United States)

    Pasqualato, A; Palombo, A; Cucina, A; Mariggiò, M A; Galli, L; Passaro, D; Dinicola, S; Proietti, S; D'Anselmi, F; Coluccia, P; Bizzarri, M

    2012-04-15

    Morphological, qualitative observations allow pathologists to correlate the shape the cells acquire with the progressive, underlying neoplastic transformation they are experienced. Cell morphology, indeed, roughly scales with malignancy. A quantitative parameter for characterizing complex irregular structures is the Normalized Bending Energy (NBE). NBE provides a global feature for shape characterization correspondent to the amount of energy needed to transform the specific shape under analysis into its lowest energy state. We hypothesized that a chemotherapy resistant cancer cell line would experience a significant change in its shape, and that such a modification might be quantified by means of NBE parameterization. We checked out the usefulness of a mathematical algorithm to distinguish wild and 5-fluorouracil (5-FU)-resistant colon cancer HCT-8 cells (HCT-8FUres). NBE values, as well as cellular and molecular parameters, were recorded in both cell populations. Results demonstrated that acquisition of drug resistance is accompanied by statistically significant morphological changes in cell membrane, as well as in biological parameters. Namely, NBE increased progressively meanwhile cells become more resistant to increasing 5-FU concentrations. These data indicate how tight the relationships between morphology and phenotype is, and they support the idea to follow a cell transition toward a drug-resistant phenotype by means of morphological monitoring.

  10. Dynamic 3D cell culture via a chemoselective photoactuated ligand.

    Science.gov (United States)

    Westcott, Nathan P; Luo, Wei; Goldstein, Jeffrey; Yousaf, Muhammad N

    2014-09-01

    A new strategy to create a dynamic scaffold for three-dimensional (3D) cell experiments based on a photo-activated cell adhesive peptide ligand is described. After polymerization, the inert matrix becomes cell adhesive by chemoselective modification through the conjugation of oxyamine-terminated ligands. Furthermore, spatial and temporal control of cell culture within the 3D matrix was achieved by the use of a biospecific photoprotected peptide and visualized by confocal microscopy.

  11. Dynamics of cell degradation. [nickel cadmium batteries

    Science.gov (United States)

    Mcdermott, P. P.

    1978-01-01

    The use of chemical and physical data as a supplement to linear regression models in the prediction of cell failure is discussed. Principal factors to be considered are the positive thickness and weight, and the negative thickness. A model for cell degradation and failure in accelerated life test cells is presented and predictions based on a teardown analysis are included.

  12. High speed optical tomography system for quantitative measurement and visualization of dynamic features in a round jet

    Science.gov (United States)

    McMackin, L.; Hugo, R. J.; Bishop, K. P.; Chen, E. Y.; Pierson, R. E.; Truman, C. R.

    An optical tomography system that is capable of operating at frame rates of up to 5 kHz has been used to obtain spatially resolved cross-sectional temperature images of a heated round jet. These tomographic images show dynamic details in the evolving vortical flow structures found in the near field of the jet that are consistent with previous studies of low speed jet flow. Reconstructions produced by the system are quantitative temperature distributions of a planar cross section of the jet measuring temperature differences with a spatial resolution of 1.4 mm.

  13. Systems analysis of quantitative shRNA-library screens identifies regulators of cell adhesion

    Directory of Open Access Journals (Sweden)

    Huang XiaoDong

    2008-06-01

    Full Text Available Abstract Background High throughput screens with RNA interference technology enable loss-of-function analyses of gene activities in mammalian cells. While the construction of genome-scale shRNA libraries has been successful, results of large-scale screening of those libraries can be difficult to analyze because of the relatively high noise levels and the fact that not all shRNAs in a library are equally effective in silencing gene expression. Results We have screened a library consisting of 43,828 shRNAs directed against 8,500 human genes for functions that are necessary in cell detachment induced by a constitutively activated c-Abl tyrosine kinase. To deal with the issues of noise and uncertainty of knockdown efficiencies, we employed an analytical strategy that combines quantitative data analysis with biological knowledge, i.e. Gene Ontology and pathway information, to increase the power of the RNAi screening technique. Using this strategy we found 16 candidate genes to be involved in Abl-induced disruption of cell adhesion, and verified that the knockdown of IL6ST is associated with enhanced cell attachment. Conclusion Our results suggest that the power of genome-wide quantitative shRNA screens can be significantly increased when analyzed using a systems biology-based approach to identify functional gene networks.

  14. Real-time quantitative fluorescence measurement of microscale cell culture analog systems

    Science.gov (United States)

    Oh, Taek-il; Kim, Donghyun; Tatosian, Daniel; Sung, Jong Hwan; Shuler, Michael

    2007-02-01

    A microscale cell culture analog (μCCA) is a cell-based lab-on-a-chip assay that, as an animal surrogate, is applied to pharmacological studies for toxicology tests. A μCCA typically comprises multiple chambers and microfluidics that connect the chambers, which represent animal organs and blood flow to mimic animal metabolism more realistically. A μCCA is expected to provide a tool for high-throughput drug discovery. Previously, a portable fluorescence detection system was investigated for a single μCCA device in real-time. In this study, we present a fluorescence-based imaging system that provides quantitative real-time data of the metabolic interactions in μCCAs with an emphasis on measuring multiple μCCA samples simultaneously for high-throughput screening. The detection system is based on discrete optics components, with a high-power LED and a charge-coupled device (CCD) camera as a light source and a detector, for monitoring cellular status on the chambers of each μCCA sample. Multiple samples are characterized mechanically on a motorized linear stage, which is fully-automated. Each μCCA sample has four chambers, where cell lines MES-SA/DX- 5, and MES-SA (tumor cells of human uterus) have been cultured. All cell-lines have been transfected to express the fusion protein H2B-GFP, which is a human histone protein fused at the amino terminus to EGFP. As a model cytotoxic drug, 10 μM doxorubicin (DOX) was used. Real-time quantitative data of the intensity loss of enhanced green fluorescent protein (EGFP) during cell death of target cells have been collected over several minutes to 40 hours. Design issues and improvements are also discussed.

  15. Cytoskeletal dynamics and supracellular organisation of cell shape fluctuations during dorsal closure.

    Science.gov (United States)

    Blanchard, Guy B; Murugesu, Sughashini; Adams, Richard J; Martinez-Arias, Alfonso; Gorfinkiel, Nicole

    2010-08-01

    Fluctuations in the shape of amnioserosa (AS) cells during Drosophila dorsal closure (DC) provide an ideal system with which to understand contractile epithelia, both in terms of the cellular mechanisms and how tissue behaviour emerges from the activity of individual cells. Using quantitative image analysis we show that apical shape fluctuations are driven by the medial cytoskeleton, with periodic foci of contractile myosin and actin travelling across cell apices. Shape changes were mostly anisotropic and neighbouring cells were often, but transiently, organised into strings with parallel deformations. During the early stages of DC, shape fluctuations with long cycle lengths produced no net tissue contraction. Cycle lengths shortened with the onset of net tissue contraction, followed by a damping of fluctuation amplitude. Eventually, fluctuations became undetectable as AS cells contracted rapidly. These transitions were accompanied by an increase in apical myosin, both at cell-cell junctions and medially, the latter ultimately forming a coherent, but still dynamic, sheet across cells. Mutants with increased myosin activity or actin polymerisation exhibited precocious cell contraction through changes in the subcellular localisation of myosin. thick veins mutant embryos, which exhibited defects in the actin cable at the leading edge, showed similar timings of fluctuation damping to the wild type, suggesting that damping is an autonomous property of the AS. Our results suggest that cell shape fluctuations are a property of cells with low and increasing levels of apical myosin, and that medial and junctional myosin populations combine to contract AS cell apices and drive DC.

  16. Human immunodeficiency virus envelope-dependent cell-cell fusion: a quantitative fluorescence cytometric assay.

    Science.gov (United States)

    Huerta, Leonor; Lamoyi, Edmundo; Báez-Saldaña, Armida; Larralde, Carlos

    2002-02-01

    In vitro fusion of transfected cells expressing the human immunodeficiency virus (HIV) envelope proteins gp120/gp41, with target cells expressing CD4, and a suitable chemokine coreceptor is used widely to investigate the mechanisms of molecular recognition and membrane fusion involved in the entry of the HIV genome into cells and in syncytia formation. We developed an assay that uses two different fluorescent lipophilic probes to single label each reacting cell population and flow cytometry to quantify the extent of cellular fusion after coculture. Fused cells are detected as double-fluorescent particles in this assay, therefore permitting measurement of their proportion in the total cell population. The time course and extent of HIV-glycoprotein-related cellular fusion, the optimal cell ratio, the size and cell composition of the fusion products, and the inhibition of fusion caused by soluble CD4 and anti-CXCR4 antibody 12G5 were determined. The assay was applied to measure fusion between gp120/gp41 and CD4-expressing cells growing as monolayers (HeLa/CHO fusion), as well as to suspension lymphocyte cultures (Jurkat/Jurkat fusion). The method's simple technical and minimal cell-invasive procedures, as well as its non-ambiguous automatic numerical quantification should be useful for the study of factors influencing cell-cell fusion. Copyright 2002 Wiley-Liss, Inc.

  17. Characterization of ubiquitination dependent dynamics in growth factor receptor signaling by quantitative proteomics

    DEFF Research Database (Denmark)

    Akimov, Vyacheslav; Rigbolt, Kristoffer T G; Nielsen, Mogens M;

    2011-01-01

    ) for selectively decoding ubiquitination-driven processes involved in the regulation of cellular signaling networks. We applied this approach to characterize the temporal dynamics of ubiquitination events accompanying epidermal growth factor receptor (EGFR) signal transduction. We used recombinant UBDs derived...

  18. Monitoring cell morphology during necrosis and apoptosis by quantitative phase imaging

    Science.gov (United States)

    Mugnano, Martina; Calabuig, Alejandro; Grilli, Simonetta; Miccio, Lisa; Ferraro, Pietro

    2015-05-01

    Cellular morphology changes and volume alterations play significant roles in many biological processes and they are mirrors of cell functions. In this paper, we propose the Digital Holographic microscope (DH) as a non-invasive imaging technique for a rapid and accurate extraction of morphological information related to cell death. In particular, we investigate the morphological variations that occur during necrosis and apoptosis. The study of necrosis is extremely important because it is often associated with unwarranted loss of cells in human pathologies such as ischemia, trauma, and some forms of neurodegeneration; therefore, a better elucidation in terms of cell morphological changes could pave the way for new treatments. Also, apoptosis is extremely important because it's involved in cancer, both in its formation and in medical treatments. Because the inability to initiate apoptosis enhances tumour formation, current cancer treatments target this pathway. Within this framework, we have developed a transmission off-axis DH apparatus integrated with a micro incubator for investigation of living cells in a temperature and CO2 controlled environment. We employ DH to analyse the necrosis cell death induced by laser light (wavelength 473 nm, light power 4 mW). We have chosen as cellular model NIH 3T3 mouse embryonic fibroblasts because their adhesive features such as morphological changes, and the time needed to adhere and spread have been well characterized in the literature. We have monitored cell volume changes and morphological alterations in real time in order to study the necrosis process accurately and quantitatively. Cell volume changes were evaluated from the measured phase changes of light transmitted through cells. Our digital holographic experiments showed that after exposure of cells to laser light for 90-120 min., they swell and then take on a balloon-like shape until the plasma membrane ruptures and finally the cell volume decreases. Furthermore, we

  19. Quantitative histopathology in the prognostic evaluation of patients with transitional cell carcinoma of the urinary bladder

    DEFF Research Database (Denmark)

    Sasaki, M; Sørensen, Flemming Brandt; Fukuzawa, S

    1993-01-01

    BACKGROUND: Morphologic grading of malignancy is considered to be of prognostic value in patients with transitional cell carcinomas of the urinary bladder (TCC). This qualitative approach is, however, associated with low reproducibility. Grading of malignancy can be carried out on a reproducible......, quantitative scale.METHODS: A retrospective, prognostic study of 110 patients treated for TCC in clinical Stages Ta-T4 (median follow-up time, 6 years) was performed, evaluating various grading techniques. Unbiased estimates of the volume-weighted mean nuclear volume (nuclear vV), nuclear volume fraction...... of nuclear vV are prognostically superior to morphologic grading of malignancy in noninvasive TCC, whereas both morphologically and quantitatively based malignancy grading are without prognostic value in invasive TCC....

  20. Quantitative histopathology in the prognostic evaluation of patients with transitional cell carcinoma of the urinary bladder

    DEFF Research Database (Denmark)

    Sasaki, M; Sørensen, Flemming Brandt; Fukuzawa, S

    1993-01-01

    BACKGROUND: Morphologic grading of malignancy is considered to be of prognostic value in patients with transitional cell carcinomas of the urinary bladder (TCC). This qualitative approach is, however, associated with low reproducibility. Grading of malignancy can be carried out on a reproducible......, quantitative scale. METHODS: A retrospective, prognostic study of 110 patients treated for TCC in clinical Stages Ta-T4 (median follow-up time, 6 years) was performed, evaluating various grading techniques. Unbiased estimates of the volume-weighted mean nuclear volume (nuclear vV), nuclear volume fraction...... of nuclear vV are prognostically superior to morphologic grading of malignancy in noninvasive TCC, whereas both morphologically and quantitatively based malignancy grading are without prognostic value in invasive TCC....

  1. Mitochondrial dynamics and morphology in beta-cells.

    Science.gov (United States)

    Stiles, Linsey; Shirihai, Orian S

    2012-12-01

    Mitochondrial dynamics contribute to the regulation of mitochondrial shape as well as various mitochondrial functions and quality control. This is of particular interest in the beta-cell because of the key role mitochondria play in the regulation of beta-cell insulin secretion function. Moreover, mitochondrial dysfunction has been suggested to contribute to the development of Type 2 Diabetes. Genetic tools that shift the balance of mitochondrial fusion and fission result in alterations to beta-cell function and viability. Additionally, conditions that induce beta-cell dysfunction, such as exposure to a high nutrient environment, disrupt mitochondrial morphology and dynamics. While it has been shown that mitochondria display a fragmented morphology in islets of diabetic patients and animal models, the mechanism behind this is currently unknown. Here, we review the current literature on mitochondrial morphology and dynamics in the beta-cell as well as some of the unanswered question in this field.

  2. RK2 plasmid dynamics in Caulobacter crescentus cells--two modes of DNA replication initiation.

    Science.gov (United States)

    Wegrzyn, Katarzyna; Witosinska, Monika; Schweiger, Pawel; Bury, Katarzyna; Jenal, Urs; Konieczny, Igor

    2013-06-01

    Undisturbed plasmid dynamics is required for the stable maintenance of plasmid DNA in bacterial cells. In this work, we analysed subcellular localization, DNA synthesis and nucleoprotein complex formation of plasmid RK2 during the cell cycle of Caulobacter crescentus. Our microscopic observations showed asymmetrical distribution of plasmid RK2 foci between the two compartments of Caulobacter predivisional cells, resulting in asymmetrical allocation of plasmids to progeny cells. Moreover, using a quantitative PCR (qPCR) method, we estimated that multiple plasmid particles form a single fluorescent focus and that the number of plasmids per focus is approximately equal in both swarmer and predivisional Caulobacter cells. Analysis of the dynamics of TrfA-oriV complex formation during the Caulobacter cell cycle revealed that TrfA binds oriV primarily during the G1 phase, however, plasmid DNA synthesis occurs during the S and G2 phases of the Caulobacter cell cycle. Both in vitro and in vivo analysis of RK2 replication initiation in C. crescentus cells demonstrated that it is independent of the Caulobacter DnaA protein in the presence of the longer version of TrfA protein, TrfA-44. However, in vivo stability tests of plasmid RK2 derivatives suggested that a DnaA-dependent mode of plasmid replication initiation is also possible.

  3. Self-referencing digital holographic microscope for dynamic imaging of living cells

    Science.gov (United States)

    Anand, Arun; Chhaniwal, Vani; Mahajan, Swapnil; Trivedi, Vismay; Singh, Amardeep; Leitgeb, Rainer; Javidi, Bahram

    2014-06-01

    Digital holographic microscope is an ideal tool for quantitative phase contrast imaging of living cells. It yields the thickness distribution of the object under investigation from a single hologram. From a series of holograms the dynamics of the cell under investigation can be obtained. But two-beam digital holographic microscopes has low temporal stability due to uncorrelated phase changes occurring in the reference and object arms. One way to overcome is to use common path techniques, in which, the reference beam is derived from the object beam itself. Both the beams travel along the same path, increasing the temporal stability of the setup. In self-referencing techniques a portion of the object beam is converted into reference beam. It could be achieved by example, using a glass plate to create two laterally sheared versions of the object beam at the sensor, which interfere to produce the holograms/interferograms. This created a common path setup, leading to high temporal stability (~0.6nm). This technique could be used to map cell membrane fluctuations with high temporal stability. Here we provide an overview of our work on the development of temporally stable quantitative phase contrast techniques for dynamic imaging of micro-objects and biological specimen including red blood cells.

  4. Cell shape dynamics: from waves to migration.

    Directory of Open Access Journals (Sweden)

    Meghan K Driscoll

    Full Text Available We observe and quantify wave-like characteristics of amoeboid migration. Using the amoeba Dictyostelium discoideum, a model system for the study of chemotaxis, we demonstrate that cell shape changes in a wave-like manner. Cells have regions of high boundary curvature that propagate from the leading edge toward the back, usually along alternating sides of the cell. Curvature waves are easily seen in cells that do not adhere to a surface, such as cells that are electrostatically repelled from surfaces or cells that extend over the edge of micro-fabricated cliffs. Without surface contact, curvature waves travel from the leading edge to the back of a cell at -35 µm/min. Non-adherent myosin II null cells do not exhibit these curvature waves. At the leading edge of adherent cells, curvature waves are associated with protrusive activity. Like regions of high curvature, protrusive activity travels along the boundary in a wave-like manner. Upon contact with a surface, the protrusions stop moving relative to the surface, and the boundary shape thus reflects the history of protrusive motion. The wave-like character of protrusions provides a plausible mechanism for the zig-zagging of pseudopods and for the ability of cells both to swim in viscous fluids and to navigate complex three dimensional topography.

  5. Quantitative evaluation of regularized phase retrieval algorithms on bone scaffolds seeded with bone cells

    Science.gov (United States)

    Weber, L.; Langer, M.; Tavella, S.; Ruggiu, A.; Peyrin, F.

    2016-05-01

    In the field of regenerative medicine, there has been a growing interest in studying the combination of bone scaffolds and cells that can maximize newly formed bone. In-line phase-contrast x-ray tomography was used to image porous bone scaffolds (Skelite©), seeded with bone forming cells. This technique allows the quantification of both mineralized and soft tissue, unlike with classical x-ray micro-computed tomography. Phase contrast images were acquired at four distances. The reconstruction is typically performed in two successive steps: phase retrieval and tomographic reconstruction. In this work, different regularization methods were applied to the phase retrieval process. The application of a priori terms for heterogeneous objects enables quantitative 3D imaging of not only bone morphology, mineralization, and soft tissue formation, but also cells trapped in the pre-bone matrix. A statistical study was performed to derive statistically significant information on the different culture conditions.

  6. On-Chip Quantitative Measurement of Mechanical Stresses During Cell Migration with Emulsion Droplets

    Science.gov (United States)

    Molino, D.; Quignard, S.; Gruget, C.; Pincet, F.; Chen, Y.; Piel, M.; Fattaccioli, J.

