Exploiting single-cell variability to infer the dynamics of immune responses

NASA Astrophysics Data System (ADS)

Höfer, Thomas

Cell division, differentiation, migration and death determine the dynamics of immune responses. These processes are regulated by a multitude of biochemical signals which, at present, cannot faithfully be reconstituted outside the living organism. However, quantitative measurements in living organisms have been limited. In recent years experimental techniques for the ``fate mapping'' of single immune cells have been developed that allow performing parallel single-cell experiments in an experimental animal. The resulting data are more informative about underlying biological processes than traditional measurements. I will show how the theory of stochastic dynamical systems can be used to infer the topology and dynamics of cell differentiation pathways from such data. The focus will be on joint theoretical and experimental work addressing: (i) the development of immune cells during hematopoiesis, and (ii) T cell responses to diverse pathogens. I will discuss questions on the nature of cellular variability that are posed by these new findings.

Cancer dormancy and criticality from a game theory perspective.

PubMed

Wu, Amy; Liao, David; Kirilin, Vlamimir; Lin, Ke-Chih; Torga, Gonzalo; Qu, Junle; Liu, Liyu; Sturm, James C; Pienta, Kenneth; Austin, Robert

2018-01-01

The physics of cancer dormancy, the time between initial cancer treatment and re-emergence after a protracted period, is a puzzle. Cancer cells interact with host cells via complex, non-linear population dynamics, which can lead to very non-intuitive but perhaps deterministic and understandable progression dynamics of cancer and dormancy. We explore here the dynamics of host-cancer cell populations in the presence of (1) payoffs gradients and (2) perturbations due to cell migration. We determine to what extent the time-dependence of the populations can be quantitively understood in spite of the underlying complexity of the individual agents and model the phenomena of dormancy.

Virtual Embryo: Cell-Agent Based Modeling of Developmental Processes and Toxicities (CSS BOSC)

EPA Science Inventory

Spatial regulation of cellular dynamics is fundamental to morphological development. As such, chemical disruption of spatial dynamics is a determinant of developmental toxicity. Incorporating spatial dynamics into AOPs for developmental toxicity is desired but constrained by the ...

Dynamic Pedagogy for Effective Training of Youths in Cell Phone Maintenance

ERIC Educational Resources Information Center

Ogbuanya, T. C.; Jimoh, Bakare

2015-01-01

The study determined dynamic pedagogies for effective training of youths in cell phone maintenance. The study was conducted in Enugu State of Nigeria. Four research questions were developed while four null hypotheses formulated were tested at 0.05 level of significance. A survey research design was adopted for the study. The population for the…

In vivo imaging of emerging endocrine cells reveals a requirement for PI3K-regulated motility in pancreatic islet morphogenesis

PubMed Central

Freudenblum, Julia; Iglesias, José A.; Hermann, Martin; Walsen, Tanja; Wilfinger, Armin; Meyer, Dirk

2018-01-01

ABSTRACT The three-dimensional architecture of the pancreatic islet is integral to beta cell function, but the process of islet formation remains poorly understood due to the difficulties of imaging internal organs with cellular resolution. Within transparent zebrafish larvae, the developing pancreas is relatively superficial and thus amenable to live imaging approaches. We performed in vivo time-lapse and longitudinal imaging studies to follow islet development, visualizing both naturally occurring islet cells and cells arising with an accelerated timecourse following an induction approach. These studies revealed previously unappreciated fine dynamic protrusions projecting between neighboring and distant endocrine cells. Using pharmacological compound and toxin interference approaches, and single-cell analysis of morphology and cell dynamics, we determined that endocrine cell motility is regulated by phosphoinositide 3-kinase (PI3K) and G-protein-coupled receptor (GPCR) signaling. Linking cell dynamics to islet formation, perturbation of protrusion formation disrupted endocrine cell coalescence, and correlated with decreased islet cell differentiation. These studies identified novel cell behaviors contributing to islet morphogenesis, and suggest a model in which dynamic exploratory filopodia establish cell-cell contacts that subsequently promote cell clustering. PMID:29386244

Reconstructing the regulatory circuit of cell fate determination in yeast mating response.

PubMed

Shao, Bin; Yuan, Haiyu; Zhang, Rongfei; Wang, Xuan; Zhang, Shuwen; Ouyang, Qi; Hao, Nan; Luo, Chunxiong

2017-07-01

Massive technological advances enabled high-throughput measurements of proteomic changes in biological processes. However, retrieving biological insights from large-scale protein dynamics data remains a challenging task. Here we used the mating differentiation in yeast Saccharomyces cerevisiae as a model and developed integrated experimental and computational approaches to analyze the proteomic dynamics during the process of cell fate determination. When exposed to a high dose of mating pheromone, the yeast cell undergoes growth arrest and forms a shmoo-like morphology; however, at intermediate doses, chemotropic elongated growth is initialized. To understand the gene regulatory networks that control this differentiation switch, we employed a high-throughput microfluidic imaging system that allows real-time and simultaneous measurements of cell growth and protein expression. Using kinetic modeling of protein dynamics, we classified the stimulus-dependent changes in protein abundance into two sources: global changes due to physiological alterations and gene-specific changes. A quantitative framework was proposed to decouple gene-specific regulatory modes from the growth-dependent global modulation of protein abundance. Based on the temporal patterns of gene-specific regulation, we established the network architectures underlying distinct cell fates using a reverse engineering method and uncovered the dose-dependent rewiring of gene regulatory network during mating differentiation. Furthermore, our results suggested a potential crosstalk between the pheromone response pathway and the target of rapamycin (TOR)-regulated ribosomal biogenesis pathway, which might underlie a cell differentiation switch in yeast mating response. In summary, our modeling approach addresses the distinct impacts of the global and gene-specific regulation on the control of protein dynamics and provides new insights into the mechanisms of cell fate determination. We anticipate that our integrated experimental and modeling strategies could be widely applicable to other biological systems.

On-chip dynamic stress control for cancer cell evolution study

NASA Astrophysics Data System (ADS)

Liu, Liyu; Austin, Robert

2010-03-01

The growth and spreading of cancer in host organisms is an evolutionary process. Cells accumulate mutations that help them adapt to changing environments and to obtain survival fitness. However, all cancer--promoting mutations do not occur at once. Cancer cells face selective environmental pressures that drive their evolution in stages. In traditional cancer studies, environmental stress is usually homogenous in space and difficult to change in time. Here, we propose a microfluidic chip employing embedded dynamic traps to generate dynamic heterogeneous microenvironments for cancer cells in evolution studies. Based on polydimethylsiloxane (PDMS) flexible diaphragms, these traps are able to enclose and shield cancer cells or expose them to external environmental stress. Digital controls for each trap determine the nutrition, antibiotics, CO2/O2 conditions, and temperatures to which trapped cells are subjected. Thus, the stress applied to cells can be varied in intensity and duration in each trap independently. The chip can also output cells from specific traps for sequencing and other biological analysis. Hence our design simultaneously monitors and analyzes cell evolution behaviors under dynamic stresses.

Live Imaging to Study Microtubule Dynamic Instability in Taxane-resistant Breast Cancers.

PubMed

Wang, Richard; Wang, Harris; Wang, Zhixiang

2017-02-20

Taxanes such as docetaxel belong to a group of microtubule-targeting agents (MTAs) that are commonly relied upon to treat cancer. However, taxane resistance in cancerous cells drastically reduces the effectiveness of the drugs' long-term usage. Accumulated evidence suggests that the mechanisms underlying taxane resistance include both general mechanisms, such as the development of multidrug resistance due to the overexpression of drug-efflux proteins, and taxane-specific mechanisms, such as those that involve microtubule dynamics. Because taxanes target cell microtubules, measuring microtubule dynamic instability is an important step in determining the mechanisms of taxane resistance and provides insight into how to overcome this resistance. In the experiment, an in vivo method was used to measure microtubule dynamic instability. GFP-tagged α-tubulin was expressed and incorporated into microtubules in MCF-7 cells, allowing for the recording of the microtubule dynamics by time lapse using a sensitive camera. The results showed that, as opposed to the non-resistant parental MCF-7CC cells, the microtubule dynamics of docetaxel-resistant MCF-7TXT cells are insensitive to docetaxel treatment, which causes the resistance to docetaxel-induced mitotic arrest and apoptosis. This paper will outline this in vivo method of measuring microtubule dynamic instability.

Collective Calcium Signaling of Defective Multicellular Networks

NASA Astrophysics Data System (ADS)

Potter, Garrett; Sun, Bo

2015-03-01

A communicating multicellular network processes environmental cues into collective cellular dynamics. We have previously demonstrated that, when excited by extracellular ATP, fibroblast monolayers generate correlated calcium dynamics modulated by both the stimuli and gap junction communication between the cells. However, just as a well-connected neural network may be compromised by abnormal neurons, a tissue monolayer can also be defective with cancer cells, which typically have down regulated gap junctions. To understand the collective cellular dynamics in a defective multicellular network we have studied the calcium signaling of co-cultured breast cancer cells and fibroblast cells in various concentrations of ATP delivered through microfluidic devices. Our results demonstrate that cancer cells respond faster, generate singular spikes, and are more synchronous across all stimuli concentrations. Additionally, fibroblast cells exhibit persistent calcium oscillations that increase in regularity with greater stimuli. To interpret these results we quantitatively analyzed the immunostaining of purigenic receptors and gap junction channels. The results confirm our hypothesis that collective dynamics are mainly determined by the availability of gap junction communications.

Cell wall elongation mode in Gram-negative bacteria is determined by peptidoglycan architecture.

PubMed

Turner, Robert D; Hurd, Alexander F; Cadby, Ashley; Hobbs, Jamie K; Foster, Simon J

2013-01-01

Cellular integrity and morphology of most bacteria is maintained by cell wall peptidoglycan, the target of antibiotics essential in modern healthcare. It consists of glycan strands, cross-linked by peptides, whose arrangement determines cell shape, prevents lysis due to turgor pressure and yet remains dynamic to allow insertion of new material, and hence growth. The cellular architecture and insertion pattern of peptidoglycan have remained elusive. Here we determine the peptidoglycan architecture and dynamics during growth in rod-shaped Gram-negative bacteria. Peptidoglycan is made up of circumferentially oriented bands of material interspersed with a more porous network. Super-resolution fluorescence microscopy reveals an unexpected discontinuous, patchy synthesis pattern. We present a consolidated model of growth via architecture-regulated insertion, where we propose only the more porous regions of the peptidoglycan network that are permissive for synthesis.

Characterization of Solid Polymers, Ceramic Gap Filler, and Closed-Cell Polymer Foam Using Low-Load Test Methods

NASA Technical Reports Server (NTRS)

Herring, Helen M.

2008-01-01

Various solid polymers, polymer-based composites, and closed-cell polymer foam are being characterized to determine their mechanical properties, using low-load test methods. The residual mechanical properties of these materials after environmental exposure or extreme usage conditions determines their value in aerospace structural applications. In this experimental study, four separate polymers were evaluated to measure their individual mechanical responses after thermal aging and moisture exposure by dynamic mechanical analysis. A ceramic gap filler, used in the gaps between the tiles on the Space Shuttle, was also tested, using dynamic mechanical analysis to determine material property limits during flight. Closed-cell polymer foam, used for the Space Shuttle External Tank insulation, was tested under low load levels to evaluate how the foam's mechanical properties are affected by various loading and unloading scenarios.

Coherent organization in gene regulation: a study on six networks

NASA Astrophysics Data System (ADS)

Aral, Neşe; Kabakçıoğlu, Alkan

2016-04-01

Structural and dynamical fingerprints of evolutionary optimization in biological networks are still unclear. Here we analyze the dynamics of genetic regulatory networks responsible for the regulation of cell cycle and cell differentiation in three organisms or cell types each, and show that they follow a version of Hebb's rule which we have termed coherence. More precisely, we find that simultaneously expressed genes with a common target are less likely to act antagonistically at the attractors of the regulatory dynamics. We then investigate the dependence of coherence on structural parameters, such as the mean number of inputs per node and the activatory/repressory interaction ratio, as well as on dynamically determined quantities, such as the basin size and the number of expressed genes.

Singularity and stability in a periodic system of particle accelerators

NASA Astrophysics Data System (ADS)

Cai, Yunhai

2018-05-01

We study the single-particle dynamics in a general and parametrized alternating-gradient cell with zero chromaticity using the Lie algebra method. To our surprise, the first-order perturbation of the sextupoles largely determines the dynamics away from the major resonances. The dynamic aperture can be estimated from the topology and geometry of the phase space. In the linearly normalized phase space, it is scaled according to A ¯ ∝ϕ √{L } , where ϕ is the bending angle and L the length of the cell. For the 2 degrees of freedom with equal betatron tunes, the analytical perturbation theory leads us to the invariant or quasi-invariant tori, which play an important role in determining the stable volume in the four-dimensional phase space.

HIV dynamics with multiple infections of target cells.

PubMed

Dixit, Narendra M; Perelson, Alan S

2005-06-07

The high incidence of multiple infections of cells by HIV sets the stage for rapid HIV evolution by means of recombination. Yet how HIV dynamics proceeds with multiple infections remains poorly understood. Here, we present a mathematical model that describes the dynamics of viral, target cell, and multiply infected cell subpopulations during HIV infection. Model calculations reproduce several experimental observations and provide key insights into the influence of multiple infections on HIV dynamics. We find that the experimentally observed scaling law, that the number of cells coinfected with two distinctly labeled viruses is proportional to the square of the total number of infected cells, can be generalized so that the number of triply infected cells is proportional to the cube of the number of infected cells, etc. Despite the expectation from Poisson statistics, we find that this scaling relationship only holds under certain conditions, which we predict. We also find that multiple infections do not influence viral dynamics when the rate of viral production from infected cells is independent of the number of times the cells are infected, a regime expected when viral production is limited by cellular rather than viral factors. This result may explain why extant models, which ignore multiple infections, successfully describe viral dynamics in HIV patients. Inhibiting CD4 down-modulation increases the average number of infections per cell. Consequently, altering CD4 down-modulation may allow for an experimental determination of whether viral or cellular factors limit viral production.

HIV dynamics with multiple infections of target cells

PubMed Central

Dixit, Narendra M.; Perelson, Alan S.

2005-01-01

The high incidence of multiple infections of cells by HIV sets the stage for rapid HIV evolution by means of recombination. Yet how HIV dynamics proceeds with multiple infections remains poorly understood. Here, we present a mathematical model that describes the dynamics of viral, target cell, and multiply infected cell subpopulations during HIV infection. Model calculations reproduce several experimental observations and provide key insights into the influence of multiple infections on HIV dynamics. We find that the experimentally observed scaling law, that the number of cells coinfected with two distinctly labeled viruses is proportional to the square of the total number of infected cells, can be generalized so that the number of triply infected cells is proportional to the cube of the number of infected cells, etc. Despite the expectation from Poisson statistics, we find that this scaling relationship only holds under certain conditions, which we predict. We also find that multiple infections do not influence viral dynamics when the rate of viral production from infected cells is independent of the number of times the cells are infected, a regime expected when viral production is limited by cellular rather than viral factors. This result may explain why extant models, which ignore multiple infections, successfully describe viral dynamics in HIV patients. Inhibiting CD4 down-modulation increases the average number of infections per cell. Consequently, altering CD4 down-modulation may allow for an experimental determination of whether viral or cellular factors limit viral production. PMID:15928092

Monomer–dimer dynamics and distribution of GPI-anchored uPAR are determined by cell surface protein assemblies

PubMed Central

Caiolfa, Valeria R.; Zamai, Moreno; Malengo, Gabriele; Andolfo, Annapaola; Madsen, Chris D.; Sutin, Jason; Digman, Michelle A.; Gratton, Enrico; Blasi, Francesco; Sidenius, Nicolai

2007-01-01

To search for functional links between glycosylphosphatidylinositol (GPI) protein monomer–oligomer exchange and membrane dynamics and confinement, we studied urokinase plasminogen activator (uPA) receptor (uPAR), a GPI receptor involved in the regulation of cell adhesion, migration, and proliferation. Using a functionally active fluorescent protein–uPAR in live cells, we analyzed the effect that extracellular matrix proteins and uPAR ligands have on uPAR dynamics and dimerization at the cell membrane. Vitronectin directs the recruitment of dimers and slows down the diffusion of the receptors at the basal membrane. The commitment to uPA–plasminogen activator inhibitor type 1–mediated endocytosis and recycling modifies uPAR diffusion and induces an exchange between uPAR monomers and dimers. This exchange is fully reversible. The data demonstrate that cell surface protein assemblies are important in regulating the dynamics and localization of uPAR at the cell membrane and the exchange of monomers and dimers. These results also provide a strong rationale for dynamic studies of GPI-anchored molecules in live cells at steady state and in the absence of cross-linker/clustering agents. PMID:18056417

Chaotic electrical activity of living β-cells in the mouse pancreatic islet

NASA Astrophysics Data System (ADS)

Kanno, Takahiro; Miyano, Takaya; Tokuda, Isao; Galvanovskis, Juris; Wakui, Makoto

2007-02-01

To test for chaotic dynamics of the insulin producing β-cell and explore its biological role, we observed the action potentials with the perforated patch clamp technique, for isolated cells as well as for intact cells of the mouse pancreatic islet. The time series obtained were analyzed using nonlinear diagnostic algorithms associated with the surrogate method. The isolated cells exhibited short-term predictability and visible determinism, in the steady state response to 10 mM glucose, while the intact cells did not. In the latter case, determinism became visible after the application of a gap junction inhibitor. This tendency was enhanced by the stimulation with tolbutamide. Our observations suggest that, thanks to the integration of individual chaotic dynamics via gap junction coupling, the β-cells will lose memory of fluctuations occurring at any instant in their electrical activity more rapidly with time. This is likely to contribute to the functional stability of the islet against uncertain perturbations.

A computational approach for inferring the cell wall properties that govern guard cell dynamics.

PubMed

Woolfenden, Hugh C; Bourdais, Gildas; Kopischke, Michaela; Miedes, Eva; Molina, Antonio; Robatzek, Silke; Morris, Richard J

2017-10-01

Guard cells dynamically adjust their shape in order to regulate photosynthetic gas exchange, respiration rates and defend against pathogen entry. Cell shape changes are determined by the interplay of cell wall material properties and turgor pressure. To investigate this relationship between turgor pressure, cell wall properties and cell shape, we focused on kidney-shaped stomata and developed a biomechanical model of a guard cell pair. Treating the cell wall as a composite of the pectin-rich cell wall matrix embedded with cellulose microfibrils, we show that strong, circumferentially oriented fibres are critical for opening. We find that the opening dynamics are dictated by the mechanical stress response of the cell wall matrix, and as the turgor rises, the pectinaceous matrix stiffens. We validate these predictions with stomatal opening experiments in selected Arabidopsis cell wall mutants. Thus, using a computational framework that combines a 3D biomechanical model with parameter optimization, we demonstrate how to exploit subtle shape changes to infer cell wall material properties. Our findings reveal that proper stomatal dynamics are built on two key properties of the cell wall, namely anisotropy in the form of hoop reinforcement and strain stiffening. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd and Society for Experimental Biology.

Flow cells for bioanalytical and bioprocess applications with optimized dynamic response and flow characteristics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lancaster, V.R.; Modlin, D.N.

1994-12-31

In this study, the authors present a method for design and characterization of flow cells developed for minimum flow volume and optimal dynamic response with a given central observation area. The dynamic response of a circular shaped dual ported flow cell was compared to that obtained from a flow cell whose optimized shape was determined using this method. In the optimized flow cell design, the flow rate at the nominal operating pressure increased by 50% whereas the flow cell volume was reduced by 70%. In addition, the dynamic response of the new flow cell was found to be 200% fastermore » than the circular flow cell. The fluid dynamic analysis included simple graphical techniques utilizing free stream vorticity functions and Hagen-Poiseuille relationships. The flow cell dynamic response was measured using a fluorescence detection system. The fluoresce in emission from a 400{micro}m spot located at the exit port was measured as a function of time after switching the input to the flow cell between fluorescent and non-fluorescent solutions. Analysis of results revealed the system could be reasonably characterized as a first order dynamic system. Although some evidence of second order behavior was also observed, it is reasonable to assume that a first order model will provide adequate predictive capability for many real world applications. Given a set of flow cell requirements, the methods presented in this study can be used to design and characterize flow cells with lower reagent consumption and reduced purging times. These improvements can be readily translated into reduced process times and/or lower usage of high cost reagents.« less

Determination of Orbiter and Carrier Aerodynamic Coefficients from Load Cell Measurements

NASA Technical Reports Server (NTRS)

Glenn, G. M.

1976-01-01

A method of determining orbiter and carrier total aerodynamic coefficients from load cell measurements is required to support the inert and the captive active flights of the ALT program. A set of equations expressing the orbiter and carrier total aerodynamic coefficients in terms of the load cell measurements, the sensed dynamics of the Boeing 747 (carrier) aircraft, and the relative geometry of the orbiter/carrier is derived.

The dynamic and geometric phase transition in the cellular network of pancreatic islet

NASA Astrophysics Data System (ADS)

Wang, Xujing

2013-03-01

The pancreatic islet is a micro-organ that contains several thousands of endocrine cells, majority of which being the insulin releasing β - cells . - cellsareexcitablecells , andarecoupledtoeachother through gap junctional channels. Here, using percolation theory, we investigate the role of network structure in determining the dynamics of the β-cell network. We show that the β-cell synchronization depends on network connectivity. More specifically, as the site occupancy is reducing, initially the β-cell synchronization is barely affected, until it reaches around a critical value, where the synchronization exhibit a sudden rapid decline, followed by an slow exponential tail. This critical value coincides with the critical site open probability for percolation transition. The dependence over bond strength is similar, exhibiting critical-behavior like dependence around a certain value of bond strength. These results suggest that the β-cell network undergoes a dynamic phase transition when the network is percolated. We further apply the findings to study diabetes. During the development of diabetes, the β - cellnetworkconnectivitydecreases . Siteoccupancyreducesfromthe reducing β-cell mass, and the bond strength is increasingly impaired from β-cell stress and chronic hyperglycemia. We demonstrate that the network dynamics around the percolation transition explain the disease dynamics around onset, including a long time mystery in diabetes, the honeymoon phenomenon.

Isotope labeling to determine the dynamics of metabolic response in CHO cell perfusion bioreactors using MALDI-TOF-MS.

PubMed

Karst, Daniel J; Steinhoff, Robert F; Kopp, Marie R G; Soos, Miroslav; Zenobi, Renato; Morbidelli, Massimo

2017-11-01

The steady-state operation of Chinese hamster ovary (CHO) cells in perfusion bioreactors requires the equilibration of reactor dynamics and cell metabolism. Accordingly, in this work we investigate the transient cellular response to changes in its environment and their interactions with the bioreactor hydrodynamics. This is done in a benchtop perfusion bioreactor using MALDI-TOF MS through isotope labeling of complex intracellular nucleotides (ATP, UTP) and nucleotide sugars (UDP-Hex, UDP-HexNAc). By switching to a 13 C 6 glucose containing feed media during constant operation at 20 × 10 6 cells and a perfusion rate of 1 reactor volume per day, isotopic steady state was studied. A step change to the 13 C 6 glucose medium in spin tubes allowed the determination of characteristic times for the intracellular turnover of unlabeled metabolites pools, τST (≤0.56 days), which were confirmed in the bioreactor. On the other hand, it is shown that the reactor residence time τR (1 day) and characteristic time for glucose uptake τGlc (0.33 days), representative of the bioreactor dynamics, delayed the consumption of 13 C 6 glucose in the bioreactor and thus the intracellular 13 C enrichment. The proposed experimental approach allowed the decoupling of bioreactor hydrodynamics and intrinsic dynamics of cell metabolism in response to a change in the cell culture environment. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1630-1639, 2017. © 2017 American Institute of Chemical Engineers.

Dynamics of human T-cell lymphotropic virus I (HTLV-I) infection of CD4+ T-cells.

PubMed

Katri, Patricia; Ruan, Shigui

2004-11-01

Stilianakis and Seydel (Bull. Math. Biol., 1999) proposed an ODE model that describes the T-cell dynamics of human T-cell lymphotropic virus I (HTLV-I) infection and the development of adult T-cell leukemia (ATL). Their model consists of four components: uninfected healthy CD4+ T-cells, latently infected CD4+ T-cells, actively infected CD4+ T-cells, and ATL cells. Mathematical analysis that completely determines the global dynamics of this model has been done by Wang et al. (Math. Biosci., 2002). In this note, we first modify the parameters of the model to distinguish between contact and infectivity rates. Then we introduce a discrete time delay to the model to describe the time between emission of contagious particles by active CD4+ T-cells and infection of pure cells. Using the results in Culshaw and Ruan (Math. Biosci., 2000) in the analysis of time delay with respect to cell-free viral spread of HIV, we study the effect of time delay on the stability of the endemically infected equilibrium. Numerical simulations are presented to illustrate the results.

Initiation of DNA replication requires actin dynamics and formin activity.

PubMed

Parisis, Nikolaos; Krasinska, Liliana; Harker, Bethany; Urbach, Serge; Rossignol, Michel; Camasses, Alain; Dewar, James; Morin, Nathalie; Fisher, Daniel

2017-11-02

Nuclear actin regulates transcriptional programmes in a manner dependent on its levels and polymerisation state. This dynamics is determined by the balance of nucleocytoplasmic shuttling, formin- and redox-dependent filament polymerisation. Here, using Xenopus egg extracts and human somatic cells, we show that actin dynamics and formins are essential for DNA replication. In proliferating cells, formin inhibition abolishes nuclear transport and initiation of DNA replication, as well as general transcription. In replicating nuclei from transcriptionally silent Xenopus egg extracts, we identified numerous actin regulators, and disruption of actin dynamics abrogates nuclear transport, preventing NLS (nuclear localisation signal)-cargo release from RanGTP-importin complexes. Nuclear formin activity is further required to promote loading of cyclin-dependent kinase (CDK) and proliferating cell nuclear antigen (PCNA) onto chromatin, as well as initiation and elongation of DNA replication. Therefore, actin dynamics and formins control DNA replication by multiple direct and indirect mechanisms. © 2017 The Authors.

Development and Characterization of a Parallelizable Perfusion Bioreactor for 3D Cell Culture.

PubMed

Egger, Dominik; Fischer, Monica; Clementi, Andreas; Ribitsch, Volker; Hansmann, Jan; Kasper, Cornelia

2017-05-25

The three dimensional (3D) cultivation of stem cells in dynamic bioreactor systems is essential in the context of regenerative medicine. Still, there is a lack of bioreactor systems that allow the cultivation of multiple independent samples under different conditions while ensuring comprehensive control over the mechanical environment. Therefore, we developed a miniaturized, parallelizable perfusion bioreactor system with two different bioreactor chambers. Pressure sensors were also implemented to determine the permeability of biomaterials which allows us to approximate the shear stress conditions. To characterize the flow velocity and shear stress profile of a porous scaffold in both bioreactor chambers, a computational fluid dynamics analysis was performed. Furthermore, the mixing behavior was characterized by acquisition of the residence time distributions. Finally, the effects of the different flow and shear stress profiles of the bioreactor chambers on osteogenic differentiation of human mesenchymal stem cells were evaluated in a proof of concept study. In conclusion, the data from computational fluid dynamics and shear stress calculations were found to be predictable for relative comparison of the bioreactor geometries, but not for final determination of the optimal flow rate. However, we suggest that the system is beneficial for parallel dynamic cultivation of multiple samples for 3D cell culture processes.

Development and Characterization of a Parallelizable Perfusion Bioreactor for 3D Cell Culture

PubMed Central

Egger, Dominik; Fischer, Monica; Clementi, Andreas; Ribitsch, Volker; Hansmann, Jan; Kasper, Cornelia

2017-01-01

The three dimensional (3D) cultivation of stem cells in dynamic bioreactor systems is essential in the context of regenerative medicine. Still, there is a lack of bioreactor systems that allow the cultivation of multiple independent samples under different conditions while ensuring comprehensive control over the mechanical environment. Therefore, we developed a miniaturized, parallelizable perfusion bioreactor system with two different bioreactor chambers. Pressure sensors were also implemented to determine the permeability of biomaterials which allows us to approximate the shear stress conditions. To characterize the flow velocity and shear stress profile of a porous scaffold in both bioreactor chambers, a computational fluid dynamics analysis was performed. Furthermore, the mixing behavior was characterized by acquisition of the residence time distributions. Finally, the effects of the different flow and shear stress profiles of the bioreactor chambers on osteogenic differentiation of human mesenchymal stem cells were evaluated in a proof of concept study. In conclusion, the data from computational fluid dynamics and shear stress calculations were found to be predictable for relative comparison of the bioreactor geometries, but not for final determination of the optimal flow rate. However, we suggest that the system is beneficial for parallel dynamic cultivation of multiple samples for 3D cell culture processes. PMID:28952530

Nonlinear dynamics and damage induced properties of soft matter with application in oncology

NASA Astrophysics Data System (ADS)

Naimark, O.