    2016-07-01

    The ability of immune cells to migrate within narrow and crowded spaces is a critical feature involved in various physiological processes from immune response to metastasis. Several in-vitro techniques have been developed so far to study the behaviour of migrating cells, the most recent being based on the fabrication of microchannels within which cells move. To address the question of the mechanical stress a cell is able to produce during the encounter of an obstacle while migrating, we developed a hybrid microchip made of parallel PDMS channels in which oil droplets are sparsely distributed and serve as deformable obstacles. We thus show that cells strongly deform droplets while passing them. Then, we show that the microdevice can be used to study the influence of drugs on migration at the population level. Finally, we describe a quantitative analysis method of the droplet deformation that allows measuring in real-time the mechanical stress exerted by a single cell. The method presented herein thus constitutes a powerful analytical tool for cell migration studies under confinement.

  7. On-Chip Quantitative Measurement of Mechanical Stresses During Cell Migration with Emulsion Droplets

    Science.gov (United States)

    Molino, D.; Quignard, S.; Gruget, C.; Pincet, F.; Chen, Y.; Piel, M.; Fattaccioli, J.

    2016-01-01

    The ability of immune cells to migrate within narrow and crowded spaces is a critical feature involved in various physiological processes from immune response to metastasis. Several in-vitro techniques have been developed so far to study the behaviour of migrating cells, the most recent being based on the fabrication of microchannels within which cells move. To address the question of the mechanical stress a cell is able to produce during the encounter of an obstacle while migrating, we developed a hybrid microchip made of parallel PDMS channels in which oil droplets are sparsely distributed and serve as deformable obstacles. We thus show that cells strongly deform droplets while passing them. Then, we show that the microdevice can be used to study the influence of drugs on migration at the population level. Finally, we describe a quantitative analysis method of the droplet deformation that allows measuring in real-time the mechanical stress exerted by a single cell. The method presented herein thus constitutes a powerful analytical tool for cell migration studies under confinement. PMID:27373558

  8. Quantitative proteomics reveals differential regulation of protein expression in recipient myocardium after trilineage cardiovascular cell transplantation.

    Science.gov (United States)

    Chang, Ying-Hua; Ye, Lei; Cai, Wenxuan; Lee, Yoonkyu; Guner, Huseyin; Lee, Youngsook; Kamp, Timothy J; Zhang, Jianyi; Ge, Ying

    2015-08-01

    Intramyocardial transplantation of cardiomyocytes (CMs), endothelial cells (ECs), and smooth muscle cells (SMCs) derived from human induced pluripotent stem cells (hiPSCs) has beneficial effects on the post-infarction heart. However, the mechanisms underlying the functional improvements remain undefined. We employed large-scale label-free quantitative proteomics to identify proteins that were differentially regulated following cellular transplantation in a swine model of myocardial infarction (MI). We identified 22 proteins that were significantly up-regulated after trilineage cell transplantation compared to both MI and Sham groups. Among them, 12 proteins, including adenylyl cyclase-associated protein 1 and tropomodulin-1, are associated with positive regulation of muscular contraction whereas 11 proteins, such as desmoplakin and zyxin, are involved in embryonic and muscular development and regeneration. Moreover, we identified 21 proteins up-regulated and another 21 down-regulated in MI, but reversed after trilineage cell transplantation. Proteins up-regulated after MI but reversed by transplantation are related to fibrosis and apoptosis. Conversely, proteins down-regulated in MI but restored after cell therapy are regulators of protein nitrosylation. Our results show that the functionally beneficial effects of trilineage cell therapy are accompanied by differential regulation of protein expression in the recipient myocardium, which may contribute to the improved cardiac function.

  9. Quantitative and integrative analysis of paracrine hepatocyte activation by nonparenchymal cells upon lipopolysaccharide induction.

    Science.gov (United States)

    Beuke, Katharina; Schildberg, Frank A; Pinna, Federico; Albrecht, Ute; Liebe, Roman; Bissinger, Michaela; Schirmacher, Peter; Dooley, Steven; Bode, Johannes G; Knolle, Percy A; Kummer, Ursula; Breuhahn, Kai; Sahle, Sven

    2017-03-01

    Gut-derived bacterial lipopolysaccharides (LPS) stimulate the secretion of tumour necrosis factor (TNF) from liver macrophages (MCs), liver sinusoidal endothelial cells (LSECs) and hepatic stellate cells (HSCs), which control the acute phase response in hepatocytes through activation of the NF-κB pathway. The individual and cooperative impact of nonparenchymal cells on this clinically relevant response has not been analysed in detail due to technical limitations. To gain an integrative view on this complex inter- and intracellular communication, we combined a multiscale mathematical model with quantitative, time-resolved experimental data of different primary murine liver cell types. We established a computational model for TNF-induced NF-κB signalling in hepatocytes, accurately describing dose-responsiveness for physiologically relevant cytokine concentrations. TNF secretion profiles were quantitatively measured for all nonparenchymal cell types upon LPS stimulation. This novel approach allowed the analysis of individual and collective paracrine TNF-mediated NF-κB induction in hepatocytes, revealing strongest effects of MCs and LSECs on hepatocellular NF-κB signalling. Simulations suggest that both cell types act together to maximize the NF-κB pathway response induced by low LPS concentrations (0.1 and 1 ng/mL). Higher LPS concentrations (≥ 5 ng/mL) induced sufficient TNF levels from MCs or LSECs to induce a strong and nonadjustable pathway response. Importantly, these simulations also revealed that the initial cytokine secretion (1-2 h after stimulation) rather than final TNF level (10 h after stimulation) defines the hepatocellular NF-κB response. This raises the question whether the current experimental standard of single high-dose cytokine administration is suitable to mimic in vivo cytokine exposure.

  10. Neuronal hypertrophy and mast cells in histologically negative, clinically diagnosed acute appendicitis: a quantitative immunophenotypical analysis.

    Science.gov (United States)

    Amber, Safeena; Mathai, Alka Mary; Naik, Ramadas; Pai, Muktha R; Kumar, Suneet; Prasad, Keerthana

    2010-03-01

    In about 20-25% of appendicectomies performed for clinically suspected acute appendicitis, definite morphological changes are lacking on histopathological examination. The present study was done to investigate whether any changes in neurons and mast cells could be detected in patients presenting with clinical acute appendicitis but found to have normal appendix at histopathology. A descriptive study was conducted on 50 appendix specimens which were categorized as histology-positive acute appendicitis (HPAA), clinically acute appendicitis but histologically negative (HNAA), appendices resected for other causes and appendices from forensic autopsy. A morphometric and quantitative evaluation of nerve fibers and ganglion plexus and its relation to mast cell density were studied. All sections were subjected to hematoxylin and eosin stain, toluidine blue stain, S 100 protein and neuron specific enolase (NSE) immunostaining and a quantitative image analysis system. Mucosal and submucosal neuronal components highlighted by NSE and S100 immunostaining observed in cases of HNAA were comparable to cases of HPAA. With S 100 immunostaining in HNAA cases, the increase in number and size of myentric neuronal plexus were mild in 40% (10/25) cases, moderate in 40% (10/25) and marked in 20% (5/25) cases as compared to 66.7% (10/15) cases of HPAA showing moderate and 33.3% (5/15) cases showing marked increase (p = 0.018). The mean mast cell count was highest in the HNAA cases (2.74) in all the four layers as compared to the HPAA (1.85) and control group (2.05). There was no difference in the relationship of the size of ganglion cells and the mast cell concentration. Neuronal hypertrophy and mast cells may play a role in the pathogenesis of appendicitis-like pain in patients with histologically normal appendices.

  11. Stem-cell dynamics and lineage topology from in vivo fate mapping in the hematopoietic system.

    Science.gov (United States)

    Höfer, Thomas; Barile, Melania; Flossdorf, Michael

    2016-06-01

    In recent years, sophisticated fate-mapping tools have been developed to study the behavior of stem cells in the intact organism. These experimental approaches are beginning to yield a quantitative picture of how cell numbers are regulated during steady state and in response to challenges. Focusing on hematopoiesis and immune responses, we discuss how novel mathematical approaches driven by these fate-mapping data have provided insights into the dynamics and topology of cellular differentiation pathways in vivo. The combination of experiment and theory has allowed to quantify the degree of self-renewal in stem and progenitor cells, shown how native hematopoiesis differs fundamentally from post-transplantation hematopoiesis, and uncovered that the diversification of T lymphocytes during immune responses resembles tissue renewal driven by stem cells.

  12. Local Nucleosome Dynamics Facilitate Chromatin Accessibility in Living Mammalian Cells

    Directory of Open Access Journals (Sweden)

    Saera Hihara

    2012-12-01

    Full Text Available Genome information, which is three-dimensionally organized within cells as chromatin, is searched and read by various proteins for diverse cell functions. Although how the protein factors find their targets remains unclear, the dynamic and flexible nature of chromatin is likely crucial. Using a combined approach of fluorescence correlation spectroscopy, single-nucleosome imaging, and Monte Carlo computer simulations, we demonstrate local chromatin dynamics in living mammalian cells. We show that similar to interphase chromatin, dense mitotic chromosomes also have considerable chromatin accessibility. For both interphase and mitotic chromatin, we observed local fluctuation of individual nucleosomes (∼50 nm movement/30 ms, which is caused by confined Brownian motion. Inhibition of these local dynamics by crosslinking impaired accessibility in the dense chromatin regions. Our findings show that local nucleosome dynamics drive chromatin accessibility. We propose that this local nucleosome fluctuation is the basis for scanning genome information.

  13. Quantitative analysis of particles, genomes and infectious particles in supernatants of haemorrhagic fever virus cell cultures

    Directory of Open Access Journals (Sweden)

    Hedlund Kjell-Olof

    2011-02-01

    Full Text Available Abstract Information on the replication of viral haemorrhagic fever viruses is not readily available and has never been analysed in a comparative approach. Here, we compared the cell culture growth characteristics of haemorrhagic fever viruses (HFV, of the Arenaviridae, Filoviridae, Bunyaviridae, and Flavivridae virus families by performing quantitative analysis of cell culture supernatants by (i electron microscopy for the quantification of virus particles, (ii quantitative real time PCR for the quantification of genomes, and (iii determination of focus forming units by coating fluorescent antibodies to infected cell monolayers for the quantification of virus infectivity. The comparative analysis revealed that filovirus and RVFV replication results in a surplus of genomes but varying degrees of packaging efficiency and infectious particles. More efficient replication and packaging was observed for Lassa virus, and Dengue virus resulting in a better yield of infectious particles while, YFV turned out to be most efficient with only 4 particles inducing one FFU. For Crimean-Congo haemorrhagic fever virus (CCHFV a surplus of empty shells was observed with only one in 24 particles equipped with a genome. The complete particles turned out to be extraordinarily infectious.

  14. A simple method for unbiased quantitation of adoptively transferred cells in solid tissues

    DEFF Research Database (Denmark)

    Petersen, Mikkel; Petersen, Charlotte Christie; Agger, Ralf

    2006-01-01

    In a mouse model, we demonstrate how to obtain a direct, unbiased estimate of the total number of adoptively transferred cells in a variety of organs at different time points. The estimate is obtained by a straightforward method based on the optical fractionator principle. Specifically, non-stimu......, the samples were chosen and prepared in accordance with the optical fractionator principle. We demonstrate that the method is simple, precise, and well suited for quantitative immunological studies.......In a mouse model, we demonstrate how to obtain a direct, unbiased estimate of the total number of adoptively transferred cells in a variety of organs at different time points. The estimate is obtained by a straightforward method based on the optical fractionator principle. Specifically, non...... node at six different time points following adoptive transfer (from 60 s to 1 week), providing a quantitative estimate of the organ distribution of the transferred cells over time. These estimates were obtained by microscopy of uniform samples of thick sections from the respective organs. Importantly...

  15. Qualitative and quantitative characteristics of the extracellular DNA delivered to the nucleus of a living cell

    Directory of Open Access Journals (Sweden)

    Bogachev Sergei S

    2006-10-01

    Full Text Available Abstract Background The blood plasma and other intertissue fluids usually contain a certain amount of DNA, getting there due to a natural cell death in the organism. Cells of this organism can capture the extracellular DNA, whereupon it is delivered to various cell compartments. It is hypothesized that the extracellular DNA is involved in the transfer of genetic information and its fixation in the genome of recipient cell. Results The existence of an active flow of extracellular DNA into the cell is demonstrated using human breast adenocarcinoma (MCF-7 cells as a recipient culture. The qualitative state of the DNA fragments delivered to the main cell compartments (cytoplasm and interchromosomal fraction was assessed. The extracellular DNA delivered to the cell is characterized quantitatively. Conclusion It is demonstrated that the extracellular DNA fragments in several minutes reach the nuclear space, where they are processed so that their linear size increases from about 500 bp to 10,000 bp. The amount of free extracellular DNA fragments simultaneously present in the nuclear space may reach up to 2% of the haploid genome. Using individual DNA fragments with a known molecular weight and sequence as an extracellular DNA, it is found that these fragments degrade instantly in the culture liquid in the absence of a competitor DNA and are delivered into the cell as degradants. When adding a sufficient amount of competitor DNA, the initial undegraded molecules of the DNA fragments with the known molecular weight and sequence are detectable both in the cytoplasm and nuclear space only at the zero point of experiments. The labeled precursor α-dNTP*, added to culture medium, was undetectable inside the cell in all the experiments.

  16. Wavelength-dependent backscattering measurements for quantitative real-time monitoring of apoptosis in living cells

    Science.gov (United States)

    Mulvey, Christine S.; Sherwood, Carly A.; Bigio, Irving J.

    2009-11-01

    is the first report of the use of backscattering spectral measurements to quantitatively monitor apoptosis in viable cell cultures in vitro.

  17. A quantitative proteomic analysis of cellular responses to high glucose media in Chinese hamster ovary cells.

    Science.gov (United States)

    Liu, Zhenke; Dai, Shujia; Bones, Jonathan; Ray, Somak; Cha, Sangwon; Karger, Barry L; Li, Jingyi Jessica; Wilson, Lee; Hinckle, Greg; Rossomando, Anthony

    2015-01-01

    A goal in recombinant protein production using Chinese hamster ovary (CHO) cells is to achieve both high specific productivity and high cell density. Addition of glucose to the culture media is necessary to maintain both cell growth and viability. We varied the glucose concentration in the media from 5 to 16 g/L and found that although specific productivity of CHO-DG44 cells increased with the glucose level, the integrated viable cell density decreased. To examine the biological basis of these results, we conducted a discovery proteomic study of CHO-DG44 cells grown under batch conditions in normal (5 g/L) or high (15 g/L) glucose over 3, 6, and 9 days. Approximately 5,000 proteins were confidently identified against an mRNA-based CHO-DG44 specific proteome database, with 2,800 proteins quantified with at least two peptides. A self-organizing map algorithm was used to deconvolute temporal expression profiles of quantitated proteins. Functional analysis of altered proteins suggested that differences in growth between the two glucose levels resulted from changes in crosstalk between glucose metabolism, recombinant protein expression, and cell death, providing an overall picture of the responses to high glucose environment. The high glucose environment may enhance recombinant dihydrofolate reductase in CHO cells by up-regulating NCK1 and down-regulating PRKRA, and may lower integrated viable cell density by activating mitochondrial- and endoplasmic reticulum-mediated cell death pathways by up-regulating HtrA2 and calpains. These proteins are suggested as potential targets for bioengineering to enhance recombinant protein production.

  18. Cell wall staining with Trypan blue enables quantitative analysis of morphological changes in yeast cells

    DEFF Research Database (Denmark)

    Liesche, Johannes; Marek, Magdalena; Günther-Pomorski, Thomas

    2015-01-01

    with Trypan Blue, which emits strong red fluorescence upon binding to chitin and yeast glucan; thereby, it facilitates cell wall analysis by confocal and super-resolution microscopy. The staining pattern of Trypan Blue was similar to that of the widely used UV-excitable, blue fluorescent cell wall stain...... Calcofluor White. Trypan Blue staining facilitated quantification of cell size and cell wall volume when utilizing the optical sectioning capacity of a confocal microscope. This enabled the quantification of morphological changes during growth under anaerobic conditions and in the presence of chemicals...

  19. Machine learning-based kinetic modeling: a robust and reproducible solution for quantitative analysis of dynamic PET data.

    Science.gov (United States)

    Pan, Leyun; Cheng, Caixia; Haberkorn, Uwe; Dimitrakopoulou-Strauss, Antonia

    2017-05-07

    A variety of compartment models are used for the quantitative analysis of dynamic positron emission tomography (PET) data. Traditionally, these models use an iterative fitting (IF) method to find the least squares between the measured and calculated values over time, which may encounter some problems such as the overfitting of model parameters and a lack of reproducibility, especially when handling noisy data or error data. In this paper, a machine learning (ML) based kinetic modeling method is introduced, which can fully utilize a historical reference database to build a moderate kinetic model directly dealing with noisy data but not trying to smooth the noise in the image. Also, due to the database, the presented method is capable of automatically adjusting the models using a multi-thread grid parameter searching technique. Furthermore, a candidate competition concept is proposed to combine the advantages of the ML and IF modeling methods, which could find a balance between fitting to historical data and to the unseen target curve. The machine learning based method provides a robust and reproducible solution that is user-independent for VOI-based and pixel-wise quantitative analysis of dynamic PET data.

  20. Machine learning-based kinetic modeling: a robust and reproducible solution for quantitative analysis of dynamic PET data

    Science.gov (United States)

    Pan, Leyun; Cheng, Caixia; Haberkorn, Uwe; Dimitrakopoulou-Strauss, Antonia

    2017-05-01

    A variety of compartment models are used for the quantitative analysis of dynamic positron emission tomography (PET) data. Traditionally, these models use an iterative fitting (IF) method to find the least squares between the measured and calculated values over time, which may encounter some problems such as the overfitting of model parameters and a lack of reproducibility, especially when handling noisy data or error data. In this paper, a machine learning (ML) based kinetic modeling method is introduced, which can fully utilize a historical reference database to build a moderate kinetic model directly dealing with noisy data but not trying to smooth the noise in the image. Also, due to the database, the presented method is capable of automatically adjusting the models using a multi-thread grid parameter searching technique. Furthermore, a candidate competition concept is proposed to combine the advantages of the ML and IF modeling methods, which could find a balance between fitting to historical data and to the unseen target curve. The machine learning based method provides a robust and reproducible solution that is user-independent for VOI-based and pixel-wise quantitative analysis of dynamic PET data.

  1. Quantitative Imaging of Turbulent Mixing Dynamics in High-Pressure Fuel Injection to Enable Predictive Simulations of Engine Combustion

    Energy Technology Data Exchange (ETDEWEB)

    Frank, Jonathan H. [Sandia National Lab. (SNL-CA), Livermore, CA (United States). Reacting Flows Dept.; Pickett, Lyle M. [Sandia National Lab. (SNL-CA), Livermore, CA (United States). Engine Combustion Dept.; Bisson, Scott E. [Sandia National Lab. (SNL-CA), Livermore, CA (United States). Remote Sensing and Energetic Materials Dept.; Patterson, Brian D. [Sandia National Lab. (SNL-CA), Livermore, CA (United States). combustion Chemistry Dept.; Ruggles, Adam J. [Sandia National Lab. (SNL-CA), Livermore, CA (United States). Reacting Flows Dept.; Skeen, Scott A. [Sandia National Lab. (SNL-CA), Livermore, CA (United States). Engine Combustion Dept.; Manin, Julien Luc [Sandia National Lab. (SNL-CA), Livermore, CA (United States). Engine Combustion Dept.; Huang, Erxiong [Sandia National Lab. (SNL-CA), Livermore, CA (United States). Reacting Flows Dept.; Cicone, Dave J. [Sandia National Lab. (SNL-CA), Livermore, CA (United States). Engine Combustion Dept.; Sphicas, Panos [Sandia National Lab. (SNL-CA), Livermore, CA (United States). Engine Combustion Dept.