2017-09-01

Molecular-morphological signs of oncogenesis could be linked to multiscale collective effects in molecular, cell and tissue related to defects (damage) dynamics. It was shown that nonlinear behavior of biological systems can be linked to the existence of characteristic collective open state modes providing the coherent expression dynamics. New type of criticality in nonequilibrium systems with defects—structural-scaling transition allows the definition of the `driving force' for a biological soft matter related to consolidated open states. The set of collective open states (breathers, autosolitons and blow-up modes) in the molecular ensembles provides the collective expression dynamics to attract the entire system (cell, tissue) toward a few preferred global states. The co-existence of three types of collective modes determines the multifractal scenario of biological soft matter dynamics. The appearance of `globally convergent' dynamics corresponding to the coherent behavior of multiscale blow-up open states (blow-up gene expression) leads to anomalous localized softening (blow-up localized damage) and the subjection of the cells (or tissue) to monofractal dynamics. This dynamics can be associated with cancer progression.

A note on the effects of replenishment of depleted cells on HIV infection dynamics: A graph-theoretic approach

NASA Astrophysics Data System (ADS)

Mukwembi, Simon

2008-02-01

We study the effects of the rate of replacement of dead cells by either healthy cells or by infected cells on HIV infection dynamics through a graph-theoretic approach. Our framework takes into account a reasonable amount of the immune action to any pathogen and the local cell interactions that occur in the lymph nodes. Our results, in an extremal case where dead cells are highly likely to be replaced by healthy cells, show that all cells become healthy in a finite number of steps of given order and infection stops propagating. Further, for this extremal case, we give an algebraic formula for the number of infected cells at any given time in the HIV progression. We also find a sufficient condition, determined by dead cell replacement rate, which guarantees that an infected patient is continually positive, and give bounds on the number of infected, healthy and dead cells at any given time. We apply our theoretical results to a recently proposed model of the HIV infection dynamics.

Retinal Determination genes function along with cell-cell signals to regulate Drosophila eye development: examples of multi-layered regulation by Master Regulators

PubMed Central

Baker, Nicholas E.; Firth, Lucy C.

2015-01-01

It is thought that Retinal Determination gene products define the response made to cell-cell signals within the eye developmental field by binding to enhancers of genes that are also regulated by cell-cell signaling pathways. In Drosophila, Retinal Determination genes including Eyeless, teashirt, eyes absent, dachsous and sine oculis, are required for normal eye development and can induce ectopic eyes when mis-expressed. Characterization of the enhancers responsible for eye expression of the hedgehog, shaven, and atonal genes, as well as the dynamics of Retinal Determination gene expression themselves, now suggest a multilayered network whereby transcriptional regulation by either Retinal Determination genes or cell-cell signaling pathways can sometimes be indirect and mediated by other transcription factor intermediates. In this updated view of the interaction between extracellular information and cell intrinsic programs during development, regulation of individual genes might sometimes be several steps removed from either the Retinal Determination genes or cell-cell signaling pathways that nevertheless govern their expression. PMID:21607995

Coarse-grained hydrodynamics from correlation functions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Palmer, Bruce

This paper will describe a formalism for using correlation functions between different grid cells as the basis for determining coarse-grained hydrodynamic equations for modeling the behavior of mesoscopic fluid systems. Configuration from a molecular dynamics simulation are projected onto basis functions representing grid cells in a continuum hydrodynamic simulation. Equilbrium correlation functions between different grid cells are evaluated from the molecular simulation and used to determine the evolution operator for the coarse-grained hydrodynamic system. The formalism is applied to some simple hydrodynamic cases to determine the feasibility of applying this to realistic nanoscale systems.

Analysis of population structures of the microalga Acutodesmus obliquus during lipid production using multi-dimensional single-cell analysis.

PubMed

Sandmann, Michael; Schafberg, Michaela; Lippold, Martin; Rohn, Sascha

2018-04-19

Microalgae bear a great potential to produce lipids for biodiesel, feed, or even food applications. To understand the still not well-known single-cell dynamics during lipid production in microalgae, a novel single-cell analytical technology was applied to study a well-established model experiment. Multidimensional single-cell dynamics were investigated with a non-supervised image analysis technique that utilizes data from epi-fluorescence microscopy. Reliability of this technique was successfully proven via reference analysis. The technique developed was used to determine cell size, chlorophyll amount, neutral lipid amount, and deriving properties on a single-cellular level in cultures of the biotechnologically promising alga Acutodesmus obliquus. The results illustrated a high correlation between cell size and chlorophyll amount, but a very low and dynamic correlation between cell size, lipid amount, and lipid density. During growth conditions under nitrogen starvation, cells with low chlorophyll content tend to start the lipid production first and the cell suspension differentiated in two subpopulations with significantly different lipid contents. Such quantitative characterization of single-cell dynamics of lipid synthesizing algae was done for the first time and the potential of such simple technology is highly relevant to other biotechnological applications and to deeper investigate the process of microalgal lipid accumulation.

Development of a new type of high pressure calorimetric cell, mechanically agitated and equipped with a dynamic pressure control system: Application to the characterization of gas hydrates

NASA Astrophysics Data System (ADS)

Plantier, F.; Marlin, L.; Missima, D.; Torré, J.-P.

2013-12-01

A novel prototype of calorimetric cell has been developed allowing experiments under pressure with an in situ agitation system and a dynamic control of the pressure inside the cell. The use of such a system opens a wide range of potential practical applications for determining properties of complex fluids in both pressurized and agitated conditions. The technical details of this prototype and its calibration procedure are described, and an application devoted to the determination of phase equilibrium and phase change enthalpy of gas hydrates is presented. Our results, obtained with a good precision and reproducibility, were found in fairly good agreement with those found in literature, illustrate the various interests to use this novel apparatus.

Probing the stochastic property of endoreduplication in cell size determination of Arabidopsis thaliana leaf epidermal tissue

PubMed Central

2017-01-01

Cell size distribution is highly reproducible, whereas the size of individual cells often varies greatly within a tissue. This is obvious in a population of Arabidopsis thaliana leaf epidermal cells, which ranged from 1,000 to 10,000 μm2 in size. Endoreduplication is a specialized cell cycle in which nuclear genome size (ploidy) is doubled in the absence of cell division. Although epidermal cells require endoreduplication to enhance cellular expansion, the issue of whether this mechanism is sufficient for explaining cell size distribution remains unclear due to a lack of quantitative understanding linking the occurrence of endoreduplication with cell size diversity. Here, we addressed this question by quantitatively summarizing ploidy profile and cell size distribution using a simple theoretical framework. We first found that endoreduplication dynamics is a Poisson process through cellular maturation. This finding allowed us to construct a mathematical model to predict the time evolution of a ploidy profile with a single rate constant for endoreduplication occurrence in a given time. We reproduced experimentally measured ploidy profile in both wild-type leaf tissue and endoreduplication-related mutants with this analytical solution, further demonstrating the probabilistic property of endoreduplication. We next extended the mathematical model by incorporating the element that cell size is determined according to ploidy level to examine cell size distribution. This analysis revealed that cell size is exponentially enlarged 1.5 times every endoreduplication round. Because this theoretical simulation successfully recapitulated experimentally observed cell size distributions, we concluded that Poissonian endoreduplication dynamics and exponential size-boosting are the sources of the broad cell size distribution in epidermal tissue. More generally, this study contributes to a quantitative understanding whereby stochastic dynamics generate steady-state biological heterogeneity. PMID:28926847

Tracking molecular dynamics without tracking: image correlation of photo-activation microscopy

NASA Astrophysics Data System (ADS)

Pandžić, Elvis; Rossy, Jérémie; Gaus, Katharina

2015-03-01

Measuring protein dynamics in the plasma membrane can provide insights into the mechanisms of receptor signaling and other cellular functions. To quantify protein dynamics on the single molecule level over the entire cell surface, sophisticated approaches such as single particle tracking (SPT), photo-activation localization microscopy (PALM) and fluctuation-based analysis have been developed. However, analyzing molecular dynamics of fluorescent particles with intermittent excitation and low signal-to-noise ratio present at high densities has remained a challenge. We overcame this problem by applying spatio-temporal image correlation spectroscopy (STICS) analysis to photo-activated (PA) microscopy time series. In order to determine under which imaging conditions this approach is valid, we simulated PA images of diffusing particles in a homogeneous environment and varied photo-activation, reversible blinking and irreversible photo-bleaching rates. Further, we simulated data with high particle densities that populated mobile objects (such as adhesions and vesicles) that often interfere with STICS and fluctuation-based analysis. We demonstrated in experimental measurements that the diffusion coefficient of the epidermal growth factor receptor (EGFR) fused to PAGFP in live COS-7 cells can be determined in the plasma membrane and revealed differences in the time-dependent diffusion maps between wild-type and mutant Lck in activated T cells. In summary, we have developed a new analysis approach for live cell photo-activation microscopy data based on image correlation spectroscopy to quantify the spatio-temporal dynamics of single proteins.

Tracking molecular dynamics without tracking: image correlation of photo-activation microscopy.

PubMed

Pandžić, Elvis; Rossy, Jérémie; Gaus, Katharina

2015-03-09

Measuring protein dynamics in the plasma membrane can provide insights into the mechanisms of receptor signaling and other cellular functions. To quantify protein dynamics on the single molecule level over the entire cell surface, sophisticated approaches such as single particle tracking (SPT), photo-activation localization microscopy (PALM) and fluctuation-based analysis have been developed. However, analyzing molecular dynamics of fluorescent particles with intermittent excitation and low signal-to-noise ratio present at high densities has remained a challenge. We overcame this problem by applying spatio-temporal image correlation spectroscopy (STICS) analysis to photo-activated (PA) microscopy time series. In order to determine under which imaging conditions this approach is valid, we simulated PA images of diffusing particles in a homogeneous environment and varied photo-activation, reversible blinking and irreversible photo-bleaching rates. Further, we simulated data with high particle densities that populated mobile objects (such as adhesions and vesicles) that often interfere with STICS and fluctuation-based analysis. We demonstrated in experimental measurements that the diffusion coefficient of the epidermal growth factor receptor (EGFR) fused to PAGFP in live COS-7 cells can be determined in the plasma membrane and revealed differences in the time-dependent diffusion maps between wild-type and mutant Lck in activated T cells. In summary, we have developed a new analysis approach for live cell photo-activation microscopy data based on image correlation spectroscopy to quantify the spatio-temporal dynamics of single proteins.

Cellular Biotechnology Operations Support System Fluid Dynamics Investigation

NASA Technical Reports Server (NTRS)

2003-01-01

Aboard the International Space Station (ISS), the Tissue Culture Medium (TCM) is the bioreactor vessel in which cell cultures are grown. With its two syringe ports, it is much like a bag used to administer intravenous fluid, except it allows gas exchange needed for life. The TCM contains cell culture medium, and when frozen cells are flown to the ISS, they are thawed and introduced to the TCM through the syringe ports. In the Cellular Biotechnology Operations Support System-Fluid Dynamics Investigation (CBOSS-FDI) experiment, several mixing procedures are being assessed to determine which method achieves the most uniform mixing of growing cells and culture medium.

Predicting human blood viscosity in silico

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fedosov, Dmitry A.; Pan, Wenxiao; Caswell, Bruce

2011-07-05

Cellular suspensions such as blood are a part of living organisms and their rheological and flow characteristics determine and affect majority of vital functions. The rheological and flow properties of cell suspensions are determined by collective dynamics of cells, their structure or arrangement, cell properties and interactions. We study these relations for blood in silico using a mesoscopic particle-based method and two different models (multi-scale/low-dimensional) of red blood cells. The models yield accurate quantitative predictions of the dependence of blood viscosity on shear rate and hematocrit. We explicitly model cell aggregation interactions and demonstrate the formation of reversible rouleaux structuresmore » resulting in a tremendous increase of blood viscosity at low shear rates and yield stress, in agreement with experiments. The non-Newtonian behavior of such cell suspensions (e.g., shear thinning, yield stress) is analyzed and related to the suspension’s microstructure, deformation and dynamics of single cells. We provide the flrst quantitative estimates of normal stress differences and magnitude of aggregation forces in blood. Finally, the flexibility of the cell models allows them to be employed for quantitative analysis of a much wider class of complex fluids including cell, capsule, and vesicle suspensions.« less

The role of telomere dynamics in aging and cancer

NASA Astrophysics Data System (ADS)

Blagoev, Krastan; Goodwin, Edwin

2006-03-01

Telomere length changes are far more dynamic than previously thought. In addition to a gradual loss of ˜100 base pairs per telomere in each cell division, losses as well as gains may occur within a single cell cycle. We are investigating how telomere exchange, extension, and deletion affect the proliferative potential of telomerase-negative somatic cells. Experimental techniques are being devised to detect dynamic telomere processes and quantify both the frequency and length changes of each. In parallel, a ``dynamic telomere model'' is being used that incorporates telomere dynamics to study how the telomere size distribution evolves with time. This is an essential step towards understanding the role that telomere dynamics play in the normal aging of tissues and organisms. The model casts light on relationships not otherwise easily explained by a deterministic ``mitotic clock,'' or to what extent the shortest initial telomere determines the onset of senescence. We also expect to identify biomarkers that will correlate with aging better than average telomere length and to shed light on the transition to unlimited growth found in telomerase-negative tumor cells having the ALT (alternative lengthening of telomeres) phenotype, and to evaluate strategies to suppress the growth of these tumors.

A high-throughput AO/PI-based cell concentration and viability detection method using the Celigo image cytometry.

PubMed

Chan, Leo Li-Ying; Smith, Tim; Kumph, Kendra A; Kuksin, Dmitry; Kessel, Sarah; Déry, Olivier; Cribbes, Scott; Lai, Ning; Qiu, Jean

2016-10-01

To ensure cell-based assays are performed properly, both cell concentration and viability have to be determined so that the data can be normalized to generate meaningful and comparable results. Cell-based assays performed in immuno-oncology, toxicology, or bioprocessing research often require measuring of multiple samples and conditions, thus the current automated cell counter that uses single disposable counting slides is not practical for high-throughput screening assays. In the recent years, a plate-based image cytometry system has been developed for high-throughput biomolecular screening assays. In this work, we demonstrate a high-throughput AO/PI-based cell concentration and viability method using the Celigo image cytometer. First, we validate the method by comparing directly to Cellometer automated cell counter. Next, cell concentration dynamic range, viability dynamic range, and consistency are determined. The high-throughput AO/PI method described here allows for 96-well to 384-well plate samples to be analyzed in less than 7 min, which greatly reduces the time required for the single sample-based automated cell counter. In addition, this method can improve the efficiency for high-throughput screening assays, where multiple cell counts and viability measurements are needed prior to performing assays such as flow cytometry, ELISA, or simply plating cells for cell culture.

Visualization of Membrane Pore in Live Cells Reveals a Dynamic-Pore Theory Governing Fusion and Endocytosis.

PubMed

Shin, Wonchul; Ge, Lihao; Arpino, Gianvito; Villarreal, Seth A; Hamid, Edaeni; Liu, Huisheng; Zhao, Wei-Dong; Wen, Peter J; Chiang, Hsueh-Cheng; Wu, Ling-Gang

2018-05-03

Fusion is thought to open a pore to release vesicular cargoes vital for many biological processes, including exocytosis, intracellular trafficking, fertilization, and viral entry. However, fusion pores have not been observed and thus proved in live cells. Its regulatory mechanisms and functions remain poorly understood. With super-resolution STED microscopy, we observed dynamic fusion pore behaviors in live (neuroendocrine) cells, including opening, expansion, constriction, and closure, where pore size may vary between 0 and 490 nm within 26 milliseconds to seconds (vesicle size: 180-720 nm). These pore dynamics crucially determine the efficiency of vesicular cargo release and vesicle retrieval. They are generated by competition between pore expansion and constriction. Pharmacology and mutation experiments suggest that expansion and constriction are mediated by F-actin-dependent membrane tension and calcium/dynamin, respectively. These findings provide the missing live-cell evidence, proving the fusion-pore hypothesis, and establish a live-cell dynamic-pore theory accounting for fusion, fission, and their regulation. Published by Elsevier Inc.

Cell Surface Mechanochemistry and the Determinants of Bleb Formation, Healing, and Travel Velocity

PubMed Central

Manakova, Kathryn; Yan, Huaming; Lowengrub, John; Allard, Jun

2016-01-01

Blebs are pressure-driven cell protrusions implicated in cellular functions such as cell division, apoptosis, and cell motility, including motility of protease-inhibited cancer cells. Because of their mechanical nature, blebs inform us about general cell-surface mechanics, including membrane dynamics, pressure propagation throughout the cytoplasm, and the architecture and dynamics of the actin cortex. Mathematical models including detailed fluid dynamics have previously been used to understand bleb expansion. Here, we develop mathematical models in two and three dimensions on longer timescales that recapitulate the full bleb life cycle, including both expansion and healing by cortex reformation, in terms of experimentally accessible biophysical parameters such as myosin contractility, osmotic pressure, and turnover of actin and ezrin. The model provides conditions under which blebbing occurs, and naturally gives rise to traveling blebs. The model predicts conditions under which blebs travel or remain stationary, as well as the bleb traveling velocity, a quantity that has remained elusive in previous models. As previous studies have used blebs as reporters of membrane tension and pressure dynamics within the cell, we have used our system to investigate various pressure equilibration models and dynamic, nonuniform membrane tension to account for the shape of a traveling bleb. We also find that traveling blebs tend to expand in all directions unless otherwise constrained. One possible constraint could be provided by spatial heterogeneity in, for example, adhesion density. PMID:27074688

Cortical actin nanodynamics determines nitric oxide release in vascular endothelium.

PubMed

Fels, Johannes; Jeggle, Pia; Kusche-Vihrog, Kristina; Oberleithner, Hans

2012-01-01

The release of the main vasodilator nitric oxide (NO) by the endothelial NO synthase (eNOS) is a hallmark of endothelial function. We aim at elucidating the underlying mechanism how eNOS activity depends on cortical stiffness (К(cortex)) of living endothelial cells. It is hypothesized that cortical actin dynamics determines К(cortex) and directly influences eNOS activity. By combined atomic force microscopy and fluorescence imaging we generated mechanical and optical sections of single living cells. This approach allows the discrimination between К(cortex) and bulk cell stiffness (К(bulk)) and, additionally, the simultaneous analysis of submembranous actin web dynamics. We show that К(cortex) softens when cortical F-actin depolymerizes and that this shift from a gel-like stiff cortex to a soft G-actin rich layer, triggers the stiffness-sensitive eNOS activity. The results implicate that stiffness changes in the ∼100 nm phase of the submembranous actin web, without affecting К(bulk), regulate NO release and thus determines endothelial function.

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions.

PubMed

Saha, Tanumoy; Rathmann, Isabel; Galic, Milos

2017-07-11

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

Sensing dynamic cytoplasm refractive index changes of adherent cells with quantitative phase microscopy using incorporated microspheres as optical probes.

PubMed

Przibilla, Sabine; Dartmann, Sebastian; Vollmer, Angelika; Ketelhut, Steffi; Greve, Burkhard; von Bally, Gert; Kemper, Björn

2012-09-01

The intracellular refractive index is an important parameter that describes the optical density of the cytoplasm and the concentration of the intracellular solutes. The refractive index of adherently grown cells is difficult to access. We present a method in which silica microspheres in living cells are used to determine the cytoplasm refractive index with quantitative phase microscopy. The reliability of our approach for refractive index retrieval is shown by data from a comparative study on osmotically stimulated adherent and suspended human pancreatic tumor cells. Results from adherent human fibro sarcoma cells demonstrate the capability of the method for sensing of dynamic refractive index changes and its usage with microfluidics.

Concise Review: Stem Cell Population Biology: Insights from Hematopoiesis.

PubMed

MacLean, Adam L; Lo Celso, Cristina; Stumpf, Michael P H

2017-01-01

Stem cells are fundamental to human life and offer great therapeutic potential, yet their biology remains incompletely-or in cases even poorly-understood. The field of stem cell biology has grown substantially in recent years due to a combination of experimental and theoretical contributions: the experimental branch of this work provides data in an ever-increasing number of dimensions, while the theoretical branch seeks to determine suitable models of the fundamental stem cell processes that these data describe. The application of population dynamics to biology is amongst the oldest applications of mathematics to biology, and the population dynamics perspective continues to offer much today. Here we describe the impact that such a perspective has made in the field of stem cell biology. Using hematopoietic stem cells as our model system, we discuss the approaches that have been used to study their key properties, such as capacity for self-renewal, differentiation, and cell fate lineage choice. We will also discuss the relevance of population dynamics in models of stem cells and cancer, where competition naturally emerges as an influential factor on the temporal evolution of cell populations. Stem Cells 2017;35:80-88. © 2016 AlphaMed Press.

Self similarities in desalination dynamics and performance using capacitive deionization.

PubMed

Ramachandran, Ashwin; Hemmatifar, Ali; Hawks, Steven A; Stadermann, Michael; Santiago, Juan G

2018-09-01

Charge transfer and mass transport are two underlying mechanisms which are coupled in desalination dynamics using capacitive deionization (CDI). We developed simple reduced-order models based on a mixed reactor volume principle which capture the coupled dynamics of CDI operation using closed-form semi-analytical and analytical solutions. We use the models to identify and explore self-similarities in the dynamics among flow rate, current, and voltage for CDI cell operation including both charging and discharging cycles. The similarity approach identifies the specific combination of cell (e.g. capacitance, resistance) and operational parameters (e.g. flow rate, current) which determine a unique effluent dynamic response. We here demonstrate self-similarity using a conventional flow between CDI (fbCDI) architecture, and we hypothesize that our similarity approach has potential application to a wide range of designs. We performed an experimental study of these dynamics and used well-controlled experiments of CDI cell operation to validate and explore limits of the model. For experiments, we used a CDI cell with five electrode pairs and a standard flow between (electrodes) architecture. Guided by the model, we performed a series of experiments that demonstrate natural response of the CDI system. We also identify cell parameters and operation conditions which lead to self-similar dynamics under a constant current forcing function and perform a series of experiments by varying flowrate, currents, and voltage thresholds to demonstrate self-similarity. Based on this study, we hypothesize that the average differential electric double layer (EDL) efficiency (a measure of ion adsorption rate to EDL charging rate) is mainly dependent on user-defined voltage thresholds, whereas flow efficiency (measure of how well desalinated water is recovered from inside the cell) depends on cell volumes flowed during charging, which is determined by flowrate, current and voltage thresholds. Results of experiments strongly support this hypothesis. Results show that cycle efficiency and salt removal for a given flowrate and current are maximum when average EDL and flow efficiencies are approximately equal. We further explored a range of CC operations with varying flowrates, currents, and voltage thresholds using our similarity variables to highlight trade-offs among salt removal, energy, and throughput performance. Copyright © 2018 Elsevier Ltd. All rights reserved.

System dynamics of subcellular transport.

PubMed

Chen, Vivien Y; Khersonsky, Sonya M; Shedden, Kerby; Chang, Young Tae; Rosania, Gus R

2004-01-01

In pharmacokinetic experiments, interpretations often hinge on treating cells as a "black box": a single, lumped compartment or boundary. Here, a combinatorial library of fluorescent small molecules was used to visualize subcellular transport pathways in living cells, using a kinetic, high content imaging system to monitor spatiotemporal variations of intracellular probe distribution. Most probes accumulate in cytoplasmic vesicles and probe kinetics conform to a nested, two-compartment dynamical system. At steady state, probes preferentially partition from the extracellular medium to the cytosol, and from the cytosol to cytoplasmic vesicles, with hydrophobic molecules favoring sequestration. Altogether, these results point to a general organizing principle underlying the system dynamics of subcellular, small molecule transport. In addition to plasma membrane permeability, subcellular transport phenomena can determine the active concentration of small molecules in the cytosol and the efflux of small molecules from cells. Fundamentally, direct observation of intracellular probe distribution challenges the simple boundary model of classical pharmacokinetics, which considers cells as static permeability barriers.

A central role for vesicle trafficking in epithelial neoplasia: Intracellular highways to carcinogenesis

PubMed Central

Goldenring, James R.

2014-01-01

Epithelial cell carcinogenesis involves the loss of polarity, alteration of polarized protein presentation, dynamic cell morphology changes, increased proliferation and increased cell motility and invasion. Elements of membrane vesicle trafficking underlie all of these processes. Specific membrane trafficking regulators, including Rab small GTPases, through the coordinated dynamics of intracellular trafficking along cytoskeletal pathways, determine cell surface presentation of proteins and overall function of both differentiated and neoplastic cells. While mutations in vesicle trafficking proteins may not be direct drivers of transformation, elements of the machinery of vesicle movement play critical roles in the phenotypes of neoplastic cells. Therefore, the regulators of membrane vesicle trafficking decisions are critical mediators of the full spectrum of cell physiologies driving cancer cell biology, including initial loss of polarity, invasion and metastasis. Targeting of these fundamental intracellular processes may provide important points for manipulation of cancer cell behaviour. PMID:24108097

Analyzing the dynamics of DNA replication in Mammalian cells using DNA combing.

PubMed

Bialic, Marta; Coulon, Vincent; Drac, Marjorie; Gostan, Thierry; Schwob, Etienne

2015-01-01

How cells duplicate their chromosomes is a key determinant of cell identity and genome stability. DNA replication can initiate from more than 100,000 sites distributed along mammalian chromosomes, yet a given cell uses only a subset of these origins due to inefficient origin activation and regulation by developmental or environmental cues. An impractical consequence of cell-to-cell variations in origin firing is that population-based techniques do not accurately describe how chromosomes are replicated in single cells. DNA combing is a biophysical DNA fiber stretching method which permits visualization of ongoing DNA synthesis along Mb-sized single-DNA molecules purified from cells that were previously pulse-labeled with thymidine analogues. This allows quantitative measurements of several salient features of chromosome replication dynamics, such as fork velocity, fork asymmetry, inter-origin distances, and global instant fork density. In this chapter we describe how to obtain this information from asynchronous cultures of mammalian cells.

Towards Predicting the Response of a Solid Tumour to Chemotherapy and Radiotherapy Treatments: Clinical Insights from a Computational Model

PubMed Central

Powathil, Gibin G.; Adamson, Douglas J. A.; Chaplain, Mark A. J.

2013-01-01

In this paper we use a hybrid multiscale mathematical model that incorporates both individual cell behaviour through the cell-cycle and the effects of the changing microenvironment through oxygen dynamics to study the multiple effects of radiation therapy. The oxygenation status of the cells is considered as one of the important prognostic markers for determining radiation therapy, as hypoxic cells are less radiosensitive. Another factor that critically affects radiation sensitivity is cell-cycle regulation. The effects of radiation therapy are included in the model using a modified linear quadratic model for the radiation damage, incorporating the effects of hypoxia and cell-cycle in determining the cell-cycle phase-specific radiosensitivity. Furthermore, after irradiation, an individual cell's cell-cycle dynamics are intrinsically modified through the activation of pathways responsible for repair mechanisms, often resulting in a delay/arrest in the cell-cycle. The model is then used to study various combinations of multiple doses of cell-cycle dependent chemotherapies and radiation therapy, as radiation may work better by the partial synchronisation of cells in the most radiosensitive phase of the cell-cycle. Moreover, using this multi-scale model, we investigate the optimum sequencing and scheduling of these multi-modality treatments, and the impact of internal and external heterogeneity on the spatio-temporal patterning of the distribution of tumour cells and their response to different treatment schedules. PMID:23874170

Temporal competition between differentiation programs determines cell fate choice

NASA Astrophysics Data System (ADS)

Kuchina, Anna; Espinar, Lorena; Cagatay, Tolga; Balbin, Alejandro; Alvarado, Alma; Garcia-Ojalvo, Jordi; Suel, Gurol

2011-03-01

During pluripotent differentiation, cells adopt one of several distinct fates. The dynamics of this decision-making process are poorly understood, since cell fate choice may be governed by interactions between differentiation programs that are active at the same time. We studied the dynamics of decision-making in the model organism Bacillus subtilis by simultaneously measuring the activities of competing differentiation programs (sporulation and competence) in single cells. We discovered a precise switch-like point of cell fate choice previously hidden by cell-cell variability. Engineered artificial crosslinks between competence and sporulation circuits revealed that the precision of this choice is generated by temporal competition between the key players of two differentiation programs. Modeling suggests that variable progression towards a switch-like decision might represent a general strategy to maximize adaptability and robustness of cellular decision-making.

Global asymptotic stability and hopf bifurcation for a blood cell production model.

PubMed

Crauste, Fabien

2006-04-01

We analyze the asymptotic stability of a nonlinear system of two differential equations with delay, describing the dynamics of blood cell produc- tion. This process takes place in the bone marrow, where stem cells differen- tiate throughout division in blood cells. Taking into account an explicit role of the total population of hematopoietic stem cells in the introduction of cells in cycle, we are led to study a characteristic equation with delay-dependent coefficients. We determine a necessary and sufficient condition for the global stability of the first steady state of our model, which describes the popula- tion's dying out, and we obtain the existence of a Hopf bifurcation for the only nontrivial positive steady state, leading to the existence of periodic solutions. These latter are related to dynamical diseases affecting blood cells known for their cyclic nature.

Notch1-Dll4 signaling and mechanical force regulate leader cell formation during collective cell migration

PubMed Central

Riahi, Reza; Sun, Jian; Wang, Shue; Long, Min; Zhang, Donna D.; Wong, Pak Kin

2015-01-01

At the onset of collective cell migration, a subset of cells within an initially homogenous population acquires a distinct “leader” phenotype with characteristic morphology and motility. However, the factors driving leader cell formation as well as the mechanisms regulating leader cell density during the migration process remain to be determined. Here, we use single cell gene expression analysis and computational modeling to show that leader cell identity is dynamically regulated by Dll4 signaling through both Notch1 and cellular stress in a migrating epithelium. Time-lapse microscopy reveals that Dll4 is induced in leader cells after the creation of the cell-free region and leader cells are regulated via Notch1-Dll4 lateral inhibition. Furthermore, mechanical stress inhibits Dll4 expression and leader cell formation in the monolayer. Collectively, our findings suggest that a reduction of mechanical force near the boundary promotes Notch1-Dll4 signaling to dynamically regulate the density of leader cells during collective cell migration. PMID:25766473

Application of Fourier transform infrared (FT-IR) spectroscopy in determination of microalgal compositions.

PubMed

Meng, Yingying; Yao, Changhong; Xue, Song; Yang, Haibo

2014-01-01

Fourier transform infrared spectroscopy (FT-IR) was applied in algal strain screening and monitoring cell composition dynamics in a marine microalga Isochrysis zhangjiangensis during algal cultivation. The content of lipid, carbohydrate and protein of samples determined by traditional methods had validated the accuracy of FT-IR method. For algal screening, the band absorption ratios of lipid/amide I and carbo/amide I from FT-IR measurements allowed for the selection of Isochrysis sp. and Tetraselmis subcordiformis as the most potential lipid and carbohydrate producers, respectively. The cell composition dynamics of I. zhangjiangensis measured by FT-IR revealed the diversion of carbon allocation from protein to carbohydrate and neutral lipid when nitrogen-replete cells were subjected to nitrogen limitation. The carbo/amide I band absorption ratio had also been demonstrated to depict physiological status under nutrient stress in T. subcordiformis. FT-IR serves as a tool for the simultaneous measurement of lipid, carbohydrate, and protein content in cell. Copyright © 2013 Elsevier Ltd. All rights reserved.

Digital signaling decouples activation probability and population heterogeneity.

PubMed

Kellogg, Ryan A; Tian, Chengzhe; Lipniacki, Tomasz; Quake, Stephen R; Tay, Savaş

2015-10-21

Digital signaling enhances robustness of cellular decisions in noisy environments, but it is unclear how digital systems transmit temporal information about a stimulus. To understand how temporal input information is encoded and decoded by the NF-κB system, we studied transcription factor dynamics and gene regulation under dose- and duration-modulated inflammatory inputs. Mathematical modeling predicted and microfluidic single-cell experiments confirmed that integral of the stimulus (or area, concentration × duration) controls the fraction of cells that activate NF-κB in the population. However, stimulus temporal profile determined NF-κB dynamics, cell-to-cell variability, and gene expression phenotype. A sustained, weak stimulation lead to heterogeneous activation and delayed timing that is transmitted to gene expression. In contrast, a transient, strong stimulus with the same area caused rapid and uniform dynamics. These results show that digital NF-κB signaling enables multidimensional control of cellular phenotype via input profile, allowing parallel and independent control of single-cell activation probability and population heterogeneity.

Dynamic modeling, experimental evaluation, optimal design and control of integrated fuel cell system and hybrid energy systems for building demands

NASA Astrophysics Data System (ADS)

Nguyen, Gia Luong Huu

Fuel cells can produce electricity with high efficiency, low pollutants, and low noise. With the advent of fuel cell technologies, fuel cell systems have since been demonstrated as reliable power generators with power outputs from a few watts to a few megawatts. With proper equipment, fuel cell systems can produce heating and cooling, thus increased its overall efficiency. To increase the acceptance from electrical utilities and building owners, fuel cell systems must operate more dynamically and integrate well with renewable energy resources. This research studies the dynamic performance of fuel cells and the integration of fuel cells with other equipment in three levels: (i) the fuel cell stack operating on hydrogen and reformate gases, (ii) the fuel cell system consisting of a fuel reformer, a fuel cell stack, and a heat recovery unit, and (iii) the hybrid energy system consisting of photovoltaic panels, fuel cell system, and energy storage. In the first part, this research studied the steady-state and dynamic performance of a high temperature PEM fuel cell stack. Collaborators at Aalborg University (Aalborg, Denmark) conducted experiments on a high temperature PEM fuel cell short stack at steady-state and transients. Along with the experimental activities, this research developed a first-principles dynamic model of a fuel cell stack. The dynamic model developed in this research was compared to the experimental results when operating on different reformate concentrations. Finally, the dynamic performance of the fuel cell stack for a rapid increase and rapid decrease in power was evaluated. The dynamic model well predicted the performance of the well-performing cells in the experimental fuel cell stack. The second part of the research studied the dynamic response of a high temperature PEM fuel cell system consisting of a fuel reformer, a fuel cell stack, and a heat recovery unit with high thermal integration. After verifying the model performance with the obtained experimental data, the research studied the control of airflow to regulate the temperature of reactors within the fuel processor. The dynamic model provided a platform to test the dynamic response for different control gains. With sufficient sensing and appropriate control, a rapid response to maintain the temperature of the reactor despite an increase in power was possible. The third part of the research studied the use of a fuel cell in conjunction with photovoltaic panels, and energy storage to provide electricity for buildings. This research developed an optimization framework to determine the size of each device in the hybrid energy system to satisfy the electrical demands of buildings and yield the lowest cost. The advantage of having the fuel cell with photovoltaic and energy storage was the ability to operate the fuel cell at baseload at night, thus reducing the need for large battery systems to shift the solar power produced in the day to the night. In addition, the dispatchability of the fuel cell provided an extra degree of freedom necessary for unforeseen disturbances. An operation framework based on model predictive control showed that the method is suitable for optimizing the dispatch of the hybrid energy system.

Structural Fluctuations of the Chromatin Fiber within Topologically Associating Domains.

PubMed

Tiana, Guido; Amitai, Assaf; Pollex, Tim; Piolot, Tristan; Holcman, David; Heard, Edith; Giorgetti, Luca

2016-03-29

Experiments based on chromosome conformation capture have shown that mammalian genomes are partitioned into topologically associating domains (TADs), within which the chromatin fiber preferentially interacts. TADs may provide three-dimensional scaffolds allowing genes to contact their appropriate distal regulatory DNA sequences (e.g., enhancers) and thus to be properly regulated. Understanding the cell-to-cell and temporal variability of the chromatin fiber within TADs, and what determines them, is thus of great importance to better understand transcriptional regulation. We recently described an equilibrium polymer model that can accurately predict cell-to-cell variation of chromosome conformation within single TADs, from chromosome conformation capture-based data. Here we further analyze the conformational and energetic properties of our model. We show that the chromatin fiber within TADs can easily fluctuate between several conformational states, which are hierarchically organized and are not separated by important free energy barriers, and that this is facilitated by the fact that the chromatin fiber within TADs is close to the onset of the coil-globule transition. We further show that in this dynamic state the properties of the chromatin fiber, and its contact probabilities in particular, are determined in a nontrivial manner not only by site-specific interactions between strongly interacting loci along the fiber, but also by nonlocal correlations between pairs of contacts. Finally, we use live-cell experiments to measure the dynamics of the chromatin fiber in mouse embryonic stem cells, in combination with dynamical simulations, and predict that conformational changes within one TAD are likely to occur on timescales that are much shorter than the duration of one cell cycle. This suggests that genes and their regulatory elements may come together and disassociate several times during a cell cycle. These results have important implications for transcriptional regulation as they support the concept of highly dynamic interactions driven by a complex interplay between site-specific interactions and the intrinsic biophysical properties of the chromatin fiber. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Intracellular Pressure Dynamics in Blebbing Cells

PubMed Central

Strychalski, Wanda; Guy, Robert D.

2016-01-01

Blebs are pressure-driven protrusions that play an important role in cell migration, particularly in three-dimensional environments. A bleb is initiated when the cytoskeleton detaches from the cell membrane, resulting in the pressure-driven flow of cytosol toward the area of detachment and local expansion of the cell membrane. Recent experiments involving blebbing cells have led to conflicting hypotheses regarding the timescale of intracellular pressure propagation. The interpretation of one set of experiments supports a poroelastic model of the cytoplasm that leads to slow pressure equilibration when compared to the timescale of bleb expansion. A different study concludes that pressure equilibrates faster than the timescale of bleb expansion. To address this discrepancy, a dynamic computational model of the cell was developed that includes mechanics of and the interactions among the cytoplasm, the actin cortex, the cell membrane, and the cytoskeleton. The model results quantify the relationship among cytoplasmic rheology, pressure, and bleb expansion dynamics, and provide a more detailed picture of intracellular pressure dynamics. This study shows the elastic response of the cytoplasm relieves pressure and limits bleb size, and that both permeability and elasticity of the cytoplasm determine bleb expansion time. Our model with a poroelastic cytoplasm shows that pressure disturbances from bleb initiation propagate faster than the timescale of bleb expansion and that pressure equilibrates slower than the timescale of bleb expansion. The multiple timescales in intracellular pressure dynamics explain the apparent discrepancy in the interpretation of experimental results. PMID:26958893

Persistence of Rift Valley fever virus in East Africa

NASA Astrophysics Data System (ADS)

Gachohi, J.; Hansen, F.; Bett, B.; Kitala, P.

2012-04-01

Rift Valley fever virus (RVFv) is a mosquito-borne pathogen of livestock, wildlife and humans that causes severe outbreaks in intervals of several years. One of the open questions is how the virus persists between outbreaks. We developed a spatially-explicit, individual-based simulation model of the RVFv transmission dynamics to investigate this question. The model, is based on livestock and mosquito population dynamics. Spatial aspects are explicitly represented by a set of grid cells that represent mosquito breeding sites. A grid cell measures 500 by 500m and the model considers a grid of 100 by 100 grid cells; the model thus operates on the regional scale of 2500km2. Livestock herds move between grid cells, and provide connectivity between the cells. The model is used to explore the spatio-temporal dynamics of RVFv persistence in absence of a wildlife reservoir in an east African semi-arid context. Specifically, the model assesses the importance of local virus persistence in mosquito breeding sites relative to global virus persistence mitigated by movement of hosts. Local persistence is determined by the length of time the virus remains in a mosquito breeding site once introduced. In the model, this is a function of the number of mosquitoes that emerge infected and their lifespan. Global persistence is determined by the level of connectivity between isolated grid cells. Our work gives insights into the ecological and epidemiological conditions under which RVFv persists. The implication for disease surveillance and management are discussed.

Computational Analysis of AMPK-Mediated Neuroprotection Suggests Acute Excitotoxic Bioenergetics and Glucose Dynamics Are Regulated by a Minimal Set of Critical Reactions.

PubMed

Connolly, Niamh M C; D'Orsi, Beatrice; Monsefi, Naser; Huber, Heinrich J; Prehn, Jochen H M

2016-01-01

Loss of ionic homeostasis during excitotoxic stress depletes ATP levels and activates the AMP-activated protein kinase (AMPK), re-establishing energy production by increased expression of glucose transporters on the plasma membrane. Here, we develop a computational model to test whether this AMPK-mediated glucose import can rapidly restore ATP levels following a transient excitotoxic insult. We demonstrate that a highly compact model, comprising a minimal set of critical reactions, can closely resemble the rapid dynamics and cell-to-cell heterogeneity of ATP levels and AMPK activity, as confirmed by single-cell fluorescence microscopy in rat primary cerebellar neurons exposed to glutamate excitotoxicity. The model further correctly predicted an excitotoxicity-induced elevation of intracellular glucose, and well resembled the delayed recovery and cell-to-cell heterogeneity of experimentally measured glucose dynamics. The model also predicted necrotic bioenergetic collapse and altered calcium dynamics following more severe excitotoxic insults. In conclusion, our data suggest that a minimal set of critical reactions may determine the acute bioenergetic response to transient excitotoxicity and that an AMPK-mediated increase in intracellular glucose may be sufficient to rapidly recover ATP levels following an excitotoxic insult.

Computational Analysis of AMPK-Mediated Neuroprotection Suggests Acute Excitotoxic Bioenergetics and Glucose Dynamics Are Regulated by a Minimal Set of Critical Reactions

PubMed Central

Connolly, Niamh M. C.; D’Orsi, Beatrice; Monsefi, Naser; Huber, Heinrich J.; Prehn, Jochen H. M.

2016-01-01

Loss of ionic homeostasis during excitotoxic stress depletes ATP levels and activates the AMP-activated protein kinase (AMPK), re-establishing energy production by increased expression of glucose transporters on the plasma membrane. Here, we develop a computational model to test whether this AMPK-mediated glucose import can rapidly restore ATP levels following a transient excitotoxic insult. We demonstrate that a highly compact model, comprising a minimal set of critical reactions, can closely resemble the rapid dynamics and cell-to-cell heterogeneity of ATP levels and AMPK activity, as confirmed by single-cell fluorescence microscopy in rat primary cerebellar neurons exposed to glutamate excitotoxicity. The model further correctly predicted an excitotoxicity-induced elevation of intracellular glucose, and well resembled the delayed recovery and cell-to-cell heterogeneity of experimentally measured glucose dynamics. The model also predicted necrotic bioenergetic collapse and altered calcium dynamics following more severe excitotoxic insults. In conclusion, our data suggest that a minimal set of critical reactions may determine the acute bioenergetic response to transient excitotoxicity and that an AMPK-mediated increase in intracellular glucose may be sufficient to rapidly recover ATP levels following an excitotoxic insult. PMID:26840769

A mix-and-measure assay for determining the activation status of endogenous Cdc42 in cytokine-stimulated macrophage cell lysates.

PubMed

Miskolci, Veronika; Spiering, Désirée; Cox, Dianne; Hodgson, Louis

2014-01-01

Cytokine stimulations of leukocytes many times result in transient activation of the p21 Rho family of small GTPases. The role of these molecules during cell migration and chemotaxis is well established. The traditional approach to study the activation dynamics of these proteins involves affinity pull-downs that are often cumbersome and prone to errors. Here, we describe a reagent and a method of simple "mix-and-measure" approach useful for determining the activation status of endogenous Cdc42 GTPase from cell lysates.

Computational spatiotemporal analysis identifies WAVE2 and Cofilin as joint regulators of costimulation-mediated T cell actin dynamics

PubMed Central

Roybal, Kole T.; Buck, Taráz E.; Ruan, Xiongtao; Cho, Baek Hwan; Clark, Danielle J.; Ambler, Rachel; Tunbridge, Helen M.; Zhang, Jianwei; Verkade, Paul; Wülfing, Christoph; Murphy, Robert F.

2016-01-01

Fluorescence microscopy is one of the most important tools in cell biology research and it provides spatial and temporal information to investigate regulatory systems inside cells. This technique can generate data in the form of signal intensities at thousands of positions resolved inside individual live cells; however, given extensive cell-to-cell variation, methods do not currently exist to assemble these data into three- or four-dimensional maps of protein concentration that can be compared across different cells and conditions. Here, we have developed one such method and applied it to investigate actin dynamics in T cell activation. Antigen recognition in T cells by the T cell receptor (TCR) is amplified by engagement of the costimulatory receptor CD28 and we have determined how CD28 modulates actin dynamics. We imaged actin and eight core actin regulators under conditions where CD28 in the context of a strong TCR signal was engaged or blocked to yield over a thousand movies. Our computational analysis identified diminished recruitment of the activator of actin nucleation WAVE2 and the actin severing protein cofilin to F-actin as the dominant difference upon costimulation blockade. Reconstitution of WAVE2 and cofilin activity restored the defect in actin signaling dynamics upon costimulation blockade. Thus we have developed and validated an approach to quantify protein distributions in time and space for analysis of complex regulatory systems. PMID:27095595

An adaptive molecular timer in p53-meidated cell fate decision

NASA Astrophysics Data System (ADS)

Zhang, Xiao-Peng; Wang, Ping; Liu, Feng; Wang, Wei

The tumor suppressor p53 decides cellular outcomes in the DNA damage response. It is intriguing to explore the link between p53 dynamics and cell fates. We developed a theoretical model of p53 signaling network to clarify the mechanism of cell fate decision mediated by its dynamics. We found that the interplay between p53-Mdm2 negative feedback loop and p53-PTEN-Mdm2 positive feedback loop shapes p53 dynamics. Depending on the intensity of DNA damage, p53 shows three modes of dynamics: persistent pulses, two-phase dynamics with pulses followed by sustained high levels and straightforward high levels. Especially, p53 shows two-phase dynamics upon moderated damage and the required number of p53 pulses before apoptosis induction decreases with increasing DNA damage. Our results suggested there exists an adaptive molecular timer that determines whether and when the apoptosis switch should be triggered. We clarified the mechanism behind the switching of p53 dynamical modes by bifurcation analysis. Moreover, we reproduced the experimental results that drug additions alter p53 pulses to sustained p53 activation and leads to senescence. Our work may advance the understanding the significance of p53 dynamics in tumor suppression. This work was supported by National Natural Science Foundation of China (Nos. 11175084, 11204126 and 31361163003).

The impact of cell regeneration on the dynamics of viral coinfection

NASA Astrophysics Data System (ADS)

Pinky, Lubna; Dobrovolny, Hana M.

2017-06-01

Many mathematical models of respiratory viral infections do not include regeneration of cells within the respiratory tract, arguing that the infection is resolved before there is significant cellular regeneration. However, recent studies have found that ˜40% of patients hospitalized with influenza-like illness are infected with at least two different viruses, which could potentially lead to longer-lasting infections. In these longer infections, cell regeneration might affect the infection dynamics, in particular, allowing for the possibility of chronic coinfections. Several mathematical models have been used to describe cell regeneration in infection models, though the effect of model choice on the predicted time course of viral coinfections is not clear. We investigate four mathematical models incorporating different mechanisms of cell regeneration during respiratory viral coinfection to determine the effect of cell regeneration on infection dynamics. We perform linear stability analysis for each of the models and find the steady states analytically. The analysis suggests that chronic illness is possible but only with one viral species; chronic coexistence of two different viral species is not possible with the regeneration models considered here.

Adaptation of pancreatic islet cyto-architecture during development

NASA Astrophysics Data System (ADS)

Striegel, Deborah A.; Hara, Manami; Periwal, Vipul

2016-04-01

Plasma glucose in mammals is regulated by hormones secreted by the islets of Langerhans embedded in the exocrine pancreas. Islets consist of endocrine cells, primarily α, β, and δ cells, which secrete glucagon, insulin, and somatostatin, respectively. β cells form irregular locally connected clusters within islets that act in concert to secrete insulin upon glucose stimulation. Varying demands and available nutrients during development produce changes in the local connectivity of β cells in an islet. We showed in earlier work that graph theory provides a framework for the quantification of the seemingly stochastic cyto-architecture of β cells in an islet. To quantify the dynamics of endocrine connectivity during development requires a framework for characterizing changes in the probability distribution on the space of possible graphs, essentially a Fokker-Planck formalism on graphs. With large-scale imaging data for hundreds of thousands of islets containing millions of cells from human specimens, we show that this dynamics can be determined quantitatively. Requiring that rearrangement and cell addition processes match the observed dynamic developmental changes in quantitative topological graph characteristics strongly constrained possible processes. Our results suggest that there is a transient shift in preferred connectivity for β cells between 1-35 weeks and 12-24 months.

New insights into mechanisms of stem cell daughter fate determination in regenerative tissues.

PubMed

Sada, Aiko; Tumbar, Tudorita

2013-01-01

Stem cells can self-renew and differentiate over extended periods of time. Understanding how stem cells acquire their fates is a central question in stem cell biology. Early work in Drosophila germ line and neuroblast showed that fate choice is achieved by strict asymmetric divisions that can generate each time one stem and one differentiated cell. More recent work suggests that during homeostasis, some stem cells can divide symmetrically to generate two differentiated cells or two identical stem cells to compensate for stem cell loss that occurred by direct differentiation or apoptosis. The interplay of all these factors ensures constant tissue regeneration and the maintenance of stem cell pool size. This interplay can be modeled as a population-deterministic dynamics that, at least in some systems, may be described as stochastic behavior. Here, we overview recent progress made on the characterization of stem cell dynamics in regenerative tissues. Copyright © 2013 Elsevier Inc. All rights reserved.

The unforeseen challenge: from genotype-to-phenotype in cell populations

NASA Astrophysics Data System (ADS)

Braun, Erez

2015-02-01

Biological cells present a paradox, in that they show simultaneous stability and flexibility, allowing them to adapt to new environments and to evolve over time. The emergence of stable cell states depends on genotype-to-phenotype associations, which essentially reflect the organization of gene regulatory modes. The view taken here is that cell-state organization is a dynamical process in which the molecular disorder manifests itself in a macroscopic order. The genome does not determine the ordered cell state; rather, it participates in this process by providing a set of constraints on the spectrum of regulatory modes, analogous to boundary conditions in physical dynamical systems. We have developed an experimental framework, in which cell populations are exposed to unforeseen challenges; novel perturbations they had not encountered before along their evolutionary history. This approach allows an unbiased view of cell dynamics, uncovering the potential of cells to evolve and develop adapted stable states. In the last decade, our experiments have revealed a coherent set of observations within this framework, painting a picture of the living cell that in many ways is not aligned with the conventional one. Of particular importance here, is our finding that adaptation of cell-state organization is essentially an efficient exploratory dynamical process rather than one founded on random mutations. Based on our framework, a set of concepts underlying cell-state organization—exploration evolving by global, non-specific, dynamics of gene activity—is presented here. These concepts have significant consequences for our understanding of the emergence and stabilization of a cell phenotype in diverse biological contexts. Their implications are discussed for three major areas of biological inquiry: evolution, cell differentiation and cancer. There is currently no unified theoretical framework encompassing the emergence of order, a stable state, in the living cell. Hopefully, the integrated picture described here will provide a modest contribution towards a physics theory of the cell.

Dynamic focus optical coherence tomography: feasibility for improved basal cell carcinoma investigation

NASA Astrophysics Data System (ADS)

Nasiri-Avanaki, M. R.; Aber, Ahmed; Hojjatoleslami, S. A.; Sira, Mano; Schofield, John B.; Jones, Carole; Podoleanu, A. Gh.

2012-03-01

Basal cell carcinoma (BCC) is the most common form of skin cancer. To improve the diagnostic accuracy, additional non-invasive methods of making a preliminary diagnosis have been sought. We have implemented an En-Face optical coherence tomography (OCT) for this study in which the dynamic focus was integrated into it. With the dynamic focus scheme, the coherence gate moves synchronously with the peak of confocal gate determined by the confocal interface optics. The transversal resolution is then conserved throughout the depth range and an enhanced signal is returned from all depths. The Basal Cell Carcinoma specimens were obtained from the eyelid a patient. The specimens under went analysis by DF-OCT imaging. We searched for remarkable features that were visualized by OCT and compared these findings with features presented in the histology slices.

Swimming Dynamics of the Lyme Disease Spirochete

NASA Astrophysics Data System (ADS)

Vig, Dhruv K.; Wolgemuth, Charles W.

2012-11-01

The Lyme disease spirochete, Borrelia burgdorferi, swims by undulating its cell body in the form of a traveling flat wave, a process driven by rotating internal flagella. We study B. burgdorferi’s swimming by treating the cell body and flagella as linearly elastic filaments. The dynamics of the cell are then determined from the balance between elastic and resistive forces and moments. We find that planar, traveling waves only exist when the flagella are effectively anchored at both ends of the bacterium and that these traveling flat waves rotate as they undulate. The model predicts how the undulation frequency is related to the torque from the flagellar motors and how the stiffness of the cell body and flagella affect the undulations and morphology.

Swimming dynamics of the lyme disease spirochete.

PubMed

Vig, Dhruv K; Wolgemuth, Charles W

2012-11-21

The Lyme disease spirochete, Borrelia burgdorferi, swims by undulating its cell body in the form of a traveling flat wave, a process driven by rotating internal flagella. We study B. burgdorferi's swimming by treating the cell body and flagella as linearly elastic filaments. The dynamics of the cell are then determined from the balance between elastic and resistive forces and moments. We find that planar, traveling waves only exist when the flagella are effectively anchored at both ends of the bacterium and that these traveling flat waves rotate as they undulate. The model predicts how the undulation frequency is related to the torque from the flagellar motors and how the stiffness of the cell body and flagella affect the undulations and morphology.

Comprehensive analysis of the dynamic structure of nuclear localization signals.

PubMed

Yamagishi, Ryosuke; Okuyama, Takahide; Oba, Shuntaro; Shimada, Jiro; Chaen, Shigeru; Kaneko, Hiroki

2015-12-01

Most transcription and epigenetic factors in eukaryotic cells have nuclear localization signals (NLSs) and are transported to the nucleus by nuclear transport proteins. Understanding the features of NLSs and the mechanisms of nuclear transport might help understand gene expression regulation, somatic cell reprogramming, thus leading to the treatment of diseases associated with abnormal gene expression. Although many studies analyzed the amino acid sequence of NLSs, few studies investigated their three-dimensional structure. Therefore, we conducted a statistical investigation of the dynamic structure of NLSs by extracting the conformation of these sequences from proteins examined by X-ray crystallography and using a quantity defined as conformational determination rate (a ratio between the number of amino acids determining the conformation and the number of all amino acids included in a certain region). We found that determining the conformation of NLSs is more difficult than determining the conformation of other regions and that NLSs may tend to form more heteropolymers than monomers. Therefore, these findings strongly suggest that NLSs are intrinsically disordered regions.

In vivo imaging of the neurovascular unit in CNS disease

PubMed Central

Merlini, Mario; Davalos, Dimitrios; Akassoglou, Katerina

2014-01-01

The neurovascular unit—comprised of glia, pericytes, neurons and cerebrovasculature—is a dynamic interface that ensures physiological central nervous system (CNS) functioning. In disease dynamic remodeling of the neurovascular interface triggers a cascade of responses that determine the extent of CNS degeneration and repair. The dynamics of these processes can be adequately captured by imaging in vivo, which allows the study of cellular responses to environmental stimuli and cell-cell interactions in the living brain in real time. This perspective focuses on intravital imaging studies of the neurovascular unit in stroke, multiple sclerosis (MS) and Alzheimer disease (AD) models and discusses their potential for identifying novel therapeutic targets. PMID:25197615

Measuring In Vivo Protein Dynamics Throughout the Cell Cycle Using Microfluidics.