    2015-09-01

    In this LDRD project, we developed a capability for quantitative high - speed imaging measurements of high - pressure fuel injection dynamics to advance understanding of turbulent mixing in transcritical flows, ignition, and flame stabilization mechanisms, and to provide e ssential validation data for developing predictive tools for engine combustion simulations. Advanced, fuel - efficient engine technologies rely on fuel injection into a high - pressure, high - temperature environment for mixture preparation and com bustion. Howe ver, the dynamics of fuel injection are not well understood and pose significant experimental and modeling challenges. To address the need for quantitative high - speed measurements, we developed a Nd:YAG laser that provides a 5ms burst of pulses at 100 kHz o n a robust mobile platform . Using this laser, we demonstrated s patially and temporally resolved Rayleigh scattering imaging and particle image velocimetry measurements of turbulent mixing in high - pressure gas - phase flows and vaporizing sprays . Quantitativ e interpretation of high - pressure measurements was advanced by reducing and correcting interferences and imaging artifacts.

  2. Fluids as Dynamic Templates for Cytoskeletal Proteins in Plant Cells

    CERN Document Server

    Lofthouse, J T

    2008-01-01

    The Dynamic Template model of biological cell membranes and the cytoplasm as spatially organised fluid layers is extended to plant cells, and is shown to offer a feasible shear driven mechanism for the co-alignment of internal and external fibres observed during growth and tropic responses

  3. The effect of EIF dynamics on the cryopreservation process of a size distributed cell population.

    Science.gov (United States)

    Fadda, S; Briesen, H; Cincotti, A

    2011-06-01

    Typical mathematical modeling of cryopreservation of cell suspensions assumes a thermodynamic equilibrium between the ice and liquid water in the extracellular solution. This work investigates the validity of this assumption by introducing a population balance approach for dynamic extracellular ice formation (EIF) in the absence of any cryo-protectant agent (CPA). The population balance model reflects nucleation and diffusion-limited growth in the suspending solution whose driving forces are evaluated in the relevant phase diagram. This population balance description of the extracellular compartment has been coupled to a model recently proposed in the literature [Fadda et al., AIChE Journal, 56, 2173-2185, (2010)], which is capable of quantitatively describing and predicting internal ice formation (IIF) inside the cells. The cells are characterized by a size distribution (i.e. through another population balance), thus overcoming the classic view of a population of identically sized cells. From the comparison of the system behavior in terms of the dynamics of the cell size distribution it can be concluded that the assumption of a thermodynamic equilibrium in the extracellular compartment is not always justified. Depending on the cooling rate, the dynamics of EIF needs to be considered. Copyright © 2011 Elsevier Inc. All rights reserved.

  4. Nanowires: Quantitative Probing of Cu(2+) Ions Naturally Present in Single Living Cells (Adv. Mater. 21/2016).

    Science.gov (United States)

    Lee, Junho; Lee, Hwa-Rim; Pyo, Jaeyeon; Jung, Youngseob; Seo, Ji-Young; Ryu, Hye Guk; Kim, Kyong-Tai; Je, Jung Ho

    2016-06-01

    Quantitative probing of the Cu(2+) ions naturally present in single living cells is accomplished by a probe made from a quantum-dot-embedded-nanowire waveguide. After inserting the active nanowire-based waveguide probe into single living cells, J. H. Je and co-workers directly observe photoluminescence (PL) quenching of the embedded quantum dots by the Cu(2+) ions diffused into the probe as described on page 4071. This results in quantitative measurement of intracellular Cu(2+) ions.

  5. The Molecular Architecture of Cell Adhesion: Dynamic Remodeling Revealed by Videonanoscopy

    Directory of Open Access Journals (Sweden)

    Arnauld eSergé

    2016-05-01

    Full Text Available The plasma membrane delimits the cell, which is the basic unit of living organisms, and is also a privileged site for cell communication with the environment. Cell adhesion can occur through cell-cell and cell-matrix contacts. Adhesion proteins such as integrins and cadherins also constitute receptors for inside-out and outside-in signaling within proteolipidic platforms. Adhesion molecule targeting and stabilization relies on specific features such as preferential segregation by the sub-membrane cytoskeleton meshwork and within membrane proteolipidic microdomains. This review presents an overview of the recent insights brought by the latest developments in microscopy, to unravel the molecular remodeling occurring at cell contacts. The dynamic aspect of cell adhesion was recently highlighted by super-resolution videomicroscopy, also named videonanoscopy. By circumventing the diffraction limit of light, nanoscopy has allowed the monitoring of molecular localization and behavior at the single-molecule level, on fixed and living cells. Accessing molecular-resolution details such as quantitatively monitoring components entering and leaving cell contacts by lateral diffusion and reversible association has revealed an unexpected plasticity. Adhesion structures can be highly specialized, such as focal adhesion in motile cells, as well as immune and neuronal synapses. Spatiotemporal reorganization of adhesion molecules, receptors and adaptors directly relates to structure/function modulation. Assembly of these supramolecular complexes is continuously balanced by dynamic events, remodeling adhesions on various timescales, notably by molecular conformation switches, lateral diffusion within the membrane and endo/exocytosis. Pathological alterations in cell adhesion are involved in cancer evolution, through cancer stem cell interaction with stromal niches, growth, extravasation and metastasis.

  6. The Molecular Architecture of Cell Adhesion: Dynamic Remodeling Revealed by Videonanoscopy.

    Science.gov (United States)

    Sergé, Arnauld

    2016-01-01

    The plasma membrane delimits the cell, which is the basic unit of living organisms, and is also a privileged site for cell communication with the environment. Cell adhesion can occur through cell-cell and cell-matrix contacts. Adhesion proteins such as integrins and cadherins also constitute receptors for inside-out and outside-in signaling within proteolipidic platforms. Adhesion molecule targeting and stabilization relies on specific features such as preferential segregation by the sub-membrane cytoskeleton meshwork and within membrane proteolipidic microdomains. This review presents an overview of the recent insights brought by the latest developments in microscopy, to unravel the molecular remodeling occurring at cell contacts. The dynamic aspect of cell adhesion was recently highlighted by super-resolution videomicroscopy, also named videonanoscopy. By circumventing the diffraction limit of light, nanoscopy has allowed the monitoring of molecular localization and behavior at the single-molecule level, on fixed and living cells. Accessing molecular-resolution details such as quantitatively monitoring components entering and leaving cell contacts by lateral diffusion and reversible association has revealed an unexpected plasticity. Adhesion structures can be highly specialized, such as focal adhesion in motile cells, as well as immune and neuronal synapses. Spatiotemporal reorganization of adhesion molecules, receptors, and adaptors directly relates to structure/function modulation. Assembly of these supramolecular complexes is continuously balanced by dynamic events, remodeling adhesions on various timescales, notably by molecular conformation switches, lateral diffusion within the membrane and endo/exocytosis. Pathological alterations in cell adhesion are involved in cancer evolution, through cancer stem cell interaction with stromal niches, growth, extravasation, and metastasis.

  7. A quantitative proteomics analysis of MCF7 breast cancer stem and progenitor cell populations.

    Science.gov (United States)

    Nie, Song; McDermott, Sean P; Deol, Yadwinder; Tan, Zhijing; Wicha, Max S; Lubman, David M

    2015-11-01

    Accumulating evidence has demonstrated that breast cancers are initiated and develop from a small population of stem-like cells termed cancer stem cells (CSCs). These cells are hypothesized to mediate tumor metastasis and contribute to therapeutic resistance. However, the molecular regulatory networks responsible for maintaining CSCs in an undifferentiated state have yet to be elucidated. In this study, we used CSC markers to isolate pure breast CSCs fractions (ALDH+ and CD44+CD24- cell populations) and the mature luminal cells (CD49f-EpCAM+) from the MCF7 cell line. Proteomic analysis was performed on these samples and a total of 3304 proteins were identified. A label-free quantitative method was applied to analyze differentially expressed proteins. Using the criteria of greater than twofold changes and p value analysis of differentially expressed proteins by Ingenuity Pathway Analysis (IPA) revealed potential molecular regulatory networks that may regulate CSCs. Selected differential proteins were validated by Western blot assay and immunohistochemical staining. The use of proteomics analysis may increase our understanding of the underlying molecular mechanisms of breast CSCs. This may be of importance in the future development of anti-CSC therapeutics.

  8. Microenvironments and different nanoparticle dynamics in living cells revealed by a standard nanoparticle.

    Science.gov (United States)

    Pack, Chan Gi; Song, Mi Ryoung; Tae, Eunju Lee; Hiroshima, Michio; Byun, Kyung Hee; Kim, Jun Sung; Sako, Yasushi

    2012-11-10

    For quantitative analysis of nanoparticle diffusions and submicro-environments in living cells, use of newly synthesized silica-based fluorescent nanoparticle (Si-FNP) as a standard nanoprobe is successfully demonstrated. The appropriate characteristics of a standard probe were fully analyzed in vitro by single molecule detection, transmission electron microscopy, and dynamic light scattering. Using fluorescence correlation analysis in single living cells, we quantitatively compared the diffusional properties of the standard Si-FNP with a diameter of 50 nm, peptide coated Si-FNP, streptavidin coated Qdot, and GFP molecule which have different sizes and surface properties. The result demonstrates that the standard Si-FNP without coat is minimally trapped in the vesicles in the process of cellular endocytosis. Interestingly, a large proportion of Si-FNP introduced into the cells by electroporation diffuses freely in the cells during a cell cycle suggesting free diffusing NPs are hardly trapped in the vesicles. The simple but highly sensitive method will provide insight into strategies to understanding the hydrodynamic process of nanoparticle delivery into living cells as well as the cellular microenvironment in the view of submicro-size.

  9. Quantitative assessment of T-cell repertoire recovery after hematopoietic stem cell transplantation

    Science.gov (United States)

    van Heijst, Jeroen W J; Ceberio, Izaskun; Lipuma, Lauren B; Samilo, Dane W; Wasilewski, Gloria D; Gonzales, Anne Marie R; Nieves, Jimmy L; van den Brink, Marcel R M; Perales, Miguel A; Pamer, Eric G

    2012-01-01

    Delayed T-cell recovery and restricted T-cell receptor (TCR) diversity after allogeneic hematopoietic stem cell transplantation (allo-HSCT) are associated with increased risks of infection and cancer relapse. Technical challenges have limited faithful measurement of TCR diversity following allo-HSCT. Here we combined 5′-RACE PCR with deep sequencing, to quantify TCR diversity in 28 allo-HSCT recipients using a single oligonucleotide pair. Analysis of duplicate blood samples confirmed that the frequency of individual TCRs was accurately determined. After 6 months, cord blood graft recipients approximated the TCR diversity of healthy individuals, whereas recipients of T-cell-depleted peripheral blood stem cell grafts had a 28-fold and 14-fold lower CD4+ and CD8+ T-cell diversity, respectively. After 12 months, these deficiencies had improved for the CD4+, but not the CD8+ T-cell compartment. Overall, this method provides unprecedented views of T-cell repertoire recovery after allo-HSCT and may identify patients at high risk of infection or relapse. PMID:23435170

  10. Relation Between the Cell Volume and the Cell Cycle Dynamics in Mammalian cell

    Science.gov (United States)

    Magno, A. C. G.; Oliveira, I. L.; Hauck, J. V. S.

    2016-08-01

    The main goal of this work is to add and analyze an equation that represents the volume in a dynamical model of the mammalian cell cycle proposed by Gérard and Goldbeter (2011) [1]. The cell division occurs when the cyclinB/Cdkl complex is totally degraded (Tyson and Novak, 2011)[2] and it reaches a minimum value. At this point, the cell is divided into two newborn daughter cells and each one will contain the half of the cytoplasmic content of the mother cell. The equations of our base model are only valid if the cell volume, where the reactions occur, is constant. Whether the cell volume is not constant, that is, the rate of change of its volume with respect to time is explicitly taken into account in the mathematical model, then the equations of the original model are no longer valid. Therefore, every equations were modified from the mass conservation principle for considering a volume that changes with time. Through this approach, the cell volume affects all model variables. Two different dynamic simulation methods were accomplished: deterministic and stochastic. In the stochastic simulation, the volume affects every model's parameters which have molar unit, whereas in the deterministic one, it is incorporated into the differential equations. In deterministic simulation, the biochemical species may be in concentration units, while in stochastic simulation such species must be converted to number of molecules which are directly proportional to the cell volume. In an effort to understand the influence of the new equation a stability analysis was performed. This elucidates how the growth factor impacts the stability of the model's limit cycles. In conclusion, a more precise model, in comparison to the base model, was created for the cell cycle as it now takes into consideration the cell volume variation

  11. Comprehensive quantitative comparison of the membrane proteome and PTM-ome of human embryonic stem cells and neural stem cells

    DEFF Research Database (Denmark)

    Braga, Marcella Nunes de Melo; Schulz, Melanie; Jakobsen, Lene

    Introduction: Human embryonic stem cells (hESCs) can differentiate into all three germ layers and self-renew. Due to its ability to differentiate in vitro into human neural stem cells (hNSCs), which can further be differentiated into motor neurons and dopaminergic neurons, these cells are potential...... source for treatment of neurological diseases such as Parkinson´s disease. Membrane proteins are very important in cellular signaling and they are regulated by post-translational modifications such as phosphorylation and glycosylation. In order to obtain more information about important membrane proteins...... and modification sites involved in the differentiation of hESCs to hNSCs and also investigate potential new markers for two stages, we have performed a comprehensive mass-spectrometry-based quantitative proteomics and PTMomics study. Methods: The hESC and hNSC were subject to Na2CO3 and ultracentrifugation...

  12. The Dynamics of Psychological Momentum : A Quantitative Study in Natural Sport Situations

    NARCIS (Netherlands)

    Briki, Walid; Den Hartigh, Ruud J. R.; Bakker, Frank C.; Gernigon, Christophe

    2012-01-01

    The present study examined the dynamics of competitive anxiety and self-confidence, as well as the relationships between these variables, during athletes' experiences of psychological momentum (PM). Male national level table tennis players (Study 1) and swimmers (Study 2) watched one of their recent

  13. Image Registration and Analysis for Quantitative Myocardial Perfusion: Application to Dynamic Circular Cardiac CT

    NARCIS (Netherlands)

    Isola, A.; Schmitt, H.; Van Stevendaal, U.; Begemann, P.G.C.; Coulon, P.; Boussel, L.; Grass, M.

    2012-01-01

    Large area detector computed tomography systems with fastrotating gantries enable volumetric dynamic cardiac perfusion studies. Prospectively ECG-triggered acquisitions limit the data acquisition to a predefined cardiac phase and thereby reduce X-ray dose andlimit motion artifacts. Even in the case

  14. The Dynamics of Psychological Momentum : A Quantitative Study in Natural Sport Situations

    NARCIS (Netherlands)

    Briki, Walid; Den Hartigh, Ruud J. R.; Bakker, Frank C.; Gernigon, Christophe

    2012-01-01

    The present study examined the dynamics of competitive anxiety and self-confidence, as well as the relationships between these variables, during athletes' experiences of psychological momentum (PM). Male national level table tennis players (Study 1) and swimmers (Study 2) watched one of their recent

  15. Image Registration and Analysis for Quantitative Myocardial Perfusion: Application to Dynamic Circular Cardiac CT

    NARCIS (Netherlands)

    Isola, A.; Schmitt, H.; Van Stevendaal, U.; Begemann, P.G.C.; Coulon, P.; Boussel, L.; Grass, M.

    2012-01-01

    Large area detector computed tomography systems with fastrotating gantries enable volumetric dynamic cardiac perfusion studies. Prospectively ECG-triggered acquisitions limit the data acquisition to a predefined cardiac phase and thereby reduce X-ray dose andlimit motion artifacts. Even in the case

  16. Shape-based motion correction in dynamic contrast-enhanced MRI for quantitative assessment of renal function.

    Science.gov (United States)

    Liu, Wenyang; Sung, Kyunghyun; Ruan, Dan

    2014-12-01

    To incorporate a newly developed shape-based motion estimation scheme into magnetic resonance urography (MRU) and verify its efficacy in facilitating quantitative functional analysis. The authors propose a motion compensation scheme in MRU that consists of three sequential modules: MRU image acquisition, motion compensation, and quantitative functional analysis. They designed two sets of complementary experiments to evaluate the performance of the proposed method. In the first experiment, dynamic contrast enhanced (DCE) MR images were acquired from three sedated subjects, from which clinically valid estimates were derived and served as the "ground truth." Physiologically sound motion was then simulated to synthesize image sequences influenced by respiratory motion. Quantitative assessment and comparison were performed on functional estimates of Patlak number, glomerular filtration rate, and Patlak differential renal function without and with motion compensation against the ground truth. In the second experiment, the authors acquired a temporal series of noncontrast MR images under free breathing from a healthy adult subject. The performance of the proposed method on compensating real motion was evaluated by comparing the standard deviation of the obtained temporal intensity curves before and after motion compensation. On DCE-MR images with simulated motion, the generated relative enhancement curves exhibited large perturbations and the Patlak numbers of the left and right kidney were significantly underestimated up to 35% and 34%, respectively, compared with the ground truth. After motion compensation, the relative enhancement curves exhibited much less perturbations and Patlak estimation errors reduced within 3% and 4% for the left and right kidneys, respectively. On clinical free-breathing MR images, the temporal intensity curves exhibited significantly reduced variations after motion compensation, with standard deviation decreased from 30.3 and 38.2 to 8.3 and

  17. A Quantitative Dynamic Simulation of Bremia lactucae Airborne Conidia Concentration above a Lettuce Canopy.

    Directory of Open Access Journals (Sweden)

    Mamadou Lamine Fall

    Full Text Available Lettuce downy mildew, caused by the oomycete Bremia lactucae Regel, is a major threat to lettuce production worldwide. Lettuce downy mildew is a polycyclic disease driven by airborne spores. A weather-based dynamic simulation model for B. lactucae airborne spores was developed to simulate the aerobiological characteristics of the pathogen. The model was built using the STELLA platform by following the system dynamics methodology. The model was developed using published equations describing disease subprocesses (e.g., sporulation and assembled knowledge of the interactions among pathogen, host, and weather. The model was evaluated with four years of independent data by comparing model simulations with observations of hourly and daily airborne spore concentrations. The results show an accurate simulation of the trend and shape of B. lactucae temporal dynamics of airborne spore concentration. The model simulated hourly and daily peaks in airborne spore concentrations. More than 95% of the simulation runs, the daily-simulated airborne conidia concentration was 0 when airborne conidia were not observed. Also, the relationship between the simulated and the observed airborne spores was linear. In more than 94% of the simulation runs, the proportion of the linear variation in the hourly-observed values explained by the variation in the hourly-simulated values was greater than 0.7 in all years except one. Most of the errors came from the deviation from the 1:1 line, and the proportion of errors due to the model bias was low. This model is the only dynamic model developed to mimic the dynamics of airborne inoculum and represents an initial step towards improved lettuce downy mildew understanding, forecasting and management.

  18. Single wall carbon nanotube electrode system capable of quantitative detection of CD4(+) T cells.

    Science.gov (United States)

    Kim, Joonhyub; Park, Gayoung; Lee, Seoho; Hwang, Suk-Won; Min, Namki; Lee, Kyung-Mi

    2017-04-15

    Development of CNT-based CD4(+) T cell imunosensors remains in its infancy due to the poor immobilization efficiency, lack of reproducibility, and difficulty in providing linear quantification. Here, we developed a fully-integrated single wall carbon nanotube (SWCNT)-based immunosensor capable of selective capture and linear quantification of CD4(+) T cells with greater dynamic range. By employing repeated two-step oxygen (O2) plasma treatment processes with 35 days of recovery periods, we achieved the enhanced functionalization of the CNT surface and the removal of the byproduct of spray-coated SWCNTs that hinders charge transfer and stable CD4(+) T cell sensing. As a result, a linear electrochemical signal was generated in direct proportion to the bound cells. The slope of a SWCNT electrode in a target concentration range (10(2)~10(6)cells/mL) was 4.55×10(-2)μA per concentration decade, with the lowest detection limit of 1×10(2)cells/mL. Since the reduced number of CD4(+) T cell counts in patients' peripheral blood corresponds to the progression of HIV disease, our CD4(+) T cell-immunosensor provides a simple and low-cost platform which can fulfill the requirement for the development of point-of-care (POC) diagnostic technologies for human immunodeficiency virus (HIV) patients in resource-limited countries.