PubMed

de Leeuw, Roy; Brazda, Peter; Charl Moolman, M; Kerssemakers, J W J; Solano, Belen; Dekker, Nynke H

2017-01-01

Studying the dynamics of intracellular processes and investigating the interaction of individual macromolecules in live cells is one of the main objectives of cell biology. These macromolecules move, assemble, disassemble, and reorganize themselves in distinct manners under specific physiological conditions throughout the cell cycle. Therefore, in vivo experimental methods that enable the study of individual molecules inside cells at controlled culturing conditions have proved to be powerful tools to obtain insights into the molecular roles of these macromolecules and how their individual behavior influence cell physiology. The importance of controlled experimental conditions is enhanced when the investigated phenomenon covers long time periods, or perhaps multiple cell cycles. An example is the detection and quantification of proteins during bacterial DNA replication. Wide-field microscopy combined with microfluidics is a suitable technique for this. During fluorescence experiments, microfluidics offer well-defined cellular orientation and immobilization, flow and medium interchangeability, and high-throughput long-term experimentation of cells. Here we present a protocol for the combined use of wide-field microscopy and microfluidics for the study of proteins of the Escherichia coli DNA replication process. We discuss the preparation and application of a microfluidic device, data acquisition steps, and image analysis procedures to determine the stoichiometry and dynamics of a replisome component throughout the cell cycle of live bacterial cells.

Composite regulation of ERK activity dynamics underlying tumour-specific traits in the intestine.

PubMed

Muta, Yu; Fujita, Yoshihisa; Sumiyama, Kenta; Sakurai, Atsuro; Taketo, M Mark; Chiba, Tsutomu; Seno, Hiroshi; Aoki, Kazuhiro; Matsuda, Michiyuki; Imajo, Masamichi

2018-06-05

Acting downstream of many growth factors, extracellular signal-regulated kinase (ERK) plays a pivotal role in regulating cell proliferation and tumorigenesis, where its spatiotemporal dynamics, as well as its strength, determine cellular responses. Here, we uncover the ERK activity dynamics in intestinal epithelial cells (IECs) and their association with tumour characteristics. Intravital imaging identifies two distinct modes of ERK activity, sustained and pulse-like activity, in IECs. The sustained and pulse-like activities depend on ErbB2 and EGFR, respectively. Notably, activation of Wnt signalling, the earliest event in intestinal tumorigenesis, augments EGFR signalling and increases the frequency of ERK activity pulses through controlling the expression of EGFR and its regulators, rendering IECs sensitive to EGFR inhibition. Furthermore, the increased pulse frequency is correlated with increased cell proliferation. Thus, ERK activity dynamics are defined by composite inputs from EGFR and ErbB2 signalling in IECs and their alterations might underlie tumour-specific sensitivity to pharmacological EGFR inhibition.

Timing the start of division in E. coli: a single-cell study

NASA Astrophysics Data System (ADS)

Reshes, G.; Vanounou, S.; Fishov, I.; Feingold, M.

2008-12-01

We monitor the shape dynamics of individual E. coli cells using time-lapse microscopy together with accurate image analysis. This allows measuring the dynamics of single-cell parameters throughout the cell cycle. In previous work, we have used this approach to characterize the main features of single-cell morphogenesis between successive divisions. Here, we focus on the behavior of the parameters that are related to cell division and study their variation over a population of 30 cells. In particular, we show that the single-cell data for the constriction width dynamics collapse onto a unique curve following appropriate rescaling of the corresponding variables. This suggests the presence of an underlying time scale that determines the rate at which the cell cycle advances in each individual cell. For the case of cell length dynamics a similar rescaling of variables emphasizes the presence of a breakpoint in the growth rate at the time when division starts, τc. We also find that the τc of individual cells is correlated with their generation time, τg, and inversely correlated with the corresponding length at birth, L0. Moreover, the extent of the T-period, τg - τc, is apparently independent of τg. The relations between τc, τg and L0 indicate possible compensation mechanisms that maintain cell length variability at about 10%. Similar behavior was observed for both fast-growing cells in a rich medium (LB) and for slower growth in a minimal medium (M9-glucose). To reveal the molecular mechanisms that lead to the observed organization of the cell cycle, we should further extend our approach to monitor the formation of the divisome.

Dynamic switching enables efficient bacterial colonization in flow.

PubMed

Kannan, Anerudh; Yang, Zhenbin; Kim, Minyoung Kevin; Stone, Howard A; Siryaporn, Albert

2018-05-22

Bacteria colonize environments that contain networks of moving fluids, including digestive pathways, blood vasculature in animals, and the xylem and phloem networks in plants. In these flow networks, bacteria form distinct biofilm structures that have an important role in pathogenesis. The physical mechanisms that determine the spatial organization of bacteria in flow are not understood. Here, we show that the bacterium P. aeruginosa colonizes flow networks using a cyclical process that consists of surface attachment, upstream movement, detachment, movement with the bulk flow, and surface reattachment. This process, which we have termed dynamic switching, distributes bacterial subpopulations upstream and downstream in flow through two phases: movement on surfaces and cellular movement via the bulk. The model equations that describe dynamic switching are identical to those that describe dynamic instability, a process that enables microtubules in eukaryotic cells to search space efficiently to capture chromosomes. Our results show that dynamic switching enables bacteria to explore flow networks efficiently, which maximizes dispersal and colonization and establishes the organizational structure of biofilms. A number of eukaryotic and mammalian cells also exhibit movement in two phases in flow, which suggests that dynamic switching is a modality that enables efficient dispersal for a broad range of cell types.

Control of complex networks requires both structure and dynamics

NASA Astrophysics Data System (ADS)

Gates, Alexander J.; Rocha, Luis M.

2016-04-01

The study of network structure has uncovered signatures of the organization of complex systems. However, there is also a need to understand how to control them; for example, identifying strategies to revert a diseased cell to a healthy state, or a mature cell to a pluripotent state. Two recent methodologies suggest that the controllability of complex systems can be predicted solely from the graph of interactions between variables, without considering their dynamics: structural controllability and minimum dominating sets. We demonstrate that such structure-only methods fail to characterize controllability when dynamics are introduced. We study Boolean network ensembles of network motifs as well as three models of biochemical regulation: the segment polarity network in Drosophila melanogaster, the cell cycle of budding yeast Saccharomyces cerevisiae, and the floral organ arrangement in Arabidopsis thaliana. We demonstrate that structure-only methods both undershoot and overshoot the number and which sets of critical variables best control the dynamics of these models, highlighting the importance of the actual system dynamics in determining control. Our analysis further shows that the logic of automata transition functions, namely how canalizing they are, plays an important role in the extent to which structure predicts dynamics.

Mitochondria-Division Inhibitor 1 Protects Against Amyloid-β induced Mitochondrial Fragmentation and Synaptic Damage in Alzheimer's Disease.

PubMed

Reddy, P Hemachandra; Manczak, Maria; Yin, XiangLing

2017-01-01

The purpose our study was to determine the protective effects of mitochondria division inhibitor 1 (Mdivi1) in Alzheimer's disease (AD). Mdivi1 is hypothesized to reduce excessive fragmentation of mitochondria and mitochondrial dysfunction in AD neurons. Very little is known about whether Mdivi1 can confer protective effects in AD. In the present study, we sought to determine the protective effects of Mdivi1 against amyloid-β (Aβ)- and mitochondrial fission protein, dynamin-related protein 1 (Drp1)-induced excessive fragmentation of mitochondria in AD progression. We also studied preventive (Mdivi1+Aβ42) and intervention (Aβ42+Mdivi1) effects against Aβ42 in N2a cells. Using real-time RT-PCR and immunoblotting analysis, we measured mRNA and protein levels of mitochondrial dynamics, mitochondrial biogenesis, and synaptic genes. We also assessed mitochondrial function by measuring H2O2, lipid peroxidation, cytochrome oxidase activity, and mitochondrial ATP. MTT assays were used to assess the cell viability. Aβ42 was found to impair mitochondrial dynamics, lower mitochondrial biogenesis, lower synaptic activity, and lower mitochondrial function. On the contrary, Mdivi1 enhanced mitochondrial fusion activity, lowered fission machinery, and increased biogenesis and synaptic proteins. Mitochondrial function and cell viability were elevated in Mdivi1-treated cells. Interestingly, Mdivi1 pre- and post-treated cells treated with Aβ showed reduced mitochondrial dysfunction, and maintained cell viability, mitochondrial dynamics, mitochondrial biogenesis, and synaptic activity. The protective effects of Mdivi1 were stronger in N2a+Aβ42 pre-treated with Mdivi1, than in N2a+Aβ42 cells than Mdivi1 post-treated cells, indicating that Mdivi1 works better in prevention than treatment in AD like neurons.

Integrated time-lapse and single-cell transcription studies highlight the variable and dynamic nature of human hematopoietic cell fate commitment

PubMed Central

Moussy, Alice; Cosette, Jérémie; Parmentier, Romuald; da Silva, Cindy; Corre, Guillaume; Richard, Angélique; Gandrillon, Olivier; Stockholm, Daniel

2017-01-01

Individual cells take lineage commitment decisions in a way that is not necessarily uniform. We address this issue by characterising transcriptional changes in cord blood-derived CD34+ cells at the single-cell level and integrating data with cell division history and morphological changes determined by time-lapse microscopy. We show that major transcriptional changes leading to a multilineage-primed gene expression state occur very rapidly during the first cell cycle. One of the 2 stable lineage-primed patterns emerges gradually in each cell with variable timing. Some cells reach a stable morphology and molecular phenotype by the end of the first cell cycle and transmit it clonally. Others fluctuate between the 2 phenotypes over several cell cycles. Our analysis highlights the dynamic nature and variable timing of cell fate commitment in hematopoietic cells, links the gene expression pattern to cell morphology, and identifies a new category of cells with fluctuating phenotypic characteristics, demonstrating the complexity of the fate decision process (which is different from a simple binary switch between 2 options, as it is usually envisioned). PMID:28749943

CLASP2 interacts with p120-catenin and governs microtubule dynamics at adherens junctions

PubMed Central

Shahbazi, Marta N.; Megias, Diego; Epifano, Carolina; Akhmanova, Anna; Gundersen, Gregg G.; Fuchs, Elaine

2013-01-01

Classical cadherins and their connections with microtubules (MTs) are emerging as important determinants of cell adhesion. However, the functional relevance of such interactions and the molecular players that contribute to tissue architecture are still emerging. In this paper, we report that the MT plus end–binding protein CLASP2 localizes to adherens junctions (AJs) via direct interaction with p120-catenin (p120) in primary basal mouse keratinocytes. Reductions in the levels of p120 or CLASP2 decreased the localization of the other protein to cell–cell contacts and altered AJ dynamics and stability. These features were accompanied by decreased MT density and altered MT dynamics at intercellular junction sites. Interestingly, CLASP2 was enriched at the cortex of basal progenitor keratinocytes, in close localization to p120. Our findings suggest the existence of a new mechanism of MT targeting to AJs with potential functional implications in the maintenance of proper cell–cell adhesion in epidermal stem cells. PMID:24368809

Efficient electron open boundaries for simulating electrochemical cells

NASA Astrophysics Data System (ADS)

Zauchner, Mario G.; Horsfield, Andrew P.; Todorov, Tchavdar N.

2018-01-01

Nonequilibrium electrochemistry raises new challenges for atomistic simulation: we need to perform molecular dynamics for the nuclear degrees of freedom with an explicit description of the electrons, which in turn must be free to enter and leave the computational cell. Here we present a limiting form for electron open boundaries that we expect to apply when the magnitude of the electric current is determined by the drift and diffusion of ions in a solution and which is sufficiently computationally efficient to be used with molecular dynamics. We present tight-binding simulations of a parallel-plate capacitor with nothing, a dimer, or an atomic wire situated in the space between the plates. These simulations demonstrate that this scheme can be used to perform molecular dynamics simulations when there is an applied bias between two metal plates with, at most, weak electronic coupling between them. This simple system captures some of the essential features of an electrochemical cell, suggesting this approach might be suitable for simulations of electrochemical cells out of equilibrium.

Mathematical Modelling as a Tool to Understand Cell Self-renewal and Differentiation.

PubMed

Getto, Philipp; Marciniak-Czochra, Anna

2015-01-01

Mathematical modeling is a powerful technique to address key questions and paradigms in a variety of complex biological systems and can provide quantitative insights into cell kinetics, fate determination and development of cell populations. The chapter is devoted to a review of modeling of the dynamics of stem cell-initiated systems using mathematical methods of ordinary differential equations. Some basic concepts and tools for cell population dynamics are summarized and presented as a gentle introduction to non-mathematicians. The models take into account different plausible mechanisms regulating homeostasis. Two mathematical frameworks are proposed reflecting, respectively, a discrete (punctuated by division events) and a continuous character of transitions between differentiation stages. Advantages and constraints of the mathematical approaches are presented on examples of models of blood systems and compared to patients data on healthy hematopoiesis.

Parvovirus Induced Alterations in Nuclear Architecture and Dynamics

PubMed Central

Ihalainen, Teemu O.; Niskanen, Einari A.; Jylhävä, Juulia; Paloheimo, Outi; Dross, Nicolas; Smolander, Hanna; Langowski, Jörg; Timonen, Jussi; Vihinen-Ranta, Maija

2009-01-01

The nucleus of interphase eukaryotic cell is a highly compartmentalized structure containing the three-dimensional network of chromatin and numerous proteinaceous subcompartments. DNA viruses induce profound changes in the intranuclear structures of their host cells. We are applying a combination of confocal imaging including photobleaching microscopy and computational methods to analyze the modifications of nuclear architecture and dynamics in parvovirus infected cells. Upon canine parvovirus infection, expansion of the viral replication compartment is accompanied by chromatin marginalization to the vicinity of the nuclear membrane. Dextran microinjection and fluorescence recovery after photobleaching (FRAP) studies revealed the homogeneity of this compartment. Markedly, in spite of increase in viral DNA content of the nucleus, a significant increase in the protein mobility was observed in infected compared to non-infected cells. Moreover, analyzis of the dynamics of photoactivable capsid protein demonstrated rapid intranuclear dynamics of viral capsids. Finally, quantitative FRAP and cellular modelling were used to determine the duration of viral genome replication. Altogether, our findings indicate that parvoviruses modify the nuclear structure and dynamics extensively. Intranuclear crowding of viral components leads to enlargement of the interchromosomal domain and to chromatin marginalization via depletion attraction. In conclusion, parvoviruses provide a useful model system for understanding the mechanisms of virus-induced intranuclear modifications. PMID:19536327

Division of labour between Myc and G1 cyclins in cell cycle commitment and pace control.

PubMed

Dong, Peng; Maddali, Manoj V; Srimani, Jaydeep K; Thélot, François; Nevins, Joseph R; Mathey-Prevot, Bernard; You, Lingchong

2014-09-01

A body of evidence has shown that the control of E2F transcription factor activity is critical for determining cell cycle entry and cell proliferation. However, an understanding of the precise determinants of this control, including the role of other cell-cycle regulatory activities, has not been clearly defined. Here, recognizing that the contributions of individual regulatory components could be masked by heterogeneity in populations of cells, we model the potential roles of individual components together with the use of an integrated system to follow E2F dynamics at the single-cell level and in real time. These analyses reveal that crossing a threshold amplitude of E2F accumulation determines cell cycle commitment. Importantly, we find that Myc is critical in modulating the amplitude, whereas cyclin D/E activities have little effect on amplitude but do contribute to the modulation of duration of E2F activation, thereby affecting the pace of cell cycle progression.

Live Cell Imaging and Measurements of Molecular Dynamics

PubMed Central

Frigault, M.; Lacoste, J.; Swift, J.; Brown, C.

2010-01-01

w3-2 Live cell microscopy is becoming widespread across all fields of the life sciences, as well as, many areas of the physical sciences. In order to accurately obtain live cell microscopy data, the live specimens must be properly maintained on the imaging platform. In addition, the fluorescence light path must be optimized for efficient light transmission in order to reduce the intensity of excitation light impacting the living sample. With low incident light intensities the processes under study should not be altered due to phototoxic effects from the light allowing for the long term visualization of viable living samples. Aspects for maintaining a suitable environment for the living sample, minimizing incident light and maximizing detection efficiency will be presented for various fluorescence based live cell instruments. Raster Image Correlation Spectroscopy (RICS) is a technique that uses the intensity fluctuations within laser scanning confocal images, as well as the well characterized scanning dynamics of the laser beam, to extract the dynamics, concentrations and clustering of fluorescent molecules within the cell. In addition, two color cross-correlation RICS can be used to determine protein-protein interactions in living cells without the many technical difficulties encountered in FRET based measurements. RICS is an ideal live cell technique for measuring cellular dynamics because the potentially damaging high intensity laser bursts required for photobleaching recovery measurements are not required, rather low laser powers, suitable for imaging, can be used. The RICS theory will be presented along with examples of live cell applications.

Reliable binary cell-fate decisions based on oscillations

NASA Astrophysics Data System (ADS)

Pfeuty, B.; Kaneko, K.

2014-02-01

Biological systems have often to perform binary decisions under highly dynamic and noisy environments, such as during cell-fate determination. These decisions can be implemented by two main bifurcation mechanisms based on the transitions from either monostability or oscillation to bistability. We compare these two mechanisms by using stochastic models with time-varying fields and by establishing asymptotic formulas for the choice probabilities. Different scaling laws for decision sensitivity with respect to noise strength and signal timescale are obtained, supporting a role for oscillatory dynamics in performing noise-robust and temporally tunable binary decision-making. This result provides a rationale for recent experimental evidences showing that oscillatory expression of proteins often precedes binary cell-fate decisions.

A Macro-to-Micro Interface for the Control of Cellular Organization

PubMed Central

Hui, Elliot E.; Li, Chun; Agrawal, Amit; Bhatia, Sangeeta N.

2015-01-01

The spatial organization of cellular communities plays a fundamental role in determining intercellular communication and emergent behavior. However, few tools exist to modulate tissue organization at the scale of individual cells, particularly in the case of dynamic manipulation. Micromechanical reconfigurable culture achieves dynamic control of tissue organization by culturing adherent cells on microfabricated plates that can be shifted to reorganize the arrangement of the cells. While biological studies utilizing this approach have been previously reported, this paper focuses on the engineering of the device, including the mechanism for translating manual manipulation to precise microscale position control, fault-tolerant design for manufacture, and the synthetic-to-living interface. PMID:26167106

Dexamethasone disrupts cytoskeleton organization and migration of T47D Human breast cancer cells by modulating the AKT/mTOR/RhoA pathway.

PubMed

Meng, Xian-Guo; Yue, Shou-Wei

2014-01-01

Glucocorticoids are commonly co-administered with chemotherapy to prevent drug-induced allergic reactions, nausea, and vomiting, and have anti-tumor functions clinically; however, the distinct effects of GC on subtypes of tumor cells, especially in breast cancer cells, are still not well understood. In this study, we aimed to clarify the effect of GC on subtypes of T47D breast cancer cells by focusing on apoptosis, cell organization and migration, and underluing molecular mechanisms. The cell scratch test was performed to observe the cell migration rate in T47D cells treated with dexamethasone (Dex). Hoechst and MTT assays were conducted to detect cell survival and rhodamine-labeled phalloidin staining to observe cytoskeleton dynamics. Related factors in the AKT/mTOR pathway were determined by Western blotting. Dex treatment could effectively inhibit T47D breast cancer cell migration with disruption of the cytoskeletal dynamic organization. Moreover, the effect of Dex on cell migration and cytoskeleton may be mediated by AKT/ mTOR/RhoA pathway. Although Dex inhibited T47D cell migration, it alone may not induce cell apoptosis in T47D cells. Dex in T47D human breast cancer cells could effectively inhibit cell migration by disrupting the cytoskeletal dynamic organization, which may be mediated by the AKT/mTOR/RhoA pathway. Our work suggests that glucocorticoid/Dex clinical use may prove helpful for the treatment of breast cancer metastasis.

Dynamic Cytology and Transcriptional Regulation of Rice Lamina Joint Development1[OPEN

PubMed Central

2017-01-01

Rice (Oryza sativa) leaf angle is determined by lamina joint and is an important agricultural trait determining leaf erectness and, hence, the photosynthesis efficiency and grain yield. Genetic studies reveal a complex regulatory network of lamina joint development; however, the morphological changes, cytological transitions, and underlying transcriptional programming remain to be elucidated. A systemic morphological and cytological study reveals a dynamic developmental process and suggests a common but distinct regulation of the lamina joint. Successive and sequential cell division and expansion, cell wall thickening, and programmed cell death at the adaxial or abaxial sides form the cytological basis of the lamina joint, and the increased leaf angle results from the asymmetric cell proliferation and elongation. Analysis of the gene expression profiles at four distinct developmental stages ranging from initiation to senescence showed that genes related to cell division and growth, hormone synthesis and signaling, transcription (transcription factors), and protein phosphorylation (protein kinases) exhibit distinct spatiotemporal patterns during lamina joint development. Phytohormones play crucial roles by promoting cell differentiation and growth at early stages or regulating the maturation and senescence at later stages, which is consistent with the quantitative analysis of hormones at different stages. Further comparison with the gene expression profile of leaf inclination1, a mutant with decreased auxin and increased leaf angle, indicates the coordinated effects of hormones in regulating lamina joint. These results reveal a dynamic cytology of rice lamina joint that is fine-regulated by multiple factors, providing informative clues for illustrating the regulatory mechanisms of leaf angle and plant architecture. PMID:28500269

Dynamic Cytology and Transcriptional Regulation of Rice Lamina Joint Development.

PubMed

Zhou, Li-Juan; Xiao, Lang-Tao; Xue, Hong-Wei

2017-07-01

Rice ( Oryza sativa ) leaf angle is determined by lamina joint and is an important agricultural trait determining leaf erectness and, hence, the photosynthesis efficiency and grain yield. Genetic studies reveal a complex regulatory network of lamina joint development; however, the morphological changes, cytological transitions, and underlying transcriptional programming remain to be elucidated. A systemic morphological and cytological study reveals a dynamic developmental process and suggests a common but distinct regulation of the lamina joint. Successive and sequential cell division and expansion, cell wall thickening, and programmed cell death at the adaxial or abaxial sides form the cytological basis of the lamina joint, and the increased leaf angle results from the asymmetric cell proliferation and elongation. Analysis of the gene expression profiles at four distinct developmental stages ranging from initiation to senescence showed that genes related to cell division and growth, hormone synthesis and signaling, transcription (transcription factors), and protein phosphorylation (protein kinases) exhibit distinct spatiotemporal patterns during lamina joint development. Phytohormones play crucial roles by promoting cell differentiation and growth at early stages or regulating the maturation and senescence at later stages, which is consistent with the quantitative analysis of hormones at different stages. Further comparison with the gene expression profile of leaf inclination1 , a mutant with decreased auxin and increased leaf angle, indicates the coordinated effects of hormones in regulating lamina joint. These results reveal a dynamic cytology of rice lamina joint that is fine-regulated by multiple factors, providing informative clues for illustrating the regulatory mechanisms of leaf angle and plant architecture. © 2017 American Society of Plant Biologists. All Rights Reserved.

Of extracellular matrix, scaffolds, and signaling: Tissuearchitectureregulates development, homeostasis, and cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nelson, Celeste M.; Bissell, Mina J.

2006-03-09

The microenvironment surrounding cells influences gene expression, such that a cell's behavior is largely determined by its interactions with the extracellular matrix, neighboring cells, and soluble cues released locally or by distant tissues. We describe the essential role of context and organ structure in directing mammary gland development and differentiated function, and in determining response to oncogenic insults including mutations. We expand on the concept of 'dynamic reciprocity' to present an integrated view of development, cancer, and aging, and posit that genes are like piano keys: while essential, it is the context that makes the music.

Engineering large cartilage tissues using dynamic bioreactor culture at defined oxygen conditions.

PubMed

Daly, Andrew C; Sathy, Binulal N; Kelly, Daniel J

2018-01-01

Mesenchymal stem cells maintained in appropriate culture conditions are capable of producing robust cartilage tissue. However, gradients in nutrient availability that arise during three-dimensional culture can result in the development of spatially inhomogeneous cartilage tissues with core regions devoid of matrix. Previous attempts at developing dynamic culture systems to overcome these limitations have reported suppression of mesenchymal stem cell chondrogenesis compared to static conditions. We hypothesize that by modulating oxygen availability during bioreactor culture, it is possible to engineer cartilage tissues of scale. The objective of this study was to determine whether dynamic bioreactor culture, at defined oxygen conditions, could facilitate the development of large, spatially homogeneous cartilage tissues using mesenchymal stem cell laden hydrogels. A dynamic culture regime was directly compared to static conditions for its capacity to support chondrogenesis of mesenchymal stem cells in both small and large alginate hydrogels. The influence of external oxygen tension on the response to the dynamic culture conditions was explored by performing the experiment at 20% O 2 and 3% O 2 . At 20% O 2 , dynamic culture significantly suppressed chondrogenesis in engineered tissues of all sizes. In contrast, at 3% O 2 dynamic culture significantly enhanced the distribution and amount of cartilage matrix components (sulphated glycosaminoglycan and collagen II) in larger constructs compared to static conditions. Taken together, these results demonstrate that dynamic culture regimes that provide adequate nutrient availability and a low oxygen environment can be employed to engineer large homogeneous cartilage tissues. Such culture systems could facilitate the scaling up of cartilage tissue engineering strategies towards clinically relevant dimensions.

Modeling the dynamics of chromosomal alteration progression in cervical cancer: A computational model

PubMed Central

2017-01-01

Computational modeling has been applied to simulate the heterogeneity of cancer behavior. The development of Cervical Cancer (CC) is a process in which the cell acquires dynamic behavior from non-deleterious and deleterious mutations, exhibiting chromosomal alterations as a manifestation of this dynamic. To further determine the progression of chromosomal alterations in precursor lesions and CC, we introduce a computational model to study the dynamics of deleterious and non-deleterious mutations as an outcome of tumor progression. The analysis of chromosomal alterations mediated by our model reveals that multiple deleterious mutations are more frequent in precursor lesions than in CC. Cells with lethal deleterious mutations would be eliminated, which would mitigate cancer progression; on the other hand, cells with non-deleterious mutations would become dominant, which could predispose them to cancer progression. The study of somatic alterations through computer simulations of cancer progression provides a feasible pathway for insights into the transformation of cell mechanisms in humans. During cancer progression, tumors may acquire new phenotype traits, such as the ability to invade and metastasize or to become clinically important when they develop drug resistance. Non-deleterious chromosomal alterations contribute to this progression. PMID:28723940

Noisy Oscillations in the Actin Cytoskeleton of Chemotactic Amoeba.

PubMed

Negrete, Jose; Pumir, Alain; Hsu, Hsin-Fang; Westendorf, Christian; Tarantola, Marco; Beta, Carsten; Bodenschatz, Eberhard

2016-09-30

Biological systems with their complex biochemical networks are known to be intrinsically noisy. Here we investigate the dynamics of actin polymerization of amoeboid cells, which are close to the onset of oscillations. We show that the large phenotypic variability in the polymerization dynamics can be accurately captured by a generic nonlinear oscillator model in the presence of noise. We determine the relative role of the noise with a single dimensionless, experimentally accessible parameter, thus providing a quantitative description of the variability in a population of cells. Our approach, which rests on a generic description of a system close to a Hopf bifurcation and includes the effect of noise, can characterize the dynamics of a large class of noisy systems close to an oscillatory instability.

Noisy Oscillations in the Actin Cytoskeleton of Chemotactic Amoeba

NASA Astrophysics Data System (ADS)

Negrete, Jose; Pumir, Alain; Hsu, Hsin-Fang; Westendorf, Christian; Tarantola, Marco; Beta, Carsten; Bodenschatz, Eberhard

2016-09-01

Biological systems with their complex biochemical networks are known to be intrinsically noisy. Here we investigate the dynamics of actin polymerization of amoeboid cells, which are close to the onset of oscillations. We show that the large phenotypic variability in the polymerization dynamics can be accurately captured by a generic nonlinear oscillator model in the presence of noise. We determine the relative role of the noise with a single dimensionless, experimentally accessible parameter, thus providing a quantitative description of the variability in a population of cells. Our approach, which rests on a generic description of a system close to a Hopf bifurcation and includes the effect of noise, can characterize the dynamics of a large class of noisy systems close to an oscillatory instability.

Modeling T-cell proliferation: an investigation of the consequences of the Hayflick limit.

PubMed

Pilyugin, S; Mittler, J; Antia, R

1997-05-07

Somatic cells, including immune cells such as T-cells have a limited capacity for proliferation and can only replicate for a finite number of generations (known as the Hayflick limit) before dying. In this paper we use mathematical models to investigate the consequences of introducing a Hayflick limit on the dynamics of T-cells stimulated with specific antigen. We show that while the Hayflick limit does not alter the dynamics of T-cell response to antigen over the short term, it may have a profound effect on the long-term immune response. In particular we show that over the long term the Hayflick limit may be important in determining whether an immune response can be maintained to a persistent antigen (or parasite). The eventual outcome is determined by the magnitude of the Hayflick limit, the extent to which antigen reduces the input of T-cells from the thymus, and the rate of antigen-induced proliferation of T-cells. Counter to what might be expected we show that the persistence of an immune response (immune memory) requires the density of persistent antigen to be less than a defined threshold value. If the amount of persistent antigen (or parasite) is greater than this threshold value then immune memory will be relatively short lived. The consequences of this threshold for persistent mycobacterial and HIV infections and for the generation of vaccines are discussed.

Flower Development as an Interplay between Dynamical Physical Fields and Genetic Networks

PubMed Central

Barrio, Rafael Ángel; Hernández-Machado, Aurora; Varea, C.; Romero-Arias, José Roberto; Álvarez-Buylla, Elena

2010-01-01

In this paper we propose a model to describe the mechanisms by which undifferentiated cells attain gene configurations underlying cell fate determination during morphogenesis. Despite the complicated mechanisms that surely intervene in this process, it is clear that the fundamental fact is that cells obtain spatial and temporal information that bias their destiny. Our main hypothesis assumes that there is at least one macroscopic field that breaks the symmetry of space at a given time. This field provides the information required for the process of cell differentiation to occur by being dynamically coupled to a signal transduction mechanism that, in turn, acts directly upon the gene regulatory network (GRN) underlying cell-fate decisions within cells. We illustrate and test our proposal with a GRN model grounded on experimental data for cell fate specification during organ formation in early Arabidopsis thaliana flower development. We show that our model is able to recover the multigene configurations characteristic of sepal, petal, stamen and carpel primordial cells arranged in concentric rings, in a similar pattern to that observed during actual floral organ determination. Such pattern is robust to alterations of the model parameters and simulated failures predict altered spatio-temporal patterns that mimic those described for several mutants. Furthermore, simulated alterations in the physical fields predict a pattern equivalent to that found in Lacandonia schismatica, the only flowering species with central stamens surrounded by carpels. PMID:21048956

Flower development as an interplay between dynamical physical fields and genetic networks.

PubMed

Barrio, Rafael Ángel; Hernández-Machado, Aurora; Varea, C; Romero-Arias, José Roberto; Alvarez-Buylla, Elena

2010-10-27

In this paper we propose a model to describe the mechanisms by which undifferentiated cells attain gene configurations underlying cell fate determination during morphogenesis. Despite the complicated mechanisms that surely intervene in this process, it is clear that the fundamental fact is that cells obtain spatial and temporal information that bias their destiny. Our main hypothesis assumes that there is at least one macroscopic field that breaks the symmetry of space at a given time. This field provides the information required for the process of cell differentiation to occur by being dynamically coupled to a signal transduction mechanism that, in turn, acts directly upon the gene regulatory network (GRN) underlying cell-fate decisions within cells. We illustrate and test our proposal with a GRN model grounded on experimental data for cell fate specification during organ formation in early Arabidopsis thaliana flower development. We show that our model is able to recover the multigene configurations characteristic of sepal, petal, stamen and carpel primordial cells arranged in concentric rings, in a similar pattern to that observed during actual floral organ determination. Such pattern is robust to alterations of the model parameters and simulated failures predict altered spatio-temporal patterns that mimic those described for several mutants. Furthermore, simulated alterations in the physical fields predict a pattern equivalent to that found in Lacandonia schismatica, the only flowering species with central stamens surrounded by carpels.

Mathematical model for dynamic cell formation in fast fashion apparel manufacturing stage

NASA Astrophysics Data System (ADS)

Perera, Gayathri; Ratnayake, Vijitha

2018-05-01

This paper presents a mathematical programming model for dynamic cell formation to minimize changeover-related costs (i.e., machine relocation costs and machine setup cost) and inter-cell material handling cost to cope with the volatile production environments in apparel manufacturing industry. The model is formulated through findings of a comprehensive literature review. Developed model is validated based on data collected from three different factories in apparel industry, manufacturing fast fashion products. A program code is developed using Lingo 16.0 software package to generate optimal cells for developed model and to determine the possible cost-saving percentage when the existing layouts used in three factories are replaced by generated optimal cells. The optimal cells generated by developed mathematical model result in significant cost saving when compared with existing product layouts used in production/assembly department of selected factories in apparel industry. The developed model can be considered as effective in minimizing the considered cost terms in dynamic production environment of fast fashion apparel manufacturing industry. Findings of this paper can be used for further researches on minimizing the changeover-related costs in fast fashion apparel production stage.

An experimental and theoretical investigation of the liquefaction dynamics of a phase change material in a normal gravity environment

NASA Technical Reports Server (NTRS)

Bain, R. L.; Stermole, F. J.; Golden, J. O.

1972-01-01

Experimental and theoretical investigations were undertaken to determine the role of gravity-induced free convection upon the liquefaction dynamics of a cylindrical paraffin slab under normal gravity conditions. The experimental equipment consisted of a test cell, a fluid-loop heating system, and a multipoint recorder. The test chamber was annular in shape with an effective radius of 1.585 cm and a length of 5.08 cm. The heating chamber was a 1.906 cm diameter tube going through the center of the test chamber, and connected to the fluid loop heating system. All experimental runs were made with the longitudinal axis of the test cell in the vertical direction to insure that convection was not a function of the angular axis of the cell. Ten melting runs were made at various hot wall temperatures. Also, two pure conduction solidification runs were made to determine an experimental latent heat of fusion.

Connecting mitochondrial dynamics and life-or-death events via Bcl-2 family proteins.

PubMed

Aouacheria, Abdel; Baghdiguian, Stephen; Lamb, Heather M; Huska, Jason D; Pineda, Fernando J; Hardwick, J Marie

2017-10-01

The morphology of a population of mitochondria is the result of several interacting dynamical phenomena, including fission, fusion, movement, elimination and biogenesis. Each of these phenomena is controlled by underlying molecular machinery, and when defective can cause disease. New understanding of the relationships between form and function of mitochondria in health and disease is beginning to be unraveled on several fronts. Studies in mammals and model organisms have revealed that mitochondrial morphology, dynamics and function appear to be subject to regulation by the same proteins that regulate apoptotic cell death. One protein family that influences mitochondrial dynamics in both healthy and dying cells is the Bcl-2 protein family. Connecting mitochondrial dynamics with life-death pathway forks may arise from the intersection of Bcl-2 family proteins with the proteins and lipids that determine mitochondrial shape and function. Bcl-2 family proteins also have multifaceted influences on cells and mitochondria, including calcium handling, autophagy and energetics, as well as the subcellular localization of mitochondrial organelles to neuronal synapses. The remarkable range of physical or functional interactions by Bcl-2 family proteins is challenging to assimilate into a cohesive understanding. Most of their effects may be distinct from their direct roles in apoptotic cell death and are particularly apparent in the nervous system. Dual roles in mitochondrial dynamics and cell death extend beyond BCL-2 family proteins. In this review, we discuss many processes that govern mitochondrial structure and function in health and disease, and how Bcl-2 family proteins integrate into some of these processes. Copyright © 2017 Elsevier Ltd. All rights reserved.

A coarse-grained model of microtubule self-assembly

NASA Astrophysics Data System (ADS)

Regmi, Chola; Cheng, Shengfeng

Microtubules play critical roles in cell structures and functions. They also serve as a model system to stimulate the next-generation smart, dynamic materials. A deep understanding of their self-assembly process and biomechanical properties will not only help elucidate how microtubules perform biological functions, but also lead to exciting insight on how microtubule dynamics can be altered or even controlled for specific purposes such as suppressing the division of cancer cells. Combining all-atom molecular dynamics (MD) simulations and the essential dynamics coarse-graining method, we construct a coarse-grained (CG) model of the tubulin protein, which is the building block of microtubules. In the CG model a tubulin dimer is represented as an elastic network of CG sites, the locations of which are determined by examining the protein dynamics of the tubulin and identifying the essential dynamic domains. Atomistic MD modeling is employed to directly compute the tubulin bond energies in the surface lattice of a microtubule, which are used to parameterize the interactions between CG building blocks. The CG model is then used to study the self-assembly pathways, kinetics, dynamics, and nanomechanics of microtubules.

The effects of dynamic compression on the development of cartilage grafts engineered using bone marrow and infrapatellar fat pad derived stem cells.

PubMed

Luo, Lu; Thorpe, Stephen D; Buckley, Conor T; Kelly, Daniel J

2015-09-21

Bioreactors that subject cell seeded scaffolds or hydrogels to biophysical stimulation have been used to improve the functionality of tissue engineered cartilage and to explore how such constructs might respond to the application of joint specific mechanical loading. Whether a particular cell type responds appropriately to physiological levels of biophysical stimulation could be considered a key determinant of its suitability for cartilage tissue engineering applications. The objective of this study was to determine the effects of dynamic compression on chondrogenesis of stem cells isolated from different tissue sources. Porcine bone marrow (BM) and infrapatellar fat pad (FP) derived stem cells were encapsulated in agarose hydrogels and cultured in a chondrogenic medium in free swelling (FS) conditions for 21 d, after which samples were subjected to dynamic compression (DC) of 10% strain (1 Hz, 1 h d(-1)) for a further 21 d. Both BM derived stem cells (BMSCs) and FP derived stem cells (FPSCs) were capable of generating cartilaginous tissues with near native levels of sulfated glycosaminoglycan (sGAG) content, although the spatial development of the engineered grafts strongly depended on the stem cell source. The mechanical properties of cartilage grafts generated from both stem cell sources also approached that observed in skeletally immature animals. Depending on the stem cell source and the donor, the application of DC either enhanced or had no significant effect on the functional development of cartilaginous grafts engineered using either BMSCs or FPSCs. BMSC seeded constructs subjected to DC stained less intensely for collagen type I. Furthermore, histological and micro-computed tomography analysis showed mineral deposition within BMSC seeded constructs was suppressed by the application of DC. Therefore, while the application of DC in vitro may only lead to modest improvements in the mechanical functionality of cartilaginous grafts, it may play an important role in the development of phenotypically stable constructs.

Increased intracellular pH is necessary for adult epithelial and embryonic stem cell differentiation

PubMed Central

Azimova, Dinara R.

2016-01-01

Despite extensive knowledge about the transcriptional regulation of stem cell differentiation, less is known about the role of dynamic cytosolic cues. We report that an increase in intracellular pH (pHi) is necessary for the efficient differentiation of Drosophila adult follicle stem cells (FSCs) and mouse embryonic stem cells (mESCs). We show that pHi increases with differentiation from FSCs to prefollicle cells (pFCs) and follicle cells. Loss of the Drosophila Na+–H+ exchanger DNhe2 lowers pHi in differentiating cells, impairs pFC differentiation, disrupts germarium morphology, and decreases fecundity. In contrast, increasing pHi promotes excess pFC cell differentiation toward a polar/stalk cell fate through suppressing Hedgehog pathway activity. Increased pHi also occurs with mESC differentiation and, when prevented, attenuates spontaneous differentiation of naive cells, as determined by expression of microRNA clusters and stage-specific markers. Our findings reveal a previously unrecognized role of pHi dynamics for the differentiation of two distinct types of stem cell lineages, which opens new directions for understanding conserved regulatory mechanisms. PMID:27821494

Virus dynamics in the presence of synaptic transmission

PubMed Central

Komarova, Natalia L.; Wodarz, Dominik

2014-01-01

Traditionally, virus dynamics models consider populations of infected and target cells, and a population of free virus that can infect susceptible cells. In recent years, however, it has become clear that direct cell-to-cell transmission can also play an important role for the in vivo spread of viruses, especially retroviruses such as human T lymphotropic virus-1 (HTLV-1) and Human immundeficeincy virus (HIV). Such cell-to-cell transmission is thought to occur through the formation of virological synapses that are formed between an infected source cell and a susceptible target cell. Here we formulate and analyze a class of virus dynamics models that include such cell-cell synaptic transmission. We explore different ”strategies” of the virus defined by the number of viruses passed per synapse, and determine how the choice of strategy influences the basic reproductive ratio, R0, of the virus and thus its ability to establish a persistent infection. We show that depending on specific assumptions about the viral kinetics, strategies with low or intermediate numbers of viruses transferred may correspond to the highest values of R0. We also explore the evolutionary competition of viruses of different strains, which differ by their synaptic strategy, and show that viruses characterized by synaptic strategies with the highest R0 win the evolutionary competition and exclude other, inferior, strains. PMID:23357287

Computational modeling of epidermal cell fate determination systems.

PubMed

Ryu, Kook Hui; Zheng, Xiaohua; Huang, Ling; Schiefelbein, John

2013-02-01

Cell fate decisions are of primary importance for plant development. Their simple 'either-or' outcome and dynamic nature has attracted the attention of computational modelers. Recent efforts have focused on modeling the determination of several epidermal cell types in the root and shoot of Arabidopsis where many molecular components have been defined. Results of integrated modeling and molecular biology experimentation in these systems have highlighted the importance of competitive positive and negative factors and interconnected feedback loops in generating flexible yet robust mechanisms for establishing distinct gene expression programs in neighboring cells. These models have proven useful in judging hypotheses and guiding future research. Copyright © 2012 Elsevier Ltd. All rights reserved.

Can human mesenchymal stem cells survive on a NiTi implant material subjected to cyclic loading?

PubMed

Habijan, T; Glogowski, T; Kühn, S; Pohl, M; Wittsiepe, J; Greulich, C; Eggeler, G; Schildhauer, T A; Köller, M

2011-06-01

Nickel-titanium shape memory alloys (NiTi-SMAs) exhibit mechanical and chemical properties which make them attractive candidate materials for various types of biomedical applications. However, the high nickel content of NiTi-SMAs may result in adverse tissue reactions, especially when they are considered for load-bearing implants. It is generally assumed that a protective titanium oxide layer separates the metallic alloy from its environment and that this explains the good biocompatibility of NiTi. Cyclic loading may result in failure of the protective oxide layer. The scientific objective of this work was to find out whether cyclic dynamic strain, in a range relevant for orthopedic implants, diminishes the biocompatibility of NiTi-SMAs. In order to analyze the biocompatibility of NiTi-SMA surfaces subjected to cyclic loading, NiTi-SMA tensile specimens were preloaded with mesenchymal stem cells, transferred to a sterile cell culture system and fixed to the pull rods of a tensile testing machine. Eighty-six thousand and four hundred strain cycles at 2% pseudoelastic strain were performed for a period of 24 h or 7 days. Cytokines (IL-6, IL-8 and VEGF) and nickel ion release were determined within the cell culture medium. Adherent cells on the tensile specimens were stained with calcein-AM and propidium iodide to determine cell viability. Dynamic loading of the tensile specimens did not influence the viability of adherent human mesenchymal stem cells (hMSCs) after 24 h or 7 days compared with the non-strained control. Dynamic cycles of loading and unloading did not affect nickel ion release from the tensile specimens. The release of IL-6 from hMSCs cultured under dynamic conditions was significantly higher after mechanical load (873 pg ml(-1)) compared with static conditions (323 pg ml(-1)). The present work demonstrates that a new type of mechanical in vitro cell culture experiment can provide information which previously could only be obtained in large animal experiments. Copyright © 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Mechanoregulation of clathrin-mediated endocytosis in isolated cells and developing tissues

NASA Astrophysics Data System (ADS)

Kural, Comert

Clathrin-coated assemblies bear the largest fraction of the endocytic load from the plasma membrane of eukaryotic cells. However, dynamics of clathrin-mediated endocytosis (CME) have not been established within tissues of multicellular organisms due to experimental and analytical bottlenecks in determining the lifespan of clathrin-coated structures. We found that clathrin coat growth rates obtained from fluorescence microscopy acquisitions can be utilized as reporters of CME dynamics. Growth rates can be assembled within time windows shorter than the average clathrin coat lifetime and, thereby, allow probing the changes in CME dynamics in real time. Furthermore, this novel approach is applicable to tissues as it is not prone to particle detection and tracking errors, which result in underestimation of the clathrin coat lifetimes. Exploiting these advantages, we detected spatial and temporal changes in CME dynamics within Drosophila amnioserosa tissues at different stages of embryo development. We also found that increased membrane tension impedes CME through inhibition of formation and dissolution of clathrin-coated structures. Therefore, the parameters defining clathrin coat dynamics (i.e., lifetime, formation density and growth rates) can be utilized to monitor the spatiotemporal gradients of the plasma membrane tension during cell migration and spreading.

Live-cell imaging of endogenous mRNAs with a small molecule.

PubMed

Sato, Shin-ichi; Watanabe, Mizuki; Katsuda, Yousuke; Murata, Asako; Wang, Dan Ohtan; Uesugi, Motonari

2015-02-02

Determination of subcellular localization and dynamics of mRNA is increasingly important to understanding gene expression. A new convenient and versatile method is reported that permits spatiotemporal imaging of specific non-engineered RNAs in living cells. The method uses transfection of a plasmid encoding a gene-specific RNA aptamer, combined with a cell-permeable synthetic small molecule, the fluorescence of which is restored only when the RNA aptamer hybridizes with its cognitive mRNA. The method was validated by live-cell imaging of the endogenous mRNA of β-actin. Application of the technology to mRNAs of a total of 84 human cytoskeletal genes allowed us to observe cellular dynamics of several endogenous mRNAs including arfaptin-2, cortactin, and cytoplasmic FMR1-interacting protein 2. The RNA-imaging technology and its further optimization might permit live-cell imaging of any RNA molecules. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The nature and nurture of cell heterogeneity: accounting for macrophage gene-environment interactions with single-cell RNA-Seq.

PubMed

Wills, Quin F; Mellado-Gomez, Esther; Nolan, Rory; Warner, Damien; Sharma, Eshita; Broxholme, John; Wright, Benjamin; Lockstone, Helen; James, William; Lynch, Mark; Gonzales, Michael; West, Jay; Leyrat, Anne; Padilla-Parra, Sergi; Filippi, Sarah; Holmes, Chris; Moore, Michael D; Bowden, Rory

2017-01-07

Single-cell RNA-Seq can be a valuable and unbiased tool to dissect cellular heterogeneity, despite the transcriptome's limitations in describing higher functional phenotypes and protein events. Perhaps the most important shortfall with transcriptomic 'snapshots' of cell populations is that they risk being descriptive, only cataloging heterogeneity at one point in time, and without microenvironmental context. Studying the genetic ('nature') and environmental ('nurture') modifiers of heterogeneity, and how cell population dynamics unfold over time in response to these modifiers is key when studying highly plastic cells such as macrophages. We introduce the programmable Polaris™ microfluidic lab-on-chip for single-cell sequencing, which performs live-cell imaging while controlling for the culture microenvironment of each cell. Using gene-edited macrophages we demonstrate how previously unappreciated knockout effects of SAMHD1, such as an altered oxidative stress response, have a large paracrine signaling component. Furthermore, we demonstrate single-cell pathway enrichments for cell cycle arrest and APOBEC3G degradation, both associated with the oxidative stress response and altered proteostasis. Interestingly, SAMHD1 and APOBEC3G are both HIV-1 inhibitors ('restriction factors'), with no known co-regulation. As single-cell methods continue to mature, so will the ability to move beyond simple 'snapshots' of cell populations towards studying the determinants of population dynamics. By combining single-cell culture, live-cell imaging, and single-cell sequencing, we have demonstrated the ability to study cell phenotypes and microenvironmental influences. It's these microenvironmental components - ignored by standard single-cell workflows - that likely determine how macrophages, for example, react to inflammation and form treatment resistant HIV reservoirs.

Polarity, cell division, and out-of-equilibrium dynamics control the growth of epithelial structures

PubMed Central

Cerruti, Benedetta; Puliafito, Alberto; Shewan, Annette M.; Yu, Wei; Combes, Alexander N.; Little, Melissa H.; Chianale, Federica; Primo, Luca; Serini, Guido; Mostov, Keith E.; Celani, Antonio

2013-01-01

The growth of a well-formed epithelial structure is governed by mechanical constraints, cellular apico-basal polarity, and spatially controlled cell division. Here we compared the predictions of a mathematical model of epithelial growth with the morphological analysis of 3D epithelial structures. In both in vitro cyst models and in developing epithelial structures in vivo, epithelial growth could take place close to or far from mechanical equilibrium, and was determined by the hierarchy of time-scales of cell division, cell–cell rearrangements, and lumen dynamics. Equilibrium properties could be inferred by the analysis of cell–cell contact topologies, and the nonequilibrium phenotype was altered by inhibiting ROCK activity. The occurrence of an aberrant multilumen phenotype was linked to fast nonequilibrium growth, even when geometric control of cell division was correctly enforced. We predicted and verified experimentally that slowing down cell division partially rescued a multilumen phenotype induced by altered polarity. These results improve our understanding of the development of epithelial organs and, ultimately, of carcinogenesis. PMID:24145168

Osteogenic Performance of Donor-Matched Human Adipose and Bone Marrow Mesenchymal Cells Under Dynamic Culture

PubMed Central

Wu, Wei; Le, Andrew V.; Mendez, Julio J.; Chang, Julie; Niklason, Laura E.

2015-01-01

Adipose-derived mesenchymal cells (ACs) and bone marrow-derived mesenchymal cells (BMCs) have been widely used for bone regeneration and can be seeded on a variety of rigid scaffolds. However, to date, a direct comparison of mesenchymal cells (MC) harvested from different tissues from the same donor and cultured in identical osteogenic conditions has not been investigated. Indeed, it is unclear whether marrow-derived or fat-derived MC possess intrinsic differences in bone-forming capabilities, since within-patient comparisons have not been previously done. This study aims at comparing ACs and BMCs from three donors ranging in age from neonatal to adult. Matched cells from each donor were studied in three distinct bioreactor settings, to determine the best method to create a viable osseous engineered construct. Human ACs and BMCs were isolated from each donor, cultured, and seeded on decellularized porcine bone (DCB) constructs. The constructs were then subjected to either static or dynamic (stirring or perfusion) bioreactor culture conditions for 7–21 days. Afterward, the constructs were analyzed for cell adhesion and distribution and osteogenic differentiation. ACs demonstrated higher seeding efficiency than BMCs. However, static and dynamic culture significantly increased BMCs proliferation more than ACs. In all conditions, BMCs demonstrated stronger osteogenic activity as compared with ACs, through higher alkaline phosphatase activity and gene expression for various bony markers. Conversely, ACs expressed more collagen I, which is a nonspecific matrix molecule in most connective tissues. Overall, dynamic bioreactor culture conditions enhanced osteogenic gene expression in both ACs and BMCs. Scaffolds seeded with BMCs in dynamic stirring culture conditions exhibit the greatest osteogenic proliferation and function in vitro, proving that marrow-derived MC have superior bone-forming potential as compared with adipose-derived cells. PMID:25668104

Investigation of polymer electrolyte membrane fuel cell internal behaviour during long term operation and its use in prognostics

NASA Astrophysics Data System (ADS)

Mao, Lei; Jackson, Lisa; Jackson, Tom

2017-09-01

This paper investigates the polymer electrolyte membrane (PEM) fuel cell internal behaviour variation at different operating condition, with characterization test data taken at predefined inspection times, and uses the determined internal behaviour evolution to predict the future PEM fuel cell performance. For this purpose, a PEM fuel cell behaviour model is used, which can be related to various fuel cell losses. By matching the model to the collected polarization curves from the PEM fuel cell system, the variation of fuel cell internal behaviour can be obtained through the determined model parameters. From the results, the source of PEM fuel cell degradation during its lifetime at different conditions can be better understood. Moreover, with determined fuel cell internal behaviour, the future fuel cell performance can be obtained by predicting the future model parameters. By comparing with prognostic results using adaptive neuro fuzzy inference system (ANFIS), the proposed prognostic analysis can provide better predictions for PEM fuel cell performance at dynamic condition, and with the understanding of variation in PEM fuel cell internal behaviour, mitigation strategies can be designed to extend the fuel cell performance.

Mathematical modeling of solid cancer growth with angiogenesis

PubMed Central

2012-01-01

Background Cancer arises when within a single cell multiple malfunctions of control systems occur, which are, broadly, the system that promote cell growth and the system that protect against erratic growth. Additional systems within the cell must be corrupted so that a cancer cell, to form a mass of any real size, produces substances that promote the growth of new blood vessels. Multiple mutations are required before a normal cell can become a cancer cell by corruption of multiple growth-promoting systems. Methods We develop a simple mathematical model to describe the solid cancer growth dynamics inducing angiogenesis in the absence of cancer controlling mechanisms. Results The initial conditions supplied to the dynamical system consist of a perturbation in form of pulse: The origin of cancer cells from normal cells of an organ of human body. Thresholds of interacting parameters were obtained from the steady states analysis. The existence of two equilibrium points determine the strong dependency of dynamical trajectories on the initial conditions. The thresholds can be used to control cancer. Conclusions Cancer can be settled in an organ if the following combination matches: better fitness of cancer cells, decrease in the efficiency of the repairing systems, increase in the capacity of sprouting from existing vascularization, and higher capacity of mounting up new vascularization. However, we show that cancer is rarely induced in organs (or tissues) displaying an efficient (numerically and functionally) reparative or regenerative mechanism. PMID:22300422

Phenotypic Plasticity and Cell Fate Decisions in Cancer: Insights from Dynamical Systems Theory.

PubMed

Jia, Dongya; Jolly, Mohit Kumar; Kulkarni, Prakash; Levine, Herbert

2017-06-22

Waddington's epigenetic landscape, a famous metaphor in developmental biology, depicts how a stem cell progresses from an undifferentiated phenotype to a differentiated one. The concept of "landscape" in the context of dynamical systems theory represents a high-dimensional space, in which each cell phenotype is considered as an "attractor" that is determined by interactions between multiple molecular players, and is buffered against environmental fluctuations. In addition, biological noise is thought to play an important role during these cell-fate decisions and in fact controls transitions between different phenotypes. Here, we discuss the phenotypic transitions in cancer from a dynamical systems perspective and invoke the concept of "cancer attractors"-hidden stable states of the underlying regulatory network that are not occupied by normal cells. Phenotypic transitions in cancer occur at varying levels depending on the context. Using epithelial-to-mesenchymal transition (EMT), cancer stem-like properties, metabolic reprogramming and the emergence of therapy resistance as examples, we illustrate how phenotypic plasticity in cancer cells enables them to acquire hybrid phenotypes (such as hybrid epithelial/mesenchymal and hybrid metabolic phenotypes) that tend to be more aggressive and notoriously resilient to therapies such as chemotherapy and androgen-deprivation therapy. Furthermore, we highlight multiple factors that may give rise to phenotypic plasticity in cancer cells, such as (a) multi-stability or oscillatory behaviors governed by underlying regulatory networks involved in cell-fate decisions in cancer cells, and (b) network rewiring due to conformational dynamics of intrinsically disordered proteins (IDPs) that are highly enriched in cancer cells. We conclude by discussing why a therapeutic approach that promotes "recanalization", i.e., the exit from "cancer attractors" and re-entry into "normal attractors", is more likely to succeed rather than a conventional approach that targets individual molecules/pathways.

Use of inert gas jets to measure the forces required for mechanical gene transfection

PubMed Central

2012-01-01

Background Transferring genes and drugs into cells is central to how we now study, identify and treat diseases. Several non-viral gene therapy methods that rely on the mechanical disruption of the plasma membrane have been proposed, but the success of these methods has been limited due to a lack of understanding of the mechanical parameters that lead to cell membrane permeability. Methods We use a simple jet of inert gas to induce local transfection of plasmid DNA both in vitro (HeLa cells) and in vivo (chicken chorioallantoic membrane). Five different capillary tube inner diameters and three different gases were used to treat the cells to understand the dependency of transfection efficiency on the dynamic parameters. Results The simple setup has the advantage of allowing us to calculate the forces acting on cells during transfection. We found permeabilization efficiency was related to the dynamic pressure of the jet. The range of dynamic pressures that led to transfection in HeLa cells was small (200 ± 20 Pa) above which cell stripping occurred. We determined that the temporary pores allow the passage of dextran up to 40 kDa and reclose in less than 5 seconds after treatment. The optimized parameters were also successfully tested in vivo using the chorioallantoic membrane of the chick embryo. Conclusions The results show that the number of cells transfected with the plasmid scales with the dynamic pressure of the jet. Our results show that mechanical methods have a very small window in which cells are permeabilized without injury (200 to 290 Pa). This simple apparatus helps define the forces needed for physical cell transfection methods. PMID:22963645

Cellular Manufacturing System with Dynamic Lot Size Material Handling

NASA Astrophysics Data System (ADS)

Khannan, M. S. A.; Maruf, A.; Wangsaputra, R.; Sutrisno, S.; Wibawa, T.