  19. Dynamic contrast optical coherence tomography: quantitative measurement of microvascular transit-time distributions in vivo (Conference Presentation)

    Science.gov (United States)

    Merkle, Conrad W.; Srinivasan, Vivek J.

    2016-03-01

    Transit time is a fundamental microcirculatory parameter that is critical in determining oxygen delivery from capillaries to surrounding tissue. Recently, it was demonstrated theoretically that capillary transit-time heterogeneity potentially leads to non-uniform oxygen extraction in micro-domains. However, in spite of its importance, capillary transit-time distribution has been challenging to quantify comprehensively and efficiently at the microscopic level. Here, we introduce a method, called Dynamic Contrast Optical Coherence Tomography (DyC-OCT), based on dynamic cross-sectional OCT imaging of the kinetics of an intravascular tracer during its passage through the field-of-view. DyC-OCT is used to quantitatively measure the transit-time distribution in microvascular networks in cross-section at the single-capillary level. Transit-time metrics are derived from analysis of the temporal characteristics of the dynamic scattering signal, related to tracer concentration, using indicator-dilution theory. Since DyC-OCT does not require calibration of the optical focus, quantitative accuracy is achieved even deep in highly scattering brain tissue where the focal spot degrades. After direct validation of DyC-OCT against the dilution curves measured using a fluorescent plasma label in the surface pial vessels of a mouse brain, imaged through a thinned-skull, glass coverslip-reinforced cranial window, the laminar transit-time distribution was investigated in microvasculature across the entire depth of the mouse somatosensory cortex. Laminar trends were identified, with the earliest transit times in the middle cortical layers, and the lowest heterogeneity in cortical layer 4. The new DyC-OCT technique affords a novel perspective of microvascular networks, with the unique capability of performing simultaneous measurements of transit-time distributions across cortical laminae.

  20. Spatial stochastic dynamics enable robust cell polarization.

    Directory of Open Access Journals (Sweden)

    Michael J Lawson

    Full Text Available Although cell polarity is an essential feature of living cells, it is far from being well-understood. Using a combination of computational modeling and biological experiments we closely examine an important prototype of cell polarity: the pheromone-induced formation of the yeast polarisome. Focusing on the role of noise and spatial heterogeneity, we develop and investigate two mechanistic spatial models of polarisome formation, one deterministic and the other stochastic, and compare the contrasting predictions of these two models against experimental phenotypes of wild-type and mutant cells. We find that the stochastic model can more robustly reproduce two fundamental characteristics observed in wild-type cells: a highly polarized phenotype via a mechanism that we refer to as spatial stochastic amplification, and the ability of the polarisome to track a moving pheromone input. Moreover, we find that only the stochastic model can simultaneously reproduce these characteristics of the wild-type phenotype and the multi-polarisome phenotype of a deletion mutant of the scaffolding protein Spa2. Significantly, our analysis also demonstrates that higher levels of stochastic noise results in increased robustness of polarization to parameter variation. Furthermore, our work suggests a novel role for a polarisome protein in the stabilization of actin cables. These findings elucidate the intricate role of spatial stochastic effects in cell polarity, giving support to a cellular model where noise and spatial heterogeneity combine to achieve robust biological function.

  1. Collective Calcium Dynamics in Networks of Communicating Cells

    Science.gov (United States)

    Byrd, Tommy; Potter, Garrett; Sun, Bo; Mugler, Andrew

    Cells can sense and encode information about their environment with remarkable precision. These properties have been studied extensively for single cells, but intercellular communication is known to be important for both single- and multicellular organisms. Here, we examine calcium dynamics of fibroblast cells exposed to external ATP stimuli, and the effects of communication and stimulus strength on cells' response. Experimental results show that increasing communication strength induces a greater fraction of cells to exhibit oscillatory calcium dynamics, but the frequencies of oscillation do not systematically shift with ATP strength. We developed a model of calcium signaling by adding noise, communication, and cell-to-cell variability to the model of Tang and Othmer. This model reproduces cells' increased tendency to oscillate as a function of communication strength, and frequency encoding is nearly removed at the global level. Our model therefore suggests that the propensity of cells to oscillate, rather than frequency encoding, determines the response to external ATP. These results suggest that the system lies near a critical boundary separating non-oscillatory and oscillatory calcium dynamics.

  2. Semi-quantitative model of the gating of KcsA ion channel. 2. Dynamic self-organization model of the gating

    Directory of Open Access Journals (Sweden)

    Boiteux C.

    2009-12-01

    Full Text Available The aim of this series of papers is to develop the semi-quantitative theory of the gating of KcsA channel. Methods. For this purpose available structural and electrophysiological data and the results of molecular dynamics simulations were used in the context of the concept of dynamical self-organization. In the second paper we describe the principles of dynamic self-organization and develop the theory of KcsA channel gating based on this concept. Conclusions. Present work is the first successful attempt of combining the structure and dynamics of real protein and the general concept of dynamic self-organization

  3. Multiscale modeling of bacterial colonies: how pili mediate the dynamics of single cells and cellular aggregates

    Science.gov (United States)

    Pönisch, Wolfram; Weber, Christoph A.; Juckeland, Guido; Biais, Nicolas; Zaburdaev, Vasily

    2017-01-01

    Neisseria gonorrhoeae is the causative agent of one of the most common sexually transmitted diseases, gonorrhea. Over the past two decades there has been an alarming increase of reported gonorrhea cases where the bacteria were resistant to the most commonly used antibiotics thus prompting for alternative antimicrobial treatment strategies. The crucial step in this and many other bacterial infections is the formation of microcolonies, agglomerates consisting of up to several thousands of cells. The attachment and motility of cells on solid substrates as well as the cell-cell interactions are primarily mediated by type IV pili, long polymeric filaments protruding from the surface of cells. While the crucial role of pili in the assembly of microcolonies has been well recognized, the exact mechanisms of how they govern the formation and dynamics of microcolonies are still poorly understood. Here, we present a computational model of individual cells with explicit pili dynamics, force generation and pili-pili interactions. We employ the model to study a wide range of biological processes, such as the motility of individual cells on a surface, the heterogeneous cell motility within the large cell aggregates, and the merging dynamics and the self-assembly of microcolonies. The results of numerical simulations highlight the central role of pili generated forces in the formation of bacterial colonies and are in agreement with the available experimental observations. The model can quantify the behavior of multicellular bacterial colonies on biologically relevant temporal and spatial scales and can be easily adjusted to include the geometry and pili characteristics of various bacterial species. Ultimately, the combination of the microbiological experimental approach with the in silico model of bacterial colonies might provide new qualitative and quantitative insights on the development of bacterial infections and thus pave the way to new antimicrobial treatments.

  4. Entrainment of cell division in phytoplankton with dynamic energy budgets

    Science.gov (United States)

    Muller, Erik B.; Ananthasubramaniam, Bharath; Klanjšček, Tin; Nisbet, Roger M.

    2011-11-01

    We explore the entrainment behavior of cell division in phytoplankton in the context of Dynamic Energy Budget (DEB) theory. In particular, we explore the range of DEB and environmental parameter values within which a cell divides at regular intervals in a periodic light environment with abundant nutrients and investigate the impact of parameter values on the phase of cell division. We consider three types of cells that differ in the evolution of surface area to volume ratio during the cell cycle: cells with a constant shape (isomorphs), cells with a constant surface area (V0-morphs) and cells with a constant surface area to volume ratio (V1-morphs), the latter being the default choice in studies on the population dynamics of unicellular organisms because of its desirable mathematical implications. Only in isomorphs and V0-morphs, however, cell division can be entrained to a periodic light. Regular cell division in V1 is purely coincidental, as it depends on exact choices for parameter values. We attribute this to the fact that V1-morphs lack the negative feedback of size on the dynamics of reserves in V0-morphs and isomorphs. Because entrained isomorphs and V0-morphs divide during the dark hours in our simulations, these two shapes can represent the division behavior of phytoplankton species that complete the cell cycle during the night, such as dinoflagellates and coccolithophores. A description of the division behavior of species completing the cell cycle during the day, such as silicon dependent diatoms and cyanobacteria, requires a more complex model than used in this paper. Furthermore, we explore the robustness of our findings by randomizing model parameters and introducing unevenness in biomass separation between daughter cells during cell division. We conclude that especially the entrainment in V0-morphs is relatively insensitive to perturbations.

  5. Quantitative proteome changes in Arabidopsis thaliana suspension-cultured cells in response to plant natriuretic peptides

    KAUST Repository

    Turek, Ilona

    2015-06-30

    Proteome changes in the Arabidopsis thaliana suspension cells in response to the A. thaliana plant natriuretic peptide (PNP), AtPNP-A (At2g18660) were assessed using quantitative proteomics employing tandem mass tag (TMT) labeling and tandem mass spectrometry (LC–MS/MS). In this study, we characterized temporal responses of suspension-cultured cells to 1 nM and 10 pM AtPNP-A at 0, 10 and 30 min post-treatment. Both concentrations we found to yield a distinct differential proteome signature. The data shown in this article are associated with the article “Plant natriuretic peptides induce a specific set of proteins diagnostic for an adaptive response to abiotic stress” by Turek et al. (Front. Plant Sci. 5 (2014) 661) and have been deposited to the ProteomeXchange with identifier PXD001386.

  6. Nanoparticle interactions with live cells: Quantitative fluorescence microscopy of nanoparticle size effects

    Directory of Open Access Journals (Sweden)

    Li Shang

    2014-12-01

    Full Text Available Engineered nanomaterials are known to enter human cells, often via active endocytosis. Mechanistic details of the interactions between nanoparticles (NPs with cells are still not well enough understood. NP size is a key parameter that controls the endocytic mechanism and affects the cellular uptake yield. Therefore, we have systematically analyzed the cellular uptake of fluorescent NPs in the size range of 3.3–100 nm (diameter by live cells. By using spinning disk confocal microscopy in combination with quantitative image analysis, we studied the time courses of NP association with the cell membrane and subsequent internalization. NPs with diameters of less than 10 nm were observed to accumulate at the plasma membrane before being internalized by the cells. In contrast, larger NPs (100 nm were directly internalized without prior accumulation at the plasma membrane, regardless of their surface charges. We attribute this distinct size dependence to the requirement of a sufficiently strong local interaction of the NPs with the endocytic machinery in order to trigger the subsequent internalization.

  7. Quantitative Analysis of Exosomes From Murine Lung Cancer Cells by Flow Cytometry

    Science.gov (United States)

    Rim, Kyung-Taek; Kim, Soo-Jin

    2016-01-01

    In vivo studies regarding biochemical, molecular biological, and histopathological changes in cancer tissues have been widely performed by the administration of carcinogens in rodents. In these established methods, dissection of the animal following sacrifice must be carried out. Exosomes are cell-derived vesicles that are present in all body fluids and these vesicles have specific roles within cells. Thus, much attention is given to the clinical application of exosomes that can possibly be used for prediction and therapy and as biomarkers related to cancer. To develop a new tool for monitoring in vivo genetic alterations, as a result of carcinogenesis, without the need for frequent euthanasia, we performed quantitative measurement of exosomes in Mlg2908 murine lung fibroblasts and LA-4 and KLN 205 murine lung cancer cells using fluorescence-activated cell sorting. We detected an increase in CD63-specific exosomes in LA-4 lung cancer cells. This result is able to be applied to the classification of cancer-specific proteins and miRNA as diagnostic markers. PMID:27722146

  8. A Caco-2 cell-based quantitative antioxidant activity assay for antioxidants.

    Science.gov (United States)

    Wan, Hongxia; Liu, Dong; Yu, Xiangying; Sun, Haiyan; Li, Yan

    2015-05-15

    A Caco-2 cell-based antioxidant activity (CAA) assay for quantitative evaluation of antioxidants was developed by optimizing seeding density and culture time of Caco-2 cells, incubation time and concentration of fluorescent probe (2',7'-dichlorofluorescin diacetate, DCFH-DA), incubation way and incubation time of antioxidants (pure phytochemicals) and DCFH-DA with cells, and detection time of fluorescence. Results showed that the CAA assay was of good reproducibility and could be used to evaluate the antioxidant activity of antioxidants at the following conditions: seeding density of 5 × 10(4)/well, cell culture time of 24h, co-incubation of 60 μM DCFH-DA and pure phytochemicals with Caco-2 cells for 20 min and fluorescence recorded for 90 min. Additionally, a significant correlation was observed between CAA values and rat plasma ORAC values following the intake of antioxidants for selected pure phytochemicals (R(2) = 0.815, p < 0.01), demonstrating the good biological relevance of CAA assay. Copyright © 2014 Elsevier Ltd. All rights reserved.

  9. Micro-PIXE for the quantitative imaging of chemical elements in single cells

    Energy Technology Data Exchange (ETDEWEB)

    Ortega, R. [Univ. Bordeaux, CENBG, Gradignan (France); CNRS, IN2P3, CENBG, Gradignan (France)

    2013-07-01

    Full text: The knowledge of the intracellular distribution of biological relevant metals is important to understand their mechanisms of action in cells, either for physiological, toxicological or pathological processes. However, the direct detection of trace metals in single cells is a challenging task that requires sophisticated analytical developments. The aim of this seminar will be to present the recent achievements in this field using micro-PIXE analysis. The combination of micro-PIXE with RBS (Rutherford Backscattering Spectrometry) and STIM (Scanning Transmission lon Microscopy) allows the quantitative determination of trace metal content within sub-cellular compartments. The application of STlM analysis will be more specifically highlighted as it provides high spatial resolution imaging (<200 nm) and excellent mass sensitivity (<0.1 ng). Application of the STIM-PIXE-RBS methodology is absolutely needed when organic mass loss appears during PIXE-RBS irradiation. This combination of STIM-PIXE-RBS provides fully quantitative determination of trace element content, expressed in μg/g, which is a quite unique capability for micro-PIXE compared to other micro-analytical methods such as the electron and synchrotron X-ray fluorescence or the techniques based on mass spectrometry. Examples of micro-PIXE studies for subcellular imaging of trace elements in the various fields of interest will be presented such as metal-based toxicology, pharmacology, and neuro degeneration [1] R. Ortega, G. Devés, A. Carmona. J. R. Soc. Interface, 6, (2009) S649-S658. (author)

  10. Quantitative Evaluation of Recombinant Protein Packaged into Outer Membrane Vesicles of Escherichia coli Cells.

    Science.gov (United States)

    Ojima, Yoshihiro; Yamaguchi, Kyota; Taya, Masahito

    2017-08-08

    Outer membrane vesicles (OMVs) are spherical bilayered proteolipids released from the cell surfaces of bacteria, which have gained traction in the biotechnology fields. Bacterial cellular machinery can be genetically engineered to produce and package heterologous enzymes into OMVs, producing nanocarriers and nanoparticle catalysts. However, the productivity or efficiency of packaging the target protein into OMVs has not been quantitatively evaluated. In this study, we packaged green fluorescence protein (GFP) into the OMVs of Escherichia coli through N-terminal fused expression to outer membrane protein W (OmpW). The OMV productivity and amount of OmpW-GFP packaged in the OMVs were quantitatively compared between two hypervesiculating mutant strains ΔnlpI and ΔdegP. Both strains increased the OMV production, but the ΔnlpI strain additionally enhanced the packaging of OmpW-GFP into OMVs. It was further confirmed that Spr, a peptidoglycan endopeptidase, plays an important role in the enhanced packaging of OmpW-GFP into OMVs through the increased OmpW-GFP expression on the ΔnlpI cells. Finally, the amount of OmpW-GFP released in the OMV fraction of both mutants was determined in terms of the OMV productivity and the packaging efficiency of OmpW-GFP into OMVs. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017. © 2017 American Institute of Chemical Engineers.

  11. Quantitative ultrasound characterization of tumor cell death: ultrasound-stimulated microbubbles for radiation enhancement.

    Directory of Open Access Journals (Sweden)

    Hyunjung Christina Kim

    Full Text Available The aim of this study was to assess the efficacy of quantitative ultrasound imaging in characterizing cancer cell death caused by enhanced radiation treatments. This investigation focused on developing this ultrasound modality as an imaging-based non-invasive method that can be used to monitor therapeutic ultrasound and radiation effects. High-frequency (25 MHz ultrasound was used to image tumor responses caused by ultrasound-stimulated microbubbles in combination with radiation. Human prostate xenografts grown in severe combined immunodeficiency (SCID mice were treated using 8, 80, or 1000 µL/kg of microbubbles stimulated with ultrasound at 250, 570, or 750 kPa, and exposed to 0, 2, or 8 Gy of radiation. Tumors were imaged prior to treatment and 24 hours after treatment. Spectral analysis of images acquired from treated tumors revealed overall increases in ultrasound backscatter intensity and the spectral intercept parameter. The increase in backscatter intensity compared to the control ranged from 1.9±1.6 dB for the clinical imaging dose of microbubbles (8 µL/kg, 250 kPa, 2 Gy to 7.0±4.1 dB for the most extreme treatment condition (1000 µL/kg, 750 kPa, 8 Gy. In parallel, in situ end-labelling (ISEL staining, ceramide, and cyclophilin A staining demonstrated increases in cell death due to DNA fragmentation, ceramide-mediated apoptosis, and release of cyclophilin A as a result of cell membrane permeabilization, respectively. Quantitative ultrasound results indicated changes that paralleled increases in cell death observed from histology analyses supporting its use for non-invasive monitoring of cancer treatment outcomes.

  12. Quantitative analysis of proteome and lipidome dynamics reveals functional regulation of global lipid metabolism

    DEFF Research Database (Denmark)

    Casanovas, Albert; Sprenger, Richard R; Tarasov, Kirill

    2015-01-01

    architecture and processes during physiological adaptations in yeast. Our results reveal that activation of cardiolipin synthesis and remodeling supports mitochondrial biogenesis in the transition from fermentative to respiratory metabolism, that down-regulation of de novo sterol synthesis machinery prompts......Elucidating how and to what extent lipid metabolism is remodeled under changing conditions is essential for understanding cellular physiology. Here, we analyzed proteome and lipidome dynamics to investigate how regulation of lipid metabolism at the global scale supports remodeling of cellular...

  13. Dynamics of Vibrio cholerae abundance in Austrian saline lakes, assessed with quantitative solid-phase cytometry

    OpenAIRE

    2015-01-01

    International audience; In order to elucidate the main predictors of Vibrio cholerae dynamics and to estimate the risk of Vibrio cholera-related diseases, a recently developed direct detection approach based on fluorescence in situ hybridization and solid-phase cytometry (CARD-FISH/ SPC) was applied in comparison to cultivation for water samples from the lake Neusiedler See, Austria and three shallow alkaline lakes over a period of 20 months. Vibrio cholerae attached to crustacean zoo-plankto...

  14. Heteronuclear Adiabatic Relaxation Dispersion (HARD) for quantitative analysis of conformational dynamics in proteins.