2016-02-01

Material Handling take as important role in Cellular Manufacturing System (CMS) design. In several study at CMS design material handling was assumed per pieces or with constant lot size. In real industrial practice, lot size may change during rolling period to cope with demand changes. This study develops CMS Model with Dynamic Lot Size Material Handling. Integer Linear Programming is used to solve the problem. Objective function of this model is minimizing total expected cost consisting machinery depreciation cost, operating costs, inter-cell material handling cost, intra-cell material handling cost, machine relocation costs, setup costs, and production planning cost. This model determines optimum cell formation and optimum lot size. Numerical examples are elaborated in the paper to ilustrate the characterictic of the model.

Photocrosslinking approaches to interactome mapping

PubMed Central

Pham, Nam D.; Parker, Randy B.; Kohler, Jennifer J.

2012-01-01

Photocrosslinking approaches can be used to map interactome networks within the context of living cells. Photocrosslinking methods rely on use of metabolic engineering or genetic code expansion to incorporate photocrosslinking analogs of amino acids or sugars into cellular biomolecules. Immunological and mass spectrometry techniques are used to analyze crosslinked complexes, thereby defining specific interactomes. Because photocrosslinking can be conducted in native, cellular settings, it can be used to define context-dependent interactions. Photocrosslinking methods are also ideally suited for determining interactome dynamics, mapping interaction interfaces, and identifying transient interactions in which intrinsically disordered proteins and glycoproteins engage. Here we discuss the application of cell-based photocrosslinking to the study of specific problems in immune cell signaling, transcription, membrane protein dynamics, nucleocytoplasmic transport, and chaperone-assisted protein folding. PMID:23149092

Mechanical influences in bacterial morphogenesis and cell division

NASA Astrophysics Data System (ADS)

Sun, Sean

2010-03-01

Bacterial cells utilize a ring-like organelle (the Z-ring) to accomplish cell division. The Z-ring actively generates a contractile force and influences cell wall growth. We will discuss a general model of bacterial morphogenesis where mechanical forces are coupled to the growth dynamics of the cell wall. The model suggests a physical mechanism that determines the shapes of bacteria cells. The roles of several bacterial cytoskeletal proteins and the Z-ring are discussed. We will also explore molecular mechanisms of force generation by the Z-ring and how cells can generate mechanical forces without molecular motors.

Identifying niche-mediated regulatory factors of stem cell phenotypic state: a systems biology approach.

PubMed

Ravichandran, Srikanth; Del Sol, Antonio

2017-02-01

Understanding how the cellular niche controls the stem cell phenotype is often hampered due to the complexity of variegated niche composition, its dynamics, and nonlinear stem cell-niche interactions. Here, we propose a systems biology view that considers stem cell-niche interactions as a many-body problem amenable to simplification by the concept of mean field approximation. This enables approximation of the niche effect on stem cells as a constant field that induces sustained activation/inhibition of specific stem cell signaling pathways in all stem cells within heterogeneous populations exhibiting the same phenotype (niche determinants). This view offers a new basis for the development of single cell-based computational approaches for identifying niche determinants, which has potential applications in regenerative medicine and tissue engineering. © 2017 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

How cells explore shape space: a quantitative statistical perspective of cellular morphogenesis.

PubMed

Yin, Zheng; Sailem, Heba; Sero, Julia; Ardy, Rico; Wong, Stephen T C; Bakal, Chris

2014-12-01

Through statistical analysis of datasets describing single cell shape following systematic gene depletion, we have found that the morphological landscapes explored by cells are composed of a small number of attractor states. We propose that the topology of these landscapes is in large part determined by cell-intrinsic factors, such as biophysical constraints on cytoskeletal organization, and reflects different stable signaling and/or transcriptional states. Cell-extrinsic factors act to determine how cells explore these landscapes, and the topology of the landscapes themselves. Informational stimuli primarily drive transitions between stable states by engaging signaling networks, while mechanical stimuli tune, or even radically alter, the topology of these landscapes. As environments fluctuate, the topology of morphological landscapes explored by cells dynamically adapts to these fluctuations. Finally we hypothesize how complex cellular and tissue morphologies can be generated from a limited number of simple cell shapes. © 2014 WILEY Periodicals, Inc.

Effects of fullerene on lipid bilayers displaying different liquid ordering: a coarse-grained molecular dynamics study.

PubMed

Sastre, Judit; Mannelli, Ilaria; Reigada, Ramon

2017-11-01

The toxic effects and environmental impact of nanomaterials, and in particular of Fullerene particles, are matters of serious concern. It has been reported that fullerene molecules enter the cell membrane and occupy its hydrophobic region. Understanding the effects of carbon-based nanoparticles on biological membranes is therefore of critical importance to determine their exposure risks. We report on a systematic coarse-grained molecular dynamics study of the interaction of fullerene molecules with simple model cell membranes. We have analyzed bilayers consisting of lipid species with different degrees of unsaturation and a variety of cholesterol fractions. Addition of fullerene particles to phase-segregated ternary membranes is also investigated in the context of the lipid raft model for the organization of the cell membrane. Fullerene addition to lipid membranes modifies their structural properties like thickness, area and internal ordering of the lipid species, as well as dynamical aspects such as molecular diffusion and cholesterol flip-flop. Interestingly, we show that phase-segregating ternary lipid membranes accumulate fullerene molecules preferentially in the liquid-disordered domains promoting phase-segregation and domain alignment across the membrane. Lipid membrane internal ordering determines the behavior and distribution of fullerene particle, and this, in turn, determines the influence of fullerene on the membrane. Lipid membranes are good solvents of fullerene molecules, and in particular those with low internal ordering. Preference of fullerene molecules to be dissolved in the more disordered hydrophobic regions of a lipid bilayer and the consequent alteration of its phase behavior may have important consequences on the activity of biological cell membranes and on the bioconcentration of fullerene in living organisms. Copyright © 2017 Elsevier B.V. All rights reserved.

Systematic analysis of protein turnover in primary cells.

PubMed

Mathieson, Toby; Franken, Holger; Kosinski, Jan; Kurzawa, Nils; Zinn, Nico; Sweetman, Gavain; Poeckel, Daniel; Ratnu, Vikram S; Schramm, Maike; Becher, Isabelle; Steidel, Michael; Noh, Kyung-Min; Bergamini, Giovanna; Beck, Martin; Bantscheff, Marcus; Savitski, Mikhail M

2018-02-15

A better understanding of proteostasis in health and disease requires robust methods to determine protein half-lives. Here we improve the precision and accuracy of peptide ion intensity-based quantification, enabling more accurate protein turnover determination in non-dividing cells by dynamic SILAC-based proteomics. This approach allows exact determination of protein half-lives ranging from 10 to >1000 h. We identified 4000-6000 proteins in several non-dividing cell types, corresponding to 9699 unique protein identifications over the entire data set. We observed similar protein half-lives in B-cells, natural killer cells and monocytes, whereas hepatocytes and mouse embryonic neurons show substantial differences. Our data set extends and statistically validates the previous observation that subunits of protein complexes tend to have coherent turnover. Moreover, analysis of different proteasome and nuclear pore complex assemblies suggests that their turnover rate is architecture dependent. These results illustrate that our approach allows investigating protein turnover and its implications in various cell types.

Dynamic electrical response of solar cells

NASA Technical Reports Server (NTRS)

Catani, J. P.

1981-01-01

The dynamic response of a solar generator is of primary importance as much for the design and development of electrical power conditioning hardware as for the analysis of electromagnetic compatibility. A mathematical model of photo-batteries was developed on the basis of impedance measurements performed under differing conditions of temperature, light intensity, before and after irradiation. This model was compared with that derived from PN junction theory and to static measurements. These dynamic measurements enabled the refinement of an integration method capable of determining, under normal laboratory conditions, the dynamic response of a generator to operational lighting conditions.

Single-Molecule Imaging of RNA Splicing in Live Cells.

PubMed

Rino, José; Martin, Robert M; Carvalho, Célia; de Jesus, Ana C; Carmo-Fonseca, Maria

2015-01-01

Expression of genetic information in eukaryotes involves a series of interconnected processes that ultimately determine the quality and amount of proteins in the cell. Many individual steps in gene expression are kinetically coupled, but tools are lacking to determine how temporal relationships between chemical reactions contribute to the output of the final gene product. Here, we describe a strategy that permits direct measurements of intron dynamics in single pre-mRNA molecules in live cells. This approach reveals that splicing can occur much faster than previously proposed and opens new avenues for studying how kinetic mechanisms impact on RNA biogenesis. © 2015 Elsevier Inc. All rights reserved.

Hydrodynamic Determinants of Cell Necrosis and Molecular Delivery Produced by Pulsed Laser Microbeam Irradiation of Adherent Cells

PubMed Central

Compton, Jonathan L.; Hellman, Amy N.; Venugopalan, Vasan

2013-01-01

Time-resolved imaging, fluorescence microscopy, and hydrodynamic modeling were used to examine cell lysis and molecular delivery produced by picosecond and nanosecond pulsed laser microbeam irradiation in adherent cell cultures. Pulsed laser microbeam radiation at λ = 532 nm was delivered to confluent monolayers of PtK2 cells via a 40×, 0.8 NA microscope objective. Using laser microbeam pulse durations of 180–1100 ps and pulse energies of 0.5–10.5 μJ, we examined the resulting plasma formation and cavitation bubble dynamics that lead to laser-induced cell lysis, necrosis, and molecular delivery. The cavitation bubble dynamics are imaged at times of 0.5 ns to 50 μs after the pulsed laser microbeam irradiation, and fluorescence assays assess the resulting cell viability and molecular delivery of 3 kDa dextran molecules. Reductions in both the threshold laser microbeam pulse energy for plasma formation and the cavitation bubble energy are observed with decreasing pulse duration. These energy reductions provide for increased precision of laser-based cellular manipulation including cell lysis, cell necrosis, and molecular delivery. Hydrodynamic analysis reveals critical values for the shear-stress impulse generated by the cavitation bubble dynamics governs the location and spatial extent of cell necrosis and molecular delivery independent of pulse duration and pulse energy. Specifically, cellular exposure to a shear-stress impulse J≳0.1 Pa s ensures cell lysis or necrosis, whereas exposures in the range of 0.035≲J≲0.1 Pa s preserve cell viability while also enabling molecular delivery of 3 kDa dextran. Exposure to shear-stress impulses of J≲0.035 Pa s leaves the cells unaffected. Hydrodynamic analysis of these data, combined with data from studies of 6 ns microbeam irradiation, demonstrates the primacy of shear-stress impulse in determining cellular outcome resulting from pulsed laser microbeam irradiation spanning a nearly two-orders-of-magnitude range of pulse energy and pulse duration. These results provide a mechanistic foundation and design strategy applicable to a broad range of laser-based cellular manipulation procedures. PMID:24209868

SPIRAL2 Determines Plant Microtubule Organization by Modulating Microtubule Severing

PubMed Central

Wightman, Raymond; Chomicki, Guillaume; Kumar, Manoj; Carr, Paul; Turner, Simon R.

2013-01-01

Summary One of the defining characteristics of plant growth and morphology is the pivotal role of cell expansion. While the mechanical properties of the cell wall determine both the extent and direction of cell expansion, the cortical microtubule array plays a critical role in cell wall organization and, consequently, determining directional (anisotropic) cell expansion [1–6]. The microtubule-severing enzyme katanin is essential for plants to form aligned microtubule arrays [7–10]; however, increasing severing activity alone is not sufficient to drive microtubule alignment [11]. Here, we demonstrate that katanin activity depends upon the behavior of the microtubule-associated protein (MAP) SPIRAL2 (SPR2). Petiole cells in the cotyledon epidermis exhibit well-aligned microtubule arrays, whereas adjacent pavement cells exhibit unaligned arrays, even though SPR2 is found at similar levels in both cell types. In pavement cells, however, SPR2 accumulates at microtubule crossover sites, where it stabilizes these crossovers and prevents severing. In contrast, in the adjacent petiole cells, SPR2 is constantly moving along the microtubules, exposing crossover sites that become substrates for severing. Consequently, our study reveals a novel mechanism whereby microtubule organization is determined by dynamics and localization of a MAP that regulates where and when microtubule severing occurs. PMID:24055158

Adhesion kinetics of human primary monocytes, dendritic cells, and macrophages: Dynamic cell adhesion measurements with a label-free optical biosensor and their comparison with end-point assays.

PubMed

Orgovan, Norbert; Ungai-Salánki, Rita; Lukácsi, Szilvia; Sándor, Noémi; Bajtay, Zsuzsa; Erdei, Anna; Szabó, Bálint; Horvath, Robert

2016-09-01

Monocytes, dendritic cells (DCs), and macrophages (MFs) are closely related immune cells that differ in their main functions. These specific functions are, to a considerable degree, determined by the differences in the adhesion behavior of the cells. To study the inherently and essentially dynamic aspects of the adhesion of monocytes, DCs, and MFs, dynamic cell adhesion assays were performed with a high-throughput label-free optical biosensor [Epic BenchTop (BT)] on surfaces coated with either fibrinogen (Fgn) or the biomimetic copolymer PLL-g-PEG-RGD. Cell adhesion profiles typically reached their maximum at ∼60 min after cell seeding, which was followed by a monotonic signal decrease, indicating gradually weakening cell adhesion. According to the biosensor response, cell types could be ordered by increasing adherence as monocytes, MFs, and DCs. Notably, all three cell types induced a larger biosensor signal on Fgn than on PLL-g-PEG-RGD. To interpret this result, the molecular layers were characterized by further exploiting the potentials of the biosensor: by measuring the adsorption signal induced during the surface coating procedure, the authors could estimate the surface density of adsorbed molecules and, thus, the number of binding sites potentially presented for the adhesion receptors. Surfaces coated with PLL-g-PEG-RGD presented less RGD sites, but was less efficient in promoting cell spreading than those coated with Fgn; hence, other binding sites in Fgn played a more decisive role in determining cell adherence. To support the cell adhesion data obtained with the biosensor, cell adherence on Fgn-coated surfaces 30-60 min after cell seeding was measured with three complementary techniques, i.e., with (1) a fluorescence-based classical adherence assay, (2) a shear flow chamber applying hydrodynamic shear stress to wash cells away, and (3) an automated micropipette using vacuum-generated fluid flow to lift cells up. These techniques confirmed the results obtained with the high-temporal-resolution Epic BT, but could only provide end-point data. In contrast, complex, nonmonotonic cell adhesion kinetics measured by the high-throughput optical biosensor is expected to open a window on the hidden background of the immune cell-extracellular matrix interactions.

Stochastic Simulation of Biomolecular Networks in Dynamic Environments

PubMed Central

Voliotis, Margaritis; Thomas, Philipp; Grima, Ramon; Bowsher, Clive G.

2016-01-01

Simulation of biomolecular networks is now indispensable for studying biological systems, from small reaction networks to large ensembles of cells. Here we present a novel approach for stochastic simulation of networks embedded in the dynamic environment of the cell and its surroundings. We thus sample trajectories of the stochastic process described by the chemical master equation with time-varying propensities. A comparative analysis shows that existing approaches can either fail dramatically, or else can impose impractical computational burdens due to numerical integration of reaction propensities, especially when cell ensembles are studied. Here we introduce the Extrande method which, given a simulated time course of dynamic network inputs, provides a conditionally exact and several orders-of-magnitude faster simulation solution. The new approach makes it feasible to demonstrate—using decision-making by a large population of quorum sensing bacteria—that robustness to fluctuations from upstream signaling places strong constraints on the design of networks determining cell fate. Our approach has the potential to significantly advance both understanding of molecular systems biology and design of synthetic circuits. PMID:27248512

G-index: A new metric to describe dynamic refractive index effects in HPLC absorbance detection.

PubMed

Kraiczek, Karsten G; Rozing, Gerard P; Zengerle, Roland

2018-09-01

High performance liquid chromatography (HPLC) with a solvent gradient and absorbance detection is one of the most widely used methods in analytical chemistry. The observed absorbance baseline is affected by the changes in the refractive index (RI) of the mobile phase. Near the limited of detection, this complicates peak quantitation. The general aspects of these RI-induced apparent absorbance effects are discussed. Two different detectors with fundamentally different optics and flow cell concepts, a variable-wavelength detector equipped with a conventional flow cell and a diode-array detector equipped with a liquid core waveguide flow cell, are compared with respect to their RI behavior. A simple method to separate static - partly unavoidable - RI effects from dynamic RI effects is presented. It is shown that the dynamic RI behavior of an absorbance detector can be well described using a single, relatively easy-to-determine metric called the G-index. The G-index is typically in the order of a few seconds and its sign depends on the optical flow cell concept. Copyright © 2018 Elsevier B.V. All rights reserved.

Cellular context–mediated Akt dynamics regulates MAP kinase signaling thresholds during angiogenesis

PubMed Central

Hellesøy, Monica; Lorens, James B.

2015-01-01

The formation of new blood vessels by sprouting angiogenesis is tightly regulated by contextual cues that affect angiogeneic growth factor signaling. Both constitutive activation and loss of Akt kinase activity in endothelial cells impair angiogenesis, suggesting that Akt dynamics mediates contextual microenvironmental regulation. We explored the temporal regulation of Akt in endothelial cells during formation of capillary-like networks induced by cell–cell contact with vascular smooth muscle cells (vSMCs) and vSMC-associated VEGF. Expression of constitutively active Akt1 strongly inhibited network formation, whereas hemiphosphorylated Akt1 epi-alleles with reduced kinase activity had an intermediate inhibitory effect. Conversely, inhibition of Akt signaling did not affect endothelial cell migration or morphogenesis in vSMC cocultures that generate capillary-like structures. We found that endothelial Akt activity is transiently blocked by proteasomal degradation in the presence of SMCs during the initial phase of capillary-like structure formation. Suppressed Akt activity corresponded to the increased endothelial MAP kinase signaling that was required for angiogenic endothelial morphogenesis. These results reveal a regulatory principle by which cellular context regulates Akt protein dynamics, which determines MAP kinase signaling thresholds necessary drive a morphogenetic program during angiogenesis. PMID:26023089

Imaging molecular dynamics in vivo--from cell biology to animal models.

PubMed

Timpson, Paul; McGhee, Ewan J; Anderson, Kurt I

2011-09-01

Advances in fluorescence microscopy have enabled the study of membrane diffusion, cell adhesion and signal transduction at the molecular level in living cells grown in culture. By contrast, imaging in living organisms has primarily been restricted to the localization and dynamics of cells in tissues. Now, imaging of molecular dynamics is on the cusp of progressing from cell culture to living tissue. This transition has been driven by the understanding that the microenvironment critically determines many developmental and pathological processes. Here, we review recent progress in fluorescent protein imaging in vivo by drawing primarily on cancer-related studies in mice. We emphasize the need for techniques that can be easily combined with genetic models and complement fluorescent protein imaging by providing contextual information about the cellular environment. In this Commentary we will consider differences between in vitro and in vivo experimental design and argue for an approach to in vivo imaging that is built upon the use of intermediate systems, such as 3-D and explant culture models, which offer flexibility and control that is not always available in vivo. Collectively, these methods present a paradigm shift towards the molecular-level investigation of disease and therapy in animal models of disease.

A network model for the specification of vulval precursor cells and cell fusion control in Caenorhabditis elegans

PubMed Central

Weinstein, Nathan; Mendoza, Luis

2013-01-01

The vulva of Caenorhabditis elegans has been long used as an experimental model of cell differentiation and organogenesis. While it is known that the signaling cascades of Wnt, Ras/MAPK, and NOTCH interact to form a molecular network, there is no consensus regarding its precise topology and dynamical properties. We inferred the molecular network, and developed a multivalued synchronous discrete dynamic model to study its behavior. The model reproduces the patterns of activation reported for the following types of cell: vulval precursor, first fate, second fate, second fate with reversed polarity, third fate, and fusion fate. We simulated the fusion of cells, the determination of the first, second, and third fates, as well as the transition from the second to the first fate. We also used the model to simulate all possible single loss- and gain-of-function mutants, as well as some relevant double and triple mutants. Importantly, we associated most of these simulated mutants to multivulva, vulvaless, egg-laying defective, or defective polarity phenotypes. The model shows that it is necessary for RAL-1 to activate NOTCH signaling, since the repression of LIN-45 by RAL-1 would not suffice for a proper second fate determination in an environment lacking DSL ligands. We also found that the model requires the complex formed by LAG-1, LIN-12, and SEL-8 to inhibit the transcription of eff-1 in second fate cells. Our model is the largest reconstruction to date of the molecular network controlling the specification of vulval precursor cells and cell fusion control in C. elegans. According to our model, the process of fate determination in the vulval precursor cells is reversible, at least until either the cells fuse with the ventral hypoderm or divide, and therefore the cell fates must be maintained by the presence of extracellular signals. PMID:23785384

Modeling Yeast Cell Polarization Induced by Pheromone Gradients

NASA Astrophysics Data System (ADS)

Yi, Tau-Mu; Chen, Shanqin; Chou, Ching-Shan; Nie, Qing

2007-07-01

Yeast cells respond to spatial gradients of mating pheromones by polarizing and projecting up the gradient toward the source. It is thought that they employ a spatial sensing mechanism in which the cell compares the concentration of pheromone at different points on the cell surface and determines the maximum point, where the projection forms. Here we constructed the first spatial mathematical model of the yeast pheromone response that describes the dynamics of the heterotrimeric and Cdc42p G-protein cycles, which are linked in a cascade. Two key performance objectives of this system are (1) amplification—converting a shallow external gradient of ligand to a steep internal gradient of protein components and (2) tracking—following changes in gradient direction. We used simulations to investigate amplification mechanisms that allow tracking. We identified specific strategies for regulating the spatial dynamics of the protein components (i.e. their changing location in the cell) that would enable the cell to achieve both objectives.

Wavelet Imaging on Multiple Scales (WIMS) reveals focal adhesion distributions, dynamics and coupling between actomyosin bundle stability

PubMed Central

Toplak, Tim; Palmieri, Benoit; Juanes-García, Alba; Vicente-Manzanares, Miguel; Grant, Martin; Wiseman, Paul W.

2017-01-01

We introduce and use Wavelet Imaging on Multiple Scales (WIMS) as an improvement to fluorescence correlation spectroscopy to measure physical processes and features that occur across multiple length scales. In this study, wavelet transforms of cell images are used to characterize molecular dynamics at the cellular and subcellular levels (i.e. focal adhesions). We show the usefulness of the technique by applying WIMS to an image time series of a migrating osteosarcoma cell expressing fluorescently labelled adhesion proteins, which allows us to characterize different components of the cell ranging from optical resolution scale through to focal adhesion and whole cell size scales. Using WIMS we measured focal adhesion numbers, orientation and cell boundary velocities for retraction and protrusion. We also determine the internal dynamics of individual focal adhesions undergoing assembly, disassembly or elongation. Thus confirming as previously shown, WIMS reveals that the number of adhesions and the area of the protruding region of the cell are strongly correlated, establishing a correlation between protrusion size and adhesion dynamics. We also apply this technique to characterize the behavior of adhesions, actin and myosin in Chinese hamster ovary cells expressing a mutant form of myosin IIB (1935D) that displays decreased filament stability and impairs front-back cell polarity. We find separate populations of actin and myosin at each adhesion pole for both the mutant and wild type form. However, we find these populations move rapidly inwards toward one another in the mutant case in contrast to the cells that express wild type myosin IIB where those populations remain stationary. Results obtained with these two systems demonstrate how WIMS has the potential to reveal novel correlations between chosen parameters that belong to different scales. PMID:29049414

Definition and dynamic control of a continuous chromatography process independent of cell culture titer and impurities.

PubMed

Chmielowski, Rebecca A; Mathiasson, Linda; Blom, Hans; Go, Daniel; Ehring, Hanno; Khan, Heera; Li, Hong; Cutler, Collette; Lacki, Karol; Tugcu, Nihal; Roush, David

2017-12-01

Advances in cell culture technology have enabled the production of antibody titers upwards of 30g/L. These highly productive cell culture systems can potentially lead to productivity bottlenecks in downstream purification due to lower column loadings, especially in the primary capture chromatography step. Alternative chromatography solutions to help remedy this bottleneck include the utilization of continuous processing systems such as periodic counter-current chromatography (PCC). Recent studies have provided methods to optimize and improve the design of PCC for cell culture titers up to about 3g/L. This paper defines a continuous loading strategy for PCC that is independent of cell culture background and encompasses cell culture titers up to about 31g/L. Initial experimentation showed a challenge with determining a difference in change in UV280nm signal (ie. ΔUV) between cell culture feed and monoclonal antibody (mAb) concentration. Further investigation revealed UV280nm absorbance of the cell culture feedstock without antibody was outside of the linear range of detection for a given cell pathlength. Additional experimentation showed the difference in ΔUV for various cell culture feeds can be either theoretically predicted by Beer's Law given a known absorbance of the media background and impurities or experimentally determined using various UV280nm cell pathlengths. Based on these results, a 0.35mm pathlength at UV280nm was chosen for dynamic control to overcome the background signal. The pore diffusion model showed good agreement with the experimental frontal analysis data, which resulted in definition of a ΔUV setpoint range between 20 and 70% for 3C-PCC experiments. Product quality of the elution pools was acceptable between various cell culture feeds and titers up to about 41g/L. Results indicated the following ΔUV setpoints to achieve robust dynamic control and maintain 3C-PCC yield: ∼20-45% for titers greater than 10g/L depending on UV absorbance of the HCCF and ∼45-70% for titers of up to 10g/L independent of UV absorbance of the HCCF. The strategy and results presented in this paper show column loading in a continuous chromatography step can be dynamically controlled independent of the cell culture feedstock and titer, and allow for enhanced process control built into the downstream continuous operations. Copyright © 2017 Elsevier B.V. All rights reserved.

Live-cell imaging of G-actin dynamics using sequential FDAP

PubMed Central

Kiuchi, Tai; Nagai, Tomoaki; Ohashi, Kazumasa; Watanabe, Naoki; Mizuno, Kensaku

2011-01-01

Various microscopic techniques have been developed to understand the mechanisms that spatiotemporally control actin filament dynamics in live cells. Kinetic data on the processes of actin assembly and disassembly on F-actin have been accumulated. However, the kinetics of cytoplasmic G-actin, a key determinant for actin polymerization, has remained unclear because of a lack of appropriate methods to measure the G-actin concentration quantitatively. We have developed two new microscopic techniques based on the fluorescence decay after photoactivation (FDAP) time-lapse imaging of photoswitchable Dronpa-labeled actin. These techniques, sequential FDAP (s-FDAP) and multipoint FDAP, were used to measure the time-dependent changes in and spatial distribution of the G-actin concentration in live cells. Use of s-FDAP provided data on changes in the G-actin concentration with high temporal resolution; these data were useful for the model analysis of actin assembly processes in live cells. The s-FDAP analysis also provided evidence that the cytoplasmic G-actin concentration substantially decreases after cell stimulation and that the extent of stimulus-induced actin assembly and cell size extension are linearly correlated with the G-actin concentration before cell stimulation. The advantages of using s-FDAP and multipoint FDAP to measure spatiotemporal G-actin dynamics and the roles of G-actin concentration and ADF/cofilin in stimulus-induced actin assembly and lamellipodium extension in live cells are discussed. PMID:22754616

Self-Organization Processes at the Intracellular Level

NASA Astrophysics Data System (ADS)

Ponce Dawson, Silvina

2003-03-01

In spite of their relatively small sizes, cells are incredibly complex objects in which various sorts of self-organizing processes occur. Cell division is an example of a process that nearly all cells undergo in which a concerted sequence of events takes place. What are the signals that tell the cell to move along this sequence? Clearly, this is a self-organized process. Microtubules (long polymers that are part of the cytoskeleton) and calcium signals play a major role during cell division. In this course we will focus on some features of microtubule dynamics and calcium signals that are amenable to modeling. In both of these biological systems, behaviors at a single molecule level are key determinants of the self-organized dynamics that is observed at larger scales. Thus, the modeling of these systems presents interesting challenges which require novel strategies. Their study may not only provide answers for biologically motivated questions, but is also a natural setting in which the transition between particle-like and mean-field models can be explored. In this course we will first give a brief biological introduction on the structure of eukaryotic cells, microtubule dynamics and intracellular calcium signals. We will then describe some of the models that have been presented in the literature and discuss the spatio-temporal dynamics that they predict, comparing them with observed behaviors in vitro or in vivo. We will end with a discussion on the virtues and limitations of the various modeling strategies described.

Viscoelastic properties of cell walls of single living plant cells determined by dynamic nanoindentation

PubMed Central

Hayot, Céline M.; Forouzesh, Elham; Goel, Ashwani; Avramova, Zoya; Turner, Joseph A.

2012-01-01

Plant development results from controlled cell divisions, structural modifications, and reorganizations of the cell wall. Thereby, regulation of cell wall behaviour takes place at multiple length scales involving compositional and architectural aspects in addition to various developmental and/or environmental factors. The physical properties of the primary wall are largely determined by the nature of the complex polymer network, which exhibits time-dependent behaviour representative of viscoelastic materials. Here, a dynamic nanoindentation technique is used to measure the time-dependent response and the viscoelastic behaviour of the cell wall in single living cells at a micron or sub-micron scale. With this approach, significant changes in storage (stiffness) and loss (loss of energy) moduli are captured among the tested cells. The results reveal hitherto unknown differences in the viscoelastic parameters of the walls of same-age similarly positioned cells of the Arabidopsis ecotypes (Col 0 and Ws 2). The technique is also shown to be sensitive enough to detect changes in cell wall properties in cells deficient in the activity of the chromatin modifier ATX1. Extensive computational modelling of the experimental measurements (i.e. modelling the cell as a viscoelastic pressure vessel) is used to analyse the influence of the wall thickness, as well as the turgor pressure, at the positions of our measurements. By combining the nanoDMA technique with finite element simulations quantifiable measurements of the viscoelastic properties of plant cell walls are achieved. Such techniques are expected to find broader applications in quantifying the influence of genetic, biological, and environmental factors on the nanoscale mechanical properties of the cell wall. PMID:22291130

Measuring the regulation of keratin filament network dynamics

PubMed Central

Moch, Marcin; Herberich, Gerlind; Aach, Til; Leube, Rudolf E.; Windoffer, Reinhard

2013-01-01

The organization of the keratin intermediate filament cytoskeleton is closely linked to epithelial function. To study keratin network plasticity and its regulation at different levels, tools are needed to localize and measure local network dynamics. In this paper, we present image analysis methods designed to determine the speed and direction of keratin filament motion and to identify locations of keratin filament polymerization and depolymerization at subcellular resolution. Using these methods, we have analyzed time-lapse fluorescence recordings of fluorescent keratin 13 in human vulva carcinoma-derived A431 cells. The fluorescent keratins integrated into the endogenous keratin cytoskeleton, and thereby served as reliable markers of keratin dynamics. We found that increased times after seeding correlated with down-regulation of inward-directed keratin filament movement. Bulk flow analyses further revealed that keratin filament polymerization in the cell periphery and keratin depolymerization in the more central cytoplasm were both reduced. Treating these cells and other human keratinocyte-derived cells with EGF reversed all these processes within a few minutes, coinciding with increased keratin phosphorylation. These results highlight the value of the newly developed tools for identifying modulators of keratin filament network dynamics and characterizing their mode of action, which, in turn, contributes to understanding the close link between keratin filament network plasticity and epithelial physiology. PMID:23757496

Supervised chaos genetic algorithm based state of charge determination for LiFePO4 batteries in electric vehicles

NASA Astrophysics Data System (ADS)

Shen, Yanqing

2018-04-01

LiFePO4 battery is developed rapidly in electric vehicle, whose safety and functional capabilities are influenced greatly by the evaluation of available cell capacity. Added with adaptive switch mechanism, this paper advances a supervised chaos genetic algorithm based state of charge determination method, where a combined state space model is employed to simulate battery dynamics. The method is validated by the experiment data collected from battery test system. Results indicate that the supervised chaos genetic algorithm based state of charge determination method shows great performance with less computation complexity and is little influenced by the unknown initial cell state.

Dynamics of gas cell coalescence during baking expansion of leavened dough.

PubMed

Miś, Antoni; Nawrocka, Agnieszka; Lamorski, Krzysztof; Dziki, Dariusz

2018-01-01

The investigation of the dynamics of gas cell coalescence, i.e. a phenomenon that deteriorates the homogeneity of the cellular structure of bread crumb, was carried out performing simultaneously measurements of the dough volume, pressure, and viscosity. It was demonstrated that, during the baking expansion of chemically leavened wheat flour dough, the maximum growth rate of the gas cell radius determined from the ratio of pressure exerted by the expanded dough to its viscosity was on average four-fold lower than that calculated from volume changes in the gas phase of the dough. Such a high discrepancy was interpreted as a result of the course of coalescence, and a formula for determination of its rate was developed. The coalescence rate in the initial baking expansion phase had negative values, indicating nucleation of newly formed gas cells, which increased the number of gas cells even by 8%. In the next baking expansion phase, the coalescence rate started to exhibit positive values, reflecting dominance of the coalescence phenomenon over nucleation. The maximum coalescence rates indicate that, during the period of the most intensive dough expansion, the number of gas cells decreased by 2-3% within one second. At the end of the formation of bread crumb, the number of the gas cells declined by 55-67% in comparison with the initial value. The correctness of the results was positively verified using X-ray micro-computed tomography. The developed method can be a useful tool for more profound exploration of the coalescence phenomenon at various stages of evolution of the cellular structure and its determinants, which may contribute to future development of more effective methods for improving the texture and sensory quality of bread crumb. Copyright © 2017 Elsevier Ltd. All rights reserved.

Model-based cell number quantification using online single-oxygen sensor data for tissue engineering perfusion bioreactors.

PubMed

Lambrechts, T; Papantoniou, I; Sonnaert, M; Schrooten, J; Aerts, J-M

2014-10-01

Online and non-invasive quantification of critical tissue engineering (TE) construct quality attributes in TE bioreactors is indispensable for the cost-effective up-scaling and automation of cellular construct manufacturing. However, appropriate monitoring techniques for cellular constructs in bioreactors are still lacking. This study presents a generic and robust approach to determine cell number and metabolic activity of cell-based TE constructs in perfusion bioreactors based on single oxygen sensor data in dynamic perfusion conditions. A data-based mechanistic modeling technique was used that is able to correlate the number of cells within the scaffold (R(2)  = 0.80) and the metabolic activity of the cells (R(2)  = 0.82) to the dynamics of the oxygen response to step changes in the perfusion rate. This generic non-destructive measurement technique is effective for a large range of cells, from as low as 1.0 × 10(5) cells to potentially multiple millions of cells, and can open-up new possibilities for effective bioprocess monitoring. © 2014 Wiley Periodicals, Inc.

In Vivo Killing Capacity of Cytotoxic T Cells Is Limited and Involves Dynamic Interactions and T Cell Cooperativity

PubMed Central

Halle, Stephan; Keyser, Kirsten Anja; Stahl, Felix Rolf; Busche, Andreas; Marquardt, Anja; Zheng, Xiang; Galla, Melanie; Heissmeyer, Vigo; Heller, Katrin; Boelter, Jasmin; Wagner, Karen; Bischoff, Yvonne; Martens, Rieke; Braun, Asolina; Werth, Kathrin; Uvarovskii, Alexey; Kempf, Harald; Meyer-Hermann, Michael; Arens, Ramon; Kremer, Melanie; Sutter, Gerd; Messerle, Martin; Förster, Reinhold

2016-01-01

Summary According to in vitro assays, T cells are thought to kill rapidly and efficiently, but the efficacy and dynamics of cytotoxic T lymphocyte (CTL)-mediated killing of virus-infected cells in vivo remains elusive. We used two-photon microscopy to quantify CTL-mediated killing in mice infected with herpesviruses or poxviruses. On average, one CTL killed 2–16 virus-infected cells per day as determined by real-time imaging and by mathematical modeling. In contrast, upon virus-induced MHC class I downmodulation, CTLs failed to destroy their targets. During killing, CTLs remained migratory and formed motile kinapses rather than static synapses with targets. Viruses encoding the calcium sensor GCaMP6s revealed strong heterogeneity in individual CTL functional capacity. Furthermore, the probability of death of infected cells increased for those contacted by more than two CTLs, indicative of CTL cooperation. Thus, direct visualization of CTLs during killing of virus-infected cells reveals crucial parameters of CD8+ T cell immunity. PMID:26872694

The role of electron irradiation history in liquid cell transmission electron microscopy.

PubMed

Moser, Trevor H; Mehta, Hardeep; Park, Chiwoo; Kelly, Ryan T; Shokuhfar, Tolou; Evans, James E

2018-04-01

In situ liquid cell transmission electron microscopy (LC-TEM) allows dynamic nanoscale characterization of systems in a hydrated state. Although powerful, this technique remains impaired by issues of repeatability that limit experimental fidelity and hinder the identification and control of some variables underlying observed dynamics. We detail new LC-TEM devices that improve experimental reproducibility by expanding available imaging area and providing a platform for investigating electron flux history on the sample. Irradiation history is an important factor influencing LC-TEM results that has, to this point, been largely qualitatively and not quantitatively described. We use these devices to highlight the role of cumulative electron flux history on samples from both nanoparticle growth and biological imaging experiments and demonstrate capture of time zero, low-dose images on beam-sensitive samples. In particular, the ability to capture pristine images of biological samples, where the acquired image is the first time that the cell experiences significant electron flux, allowed us to determine that nanoparticle movement compared to the cell membrane was a function of cell damage and therefore an artifact rather than visualizing cell dynamics in action. These results highlight just a subset of the new science that is accessible with LC-TEM through the new multiwindow devices with patterned focusing aides.

The role of electron irradiation history in liquid cell transmission electron microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Moser, Trevor H.; Mehta, Hardeep; Park, Chiwoo

In situ liquid cell transmission electron microscopy (LC-TEM) allows dynamic nanoscale characterization of systems in a hydrated state. Although powerful, this technique remains impaired by issues of repeatability that limit experimental fidelity and hinder the identification and control of some variables underlying observed dynamics. We detail new LC- TEM devices that improve experimental reproducibility by expanding available imaging area and providing a platform for investigating electron flux history on the sample. Irradiation history is an important factor influencing LC-TEM results that has, to this point, been largely qualitatively and not quantitatively described. We use these devices to highlight the rolemore » of cumulative electron flux history on samples from both nanoparticle growth and biological imaging experiments and demonstrate capture of time zero, low-dose images on beam-sensitive samples. In particular, the ability to capture pristine images of biological samples, where the acquired image is the first time that the cell experiences significant electron flux, allowed us to determine that nanoparticle movement compared to the cell membrane was a function of cell damage and therefore an artifact rather than visualizing cell dynamics in action. These results highlight just a subset of the new science that is accessible with LC-TEM through the new multiwindow devices with patterned focusing aides.« less

The role of electron irradiation history in liquid cell transmission electron microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Moser, Trevor H.; Mehta, Hardeep; Park, Chiwoo

In situ liquid cell transmission electron microscopy (LC-TEM) allows dynamic nanoscale characterization of systems in a hydrated state. Although powerful, this technique remains impaired by issues of repeatability that limit experimental fidelity and hinder the identification and control of some variables underlying observed dynamics. We detail new LC-TEM devices that improve experimental reproducibility by expanding available imaging area and providing a platform for investigating electron flux history on the sample. Irradiation history is an important factor influencing LC-TEM results that has, to this point, been largely qualitatively and not quantitatively described. We use these devices to highlight the role ofmore » cumulative electron flux history on samples from both nanoparticle growth and biological imaging experiments and demonstrate capture of time zero, low-dose images on beam-sensitive samples. In particular, the ability to capture pristine images of biological samples, where the acquired image is the first time that the cell experiences significant electron flux, allowed us to determine that nanoparticle movement compared to the cell membrane was a function of cell damage and therefore an artifact rather than visualizing cell dynamics in action. Lastly, these results highlight just a subset of the new science that is accessible with LC-TEM through the new multiwindow devices with patterned focusing aides.« less

The role of electron irradiation history in liquid cell transmission electron microscopy

PubMed Central

Mehta, Hardeep

2018-01-01

In situ liquid cell transmission electron microscopy (LC-TEM) allows dynamic nanoscale characterization of systems in a hydrated state. Although powerful, this technique remains impaired by issues of repeatability that limit experimental fidelity and hinder the identification and control of some variables underlying observed dynamics. We detail new LC-TEM devices that improve experimental reproducibility by expanding available imaging area and providing a platform for investigating electron flux history on the sample. Irradiation history is an important factor influencing LC-TEM results that has, to this point, been largely qualitatively and not quantitatively described. We use these devices to highlight the role of cumulative electron flux history on samples from both nanoparticle growth and biological imaging experiments and demonstrate capture of time zero, low-dose images on beam-sensitive samples. In particular, the ability to capture pristine images of biological samples, where the acquired image is the first time that the cell experiences significant electron flux, allowed us to determine that nanoparticle movement compared to the cell membrane was a function of cell damage and therefore an artifact rather than visualizing cell dynamics in action. These results highlight just a subset of the new science that is accessible with LC-TEM through the new multiwindow devices with patterned focusing aides. PMID:29725619

The role of electron irradiation history in liquid cell transmission electron microscopy

DOE PAGES

Moser, Trevor H.; Mehta, Hardeep; Park, Chiwoo; ...

2018-04-20

In situ liquid cell transmission electron microscopy (LC-TEM) allows dynamic nanoscale characterization of systems in a hydrated state. Although powerful, this technique remains impaired by issues of repeatability that limit experimental fidelity and hinder the identification and control of some variables underlying observed dynamics. We detail new LC-TEM devices that improve experimental reproducibility by expanding available imaging area and providing a platform for investigating electron flux history on the sample. Irradiation history is an important factor influencing LC-TEM results that has, to this point, been largely qualitatively and not quantitatively described. We use these devices to highlight the role ofmore » cumulative electron flux history on samples from both nanoparticle growth and biological imaging experiments and demonstrate capture of time zero, low-dose images on beam-sensitive samples. In particular, the ability to capture pristine images of biological samples, where the acquired image is the first time that the cell experiences significant electron flux, allowed us to determine that nanoparticle movement compared to the cell membrane was a function of cell damage and therefore an artifact rather than visualizing cell dynamics in action. Lastly, these results highlight just a subset of the new science that is accessible with LC-TEM through the new multiwindow devices with patterned focusing aides.« less

Morphology and viscoelastic properties of sealing materials based on EPDM rubber.

PubMed

Milić, J; Aroguz, A; Budinski-Simendić, J; Radicević, R; Prendzov, S

2008-12-01

In this applicative study, the ratio of active and inactive filler loadings was the prime factor for determining the dynamic-mechanical behaviour of ethylene-propylene-diene monomer rubbers. Scanning electron microscopy was used to study the structure of reinforced dense and microcellular elastomeric materials. The effects of filler and blowing agent content on the morphology of composites were investigated. Microcellular samples cured in salt bath show smaller cells and uniform cell size compared with samples cured in hot air. Dynamic-mechanical thermal analysis showed appreciable changes in the viscoelastic properties by increasing active filler content, which could enable tailoring the material properties to suit sealing applications.

The Regulation of Nucleolin Expression in Prostrate Epithelial Cells; Possible Involvement of MYC

DTIC Science & Technology

2002-01-01

SH Yeh, NH (1993). The self-cleaving activity of nucleolin determines its molecular dynamics in relation to cell proliferation. Exp Cell Res 208: 48... complementarity were mixed with a segment encompassing the two Nucleolin domains (p50), the rate with which the strands annealed was significantly enhanced...a recent study (Zhu et al 1999) that Nucleolin with all its phosphorylation sites mutated could localize during mitosis as wild-type Nucleolin did

Determination of Dynamics of Plant Plasma Membrane Proteins with Fluorescence Recovery and Raster Image Correlation Spectroscopy.

PubMed

Laňková, Martina; Humpolíčková, Jana; Vosolsobě, Stanislav; Cit, Zdeněk; Lacek, Jozef; Čovan, Martin; Čovanová, Milada; Hof, Martin; Petrášek, Jan

2016-04-01

A number of fluorescence microscopy techniques are described to study dynamics of fluorescently labeled proteins, lipids, nucleic acids, and whole organelles. However, for studies of plant plasma membrane (PM) proteins, the number of these techniques is still limited because of the high complexity of processes that determine the dynamics of PM proteins and the existence of cell wall. Here, we report on the usage of raster image correlation spectroscopy (RICS) for studies of integral PM proteins in suspension-cultured tobacco cells and show its potential in comparison with the more widely used fluorescence recovery after photobleaching method. For RICS, a set of microscopy images is obtained by single-photon confocal laser scanning microscopy (CLSM). Fluorescence fluctuations are subsequently correlated between individual pixels and the information on protein mobility are extracted using a model that considers processes generating the fluctuations such as diffusion and chemical binding reactions. As we show here using an example of two integral PM transporters of the plant hormone auxin, RICS uncovered their distinct short-distance lateral mobility within the PM that is dependent on cytoskeleton and sterol composition of the PM. RICS, which is routinely accessible on modern CLSM instruments, thus represents a valuable approach for studies of dynamics of PM proteins in plants.

Classification of Dynamical Diffusion States in Single Molecule Tracking Microscopy

PubMed Central

Bosch, Peter J.; Kanger, Johannes S.; Subramaniam, Vinod

2014-01-01

Single molecule tracking of membrane proteins by fluorescence microscopy is a promising method to investigate dynamic processes in live cells. Translating the trajectories of proteins to biological implications, such as protein interactions, requires the classification of protein motion within the trajectories. Spatial information of protein motion may reveal where the protein interacts with cellular structures, because binding of proteins to such structures often alters their diffusion speed. For dynamic diffusion systems, we provide an analytical framework to determine in which diffusion state a molecule is residing during the course of its trajectory. We compare different methods for the quantification of motion to utilize this framework for the classification of two diffusion states (two populations with different diffusion speed). We found that a gyration quantification method and a Bayesian statistics-based method are the most accurate in diffusion-state classification for realistic experimentally obtained datasets, of which the gyration method is much less computationally demanding. After classification of the diffusion, the lifetime of the states can be determined, and images of the diffusion states can be reconstructed at high resolution. Simulations validate these applications. We apply the classification and its applications to experimental data to demonstrate the potential of this approach to obtain further insights into the dynamics of cell membrane proteins. PMID:25099798

The Roles and Regulation of Polycomb Complexes in Neural Development

PubMed Central

Corley, Matthew; Kroll, Kristen L.

2014-01-01

In the developing mammalian nervous system, common progenitors integrate both cell extrinsic and intrinsic regulatory programs to produce distinct neuronal and glial cell types as development proceeds. This spatiotemporal restriction of neural progenitor differentiation is enforced, in part, by the dynamic reorganization of chromatin into repressive domains by Polycomb Repressive Complexes, effectively limiting the expression of fate-determining genes. Here, we review distinct roles that the Polycomb Repressive Complexes play during neurogenesis and gliogenesis, while also highlighting recent work describing the molecular mechanisms that govern their dynamic activity in neural development. Further investigation of how Polycomb complexes are regulated in neural development will enable more precise manipulation of neural progenitor differentiation, facilitating the efficient generation of specific neuronal and glial cell types for many biological applications. PMID:25367430

Dispersal, density dependence, and population dynamics of a fungal microbe on leaf surfaces.

PubMed

Woody, Scott T; Ives, Anthony R; Nordheim, Erik V; Andrews, John H

2007-06-01

Despite the ubiquity and importance of microbes in nature, little is known about their natural population dynamics, especially for those that occupy terrestrial habitats. Here we investigate the dynamics of the yeast-like fungus Aureobasidium pullulans (Ap) on apple leaves in an orchard. We asked three questions. (1) Is variation in fungal population density among leaves caused by variation in leaf carrying capacities and strong density-dependent population growth that maintains densities near carrying capacity? (2) Do resident populations have competitive advantages over immigrant cells? (3) Do Ap dynamics differ at different times during the growing season? To address these questions, we performed two experiments at different times in the growing season. Both experiments used a 2 x 2 factorial design: treatment 1 removed fungal cells from leaves to reveal density-dependent population growth, and treatment 2 inoculated leaves with an Ap strain engineered to express green fluorescent protein (GFP), which made it possible to track the fate of immigrant cells. The experiments showed that natural populations of Ap vary greatly in density due to sustained differences in carrying capacities among leaves. The maintenance of populations close to carrying capacities indicates strong density-dependent processes. Furthermore, resident populations are strongly competitive against immigrants, while immigrants have little impact on residents. Finally, statistical models showed high population growth rates of resident cells in one experiment but not in the other, suggesting that Ap experiences relatively "good" and "bad" periods for population growth. This picture of Ap dynamics conforms to commonly held, but rarely demonstrated, expectations of microbe dynamics in nature. It also highlights the importance of local processes, as opposed to immigration, in determining the abundance and dynamics of microbes on surfaces in terrestrial systems.

Dynamic speckle interferometry of microscopic processes in solid state and thin biological objects

NASA Astrophysics Data System (ADS)

Vladimirov, A. P.

2015-08-01

Modernized theory of dynamic speckle interferometry is considered. It is shown that the time-average radiation intensity has the parameters characterizing the wave phase changes. It also brings forward an expression for time autocorrelation function of the radiation intensity. It is shown that with the vanishing averaging time value the formulas transform to the prior expressions. The results of experiments with high-cycle material fatigue and cell metabolism analysis conducted using the time-averaging technique are discussed. Good reproducibility of the results is demonstrated. It is specified that the upgraded technique allows analyzing accumulation of fatigue damage, detecting the crack start moment and determining its growth velocity with uninterrupted cyclic load. It is also demonstrated that in the experiments with a cell monolayer the technique allows studying metabolism change both in an individual cell and in a group of cells.

Physiological Maturation of Regenerating Hair Cells

NASA Technical Reports Server (NTRS)

Baird, Richard A.

2003-01-01

The bullfrog saccule, a sensor of gravity and substrate-borne vibration, is a model system for hair cell transduction. Saccular hair cells also increase in number throughout adult life and rapidly recover after hair cell damage, making this organ an ideal system for studying hair cell development, repair, and regeneration. We have used of hair cell and supporting cell immunocytochemical markers to identify damaged hair cells and hair cell precursors in organotypic cultures of the bullfrog saccule. We then used an innovative combination of confocal, electron, and time-lapse microscopy to study the fate of damaged hair cells and the origin of new hair cells after gentamicin ototoxicity in normal and mitotically blocked saccular cultures. These studies have shown that gentamicin ototoxicity produces both lethal and sublethal hair cell damage. They have also shown that hair cell recovery in this organ takes place by both the repair of sublethally damaged hair cells and by the replacement of lost hair cells by mitotic regeneration. In parallel studies, we have used biophysical and molecular biological techniques to study the differentiation and innervation of developing, repairing, and regenerating hair cells. More specifically, we have used RT-PCR to obtain the bullfrog homologues of L-type voltage- gated calcium (L-VGCC) and large-conductance Ca(2+)-activated potassium (BK) channel genes. We have then obtained probes for these genes and, using in situ hybridization, begun to examine their expression in the bullfrog saccule and amphibian papilla. We have also used fluorescent-labeled channel toxins and channel toxin derivatives to determine the time of appearance of L-type voltage-gated calcium (L-VGCC) and Ca(2+)-activated potassium (BK) channels and to study dynamic changes in the number, distribution, and co-localization of these proteins in developing, repairing, and regenerating hair cells. Using time-lapse microscopy, we are also studying the dynamic relationship between ion channel clustering and synaptic formation in hair cells and afferent neurons. In future studies, we will determine when hair cell precursors acquire electrical tuning, and, using whole-cell patch-clamp techniques, identify and characterize their L-VGCC and BK currents. We will also use biophysical techniques to determine the number of L-VGCC and BK channels and the size and gating kinetics of their underlying L-VGCC and BK conductances, correlating these variables with the amplitude and frequency of membrane oscillations produced by intracellular current steps. We expect these studies to determine how hair cells regulate ion channel expression to achieve specific physiological responses.

Growth-rate-dependent dynamics of a bacterial genetic oscillator

NASA Astrophysics Data System (ADS)

Osella, Matteo; Lagomarsino, Marco Cosentino

2013-01-01

Gene networks exhibiting oscillatory dynamics are widespread in biology. The minimal regulatory designs giving rise to oscillations have been implemented synthetically and studied by mathematical modeling. However, most of the available analyses generally neglect the coupling of regulatory circuits with the cellular “chassis” in which the circuits are embedded. For example, the intracellular macromolecular composition of fast-growing bacteria changes with growth rate. As a consequence, important parameters of gene expression, such as ribosome concentration or cell volume, are growth-rate dependent, ultimately coupling the dynamics of genetic circuits with cell physiology. This work addresses the effects of growth rate on the dynamics of a paradigmatic example of genetic oscillator, the repressilator. Making use of empirical growth-rate dependencies of parameters in bacteria, we show that the repressilator dynamics can switch between oscillations and convergence to a fixed point depending on the cellular state of growth, and thus on the nutrients it is fed. The physical support of the circuit (type of plasmid or gene positions on the chromosome) also plays an important role in determining the oscillation stability and the growth-rate dependence of period and amplitude. This analysis has potential application in the field of synthetic biology, and suggests that the coupling between endogenous genetic oscillators and cell physiology can have substantial consequences for their functionality.

Cytoskeletal mechanics: Structure and Dynamics

NASA Astrophysics Data System (ADS)

Bausch, Andreas

2008-03-01

The actin cytoskeleton, a dynamic network of semiflexible filaments and associated regulatory proteins, is responsible for the extraordinary viscoelastic properties of cells. Especially for cellular motility the controlled self assembly to defined structures and the dynamic reorganization on different time scales are of outstanding importance. A prominent example for the controlled self assembly are actin bundles: in many cytoskeletal processes cells rely on the tight control of the structural and mechanical properties of the actin bundles. Using an in vitro model system we show that size control relies on a mismatch between the helical structure of individual actin filaments and the packing symmetry within bundles. While such self assembled structure may evoke the picture of a static network the contrary is the case: the cytoskeleton is highly dynamic and a constant remodeling takes place in vivo. Such dynamic reorganization of the cytoskeleton relies on the non-static nature of single actin/ABP bonds. Here, we study the thermal and forced unbinding events of individual ABP in such in vitro networks. The binding kinetics of the transient crosslinkers determines the mechanical response of such networks -- in the linear as well in the non-linear regime. These effects are important prerequisites for the high adaptability of cells and at the same time might be the molecular mechanism employed by them for mechanosensing.

Geometric effects in microfluidics on heterogeneous cell stress using an Eulerian-Lagrangian approach.

PubMed

Warren, K M; Mpagazehe, J N; LeDuc, P R; Higgs, C F

2016-02-07

The response of individual cells at the micro-scale in cell mechanics is important in understanding how they are affected by changing environments. To control cell stresses, microfluidics can be implemented since there is tremendous control over the geometry of the devices. Designing microfluidic devices to induce and manipulate stress levels on biological cells can be aided by computational modeling approaches. Such approaches serve as an efficient precursor to fabricating various microfluidic geometries that induce predictable levels of stress on biological cells, based on their mechanical properties. Here, a three-dimensional, multiphase computational fluid dynamics (CFD) modeling approach was implemented for soft biological materials. The computational model incorporates the physics of the particle dynamics, fluid dynamics and solid mechanics, which allows us to study how stresses affect the cells. By using an Eulerian-Lagrangian approach to treat the fluid domain as a continuum in the microfluidics, we are conducting studies of the cells' movement and the stresses applied to the cell. As a result of our studies, we were able to determine that a channel with periodically alternating columns of obstacles was capable of stressing cells at the highest rate, and that microfluidic systems can be engineered to impose heterogenous cell stresses through geometric configuring. We found that when using controlled geometries of the microfluidics channels with staggered obstructions, we could increase the maximum cell stress by nearly 200 times over cells flowing through microfluidic channels with no obstructions. Incorporating computational modeling in the design of microfluidic configurations for controllable cell stressing could help in the design of microfludic devices for stressing cells such as cell homogenizers.