    Science.gov (United States)

    Traaseth, Nathaniel J; Chao, Fa-An; Masterson, Larry R; Mangia, Silvia; Garwood, Michael; Michaeli, Shalom; Seelig, Burckhard; Veglia, Gianluigi

    2012-06-01

    NMR relaxation methods probe biomolecular motions over a wide range of timescales. In particular, the rotating frame spin-lock R(1ρ) and Carr-Purcell-Meiboom-Gill (CPMG) R(2) experiments are commonly used to characterize μs to ms dynamics, which play a critical role in enzyme folding and catalysis. In an effort to complement these approaches, we introduced the Heteronuclear Adiabatic Relaxation Dispersion (HARD) method, where dispersion in rotating frame relaxation rate constants (longitudinal R(1ρ) and transverse R(2ρ)) is created by modulating the shape and duration of adiabatic full passage (AFP) pulses. Previously, we showed the ability of the HARD method to detect chemical exchange dynamics in the fast exchange regime (k(ex)∼10(4)-10(5) s(-1)). In this article, we show the sensitivity of the HARD method to slower exchange processes by measuring R(1ρ) and R(2ρ) relaxation rates for two soluble proteins (ubiquitin and 10C RNA ligase). One advantage of the HARD method is its nominal dependence on the applied radio frequency field, which can be leveraged to modulate the dispersion in the relaxation rate constants. In addition, we also include product operator simulations to define the dynamic range of adiabatic R(1ρ) and R(2ρ) that is valid under all exchange regimes. We conclude from both experimental observations and simulations that this method is complementary to CPMG-based and rotating frame spin-lock R(1ρ) experiments to probe conformational exchange dynamics for biomolecules. Finally, this approach is germane to several NMR-active nuclei, where relaxation rates are frequency-offset independent. Copyright © 2012 Elsevier Inc. All rights reserved.

  15. Quantitative analysis of the angular dynamics of a single spheroid in simple shear flow at moderate Reynolds numbers

    Science.gov (United States)

    Rosén, Tomas; Nordmark, Arne; Aidun, Cyrus K.; Do-Quang, Minh; Lundell, Fredrik

    2016-08-01

    A spheroidal particle in simple shear flow shows surprisingly complicated angular dynamics; caused by effects of fluid inertia (characterized by the particle Reynolds number Rep) and particle inertia (characterized by the Stokes number St). Understanding this behavior can provide important fundamental knowledge of suspension flows with spheroidal particles. Up to now only qualitative analysis has been available at moderate Rep. Rigorous analytical methods apply only to very small Rep and numerical results lack accuracy due to the difficulty in treating the moving boundary of the particle. Here we show that the dynamics of the rotational motion of a prolate spheroidal particle in a linear shear flow can be quantitatively analyzed through the eigenvalues of the log-rolling particle (particle aligned with vorticity). This analysis provides an accurate description of stable rotational states in terms of Rep,St, and particle aspect ratio (rp). Furthermore we find that the effect on the orientational dynamics from fluid inertia can be modeled with a Duffing-Van der Pol oscillator. This opens up the possibility of developing a reduced-order model that takes into account effects from both fluid and particle inertia.

  16. Comparison of Myocardial Perfusion Estimates From Dynamic Contrast-Enhanced Magnetic Resonance Imaging With Four Quantitative Analysis Methods

    Science.gov (United States)

    Pack, Nathan A.; DiBella, Edward V. R.

    2012-01-01

    Dynamic contrast-enhanced MRI has been used to quantify myocardial perfusion in recent years. Published results have varied widely, possibly depending on the method used to analyze the dynamic perfusion data. Here, four quantitative analysis methods (two-compartment modeling, Fermi function modeling, model-independent analysis, and Patlak plot analysis) were implemented and compared for quantifying myocardial perfusion. Dynamic contrast-enhanced MRI data were acquired in 20 human subjects at rest with low-dose (0.019 ± 0.005 mmol/kg) bolus injections of gadolinium. Fourteen of these subjects were also imaged at adenosine stress (0.021 ± 0.005 mmol/kg). Aggregate rest perfusion estimates were not significantly different between all four analysis methods. At stress, perfusion estimates were not significantly different between two-compartment modeling, model-independent analysis, and Patlak plot analysis. Stress estimates from the Fermi model were significantly higher (~20%) than the other three methods. Myocardial perfusion reserve values were not significantly different between all four methods. Model-independent analysis resulted in the lowest model curve-fit errors. When more than just the first pass of data was analyzed, perfusion estimates from two-compartment modeling and model-independent analysis did not change significantly, unlike results from Fermi function modeling. PMID:20577976

  17. CSF dynamics in the patients with syringomyelia associated with Chiari's malformation; Quantitative analysis on cine MRI

    Energy Technology Data Exchange (ETDEWEB)

    Kuroda, Satoshi; Matsuzawa, Hitoshi; Iwasaki, Yoshinobu; Hida, Kazutoshi; Imamura, Hiroyuki; Abe, Hiroshi (Hokkaido Univ., Sapporo (Japan). School of Medicine); Saito, Hisatoshi

    1994-01-01

    In a series of 12 patients with syringomyelia associated with Chiari's malformation, the authors quantitatively analyzed cerebrospinal fluid (CSF) dynamics in the subarachnoid space of the cranio-spinal junction, using cine MRI combined with pre-saturation method. In most of subjects, cine MRI revealed (1) decreased or increased maximum velocity of CSF in the caudal direction and (2) disturbed CSF motion in the caudal direction (delayed % cardiac cycle) in the craniospinal junction, strongly suggesting disturbed CSF dynamics in the craniospinal junction because of the tonsilar herniation. Of 12 subjects, 8 patients underwent formen magnum decompression and 4 underwent syringo-subarachnoid shunt (SS shunt). In the patients who showed marked callapse of syrinx after foramen magnum decompression, follow-up cine MRI revealed the normalization of % cardiac cycle, representing postoperative improvement of CSF dynamics in the craniospinal function. On the other hand, % cardiac cycle did not improve significantly in the patients who did not show marked collapse of the syrinx or suffered from meningitis after surgery. Significant changes were not observed in the patients who underwent SS shunt. In summary, these results suggested that cine MRI combined with pre-saturation method could detect the pathophysiological changes and evaluate the efficacy of the surgery, especially foramen magnum decompression, in the patients with syringomyelia associated with Chiari's malformation. (author).

  18. Dynamic modulation of thymidylate synthase gene expression and fluorouracil sensitivity in human colorectal cancer cells.

    Directory of Open Access Journals (Sweden)

    Kentaro Wakasa

    Full Text Available Biomarkers have revolutionized cancer chemotherapy. However, many biomarker candidates are still in debate. In addition to clinical studies, a priori experimental approaches are needed. Thymidylate synthase (TS expression is a long-standing candidate as a biomarker for 5-fluorouracil (5-FU treatment of cancer patients. Using the Tet-OFF system and a human colorectal cancer cell line, DLD-1, we first constructed an in vitro system in which TS expression is dynamically controllable. Quantitative assays have elucidated that TS expression in the transformant was widely modulated, and that the dynamic range covered 15-fold of the basal level. 5-FU sensitivity of the transformant cells significantly increased in response to downregulated TS expression, although being not examined in the full dynamic range because of the doxycycline toxicity. Intriguingly, our in vitro data suggest that there is a linear relationship between TS expression and the 5-FU sensitivity in cells. Data obtained in a mouse model using transformant xenografts were highly parallel to those obtained in vitro. Thus, our in vitro and in vivo observations suggest that TS expression is a determinant of 5-FU sensitivity in cells, at least in this specific genetic background, and, therefore, support the possibility of TS expression as a biomarker for 5-FU-based cancer chemotherapy.

  19. Ultra-deep and quantitative saliva proteome reveals dynamics of the oral microbiome

    DEFF Research Database (Denmark)

    Grassl, Niklas; Kulak, Nils Alexander; Pichler, Garwin

    2016-01-01

    , disruptions in saliva secretion and changes in the oral microbiome contribute to conditions such as tooth decay and respiratory tract infections. Here we set out to quantitatively map the saliva proteome in great depth with a rapid and in-depth mass spectrometry-based proteomics workflow. METHODS: We used...... with next-generation sequencing data from the Human Microbiome Project as well as a comparison to MALDI-TOF mass spectrometry on microbial cultures revealed strong agreement. The oral microbiome differs between individuals and changes drastically upon eating and tooth brushing. CONCLUSION: Rapid shotgun...... and robust technology can now simultaneously characterize the human and microbiome contributions to the proteome of a body fluid and is therefore a valuable complement to genomic studies. This opens new frontiers for the study of host-pathogen interactions and clinical saliva diagnostics....

  20. An Integrated Method of Multiradar Quantitative Precipitation Estimation Based on Cloud Classification and Dynamic Error Analysis

    Directory of Open Access Journals (Sweden)

    Yong Huang

    2017-01-01

    Full Text Available Relationships between radar reflectivity factor and rainfall are different in various precipitation cloud systems. In this study, the cloud systems are firstly classified into five categories with radar and satellite data to improve radar quantitative precipitation estimation (QPE algorithm. Secondly, the errors of multiradar QPE algorithms are assumed to be different in convective and stratiform clouds. The QPE data are then derived with methods of Z-R, Kalman filter (KF, optimum interpolation (OI, Kalman filter plus optimum interpolation (KFOI, and average calibration (AC based on error analysis on the Huaihe River Basin. In the case of flood on the early of July 2007, the KFOI is applied to obtain the QPE product. Applications show that the KFOI can improve precision of estimating precipitation for multiple precipitation types.

  1. Dynamic monitoring of changes in endothelial cell-substrate adhesiveness during leukocyte adhesion by microelectrical impedance assay

    Institute of Scientific and Technical Information of China (English)

    Yakun Ge; Tongle Deng; Xiaoxiang Zheng

    2009-01-01

    Adhesion of leukocytes to endothelial cells in inflammation processes leads to changes of endothelial cell-substrate adhesiveness, and understanding of such changes will provide us with important information of inflammation processes. In this study, we used a non-invasive biosensor system referred to as real-time cell electronic sensor (RT-CES) system to monitor the changes in endothelial cell-substrate adhesiveness induced by human monoblastic cell line U937 cell adhesion in a dynamic and quantitative manner. This assay, which is based on cell-substrate impedance readout, is able to monitor transient changes in cell-substrate adhesiveness as a result of U937 cell adhesion. The U937 cell adhesion to endothelial cells was induced by lipopolysaccharide (LPS) in a dose-dependent manner. Although the number of adherent U937 cells to the endothelial cells was verified by a standard assay, the adhesiveness of endothelial cells after addition of U937 cells was monitored by the RT-CES system. Furthermore, focal adhesion kinase protein decrease and F-actin rearrangement in endothelial cells were observed after addition of U937 cells. Our results indicated that the adhesion of U937 cells to LPS-treated endothelial cells reduced the cell adhesiveness to the substrate, and such reduction might facilitate infiltration of leukocytes.

  2. Real time imaging of mRNA expression dynamics in live cells using protein complementation methods

    Science.gov (United States)

    Meller, Amit

    2009-03-01

    Traditional methods for mRNA quantification in cells, such as northern blots, quantitative PCR or microarrays assays, require cell lysis and therefore do not preserve its dynamics. These methods cannot be used to probe the spatio-temporal localization of mRNA in cells, which provide useful information for a wide range biomolecular process, including RNA metabolizim, expression kinetics and RNA interference. To probe mRNA dynamics in live prokaryotic and eukaryotic cells, we develop a method, which exploit the strong affinity of the eukaryotic initiation factor 4A (eIF4A) to specific RNA aptamers. Two parts of the eIF4A are fused to a split Green Fluorescence Protein (GFP), and are expressed in the cells at high abundance. However, only when the RNA apatmer is also present, the two protein parts complement and become fluorescent. Thus, the fluorescent background remains low, allowing us to directly image the expression of mRNA molecules in live e-coli cells from its early onset, over hours. We find that the expression kinetics can be classified in one out of at least three forms, which also display distinct spatial distributions. I will discuss the possible biological origin for these distributions and their time evolution.

  3. Inverse problem of HIV cell dynamics using Genetic Algorithms

    Science.gov (United States)

    González, J. A.; Guzmán, F. S.

    2017-01-01

    In order to describe the cell dynamics of T-cells in a patient infected with HIV, we use a flavour of Perelson's model. This is a non-linear system of Ordinary Differential Equations that describes the evolution of healthy, latently infected, infected T-cell concentrations and the free viral cells. Different parameters in the equations give different dynamics. Considering the concentration of these types of cells is known for a particular patient, the inverse problem consists in estimating the parameters in the model. We solve this inverse problem using a Genetic Algorithm (GA) that minimizes the error between the solutions of the model and the data from the patient. These errors depend on the parameters of the GA, like mutation rate and population, although a detailed analysis of this dependence will be described elsewhere.

  4. Experimental validation of the AVIVET trap, a tool to quantitatively monitor the dynamics of Dermanyssus gallinae populations in laying hens.

    Science.gov (United States)

    Lammers, G A; Bronneberg, R G G; Vernooij, J C M; Stegeman, J A

    2016-12-05

    Dermanyssus gallinae (D.gallinae) infestation causes economic losses due to impaired health and production of hens and costs of parasite control across the world. Moreover, infestations are associated with reduced welfare of hens and may cause itching in humans. To effectively implement control methods it is crucially important to have high quality information about the D.gallinae populations in poultry houses in space and time. At present no validated tool is available to quantitatively monitor the dynamics of all four stages of D.gallinae (i.e., eggs, larvae, nymphs, and adults) in poultry houses.This article describes the experimental validation of the AVIVET trap, a device to quantitatively monitor dynamics of D.gallinae infestations. We used the device to study D.gallinae in fully equipped cages with two white specific pathogen free Leghorn laying hens experimentally exposed to three different infestation levels of D.gallinae (low to high).The AVIVET trap was successfully able to detect D.gallinae at high (5,000 D.gallinae), medium (2,500 D.gallinae), and low (50 D.gallinae) level of D.gallinae infestation. The linear equation Y = 10(∧)10(∧)(0.47 + 1.21X) with Y = log10 (Total number of D.gallinae nymphs and adults) in the cage and X = log10 (Total number of D.gallinae nymphs and adults) in the AVIVET trap explained 93.8% of the variation.The weight of D.gallinae in the AVIVET trap also appears to be a reliable parameter for quantifying D.gallinae infestation in a poultry house. The weight of D.gallinae in the AVIVET trap correlates 99.6% (P gallinae in the trap (i.e., eggs, larvae, nymphs, and adults) indicating that the trap is highly specific.From this experiment it can be concluded that the AVIVET trap is promising as quantitative tool for monitoring D.gallinae dynamics in a poultry house.

  5. Quantitative PCR for HTLV-1 provirus in adult T-cell leukemia/lymphoma using paraffin tumor sections.

    Science.gov (United States)

    Kato, Junki; Masaki, Ayako; Fujii, Keiichiro; Takino, Hisashi; Murase, Takayuki; Yonekura, Kentaro; Utsunomiya, Atae; Ishida, Takashi; Iida, Shinsuke; Inagaki, Hiroshi

    2016-11-01

    Detection of HTLV-1 provirus using paraffin tumor sections may assist the diagnosis of adult T-cell leukemia/lymphoma (ATLL). For the detection, non-quantitative PCR assay has been reported, but its usefulness and limitations remain unclear. To our knowledge, quantitative PCR assay using paraffin tumor sections has not been reported. Using paraffin sections from ATLLs and non-ATLL T-cell lymphomas, we first performed non-quantitative PCR for HTLV-1 provirus. Next, we determined tumor ratios and carried out quantitative PCR to obtain provirus copy numbers. The results were analyzed with a simple regression model and a novel criterion, cut-off using 95 % rejection limits. Our quantitative PCR assay showed an excellent association between tumor ratios and the copy numbers (r = 0.89, P quantitative PCR assay should be interpreted very carefully and that our quantitative PCR assay is useful to estimate the status of HTLV-1 involvement in the tumor cases. In conclusion, our quantitative PCR assay using paraffin tumor sections may be useful for the screening of ATLL cases, especially in HTLV-1 non-endemic areas where easy access to serological testing for HTLV-1 infection is limited. © 2016 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd.

  6. Quantitative evaluation of CART-containing cells in urinary bladder of rats with renovascular hypertension.

    Science.gov (United States)

    Janiuk, I; Kasacka, I

    2015-04-13

    Recent biological advances make it possible to discover new peptides associated with hypertension. The cocaine- and amphetamine-regulated transcript (CART) is a known factor in appetite and feeding behaviour. Various lines of evidence suggest that this peptide participates not only in control of feeding behaviour but also in the regulation of the cardiovascular and sympathetic systems and blood pressure. The role of CART in blood pressure regulation led us to undertake a study aimed at analysing quantitative changes in CART-containing cells in urinary bladders (UB) of rats with renovascular hypertension. We used the Goldblatt model of arterial hypertension (two-kidney, one clip) to evaluate quantitative changes. This model provides researchers with a commonly used tool to analyse the renin-angiotensin system of blood pressure control and, eventually, to develop drugs for the treatment of chronic hypertension. The study was performed on sections of urinary bladders of rats after 3-, 14-, 28-, 42 and 91 days from hypertension induction. Immunohistochemical identification of CART cells was performed on paraffin for the UBs of all the study animals. CART was detected in the endocrine cells, especially numerous in the submucosa and muscularis layers, with a few found in the transitional epithelium and only occasionally in serosa. Hypertension significantly increased the number of CART-positive cells in the rat UBs. After 3 and 42 days following the procedure, statistically significantly higher numbers of CART-positive cells were identified in comparison with the control animals. The differences between the hypertensive rats and the control animals concerned not only the number density of CART-immunoreactive cells but also their localization. After a 6-week period, each of the rats subjected to the renal artery clipping procedure developed stable hypertension. CART appeared in numerous transitional epithelium cells. As this study provides novel findings, the question

  7. Quantitative Evaluation of CART-Containing Cells in Urinary Bladder of Rats with Renovascular Hypertension

    Science.gov (United States)

    Janiuk, I.; Kasacka, I.

    2015-01-01

    Recent biological advances make it possible to discover new peptides associated with hypertension. The cocaine- and amphetamine-regulated transcript (CART) is a known factor in appetite and feeding behaviour. Various lines of evidence suggest that this peptide participates not only in control of feeding behaviour but also in the regulation of the cardiovascular and sympathetic systems and blood pressure. The role of CART in blood pressure regulation led us to undertake a study aimed at analysing quantitative changes in CART-containing cells in urinary bladders (UB) of rats with renovascular hypertension. We used the Goldblatt model of arterial hypertension (two-kidney, one clip) to evaluate quantitative changes. This model provides researchers with a commonly used tool to analyse the renin-angiotensin system of blood pressure control and, eventually, to develop drugs for the treatment of chronic hypertension. The study was performed on sections of urinary bladders of rats after 3-, 14-, 28-, 42 and 91 days from hypertension induction. Immunohistochemical identification of CART cells was performed on paraffin for the UBs of all the study animals. CART was detected in the endocrine cells, especially numerous in the submucosa and muscularis layers, with a few found in the transitional epithelium and only occasionally in serosa. Hypertension significantly increased the number of CART-positive cells in the rat UBs. After 3 and 42 days following the procedure, statistically significantly higher numbers of CART-positive cells were identified in comparison with the control animals. The differences between the hypertensive rats and the control animals concerned not only the number density of CART-immunoreactive cells but also their localization. After a 6-week period, each of the rats subjected to the renal artery clipping procedure developed stable hypertension. CART appeared in numerous transitional epithelium cells. As this study provides novel findings, the question

  8. Quantitative evaluation of CART-containing cells in urinary bladder of rats with renovascular hypertension

    Directory of Open Access Journals (Sweden)

    I. Janiuk

    2015-04-01

    Full Text Available Recent biological advances make it possible to discover new peptides associated with hypertension. The cocaine- and amphetamine-regulated transcript (CART is a known factor in appetite and feeding behaviour. Various lines of evidence suggest that this peptide participates not only in control of feeding behaviour but also in the regulation of the cardiovascular and sympathetic systems and blood pressure. The role of CART in blood pressure regulation led us to undertake a study aimed at analysing quantitative changes in CART-containing cells in urinary bladders (UB of rats with renovascular hypertension. We used the Goldblatt model of arterial hypertension (two-kidney, one clip to evaluate quantitative changes. This model provides researchers with a commonly used tool to analyse the renin-angiotensin system of blood pressure control and, eventually, to develop drugs for the treatment of chronic hypertension. The study was performed on sections of urinary bladders of rats after 3-, 14-, 28-, 42 and 91 days from hypertension induction. Immunohistochemical identification of CART cells was performed on paraffin for the UBs of all the study animals. CART was detected in the endocrine cells, especially numerous in the submucosa and muscularis layers, with a few found in the transitional epithelium and only occasionally in serosa. Hypertension significantly increased the number of CART-positive cells in the rat UBs. After 3 and 42 days following the procedure, statistically significantly higher numbers of CART-positive cells were identified in comparison with the control animals. The differences between the hypertensive rats and the control animals concerned not only the number density of CART-immunoreactive cells but also their localization. After a 6-week period, each of the rats subjected to the renal artery clipping procedure developed stable hypertension. CART appeared in numerous transitional epithelium cells. As this study provides novel findings

  9. Issues associated with modelling of proton exchange membrane fuel cell by computational fluid dynamics

    Science.gov (United States)

    Bednarek, Tomasz; Tsotridis, Georgios

    2017-03-01

    The objective of the current study is to highlight possible limitations and difficulties associated with Computational Fluid Dynamics in PEM single fuel cell modelling. It is shown that an appropriate convergence methodology should be applied for steady-state solutions, due to inherent numerical instabilities. A single channel fuel cell model has been taken as numerical example. Results are evaluated for quantitative as well qualitative points of view. The contribution to the polarization curve of the different fuel cell components such as bi-polar plates, gas diffusion layers, catalyst layers and membrane was investigated via their effects on the overpotentials. Furthermore, the potential losses corresponding to reaction kinetics, due to ohmic and mas transport limitations and the effect of the exchange current density and open circuit voltage, were also investigated. It is highlighted that the lack of reliable and robust input data is one of the issues for obtaining accurate results.

  10. Dynamic Pax6 expression during the neurogenic cell cycle influences proliferation and cell fate choices of retinal progenitors

    Directory of Open Access Journals (Sweden)

    Yang Xian-Jie

    2009-08-01

    Full Text Available Abstract Background The paired homeobox protein Pax6 is essential for proliferation and pluripotency of retinal progenitors. However, temporal changes in Pax6 protein expression associated with the generation of various retinal neurons have not been characterized with regard to the cell cycle. Here, we examine the dynamic changes of Pax6 expression among chicken retinal progenitors as they progress through the neurogenic cell cycle, and determine the effects of altered Pax6 levels on retinogenesis. Results We provide evidence that during the preneurogenic to neurogenic transition, Pax6 protein levels in proliferating progenitor cells are down-regulated. Neurogenic retinal progenitors retain a relatively low level of Pax6 protein, whereas postmitotic neurons either elevate or extinguish Pax6 expression in a cell type-specific manner. Cell imaging and cell cycle analyses show that neurogenic progenitors in the S phase of the cell cycle contain low levels of Pax6 protein, whereas a subset of progenitors exhibits divergent levels of Pax6 protein upon entering the G2 phase of the cell cycle. We also show that M phase cells contain varied levels of Pax6, and some correlate with the onset of early neuronal marker expression, forecasting cell cycle exit and cell fate commitment. Furthermore, either elevating or knocking down Pax6 attenuates cell proliferation and results in increased cell death. Reducing Pax6 decreases retinal ganglion cell genesis and enhances cone photoreceptor and amacrine interneuron production, whereas elevating Pax6 suppresses cone photoreceptor and amacrine cell fates. Conclusion These studies demonstrate for the first time quantitative changes in Pax6 protein expression during the preneurogenic to neurogenic transition and during the neurogenic cell cycle. The results indicate that Pax6 protein levels are stringently controlled in proliferating progenitors. Maintaining a relatively low Pax6 protein level is necessary for S phase

  11. Quantitative mechanical assessment of the whole prostate gland ex vivo using dynamic instrumented palpation.

    Science.gov (United States)

    Hammer, Steven J; Good, Daniel W; Scanlan, Paul; Palacio-Torralba, Javier; Phipps, Simon; Stewart, Grant D; Shu, Will; Chen, Yuhang; McNeill, S Alan; Reuben, Robert L

    2017-09-01

    An instrumented palpation sensor, designed for measuring the dynamic modulus of tissue in vivo, has been developed and trialled on ex vivo whole prostate glands. The sensor consists of a flexible membrane sensor/actuator with an embedded strain gauge and is actuated using a dynamically varying airflow at frequencies of 1 and 5 Hz. The device was calibrated using an indentation stiffness measurement rig and gelatine samples with a range of static modulus similar to that reported in the literature for prostate tissue. The glands were removed from patients with diagnosed prostate cancer scheduled for radical prostatectomy, and the stiffness was measured within 30 min of surgical removal. Each prostate was later examined histologically in a column immediately below each indentation point and graded into one of the four groups; normal, benign prostatic hyperplasia, cancerous and mixed cancer and benign prostatic hyperplasia. In total, 11 prostates were assessed using multiple point probing, and the complex modulus at 1 and 5 Hz was calculated on a point-by-point basis. The device yielded values of quasi-static modulus of 15 ± 0.5 kPa and dynamic modulus of 20 ± 0.5 kPa for whole prostates, and a sensitivity of up to 80% with slightly lower specificity was achieved on diagnosis of prostate cancer using a combination of mechanical measures. This assessment did not take into account some obvious factors such as edge effects, overlap and clinical significance of the cancer, all of which would improve performance. The device, as currently configured, is immediately deployable in vivo. A number of improvements are also identified which could improve the sensitivity and specificity in future embodiments of the probe.

  12. Multiplex Quantitative Histologic Analysis of Human Breast Cancer Cell Signaling and Cell Fate

    Science.gov (United States)

    2010-05-01

    P. Lewis , B. S. Manjunath, and S. K. Fisher, “Automated tool for the detection of cell nuclei in digital microscopic images: Application to retinal...set model and some applications to Mumford -Shah image segmentation,” IEEE Trans. Image Process., vol. 15, no. 5, pp. 1171–1181, May 2006. [37] N. R

  13. Quantitative approaches in developmental biology.

    Science.gov (United States)

    Oates, Andrew C; Gorfinkiel, Nicole; González-Gaitán, Marcos; Heisenberg, Carl-Philipp

    2009-08-01

    The tissues of a developing embryo are simultaneously patterned, moved and differentiated according to an exchange of information between their constituent cells. We argue that these complex self-organizing phenomena can only be fully understood with quantitative mathematical frameworks that allow specific hypotheses to be formulated and tested. The quantitative and dynamic imaging of growing embryos at the molecular, cellular and tissue level is the key experimental advance required to achieve this interaction between theory and experiment. Here we describe how mathematical modelling has become an invaluable method to integrate quantitative biological information across temporal and spatial scales, serving to connect the activity of regulatory molecules with the morphological development of organisms.

  14. Identification of Cell Cycle Dependent Interaction Partners of the Septins by Quantitative Mass Spectrometry.

    Directory of Open Access Journals (Sweden)

    Christian Renz

    Full Text Available The septins are a conserved family of GTP-binding proteins that, in the baker's yeast, assemble into a highly ordered array of filaments at the mother bud neck. These filaments undergo significant structural rearrangements during the cell cycle. We aimed at identifying key components that are involved in or regulate the transitions of the septins. By combining cell synchronization and quantitative affinity-purification mass-spectrometry, we performed a screen for specific interaction partners of the septins at three distinct stages of the cell cycle. A total of 83 interaction partners of the septins were assigned. Surprisingly, we detected DNA-interacting/nuclear proteins and proteins involved in ribosome biogenesis and protein synthesis predominantly present in alpha-factor arrested that do not display an assembled septin structure. Furthermore, two distinct sets of regulatory proteins that are specific for cells at S-phase with a stable septin collar or at mitosis with split septin rings were identified. Complementary methods like SPLIFF and immunoprecipitation allowed us to more exactly define the spatial and temporal characteristics of selected hits of the AP-MS screen.

  15. Quantitative kinetics analysis of BMP2 uptake into cells and its modulation by BMP antagonists.

    Science.gov (United States)

    Alborzinia, Hamed; Schmidt-Glenewinkel, Hannah; Ilkavets, Iryna; Breitkopf-Heinlein, Katja; Cheng, Xinlai; Hortschansky, Peter; Dooley, Steven; Wölfl, Stefan

    2013-01-01

    Bone morphogenetic proteins (BMPs) are members of the TGFβ family of signaling proteins and play an important role during development and in tissue formation. BMP signaling is a well-studied process, which is initiated through binding of cognate receptors and processed through activation of Smad downstream mediators. A hallmark of BMP signaling is its modulation at the extracellular level through specific antagonists. Although it had been shown that BMP and TGFβ receptors are internalized following activation, little is known about the fate of BMP ligands. We prepared biologically active fluorescently labeled BMP2 and quantitatively analyzed its binding and uptake in cells using flow cytometry and confocal microscopy. Exogenous BMP2 was rapidly bound to the cell surface and subsequently internalized in a time-dependent manner and accumulated in the cell center. Although binding to the cell surface was limited by binding sites at the beginning, internalization continously increased with time, after a short delay. Using different inhibitors we found that internalization of BMP2 through endosomal particles occurred in a clathrin-dependent pathway. Furthermore, uptake of BMP2 was modulated in strikingly different ways by BMP2 antagonists. Although Noggin and Gremlin increased BMP2 uptake, Chordin blocked BMP2 uptake, which was concentration dependent in both cases. In conclusion, our findings present interesting mechanisms for the modulation of BMP signaling by concentration gradients of BMP ligands and antagonists in a dose- and time-dependent manner, which can provide an explanation of some properties of the BMP regulatory network.

  16. Quantitative Imaging of Cell-Permeable Magnetic Resonance Contrast Agents Using X-Ray Fluorescence

    Directory of Open Access Journals (Sweden)

    Paul J. Endres

    2006-10-01

    Full Text Available The inability to transduce cellular membranes is a limitation of current magnetic resonance imaging probes used in biologic and clinical settings. This constraint confines contrast agents to extracellular and vascular regions of the body, drastically reducing their viability for investigating processes and cycles in developmental biology. Conversely, a contrast agent with the ability to permeate cell membranes could be used in visualizing cell patterning, cell fate mapping, gene therapy, and, eventually, noninvasive cancer diagnosis. Therefore, we describe the synthesis and quantitative imaging of four contrast agents with the capability to cross cell membranes in sufficient quantity for detection. Each agent is based on the conjugation of a Gd(III chelator with a cellular transduction moiety. Specifically, we coupled Gd(III–diethylenetriaminepentaacetic acid DTPA and Gd(III–1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid with an 8–amino acid polyarginine oligomer and an amphipathic stilbene molecule, 4-amino-4'-(N,N-dimethylaminostilbene. The imaging modality that provided the best sensitivity and spatial resolution for direct detection of the contrast agents is synchrotron radiation x-ray fluorescence (SR-XRF. Unlike optical microscopy, SR-XRF provides two-dimensional images with resolution 103 better than 153Gd gamma counting, without altering the agent by organic fluorophore conjugation. The transduction efficiency of the intracellular agents was evaluated by T1 analysis and inductively coupled plasma mass spectrometry to determine the efficacy of each chelate-transporter combination.

  17. Quantitative proteomics of Spodoptera frugiperda cells during growth and baculovirus infection.

    Directory of Open Access Journals (Sweden)

    Nuno Carinhas

    Full Text Available Baculovirus infection of Spodoptera frugiperda cells is a system of choice to produce a range of recombinant proteins, vaccines and, potentially, gene therapy vectors. While baculovirus genomes are well characterized, the genome of S. frugiperda is not sequenced and the virus-host molecular interplay is sparsely known. Herein, we describe the application of stable isotope labeling by amino acids in cell culture (SILAC to obtain the first comparative proteome quantitation of S. frugiperda cells during growth and early baculovirus infection. The proteome coverage was maximized by compiling a search database with protein annotations from insect species. Of interest were differentially proteins related to energy metabolism, endoplasmic reticulum and oxidative stress, yet not investigated in the scope of baculovirus infection. Further, the reduced expression of key viral-encoded proteins early in the infection cycle is suggested to be related with decreased viral replication at high cell density culture. These findings have implications for virological research and improvement of baculovirus-based bioprocesses.

  18. Quantitation of malaria parasite-erythrocyte cell-cell interactions using optical tweezers.

    Science.gov (United States)

    Crick, Alex J; Theron, Michel; Tiffert, Teresa; Lew, Virgilio L; Cicuta, Pietro; Rayner, Julian C

    2014-08-19

    Erythrocyte invasion by Plasmodium falciparum merozoites is an essential step for parasite survival and hence the pathogenesis of malaria. Invasion has been studied intensively, but our cellular understanding has been limited by the fact that it occurs very rapidly: invasion is generally complete within 1 min, and shortly thereafter the merozoites, at least in in vitro culture, lose their invasive capacity. The rapid nature of the process, and hence the narrow time window in which measurements can be taken, have limited the tools available to quantitate invasion. Here we employ optical tweezers to study individual invasion events for what we believe is the first time, showing that newly released P. falciparum merozoites, delivered via optical tweezers to a target erythrocyte, retain their ability to invade. Even spent merozoites, which had lost the ability to invade, retain the ability to adhere to erythrocytes, and furthermore can still induce transient local membrane deformations in the erythrocyte membrane. We use this technology to measure the strength of the adhesive force between merozoites and erythrocytes, and to probe the cellular mode of action of known invasion inhibitory treatments. These data add to our understanding of the erythrocyte-merozoite interactions that occur during invasion, and demonstrate the power of optical tweezers technologies in unraveling the blood-stage biology of malaria. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  19. Long term charge retention dynamics of SONOS cells

    Science.gov (United States)

    Arreghini, A.; Akil, N.; Driussi, F.; Esseni, D.; Selmi, L.; van Duuren, M. J.

    2008-09-01

    We present a model for charge retention dynamics in SONOS non volatile memory cells which accounts for the space and energy distributions of the trapped charge in the silicon nitride, self consistently with the potential. Long term retention measurements (beyond 106 s) versus temperature allowed us to decouple two charge loss mechanisms, to calibrate the model parameters and then to reproduce a large set of measurements on devices featuring different gate stacks, initial threshold voltages (including negative ones) and operation temperatures. A detailed analysis has been also carried out to compare the retention dynamics of cells featuring thin or thick tunnel oxide barriers.

  20. Quantitative Fluorescence Studies in Living Cells: Extending Fluorescence Fluctuation Spectroscopy to Peripheral Membrane Proteins

    Science.gov (United States)

    Smith, Elizabeth Myhra

    The interactions of peripheral membrane proteins with both membrane lipids and proteins are vital for many cellular processes including membrane trafficking, cellular signaling, and cell growth/regulation. Building accurate biophysical models of these processes requires quantitative characterization of the behavior of peripheral membrane proteins, yet methods to quantify their interactions inside living cells are very limited. Because peripheral membrane proteins usually exist both in membrane-bound and cytoplasmic forms, the separation of these two populations is a key challenge. This thesis aims at addressing this challenge by extending fluorescence fluctuation spectroscopy (FFS) to simultaneously measure the oligomeric state of peripheral membrane proteins in the cytoplasm and at the plasma membrane. We developed a new method based on z-scan FFS that accounts for the fluorescence contributions from cytoplasmic and membrane layers by incorporating a fluorescence intensity z-scan through the cell. H-Ras-EGFP served as a model system to demonstrate the feasibility of the technique. The resolvability and stability of z-scanning was determined as well as the oligomeric state of H-Ras-EGFP at the plasma membrane and in the cytoplasm. Further, we successfully characterized the binding affinity of a variety of proteins to the plasma membrane by quantitative analysis of the z-scan fluorescence intensity profile. This analysis method, which we refer to as z-scan fluorescence profile deconvoution, was further used in combination with dual-color competition studies to determine the lipid specificity of protein binding. Finally, we applied z-scan FFS to provide insight into the early assembly steps of the HTLV-1 retrovirus.

  1. 3D quantitative visualization of altered LV wall thickening dynamics caused by coronary microembolization

    Science.gov (United States)

    Eusemann, Christian D.; Mohlenkamp, Stefan; Ritman, Erik L.; Robb, Richard A.

    2001-05-01

    Regional heart wall dynamics has been shown to be a sensitive indicator of LV wall ischemia. Rates of local LV wall thickening during a cardiac cycle can be measured and illustrated using functional parametric mappings. This display conveys the spatial distribution of dynamic strain in the myocardium and thereby provides a rapid qualitative appreciation of the severity and extent of the ischemic region. 3D reconstructions were obtained in an anesthetized pig from 8 adjacent, shortaxis, slices of the left ventricle imaged with an Electron Beam Computer Tomograph at 11 time points through one complete cardiac cycle. The 3D reconstructions were obtained before and after injection of 100 micrometer microspheres into the Left Anterior Descending (LAD) coronary artery. This injection causes microembolization of LAD artery branches within the heart wall. The image processing involved radially dividing the tomographic images of the myocardium into small subdivisions with color encoding of the local magnitude of regional thickness or regional velocities of LV wall thickening throughout the cardiac cycle. We compared the effectiveness of animation of wall thickness encoded in color versus a static image of computed rate of wall thickness change in color. The location, extent and severity of regional wall akinesis or dyskinesis, as determined from these displays, can then be compared to the region of embolization as indicated by the distribution of altered LV wall perfusion.

  2. Quantitative adaptation analytics for assessing dynamic systems of systems: LDRD Final Report

    Energy Technology Data Exchange (ETDEWEB)

    Gauthier, John H. [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States). System Readiness & Sustainment Technologies (6133, M/S 1188); Miner, Nadine E. [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States). Military & Energy Systems Analysis (6114, M/S 1188); Wilson, Michael L. [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States). Resilience and Regulatory Effects (6921, M/S 1138); Le, Hai D. [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States). System Readiness & Sustainment Technologies (6133, M/S 1188); Kao, Gio K. [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States). Networked System Survivability & Assurance (5629, M/S 0671); Melander, Darryl J. [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States). Software Systems R& D (9525, M/S 1188); Longsine, Dennis Earl [Sandia National Laboratories, Unknown, Unknown; Vander Meer, Jr., Robert C. [SAIC, Inc., Albuquerque, NM (United States)

    2015-01-01

    Our society is increasingly reliant on systems and interoperating collections of systems, known as systems of systems (SoS). These SoS are often subject to changing missions (e.g., nation- building, arms-control treaties), threats (e.g., asymmetric warfare, terrorism), natural environments (e.g., climate, weather, natural disasters) and budgets. How well can SoS adapt to these types of dynamic conditions? This report details the results of a three year Laboratory Directed Research and Development (LDRD) project aimed at developing metrics and methodologies for quantifying the adaptability of systems and SoS. Work products include: derivation of a set of adaptability metrics, a method for combining the metrics into a system of systems adaptability index (SoSAI) used to compare adaptability of SoS designs, development of a prototype dynamic SoS (proto-dSoS) simulation environment which provides the ability to investigate the validity of the adaptability metric set, and two test cases that evaluate the usefulness of a subset of the adaptability metrics and SoSAI for distinguishing good from poor adaptability in a SoS. Intellectual property results include three patents pending: A Method For Quantifying Relative System Adaptability, Method for Evaluating System Performance, and A Method for Determining Systems Re-Tasking.

  3. Quantitative evaluation of small breast masses using a compartment model analysis on dynamic MR imaging

    Energy Technology Data Exchange (ETDEWEB)

    Ikeda, Osamu; Morishita, Shoji; Kido, Taeko; Kitajima, Mika; Okamura, Kenji; Fukuda, Seiji [Kumamoto Rosai Hospital, Yatsushiro (Japan); Yamashita, Yasuyuki; Takahashi, Mutsumasa

    1998-07-01

    To differentiate between malignant and benign breast masses using a compartmental analysis, 55 patients with breast masses (fibroadenoma, n=22; invasive ductal carcinoma, n=29; noninvasive ductal carcinoma, n=8) underwent Gd-DTPA enhanced dynamic MR imaging. Dynamic MR images obtained using two-dimensional fat-saturated fast multiplanar corrupted gradient echo technique over 10 minutes following bolus injection of Gd-DTPA. The triexponential concentration curve of Gd-DTPA was fitted to a theoretical model based on compartmental analysis. Using this method, the transfer constant (or permeability surface product per unit volume of component k) and f{sub 3}/f{sub 1}=f were measured, where f{sub 1} represents tumor vessel volume and f{sub 3} represents extracellular volume. The k value was significantly greater (p<0.01) for malignant tumors, and the k value seen in cases of noninvasive ductal carcinoma was less than that for invasive ductal carcinoma. The f value was significantly smaller (p<0.01) for malignant tumors, whereas the f value for noninvasive ductal carcinoma was not significantly different from that for invasive ductal carcinoma. We believe that this type of compartmental analysis may be of value for the evaluation of breast masses. (author)

  4. Intermediate filaments: a dynamic network that controls cell mechanics.

    Science.gov (United States)

    Gruenbaum, Yosef; Aebi, Ueli

    2014-01-01

    In humans the superfamily of intermediate filament (IF) proteins is encoded by more than 70 different genes, which are expressed in a cell- and tissue-specific manner. IFs assemble into approximately 10 nm-wide filaments that account for the principal structural elements at the nuclear periphery, nucleoplasm, and cytoplasm. They are also required for organizing the microtubule and microfilament networks. In this review, we focus on the dynamics of IFs and how modifications regulate it. We also discuss the role of nuclear IF organization in determining nuclear mechanics as well as that of cytoplasmic IFs organization in maintaining cell stiffness, formation of lamellipodia, regulation of cell migration, and permitting cell adhesion.

  5. On-Beads Digestion in Conjunction with Data-Dependent Mass Spectrometry: A Shortcut to Quantitative and Dynamic Interaction Proteomics

    Directory of Open Access Journals (Sweden)

    Benedetta Turriziani

    2014-04-01

    Full Text Available With the advent of the “-omics” era, biological research has shifted from functionally analyzing single proteins to understanding how entire protein networks connect and adapt to environmental cues. Frequently, pathological processes are initiated by a malfunctioning protein network rather than a single protein. It is therefore crucial to investigate the regulation of proteins in the context of a pathway first and signaling network second. In this study, we demonstrate that a quantitative interaction proteomic approach, combining immunoprecipitation, in-solution digestion and label-free quantification mass spectrometry, provides data of high accuracy and depth. This protocol is applicable, both to tagged, exogenous and untagged, endogenous proteins. Furthermore, it is fast, reliable and, due to a label-free quantitation approach, allows the comparison of multiple conditions. We further show that we are able to generate data in a medium throughput fashion and that we can quantify dynamic interaction changes in signaling pathways in response to mitogenic stimuli, making our approach a suitable method to generate data for system biology approaches.

  6. Quantitative measurement of the medial surface dynamics of the vocal folds using high-speed digital imaging

    Science.gov (United States)

    Doellinger, Michael; Neubauer, Juergen; Berry, David A.

    2003-10-01

    To increase our understanding of pathological and healthy voice production, the quantitative measurement of the medial surface dynamics of the vocal folds is significant, albeit rarely performed because of the inaccessibility of the vocal folds. Hence, an excised hemilarynx procedure is applied, Berry et al. recently reported such quantitative measurements along one coronal plane of the left vocal fold of a canine [J. Acoust. Soc. Am. 110, 2539-2547 (2001)]. The present work extends previous studies by capturing vibrations along the entire medial surface of the left vocal fold. The number of vertical rows of sutures used to demarcate fleshpoints was increased from one to five. An automatic algorithm for tracking vocal fold fleshpoints will be reported, along with calibration techniques, and error estimation. Preliminary results will be reported for both periodic and aperiodic vocal fold vibrations. High-speed digital imaging was performed using a Photron APX machine with a sampling frequency of 4000 Hz, a spatial resolution of 1024×512 pixels, and 256 levels of grayscale.

  7. STIM evaluation in GeoPIXE to complement the quantitative dynamic analysis

    Energy Technology Data Exchange (ETDEWEB)

    Pallon, J. [Division of Nuclear Physics, Department of Physics, Lund Institute of Technology, Lund University, P.O. Box 118, SE-221 00 Lund (Sweden)], E-mail: Jan.Pallon@nuclear.lu.se; Ryan, C.G. [CSIRO Exploration and Mining, Geosciences, Monash University, Clayton VIC 3168 (Australia); Physics Department, University of Melbourne, VIC 3010 (Australia); CODES Centre of Excellence, University of Tasmania, Hobart TAS 7001 (Australia); Arteaga Marrero, N.; Elfman, M.; Kristiansson, P.; Nilsson, E.J.C.; Nilsson, C. [Division of Nuclear Physics, Department of Physics, Lund Institute of Technology, Lund University, P.O. Box 118, SE-221 00 Lund (Sweden)

    2009-06-15

    The GeoPIXE software for quantitative PIXE trace element imaging and analysis is a well established package for evaluation of characteristic X-ray data for both PIXE and SXRF. For the case of microbeam applications on semi-thick samples knowledge of the local areal density distribution is important for precise quantification. A technique is reported to achieve this using the measurement of beam particle energy loss as it traverses the sample, as in scanning transmission ion microscopy (STIM). New functionality is added to the GeoPIXE code through integration of routines for STIM sorting of event-by-event data to create elemental maps of the mean energy after traversing the sample. Integration of stopping powers for a given sample matrix then permits the measured energy loss to be related to the local areal density. In a further step, this information is used for X-ray absorption corrections made directly to the PIXE analysis results. As a complement, user-written plugins operating on single STIM spectra have been used to compare the estimated areal density from chosen spots with the corresponding values calculated with the new GeoPIXE routines. The additions made to the code allow a more precise quantification to be done on inhomogeneous, semi-thick samples.

  8. Streaking Artifact Reduction for Quantitative Susceptibility Mapping of Sources with Large Dynamic Range

    Science.gov (United States)

    Wei, Hongjiang; Dibb, Russell; Zhou, Yan; Sun, Yawen; Xu, Jianrong; Wang, Nian; Liu, Chunlei

    2015-01-01

    Quantitative susceptibility mapping (QSM) is a novel MRI technique for measuring tissue magnetic susceptibility in 3D. While there are numerous algorithms developed to solve this ill-posed inverse problem, estimating susceptibility maps with a wide range of values is still problematic. In cases such as large veins, contrast agent uptake, and intracranial hemorrhages, extreme susceptibility values in focal areas cause severe streaking artifacts. To enable the reduction of these artifacts while preserving subtle susceptibility contrast, a two-level QSM reconstruction algorithm (STAR-QSM) was developed in this study by tuning a regularization parameter to automatically reconstruct both large and small susceptibility values. Compared to current state-of-the-art QSM methods such as iLSQR, STAR-QSM significantly reduced streaking artifacts while preserving sharp boundaries for blood vessels of mouse brains in vivo and fine anatomical details of high resolution mouse brains ex vivo. Brain image data from patients with cerebral hematoma and multiple sclerosis further illustrated the superiority of this method in reducing streaking artifacts caused by large susceptibility sources while maintaining sharp anatomical details. STAR-QSM is implemented in STI Suite, a comprehensive shareware for susceptibility imaging and quantification. PMID:26313885

  9. Quantitative ultrastructural analysis of basket and axo-axonic cell terminals in the mouse hippocampus.

    Science.gov (United States)

    Takács, Virág T; Szőnyi, András; Freund, Tamás F; Nyiri, Gábor; Gulyás, Attila I

    2015-03-01

    Three functionally different populations of perisomatic interneurons establish GABAergic synapses on hippocampal pyramidal cells: parvalbumin (PV)-containing basket cells, type 1 cannabinoid receptor (CB1)-positive basket cells both of which target somata, and PV-positive axo-axonic cells that innervate axon initial segments. Using electron microscopic reconstructions, we estimated that a pyramidal cell body receives synapses from about 60 and 140 synaptic terminals in the CA1 and CA3 area, respectively. About 60 % of these terminals were PV positive, whereas 35-40 % of them were CB1 positive. Only about 1 % (CA1) and 4 % (CA3) of the somatic boutons were negative for both markers. Using fluorescent labeling, we showed that most of the CB1-positive terminals expressed vesicular glutamate transporter 3. Reconstruction of somatic boutons revealed that although their volumes are similar, CB1-positive boutons are more flat and the total volume of their mitochondria was smaller than that of PV-positive boutons. Both types of boutons contain dense-core vesicles and frequently formed multiple release sites on their targets and innervated an additional soma or dendrite as well. PV-positive boutons possessed small, macular synapses; whereas the total synaptic area of CB1-positive boutons was larger and formed multiple irregular-shaped synapses. Axo-axonic boutons were smaller than somatic boutons, had only one synapse and their ultrastructural parameters were closer to those of PV-positive somatic boutons. Our results represent the first quantitative measurement-using a highly reliable method-of the contribution of different cell types to the perisomatic innervation of pyramidal neurons, and may help to explain functional differences in their output properties.

  10. Absolute choline concentration measured by quantitative proton MR spectroscopy correlates with cell density in meningioma

    Energy Technology Data Exchange (ETDEWEB)

    Yue, Qiang [University of Tsukuba, Department of Neurosurgery, Institute of Clinical Medicine, Tsukuba Science City, Ibaraki (Japan)]|[West China Hospital of Sichuan University, Huaxi MR Research Center, Department of Radiology, Chengdu (China); Shibata, Yasushi; Kawamura, Hiraku; Matsumura, Akira [University of Tsukuba, Department of Neurosurgery, Institute of Clinical Medicine, Tsukuba Science City, Ibaraki (Japan); Isobe, Tomonori [Kitasato University, Department of Medical Technology, School of Allied Health Sciences, Minato, Tokyo (Japan); Anno, Izumi [University of Tsukuba, Department of Radiology, Institute of Clinical Medicine, Tsukuba, Ibaraki (Japan); Gong, Qi-Yong [West China Hospital of Sichuan University, Huaxi MR Research Center, Department of Radiology, Chengdu (China)]|[University of Liverpool, Division of Medical Imaging, Faculty of Medicine, Liverpool (United Kingdom)

    2009-01-15

    This study was aimed to investigate the relationship between quantitative proton magnetic resonance spectroscopy (1H-MRS) and pathological changes in meningioma. Twenty-two meningioma cases underwent single voxel 1H-MRS (point-resolved spectroscopy sequence, repetition time/echo time = 2,000 ms/68, 136, 272 ms). Absolute choline (Cho) concentration was calculated using tissue water as the internal reference and corrected according to intra-voxel cystic/necrotic parts. Pathological specimens were stained with MIB-1 antibody to measure cell density and proliferation index. Correlation analysis was performed between absolute Cho concentration and cell density and MIB-1 labeled proliferation index. Average Cho concentration of all meningiomas before correction was 2.95 {+-} 0.86 mmol/kg wet weight. It was increased to 3.23 {+-} 1.15 mmol/kg wet weight after correction. Average cell density of all meningiomas was 333 {+-} 119 cells/HPF, and average proliferation index was 2.93 {+-} 5.72%. A linear, positive correlation between cell density and Cho concentration was observed (r = 0.650, P = 0.001). After correction of Cho concentration, the correlation became more significant (r = 0.737, P < 0.001). However, no significant correlation between Cho concentration and proliferation index was found. There seemed to be a positive correlation trend after correction of Cho concentration but did not reach significant level. Absolute Cho concentration, especially Cho concentration corrected according to intra-voxel cystic/necrotic parts, reflects cell density of meningioma. (orig.)

  11. Automated live cell imaging systems reveal dynamic cell behavior.

    Science.gov (United States)

    Chirieleison, Steven M; Bissell, Taylor A; Scelfo, Christopher C; Anderson, Jordan E; Li, Yong; Koebler, Doug J; Deasy, Bridget M

    2011-07-01

    Automated time-lapsed microscopy provides unique research opportunities to visualize cells and subcellular components in experiments with time-dependent parameters. As accessibility to these systems is increasing, we review here their use in cell science with a focus on stem cell research. Although the use of time-lapsed imaging to answer biological questions dates back nearly 150 years, only recently have the use of an environmentally controlled chamber and robotic stage controllers allowed for high-throughput continuous imaging over long periods at the cell and subcellular levels. Numerous automated imaging systems are now available from both companies that specialize in live cell imaging and from major microscope manufacturers. We discuss the key components of robots used for time-lapsed live microscopic imaging, and the unique data that can be obtained from image analysis. We show how automated features enhance experimentation by providing examples of uniquely quantified proliferation and migration live cell imaging data. In addition to providing an efficient system that drastically reduces man-hours and consumes fewer laboratory resources, this technology greatly enhances cell science by providing a unique dataset of temporal changes in cell activity. Copyright © 2011 American Institute of Chemical Engineers (AIChE).

  12. Processes on the emergent landscapes of biochemical reaction networks and heterogeneous cell population dynamics: differentiation in living matters

    Science.gov (United States)

    Li, Fangting

    2017-01-01

    The notion of an attractor has been widely employed in thinking about the nonlinear dynamics of organisms and biological phenomena as systems and as processes. The notion of a landscape with valleys and mountains encoding multiple attractors, however, has a rigorous foundation only for closed, thermodynamically non-driven, chemical systems, such as a protein. Recent advances in the theory of nonlinear stochastic dynamical systems and its applications to mesoscopic reaction networks, one reaction at a time, have provided a new basis for a landscape of open, driven biochemical reaction systems under sustained chemostat. The theory is equally applicable not only to intracellular dynamics of biochemical regulatory networks within an individual cell but also to tissue dynamics of heterogeneous interacting cell populations. The landscape for an individual cell, applicable to a population of isogenic non-interacting cells under the same environmental conditions, is defined on the counting space of intracellular chemical compositions x = (x1,x2, … ,xN) in a cell, where xℓ is the concentration of the ℓth biochemical species. Equivalently, for heterogeneous cell population dynamics xℓ is the number density of cells of the ℓth cell type. One of the insights derived from the landscape perspective is that the life history of an individual organism, which occurs on the hillsides of a landscape, is nearly deterministic and ‘programmed’, while population-wise an asynchronous non-equilibrium steady state resides mostly in the lowlands of the landscape. We argue that a dynamic ‘blue-sky’ bifurcation, as a representation of Waddington's landscape, is a more robust mechanism for a cell fate decision and subsequent differentiation than the widely pictured pitch-fork bifurcation. We revisit, in terms of the chemostatic driving forces upon active, living matter, the notions of near-equilibrium thermodynamic branches versus far-from-equilibrium states. The emergent

  13. Quantitative analysis of proteome and lipidome dynamics reveals functional regulation of global lipid metabolism.

    Science.gov (United States)

    Casanovas, Albert; Sprenger, Richard R; Tarasov, Kirill; Ruckerbauer, David E; Hannibal-Bach, Hans Kristian; Zanghellini, Jürgen; Jensen, Ole N; Ejsing, Christer S

    2015-03-19

    Elucidating how and to what extent lipid metabolism is remodeled under changing conditions is essential for understanding cellular physiology. Here, we analyzed proteome and lipidome dynamics to investigate how regulation of lipid metabolism at the global scale supports remodeling of cellular architecture and processes during physiological adaptations in yeast. Our results reveal that activation of cardiolipin synthesis and remodeling supports mitochondrial biogenesis in the transition from fermentative to respiratory metabolism, that down-regulation of de novo sterol synthesis machinery prompts differential turnover of lipid droplet-associated triacylglycerols and sterol esters during respiratory growth, that sphingolipid metabolism is regulated in a previously unrecognized growth stage-specific manner, and that endogenous synthesis of unsaturated fatty acids constitutes an in vivo upstream activator of peroxisomal biogenesis, via the heterodimeric Oaf1/Pip2 transcription factor. Our work demonstrates the pivotal role of lipid metabolism in adaptive processes and provides a resource to investigate its regulation at the cellular level.

  14. Quantitative attribution of major driving forces on soil organic carbon dynamics

    Science.gov (United States)

    Wu, Yiping; Liu, Shuguang; Tan, Zhengxi

    2015-03-01

    Soil organic carbon (SOC) storage plays a major role in the global carbon cycle and is affected by many factors including land use/management changes (e.g., biofuel production-oriented changes). However, the contributions of various factors to SOC changes are not well understood and quantified. This study was designed to investigate the impacts of changing farming practices, initial SOC levels, and biological enhancement of grain production on SOC dynamics and to attribute the relative contributions of major driving forces (CO2 enrichment and farming practices) using a fractional factorial modeling design. The case study at a crop site in Iowa in the United States demonstrated that the traditional corn-soybean (CS) rotation could still accumulate SOC over this century (from 4.2 to 6.8 kg C/m2) under the current condition; whereas the continuous-corn (CC) system might have a higher SOC sequestration potential than CS. In either case, however, residue removal could reduce the sink potential substantially. Long-term simulation results also suggested that the equilibrium SOC level may vary greatly (˜5.7 to ˜11 kg C/m2) depending on cropping systems and management practices, and projected growth enhancement could make the magnitudes higher (˜7.8 to ˜13 kg C/m2). Importantly, the factorial design analysis indicated that residue management had the most significant impact (contributing 49.4%) on SOC changes, followed by CO2 Enrichment (37%), Tillage (6.2%), the combination of CO2 Enrichment-Residue removal (5.8%), and Fertilization (1.6%). In brief, this study is valuable for understanding the major forces driving SOC dynamics of agroecosystems and informative for decision-makers when seeking the enhancement of SOC sequestration potential and sustainability of biofuel production, especially in the Corn Belt region of the United States.

  15. Quantitative single-cell motility analysis of platelet-rich plasma-treated endothelial cells in vitro.

    Science.gov (United States)

    Kawase, Tomoyuki; Tanaka, Takaaki; Okuda, Kazuhiro; Tsuchimochi, Makoto; Oda, Masafumi; Hara, Toshiaki

    2015-05-01

    Platelet-rich plasma (PRP) has been widely applied in regenerative therapy due to its high concentration of growth factors. Previous in vitro and in vivo studies have provided evidence supporting the angiogenic activity of PRP. To more directly demonstrate how PRP acts on endothelial cells, we examined the PRP-induced changes in the motility of human umbilical vein endothelial cells by examining the involvement of VEGF. Time-lapse quantitative imaging demonstrated that in the initial phase (∼2 h) of treatment, PRP substantially stimulated cell migration in a wound-healing assay. However, this effect of PRP was not sustained at significant levels beyond the initial phase. The average net distance of cell migration at 10 h was 0.45 ± 0.16 mm and 0.82 ± 0.23 mm in control and PRP-stimulated cells, respectively. This effect was also demonstrated with recombinant human VEGF and was significantly attenuated by a neutralizing anti-VEGF antibody. Immunofluorescent examination of paxillin and actin fibers demonstrated that PRP concomitantly up-regulated focal adhesion and cytoskeletal formation. Western blotting analysis of phosphorylated VEGFR2 demonstrated that PRP mainly stimulated the phosphorylation of immature VEGFR2 in a dose- and time-dependent manner, an action that was completely blocked by the neutralizing antibody. Taken together, these data suggest that PRP acts directly on endothelial cells via the activation of VEGFR2 to transiently up-regulate their motility. Thus, the possibility that PRP desensitizes target endothelial cells for a relatively long period of time after short-term activation should be considered when the controlled release system of PRP components is designed.

  16. Dynamics of Lgr6+ Progenitor Cells in the Hair Follicle, Sebaceous Gland, and Interfollicular Epidermis

    Directory of Open Access Journals (Sweden)

    Anja Füllgrabe

    2015-11-01

    Full Text Available The dynamics and interactions between stem cell pools in the hair follicle (HF, sebaceous gland (SG, and interfollicular epidermis (IFE of murine skin are still poorly understood. In this study, we used multicolor lineage tracing to mark Lgr6-expressing basal cells in the HF isthmus, SG, and IFE. We show that these Lgr6+ cells constitute long-term self-renewing populations within each compartment in adult skin. Quantitative analysis of clonal dynamics revealed that the Lgr6+ progenitor cells compete neutrally in the IFE, isthmus, and SG, indicating population asymmetry as the underlying mode of tissue renewal. Transcriptional profiling of Lgr6+ and Lgr6− cells did not reveal a distinct Lgr6-associated gene expression signature, raising the question of whether Lgr6 expression requires extrinsic niche signals. Our results elucidate the interrelation and behavior of Lgr6+ populations in the IFE, HF, and SG and suggest population asymmetry as a common mechanism for homeostasis in several epithelial skin compartments.

  17. Dynamics of cell shape and forces on micropatterned substrates predicted by a cellular Potts model.

    Science.gov (United States)

    Albert, Philipp J; Schwarz, Ulrich S

    2014-06-03

    Micropatterned substrates are often used to standardize cell experiments and to quantitatively study the relation between cell shape and function. Moreover, they are increasingly used in combination with traction force microscopy on soft elastic substrates. To predict the dynamics and steady states of cell shape and forces without any a priori knowledge of how the cell will spread on a given micropattern, here we extend earlier formulations of the two-dimensional cellular Potts model. The third dimension is treated as an area reservoir for spreading. To account for local contour reinforcement by peripheral bundles, we augment the cellular Potts model by elements of the tension-elasticity model. We first parameterize our model and show that it accounts for momentum conservation. We then demonstrate that it is in good agreement with experimental data for shape, spreading dynamics, and traction force patterns of cells on micropatterned substrates. We finally predict shapes and forces for micropatterns that have not yet been experimentally studied. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  18. Preparation of cell lines for single-cell analysis of transcriptional activation dynamics.

    Science.gov (United States)

    Rafalska-Metcalf, Ilona U; Janicki, Susan M

    2013-01-01

    Imaging molecularly defined regions of chromatin in single living cells during transcriptional activation has the potential to provide new insight into gene regulatory mechanisms. Here, we describe a method for isolating cell lines with multi-copy arrays of reporter transgenes, which can be used for real-time high-resolution imaging of transcriptional activation dynamics in single cells.

  19. Cell mass and cell cycle dynamics of an asynchronous budding yeast population: experimental observations, flow cytometry data analysis, and multi-scale modeling.

    Science.gov (United States)

    Lencastre Fernandes, Rita; Carlquist, Magnus; Lundin, Luisa; Heins, Anna-Lena; Dutta, Abhishek; Sørensen, Søren J; Jensen, Anker D; Nopens, Ingmar; Lantz, Anna Eliasson; Gernaey, Krist V

    2013-03-01

    Despite traditionally regarded as identical, cells in a microbial cultivation present a distribution of phenotypic traits, forming a heterogeneous cell population. Moreover, the degree of heterogeneity is notably enhanced by changes in micro-environmental conditions. A major development in experimental single-cell studies has taken place in the last decades. It has however not been fully accompanied by similar contributions within data analysis and mathematical modeling. Indeed, literature reporting, for example, quantitative analyses of experimental single-cell observations and validation of model predictions for cell property distributions against experimental data is scarce. This study focuses on the experimental and mathematical description of the dynamics of cell size and cell cycle position distributions, of a population of Saccharomyces cerevisiae, in response to the substrate consumption observed during batch cultivation. The good agreement between the proposed multi-scale model (a population balance model [PBM] coupled to an unstructured model) and experimental data (both the overall physiology and cell size and cell cycle distributions) indicates that a mechanistic model is a suitable tool for describing the microbial population dynamics in a bioreactor. This study therefore contributes towards the understanding of the development of heterogeneous populations during microbial cultivations. More generally, it consists of a step towards a paradigm change in the study and description of cell cultivations, where average cell behaviors observed experimentally now are interpreted as a potential joint result of various co-existing single-cell behaviors, rather than a unique response common to all cells in the cultivation.

  20. Telomerase Activity Detected by Quantitative Assay in Bladder Carcinoma and Exfoliated Cells in Urine

    Directory of Open Access Journals (Sweden)

    Roberta Fedriga

    2001-01-01

    Full Text Available Early diagnosis is one of the most determining factors for patient survival. The detection of telomerase activity is a potentially promising tool in the diagnosis of bladder and other types of cancer due to the high expression of this enzyme in tumor cells. We carried out a quantitative evaluation of telomerase activity in urine samples in an attempt to determine a cut-off capable of identifying cancer patients. Telomerase activity was quantified by fluorescence TRAP assay in urine from 50 healthy volunteers and in urine and bioptic tumor samples from 56 previously untreated bladder cancer patients and expressed in arbitrary enzymatic units (AEU. Telomerase activity in urine ranged from 0 to 106 AEU (median 0 in healthy donors and from 0 to 282 AEU (median 87 in patients with cancer. A telomerase expression higher than the cut off value determined by receiver operating characteristic (ROC analysis was observed in 78% of cases, regardless of tumor grade and in 71% (15/21 of cases of nonassessable or negative cytology. The quantitative analysis of telomerase activity in urine enabled us to define cut-off values characterized by different sensitivity and specificity. Cytologic and telomerase determination, used sequentially, enabled us to detect about 90% of tumors.

  1. Multicolor bioluminescence boosts malaria research: quantitative dual-color assay and single-cell imaging in Plasmodium falciparum parasites.

    Science.gov (United States)

    Cevenini, Luca; Camarda, Grazia; Michelini, Elisa; Siciliano, Giulia; Calabretta, Maria Maddalena; Bona, Roberta; Kumar, T R Santha; Cara, Andrea; Branchini, Bruce R; Fidock, David A; Roda, Aldo; Alano, Pietro

    2014-09-02

    New reliable and cost-effective antimalarial drug screening assays are urgently needed to identify drugs acting on different stages of the parasite Plasmodium falciparum, and particularly those responsible for human-to-mosquito transmission, that is, the P. falciparum gametocytes. Low Z' factors, narrow dynamic ranges, and/or extended assay times are commonly reported in current gametocyte assays measuring gametocyte-expressed fluorescent or luciferase reporters, endogenous ATP levels, activity of gametocyte enzymes, or redox-dependent dye fluorescence. We hereby report on a dual-luciferase gametocyte assay with immature and mature P. falciparum gametocyte stages expressing red and green-emitting luciferases from Pyrophorus plagiophthalamus under the control of the parasite sexual stage-specific pfs16 gene promoter. The assay was validated with reference antimalarial drugs and allowed to quantitatively and simultaneously measure stage-specific drug effects on parasites at different developmental stages. The optimized assay, requiring only 48 h incubation with drugs and using a cost-effective luminogenic substrate, significantly reduces assay cost and time in comparison to state-of-the-art analogous assays. The assay had a Z' factor of 0.71 ± 0.03, and it is suitable for implementation in 96- and 384-well microplate formats. Moreover, the use of a nonlysing D-luciferin substrate significantly improved the reliability of the assay and allowed one to perform, for the first time, P. falciparum bioluminescence imaging at single-cell level.

  2. Dynamic quantitative trait locus analysis of seed vigor at three maturity stages in rice.

    Science.gov (United States)

    Liu, Liangfeng; Lai, Yanyan; Cheng, Jinping; Wang, Ling; Du, Wenli; Wang, Zhoufei; Zhang, Hongsheng

    2014-01-01

    Seed vigor is an important characteristic of seed quality. In this study, one rice population of recombinant inbred lines (RILs) was used to determine the genetic characteristics of seed vigor, including the germination potential, germination rate, germination index and time for 50% of germination, at 4 (early), 5 (middle) and 6 weeks (late) after heading in two years. A total of 24 additive and 9 epistatic quantitative trait loci (QTL) for seed vigor were identified using QTL Cartographer and QTLNetwork program respectively in 2012; while 32 simple sequence repeat (SSR) markers associated with seed vigor were detected using bulked segregant analysis (BSA) in 2013. The additive, epistatic and QTL × development interaction effects regulated the dry maturity developmental process to improve seed vigor in rice. The phenotypic variation explained by each additive, epistatic QTL and QTL × development interaction ranged from 5.86 to 40.67%, 4.64 to 11.28% and 0.01 to 1.17%, respectively. The QTLs were rarely co-localized among the different maturity stages; more QTLs were expressed at the early maturity stage followed by the late and middle stages. Twenty additive QTLs were stably expressed in two years which might play important roles in establishment of seed vigor in different environments. By comparing chromosomal positions of these stably expressed additive QTLs with those previously identified, the regions of QTL for seed vigor are likely to coincide with QTL for grain size, low temperature germinability and seed dormancy; while 5 additive QTL might represent novel genes. Using four selected RILs, three cross combinations of seed vigor for the development of RIL populations were predicted; 19 elite alleles could be pyramided by each combination.

  3. Imaging of Lamb Waves in Plates for Quantitative Determination of Anisotropy using Photorefractive Dynamic Holography

    Energy Technology Data Exchange (ETDEWEB)

    Telschow, Kenneth Louis; Deason, Vance Albert; Schley, Robert Scott; Watson, Scott Marshall

    1998-06-01

    Anisotropic properties of sheet materials can be determined by measuring the propagation of Lamb waves in different directions. Electromagnetic acoustic transduction and laser ultrasonic methods provide noncontacting approaches that are often desired for application to industrial and processing environments. This paper describes a laser imaging approach utilizing the adaptive property of photorefractive materials to produce a real-time measurement of the antisymmetric Lamb wave mode in all directions simultaneously. Continuous excitation is employed enabling the data to be recorded and displayed by a CCD camera. Analysis of the image produces a direct quantitative determination of the phase velocity in all directions showing plate anisotropy in the plane. Many optical techniques for measuring ultrasonic motion at surfaces have been developed for use in applications such as vibration measurement and laser ultrasonics. Most of these methods have similar sensitivities and are based on time domain processing using homodyne, Fabry-Perot [1], and, more recently, photorefractive interferometry [2]. Generally, the methods described above do not allow measurement at more than one surface point simultaneously, requiring multiple beam movements and scanning in order to produce images of surface ultrasonic motion over a large area. Electronic speckle interferometry, including shearography, does provide images directly of vibrations over large surface areas. This method has proven very durable in the field for large displacement amplitudes of several wavelengths. In addition, a sensitivity of ë/3000 has been demonstrated under laboratory conditions [3]. Full-field imaging of traveling ultrasonic waves using digital shearography has been recently reported with sensitivity in the nanometer range [4]. With this method, optical interference occurs at the photodetector

  4. Quantitative secretome analysis reveals the interactions between epithelia and tumor cells by in vitro modulating colon cancer microenvironment.

    Science.gov (United States)

    Zeng, Xiao; Yang, Pengbo; Chen, Bing; Jin, Xuewen; Liu, Yuling; Zhao, Xia; Liang, Shufang

    2013-08-26

    In tumor microenvironment, interactions among multiple cell types are critical for cancer progression. Secreted proteins are responsible for crosstalk among these cells within tumor microenvironment. To elucidate the interactions of tumor and epithelia, we co-cultured colon cancer cell line HT29 with normal human colon mucosal epithelial cell line NCM460 to mimic tumor microenvironment in vitro and investigated the differential expression pattern of secretome. A quantitative proteomics approach based on stable isotope labeling by amino acids in cell culture (SILAC) and LC-mass spectrometry was used for secretome analysis. Totally 45 proteins were altered over 2-fold in co-cultured cellular supernatants between equal amounts of NCM460 and HT29 cells, compared with mono-cultured conditions. These differential secreted proteins involve in multiple tumor-associated biological functions. The secretion level and acting pattern of acrogranin, IGFBP6 and vimentin were changed along with different co-cultured cell number ratios between NCM460 and HT29 cells, simulating early, middle or advanced stage of colon cancer. Therefore, a quantitative secretome profiling based on a co-culture system can track secreted protein changes and their associated biological roles between tumor and epithelia, which gives a new insight on communications between tumor and epithelia as well as cancer biotherapy by inhibiting cell interactions. Tumor microenvironment is a complex system and comprised of cancer cells and host stromal cells. The growth and progression of tumor have been recognized were affected by multidirectional interactions of secreted proteins (secretome), which were produced by the cells within tumor microenvironment. Focus on general secreted molecules of living cells via proteomic tools, is promising for investigating cell communication. Stable isotope labeling by amino acids in cell culture (SILAC) is a metabolic labeling strategy for quantitative analysis, which is gaining

  5. Quantitative profiling of housekeeping and Epstein-Barr virus gene transcription in Burkitt lymphoma cell lines using an oligonucleotide microarray

    Directory of Open Access Journals (Sweden)

    Niggli Felix K

    2006-06-01

    Full Text Available Abstract Background The Epstein-Barr virus (EBV is associated with lymphoid malignancies, including Burkitt's lymphoma (BL, and can transform human B cells in vitro. EBV-harboring cell lines are widely used to investigate lymphocyte transformation and oncogenesis. Qualitative EBV gene expression has been extensively described, but knowledge of quantitative transcription is lacking. We hypothesized that transcription levels of EBNA1, the gene essential for EBV persistence within an infected cell, are similar in BL cell lines. Results To compare quantitative gene transcription in the BL cell lines Namalwa, Raji, Akata, Jijoye, and P3HR1, we developed an oligonucleotide microarray chip, including 17 housekeeping genes, six latent EBV genes (EBNA1, EBNA2, EBNA3A, EBNA3C, LMP1, LMP2, and four lytic EBV genes (BZLF1, BXLF2, BKRF2, BZLF2, and used the cell line B95.8 as a reference for EBV gene transcription. Quantitative polymerase chain reaction assays were used to validate microarray results. We found that transcription levels of housekeeping genes differed considerably among BL cell lines. Using a selection of housekeeping genes with similar quantitative transcription in the tested cell lines to normalize EBV gene transcription data, we showed that transcription levels of EBNA1 were quite similar in very different BL cell lines, in contrast to transcription levels of other EBV genes. As demonstrated with Akata cells, the chip allowed us to accurately measure EBV gene transcription changes triggered by treatment interventions. Conclusion Our results suggest uniform EBNA1 transcription levels in BL and that microarray profiling can reveal novel insights on quantitative EBV gene transcription and its impact on lymphocyte biology.

  6. Optical sensors for monitoring dynamic changes of intracellular metabolite levels in mammalian cells.

    Science.gov (United States)

    Hou, Bi-Huei; Takanaga, Hitomi; Grossmann, Guido; Chen, Li-Qing; Qu, Xiao-Qing; Jones, Alexander M; Lalonde, Sylvie; Schweissgut, Oliver; Wiechert, Wolfgang; Frommer, Wolf B

    2011-10-27

    Knowledge of the in vivo levels, distribution and flux of ions and metabolites is crucial to our understanding of physiology in both healthy and diseased states. The quantitative analysis of the dynamics of ions and metabolites with subcellular resolution in vivo poses a major challenge for the analysis of metabolic processes. Genetically encoded Förster resonance energy transfer (FRET) sensors can be used for real-time in vivo detection of metabolites. FRET sensor proteins, for example, for glucose, can be targeted genetically to any cellular compartment, or even to subdomains (e.g., a membrane surface), by adding signal sequences or fusing the sensors to specific proteins. The sensors can be used for analyses in individual mammalian cells in culture, in tissue slices and in intact organisms. Applications include gene discovery, high-throughput drug screens or systematic analysis of regulatory networks affecting uptake, efflux and metabolism. Quantitative analyses obtained with the help of FRET sensors for glucose or other ions and metabolites provide valuable data for modeling of flux. Here we provide a detailed protocol for monitoring glucose levels in the cytosol of mammalian cell cultures through the use of FRET glucose sensors; moreover, the protocol can be used for other ions and metabolites and for analyses in other organisms, as has been successfully demonstrated in bacteria, yeast and even intact plants. The whole procedure typically takes ∼4 d including seeding and transfection of mammalian cells; the FRET-based analysis of transfected cells takes ∼5 h.

  7. Simple and fast spectral domain algorithm for quantitative phase imaging of living cells with digital holographic microscopy.

    Science.gov (United States)

    Min, Junwei; Yao, Baoli; Ketelhut, Steffi; Engwer, Christian; Greve, Burkhard; Kemper, Björn

    2017-01-15

    We present a simple and fast phase aberration compensation method in digital holographic microscopy (DHM) for quantitative phase imaging of living cells. By analyzing the frequency spectrum of an off-axis hologram, phase aberrations can be compensated for automatically without fitting or pre-knowledge of the setup and/or the object. Simple and effective computation makes the method suitable for quantitative online monitoring with highly variable DHM systems. Results from automated quantitative phase imaging of living NIH-3T3 mouse fibroblasts demonstrate the effectiveness and the feasibility of the method.

  8. Integrated quantitative phase and birefringence microscopy for imaging malaria-infected red blood cells

    Science.gov (United States)

    Li, Chengshuai; Chen, Shichao; Klemba, Michael; Zhu, Yizheng

    2016-09-01

    A dual-modality birefringence/phase imaging system is presented. The system features a crystal retarder that provides polarization mixing and generates two interferometric carrier waves in a single signal spectrum. The retardation and orientation of sample birefringence can then be measured simultaneously based on spectral multiplexing interferometry. Further, with the addition of a Nomarski prism, the same setup can be used for quantitative differential interference contrast (DIC) imaging. Sample phase can then be obtained with two-dimensional integration. In addition, birefringence-induced phase error can be corrected using the birefringence data. This dual-modality approach is analyzed theoretically with Jones calculus and validated experimentally with malaria-infected red blood cells. The system generates not only corrected DIC and phase images, but a birefringence map that highlights the distribution of hemozoin crystals.

  9. Dynamic imaging and quantitative analysis of cranial neural tube closure in the mouse embryo using optical coherence tomography.

    Science.gov (United States)

    Wang, Shang; Garcia, Monica D; Lopez, Andrew L; Overbeek, Paul A; Larin, Kirill V; Larina, Irina V

    2017-01-01

    Neural tube closure is a critical feature of central nervous system morphogenesis during embryonic development. Failure of this process leads to neural tube defects, one of the most common forms of human congenital defects. Although molecular and genetic studies in model organisms have provided insights into the genes and proteins that are required for normal neural tube development, complications associated with live imaging of neural tube closure in mammals limit efficient morphological analyses. Here, we report the use of optical coherence tomography (OCT) for dynamic imaging and quantitative assessment of cranial neural tube closure in live mouse embryos in culture. Through time-lapse imaging, we captured two neural tube closure mechanisms in different cranial regions, zipper-like closure of the hindbrain region and button-like closure of the midbrain region. We also used OCT imaging for phenotypic characterization of a neural tube defect in a mouse mutant. These results suggest that the described approach is a useful tool for live dynamic analysis of normal neural tube closure and neural tube defects in the mouse model.

  10. Quantitative impedimetric NPY-receptor activation monitoring and signal pathway profiling in living cells.

    Science.gov (United States)

    te Kamp, Verena; Lindner, Ricco; Jahnke, Heinz-Georg; Krinke, Dana; Kostelnik, Katja B; Beck-Sickinger, Annette G; Robitzki, Andrea A

    2015-05-15

    Label-free and non-invasive monitoring of receptor activation and identification of the involved signal pathways in living cells is an ongoing analytic challenge and a great opportunity for biosensoric systems. In this context, we developed an impedance spectroscopy-based system for the activation monitoring of NPY-receptors in living cells. Using an optimized interdigital electrode array for sensitive detection of cellular alterations, we were able for the first time to quantitatively detect the NPY-receptor activation directly without a secondary or enhancer reaction like cAMP-stimulation by forskolin. More strikingly, we could show that the impedimetric based NPY-receptor activation monitoring is not restricted to the Y1-receptor but also possible for the Y2- and Y5-receptor. Furthermore, we could monitor the NPY-receptor activation in different cell lines that natively express NPY-receptors and proof the specificity of the observed impedimetric effect by agonist/antagonist studies in recombinant NPY-receptor expre