Large Variations in HIV-1 Viral Load Explained by Shifting-Mosaic Metapopulation Dynamics

PubMed Central

Lythgoe, Katrina A.; Blanquart, François

2016-01-01

The viral population of HIV-1, like many pathogens that cause systemic infection, is structured and differentiated within the body. The dynamics of cellular immune trafficking through the blood and within compartments of the body has also received wide attention. Despite these advances, mathematical models, which are widely used to interpret and predict viral and immune dynamics in infection, typically treat the infected host as a well-mixed homogeneous environment. Here, we present mathematical, analytical, and computational results that demonstrate that consideration of the spatial structure of the viral population within the host radically alters predictions of previous models. We study the dynamics of virus replication and cytotoxic T lymphocytes (CTLs) within a metapopulation of spatially segregated patches, representing T cell areas connected by circulating blood and lymph. The dynamics of the system depend critically on the interaction between CTLs and infected cells at the within-patch level. We show that for a wide range of parameters, the system admits an unexpected outcome called the shifting-mosaic steady state. In this state, the whole body’s viral population is stable over time, but the equilibrium results from an underlying, highly dynamic process of local infection and clearance within T-cell centers. Notably, and in contrast to previous models, this new model can explain the large differences in set-point viral load (SPVL) observed between patients and their distribution, as well as the relatively low proportion of cells infected at any one time, and alters the predicted determinants of viral load variation. PMID:27706164

Energy and structure of bonds in the interaction of organic anions with layered double hydroxide nanosheets: A molecular dynamics study

NASA Astrophysics Data System (ADS)

Tsukanov, A. A.; Psakhie, S. G.

2016-01-01

The application of hybrid and hierarchical nanomaterials based on layered hydroxides and oxyhydroxides of metals is a swiftly progressing field in biomedicine. Layered double hydroxides (LDH) possess a large specific surface area, significant surface electric charge and biocompatibility. Their physical and structural properties enable them to adsorb various kinds of anionic species and to transport them into cells. However, possible side effects resulting from the interaction of LDH with anions of the intercellular and intracellular medium need to be considered, since such interaction can potentially disrupt ion transport, signaling processes, apoptosis, nutrition and proliferation of living cells. In the present paper molecular dynamics is used to determine the energies of interaction of organic anions (aspartic acid, glutamic acid and bicarbonate) with a fragment of layered double hydroxide Mg/Al-LDH. The average number of hydrogen bonds between the anions and the hydroxide surface and characteristic binding configurations are determined. Possible effects of LDH on the cell resulting from binding of protein fragments and replacement of native intracellular anions with delivered anions are considered.

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.

PubMed

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 < 0.0001) and limits of agreement with actual po%FEV1 (non-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.

Visualization of endothelial cell cycle dynamics in mouse using the Flt-1/eGFP-anillin system.

PubMed

Herz, Katia; Becker, Alexandra; Shi, Chenyue; Ema, Masatsugo; Takahashi, Satoru; Potente, Michael; Hesse, Michael; Fleischmann, Bernd K; Wenzel, Daniela

2018-05-01

Endothelial cell proliferation is a key process during vascular growth but its kinetics could only be assessed in vitro or ex vivo so far. To enable the monitoring and quantification of cell cycle kinetics in vivo, we have generated transgenic mice expressing an eGFP-anillin construct under control of the endothelial-specific Flt-1 promoter. This construct labels the nuclei of endothelial cells in late G1, S and G2 phase and changes its localization during the different stages of M phase, thereby enabling the monitoring of EC proliferation and cytokinesis. In Flt-1/eGFP-anillin mice, we found eGFP + signals specifically in Ki67 + /PECAM + endothelial cells during vascular development. Quantification using this cell cycle reporter in embryos revealed a decline in endothelial cell proliferation between E9.5 to E12.5. By time-lapse microscopy, we determined the length of different cell cycle phases in embryonic endothelial cells in vivo and found a M phase duration of about 80 min with 2/3 covering karyokinesis and 1/3 cytokinesis. Thus, we have generated a versatile transgenic system for the accurate assessment of endothelial cell cycle dynamics in vitro and in vivo.

Cytoskeletal Reorganization Drives Mesenchymal Condensation and Regulates Downstream Molecular Signaling.

PubMed

Ray, Poulomi; Chapman, Susan C

2015-01-01

Skeletal condensation occurs when specified mesenchyme cells self-organize over several days to form a distinctive cartilage template. Here, we determine how and when specified mesenchyme cells integrate mechanical and molecular information from their environment, forming cartilage condensations in the pharyngeal arches of chick embryos. By disrupting cytoskeletal reorganization, we demonstrate that dynamic cell shape changes drive condensation and modulate the response of the condensing cells to Fibroblast Growth Factor (FGF), Bone Morphogenetic Protein (BMP) and Transforming Growth Factor beta (TGF-β) signaling pathways. Rho Kinase (ROCK)-driven actomyosin contractions and Myosin II-generated differential cell cortex tension regulate these cell shape changes. Disruption of the condensation process inhibits the differentiation of the mesenchyme cells into chondrocytes, demonstrating that condensation regulates the fate of the mesenchyme cells. We also find that dorsal and ventral condensations undergo distinct cell shape changes. BMP signaling is instructive for dorsal condensation-specific cell shape changes. Moreover, condensations exhibit ventral characteristics in the absence of BMP signaling, suggesting that in the pharyngeal arches ventral morphology is the ground pattern. Overall, this study characterizes the interplay between cytoskeletal dynamics and molecular signaling in a self-organizing system during tissue morphogenesis.

Cytoskeletal Reorganization Drives Mesenchymal Condensation and Regulates Downstream Molecular Signaling

PubMed Central

Ray, Poulomi; Chapman, Susan C.

2015-01-01

Skeletal condensation occurs when specified mesenchyme cells self-organize over several days to form a distinctive cartilage template. Here, we determine how and when specified mesenchyme cells integrate mechanical and molecular information from their environment, forming cartilage condensations in the pharyngeal arches of chick embryos. By disrupting cytoskeletal reorganization, we demonstrate that dynamic cell shape changes drive condensation and modulate the response of the condensing cells to Fibroblast Growth Factor (FGF), Bone Morphogenetic Protein (BMP) and Transforming Growth Factor beta (TGF-β) signaling pathways. Rho Kinase (ROCK)-driven actomyosin contractions and Myosin II-generated differential cell cortex tension regulate these cell shape changes. Disruption of the condensation process inhibits the differentiation of the mesenchyme cells into chondrocytes, demonstrating that condensation regulates the fate of the mesenchyme cells. We also find that dorsal and ventral condensations undergo distinct cell shape changes. BMP signaling is instructive for dorsal condensation-specific cell shape changes. Moreover, condensations exhibit ventral characteristics in the absence of BMP signaling, suggesting that in the pharyngeal arches ventral morphology is the ground pattern. Overall, this study characterizes the interplay between cytoskeletal dynamics and molecular signaling in a self-organizing system during tissue morphogenesis. PMID:26237312

Coarse-grained hydrodynamics from correlation functions

NASA Astrophysics Data System (ADS)

Palmer, Bruce

2018-02-01

This paper will describe a formalism for using correlation functions between different grid cells as the basis for determining coarse-grained hydrodynamic equations for modeling the behavior of mesoscopic fluid systems. Configurations from a molecular dynamics simulation or other atomistic simulation are projected onto basis functions representing grid cells in a continuum hydrodynamic simulation. Equilibrium correlation functions between different grid cells are evaluated from the molecular simulation and used to determine the evolution operator for the coarse-grained hydrodynamic system. The formalism is demonstrated on a discrete particle simulation of diffusion with a spatially dependent diffusion coefficient. Correlation functions are calculated from the particle simulation and the spatially varying diffusion coefficient is recovered using a fitting procedure.

Peculiarities of Vibration Characteristics of Amorphous Ices

NASA Astrophysics Data System (ADS)

Gets, Kirill V.; Subbotin, Oleg S.; Belosludov, Vladimir R.

2012-03-01

Dynamic properties of low (LDA), high (HDA) and very high (VHDA) density amorphous ices were investigated within the approach based on Lattice Dynamics simulations. In this approach, we assume that the short-range molecular order mainly determines the dynamic and thermodynamic properties of amorphous ices. Simulation cell of 512 water molecules with periodical boundary conditions and disordering allows us to study dynamical properties and dispersion curves in the Brillouin zone of pseudo-crystal. Existence of collective phenomena in amorphous ices which is usual for crystals but anomalous for disordered phase was confirmed in our simulations. Molecule amplitudes of delocalized (collective) as well as localized vibrations have been considered.

Universal Features of Electron Dynamics in Solar Cells with TiO2 Contact: From Dye Solar Cells to Perovskite Solar Cells.

PubMed

Todinova, Anna; Idígoras, Jesús; Salado, Manuel; Kazim, Samrana; Anta, Juan A

2015-10-01

The electron dynamics of solar cells with mesoporous TiO2 contact is studied by electrochemical small-perturbation techniques. The study involved dye solar cells (DSC), solid-state perovskite solar cells (SSPSC), and devices where the perovskite acts as sensitizer in a liquid-junction device. Using a transport-recombination continuity equation we found that mid-frequency time constants are proper lifetimes that determine the current-voltage curve. This is not the case for the SSPSC, where a lifetime of ∼1 μs, 1 order of magnitude longer, is required to reproduce the current-voltage curve. This mismatch is attributed to the dielectric response on the mid-frequency component. Correcting for this effect, lifetimes lie on a common exponential trend with respect to open-circuit voltage. Electron transport times share a common trend line too. This universal behavior of lifetimes and transport times suggests that the main difference between the cells is the power to populate the mesoporous TiO2 contact with electrons.

Microscopic observation of carrier-transport dynamics in quantum-structure solar cells using a time-of-flight technique

DOE Office of Scientific and Technical Information (OSTI.GOV)

Toprasertpong, Kasidit; Fujii, Hiromasa; Sugiyama, Masakazu

2015-07-27

In this study, we propose a carrier time-of-flight technique to evaluate the carrier transport time across a quantum structure in an active region of solar cells. By observing the time-resolved photoluminescence signal with a quantum-well probe inserted under the quantum structure at forward bias, the carrier transport time can be efficiently determined at room temperature. The averaged drift velocity shows linear dependence on the internal field, allowing us to estimate the quantum structure as a quasi-bulk material with low effective mobility containing the information of carrier dynamics. We show that this direct and real-time observation is more sensitive to carriermore » transport than other conventional techniques, providing better insights into microscopic carrier transport dynamics to overcome a device design difficulty.« less

Mathematical modelling of tumour volume dynamics in response to stereotactic ablative radiotherapy for non-small cell lung cancer

NASA Astrophysics Data System (ADS)

Tariq, Imran; Humbert-Vidan, Laia; Chen, Tao; South, Christopher P.; Ezhil, Veni; Kirkby, Norman F.; Jena, Rajesh; Nisbet, Andrew

2015-05-01

This paper reports a modelling study of tumour volume dynamics in response to stereotactic ablative radiotherapy (SABR). The main objective was to develop a model that is adequate to describe tumour volume change measured during SABR, and at the same time is not excessively complex as lacking support from clinical data. To this end, various modelling options were explored, and a rigorous statistical method, the Akaike information criterion, was used to help determine a trade-off between model accuracy and complexity. The models were calibrated to the data from 11 non-small cell lung cancer patients treated with SABR. The results showed that it is feasible to model the tumour volume dynamics during SABR, opening up the potential for using such models in a clinical environment in the future.

Dynamic refractometer

NASA Technical Reports Server (NTRS)

Curley, Michael J. (Inventor); Sarkisov, Sergey S. (Inventor)

2008-01-01

A refractometer computer controls the rotation of a rotary plate upon which are mounted a prism optically coupled via an optical window to a spectroscopic cell holding a resin exhibiting a dynamic refractive index during photocuring. The computer system positions the prism and spectroscopic cell relative to a visible light laser which illuminates the prism-resin interface at selected incidence angles. A photodetector mounted on the plate generates a signal to the computer proportional to intensity of an internally reflected light beam. A curing light is selectively transmitted through the prism and into the photocurable resin. The refractometer determines the intensity of the internally reflected beam a selected incidence angles and determines the effective refractive index curve of the resin at an uncured state and, optionally, at a completely cured state. Next, an amount of uncured resin and selected optical components to be joined by the resin is placed in the spectroscopic cell and irradiated with the UV light. The refractometer is fixed at a selected incidence angle and measures the intensity of an internally reflected light beam of light throughout the cure cycle. The refractometer determines the resin's refractive index of the polymeric mixture by means of extrapolation of a horizontal shift in the effective refractive index curve of the resin from an uncured state to a selected point in the cure cycle.

Optimization of a pH-shift control strategy for producing monoclonal antibodies in Chinese hamster ovary cell cultures using a pH-dependent dynamic model.

PubMed

Hogiri, Tomoharu; Tamashima, Hiroshi; Nishizawa, Akitoshi; Okamoto, Masahiro

2018-02-01

To optimize monoclonal antibody (mAb) production in Chinese hamster ovary cell cultures, culture pH should be temporally controlled with high resolution. In this study, we propose a new pH-dependent dynamic model represented by simultaneous differential equations including a minimum of six system component, depending on pH value. All kinetic parameters in the dynamic model were estimated using an evolutionary numerical optimization (real-coded genetic algorithm) method based on experimental time-course data obtained at different pH values ranging from 6.6 to 7.2. We determined an optimal pH-shift schedule theoretically. We validated this optimal pH-shift schedule experimentally and mAb production increased by approximately 40% with this schedule. Throughout this study, it was suggested that the culture pH-shift optimization strategy using a pH-dependent dynamic model is suitable to optimize any pH-shift schedule for CHO cell lines used in mAb production projects. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Measuring and Modeling Sonoporation Dynamics in Mammalian Cells via Calcium Imaging

NASA Astrophysics Data System (ADS)

Kumon, R. E.; Parikh, P.; Sabens, D.; Aehle, M.; Kourennyi, D.; Deng, C. X.

2007-05-01

In this study, calcium imaging via the fluorescent indicator Fura-2 is used to characterize the sonoporation of Chinese Hamster Ovarian (CHO) cells in the presence of Optison™ microbubbles. Evolution of the calcium concentration within cells is determined from real-time fluorescence intensity measurements before, during, and after exposure to a 1 MHz ultrasound tone burst (0.2 s, 0.45 MPa). To relate microscopic sonoporation parameters to the measurements, an analytical model that includes sonoporation and plasma membrane transport is developed, assuming rapid mixing (uniform spatial distribution) in the cell. Fitting the measured data to the model provides estimated values for the poration area as a function of poration relaxation rate as well as plasma membrane pump and leakage rates. A modified compartment model that includes the effects of sonoporation, buffering proteins, and transport across the plasma membrane, endoplasmic reticulum, and mitochondria is also investigated. Numerical 3solutions of this model show a variety of behaviors for the calcium dynamics of the cell.

RhoA, Rho kinase, JAK2, and STAT3 may be the intracellular determinants of longevity implicated in the progeric influence of obesity: Insulin, IGF-1, and leptin may all conspire to promote stem cell exhaustion.

PubMed

Tapia, Patrick C

2006-01-01

The aging process in higher mammals is increasingly being shown to feature a potentially substantial contribution from the longitudinal deterioration of normative stem cell dynamics seen with the passage of time. The precise mechanistic sequence producing this phenomenon is not entirely understood, but recent evidence has strongly implicated intracellular downstream effectors of endocrinologic pathways thought to be engaged by the obese state, specifically the insulin, IGF-1, and leptin signaling pathways. Among the intracellular effectors of these signals, a uniquely potent influence on stem cell dynamics may be attributable to Rho/ROCK, JAK kinase activity and STAT3 activity. In particular, it has already been shown that specific tyrosine kinase activities, such as that seen with Rho kinase, are presently thought to be associated with adverse health outcomes in numerous clinical contexts. Furthermore, the Rho GTPase is thought to be contributing to end-stage renal disease. However, in addition to its contribution to organ system dysfunction, the Rho/ROCK pathway has recently been shown to be activated by insulin and IGF-1, providing a tantalizing connection to nutrition and aging science. The JAK-STAT pathway, in contrast, has long been associated with pro-inflammatory cytokines, but has recently been implicated in leptin signaling as well. Importantly, JAK-STAT signaling has, similarly to Rho/ROCK signaling, been implicated as capable of accelerating stem cell proliferation. The implications of these recent determinations, in light of the recent finding of telomere attrition in humans associated with obesity, are that the intracellular determinants of aging may already be known, and the known common influence of these signaling elements on longitudinal stem cell dynamics is a pronounced induction of proliferation, an elevation that has been linked to the pathologic evolution of longitudinal organ-level dysfunction and the organismal-level physiologic decline seen with the inexorable passage of time. Besides the obvious utility for the management for human age-related dysfunction that investigation of pharmacologic inhibitors of these proteins would provide, interventions such as caloric restriction and possibly intermittent fasting may beneficially influence stem cell proliferation dynamics and reduce intracellular correlates of mitogenic drive. Integrating the findings present in the present body of research may reveal endocrinological states that are compatible with longevity, and will also provide novel insight into the specific proteomic determinants of age-related physiologic decline, ushering in a new epoch of medicine that fosters the management of the "pre-etiopathology" of chronic disease and disability of aging, therefore mitigating the suffering widely thought to be inherent in the latter stages of life.

The Z-cad dual fluorescent sensor detects dynamic changes between the epithelial and mesenchymal cellular states.

PubMed

Toneff, M J; Sreekumar, A; Tinnirello, A; Hollander, P Den; Habib, S; Li, S; Ellis, M J; Xin, L; Mani, S A; Rosen, J M

2016-06-17

The epithelial to mesenchymal transition (EMT) has been implicated in metastasis and therapy resistance of carcinomas and can endow cancer cells with cancer stem cell (CSC) properties. The ability to detect cancer cells that are undergoing or have completed EMT has typically relied on the expression of cell surface antigens that correlate with an EMT/CSC phenotype. Alternatively these cells may be permanently marked through Cre-mediated recombination or through immunostaining of fixed cells. The EMT process is dynamic, and these existing methods cannot reveal such changes within live cells. The development of fluorescent sensors that mirror the dynamic EMT state by following the expression of bona fide EMT regulators in live cells would provide a valuable new tool for characterizing EMT. In addition, these sensors will allow direct observation of cellular plasticity with respect to the epithelial/mesenchymal state to enable more effective studies of EMT in cancer and development. We generated a lentiviral-based, dual fluorescent reporter system, designated as the Z-cad dual sensor, comprising destabilized green fluorescent protein containing the ZEB1 3' UTR and red fluorescent protein driven by the E-cadherin (CDH1) promoter. Using this sensor, we robustly detected EMT and mesenchymal to epithelial transition (MET) in breast cancer cells by flow cytometry and fluorescence microscopy. Importantly, we observed dynamic changes in cellular populations undergoing MET. Additionally, we used the Z-cad sensor to identify and isolate minor subpopulations of cells displaying mesenchymal properties within a population comprising predominately epithelial-like cells. The Z-cad dual sensor identified cells with CSC-like properties more effectively than either the ZEB1 3' UTR or E-cadherin sensor alone. The Z-cad dual sensor effectively reports the activities of two factors critical in determining the epithelial/mesenchymal state of carcinoma cells. The ability of this stably integrating dual sensor system to detect dynamic fluctuations between these two states through live cell imaging offers a significant improvement over existing methods and helps facilitate the study of EMT/MET plasticity in response to different stimuli and in cancer pathogenesis. Finally, the versatile Z-cad sensor can be adapted to a variety of in vitro or in vivo systems to elucidate whether EMT/MET contributes to normal and disease phenotypes.

Interplay between chemotaxis and contact inhibition of locomotion determines exploratory cell migration

PubMed Central

Lin, Benjamin; Yin, Taofei; Wu, Yi I.; Inoue, Takanari; Levchenko, Andre

2015-01-01

Directed cell migration in native environments is influenced by multiple migratory cues. These cues may include simultaneously occurring attractive soluble growth factor gradients and repulsive effects arising from cell-cell contact, termed contact inhibition of locomotion (CIL). How single cells reconcile potentially conflicting cues remains poorly understood. Here we show that a dynamic crosstalk between epidermal growth factor (EGF) mediated chemotaxis and CIL guide metastatic breast cancer cell motility, whereby cells become progressively insensitive to CIL in a chemotactic input-dependent manner. This balance is determined via integration of protrusion-enhancing signaling from EGF gradients and protrusion-suppressing signaling induced by CIL, mediated in part through EphB. Our results further suggest that EphB and EGF signaling inputs control protrusion formation by converging onto regulation of phosphatidylinositol 3-kinase (PI3K). We propose that this intricate interplay may enhance the spread of loose cell ensembles in pathophysiological conditions such as cancer, and possibly other physiological settings. PMID:25851023

Dynamics of human cancer cell lines monitored by electrical and acoustic fluctuation analysis.

PubMed

Tarantola, Marco; Marel, Anna-Kristina; Sunnick, Eva; Adam, Holger; Wegener, Joachim; Janshoff, Andreas

2010-03-01

Early determination of the metastatic potential of cancer cells is a crucial step for successful oncological treatment. Besides the remarkable progress in molecular genomics- or proteomics-based diagnostics, there is a great demand for in vitro biosensor devices that allow rapid and selective detection of the invasive properties of tumor cells. Here, the classical cancer cell motility in vitro assays for migration and invasion relying on Boyden chambers are compared to a real-time biosensor that analyzes the dynamic properties of adherent cells electro-acoustically with a time resolution on the order of seconds. The sensor relies on the well-established quartz crystal microbalance technique (QCM) that measures the shift in resonance frequency and damping of an oscillating quartz crystal when adsorption, desorption or changes in material properties close to the quartz surface occur. In addition, the QCM is capable of detecting the rather subtle fluctuations of the cell bodies as an indicator for their micromotility. QCM-based micromotility readings of three different cancer cell lines (HT-29, HSC-4, FaDu) are compared with the well-known electrical cell-substrate impedance sensing (ECIS) revealing collective stochastic motion that corresponds to the malignancy of the cells.

Formation and organization of protein domains in the immunological synapse

NASA Astrophysics Data System (ADS)

Carlson, Andreas; Mahadevan, L.

2014-11-01

The cellular basis for the adaptive immune response during antigen recognition relies on a specialized protein interface known as the immunological synapse. Here, we propose a minimal mathematical model for the dynamics of the IS that encompass membrane mechanics, hydrodynamics and protein kinetics. Simple scaling laws describe the dynamics of protein clusters as a function of membrane stiffness, rigidity of the adhesive proteins, and fluid flow in the synaptic cleft. Numerical simulations complement the scaling laws by quantifying the nucleation, growth and stabilization of proteins domains on the size of the cell. Direct comparison with experiment suggests that passive dynamics suffices to describe the short-time formation and organization of protein clusters, while the stabilization and long time dynamics of the synapse is likely determined by active cytoskeleton processes triggered by receptor binding. Our study reveals that the fluid flow generated by the interplay between membrane deformation and protein binding kinetics can assist immune cells in regulating protein sorting.

Dynamics of the cytotoxic T cell response to a model of acute viral infection.

PubMed

DeWitt, William S; Emerson, Ryan O; Lindau, Paul; Vignali, Marissa; Snyder, Thomas M; Desmarais, Cindy; Sanders, Catherine; Utsugi, Heidi; Warren, Edus H; McElrath, Juliana; Makar, Karen W; Wald, Anna; Robins, Harlan S

2015-04-01

A detailed characterization of the dynamics and breadth of the immune response to an acute viral infection, as well as the determinants of recruitment to immunological memory, can greatly contribute to our basic understanding of the mechanics of the human immune system and can ultimately guide the design of effective vaccines. In addition to neutralizing antibodies, T cells have been shown to be critical for the effective resolution of acute viral infections. We report the first in-depth analysis of the dynamics of the CD8(+) T cell repertoire at the level of individual T cell clonal lineages upon vaccination of human volunteers with a single dose of YF-17D. This live attenuated yellow fever virus vaccine yields sterile, long-term immunity and has been previously used as a model to understand the immune response to a controlled acute viral infection. We identified and enumerated unique CD8(+) T cell clones specifically induced by this vaccine through a combined experimental and statistical approach that included high-throughput sequencing of the CDR3 variable region of the T cell receptor β-chain and an algorithm that detected significantly expanded T cell clones. This allowed us to establish that (i) on average, ∼ 2,000 CD8(+) T cell clones were induced by YF-17D, (ii) 5 to 6% of the responding clones were recruited to long-term memory 3 months postvaccination, (iii) the most highly expanded effector clones were preferentially recruited to the memory compartment, and (iv) a fraction of the YF-17D-induced clones could be identified from peripheral blood lymphocytes solely by measuring clonal expansion. The exhaustive investigation of pathogen-induced effector T cells is essential to accurately quantify the dynamics of the human immune response. The yellow fever vaccine (YFV) has been broadly used as a model to understand how a controlled, self-resolving acute viral infection induces an effective and long-term protective immune response. Here, we extend this previous work by reporting the identity of activated effector T cell clones that expand in response to the YFV 2 weeks postvaccination (as defined by their unique T cell receptor gene sequence) and by tracking clones that enter the memory compartment 3 months postvaccination. This is the first study to use high-throughput sequencing of immune cells to characterize the breadth of the antiviral effector cell response and to determine the contribution of unique virus-induced clones to the long-lived memory T cell repertoire. Thus, this study establishes a benchmark against which future vaccines can be compared to predict their efficacy. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Dynamics of the Cytotoxic T Cell Response to a Model of Acute Viral Infection

PubMed Central

DeWitt, William S.; Emerson, Ryan O.; Lindau, Paul; Vignali, Marissa; Snyder, Thomas M.; Desmarais, Cindy; Sanders, Catherine; Utsugi, Heidi; Warren, Edus H.; McElrath, Juliana; Makar, Karen W.; Wald, Anna

2015-01-01

ABSTRACT A detailed characterization of the dynamics and breadth of the immune response to an acute viral infection, as well as the determinants of recruitment to immunological memory, can greatly contribute to our basic understanding of the mechanics of the human immune system and can ultimately guide the design of effective vaccines. In addition to neutralizing antibodies, T cells have been shown to be critical for the effective resolution of acute viral infections. We report the first in-depth analysis of the dynamics of the CD8+ T cell repertoire at the level of individual T cell clonal lineages upon vaccination of human volunteers with a single dose of YF-17D. This live attenuated yellow fever virus vaccine yields sterile, long-term immunity and has been previously used as a model to understand the immune response to a controlled acute viral infection. We identified and enumerated unique CD8+ T cell clones specifically induced by this vaccine through a combined experimental and statistical approach that included high-throughput sequencing of the CDR3 variable region of the T cell receptor β-chain and an algorithm that detected significantly expanded T cell clones. This allowed us to establish that (i) on average, ∼2,000 CD8+ T cell clones were induced by YF-17D, (ii) 5 to 6% of the responding clones were recruited to long-term memory 3 months postvaccination, (iii) the most highly expanded effector clones were preferentially recruited to the memory compartment, and (iv) a fraction of the YF-17D-induced clones could be identified from peripheral blood lymphocytes solely by measuring clonal expansion. IMPORTANCE The exhaustive investigation of pathogen-induced effector T cells is essential to accurately quantify the dynamics of the human immune response. The yellow fever vaccine (YFV) has been broadly used as a model to understand how a controlled, self-resolving acute viral infection induces an effective and long-term protective immune response. Here, we extend this previous work by reporting the identity of activated effector T cell clones that expand in response to the YFV 2 weeks postvaccination (as defined by their unique T cell receptor gene sequence) and by tracking clones that enter the memory compartment 3 months postvaccination. This is the first study to use high-throughput sequencing of immune cells to characterize the breadth of the antiviral effector cell response and to determine the contribution of unique virus-induced clones to the long-lived memory T cell repertoire. Thus, this study establishes a benchmark against which future vaccines can be compared to predict their efficacy. PMID:25653453

High-efficient and high-content cytotoxic recording via dynamic and continuous cell-based impedance biosensor technology.

PubMed

Hu, Ning; Fang, Jiaru; Zou, Ling; Wan, Hao; Pan, Yuxiang; Su, Kaiqi; Zhang, Xi; Wang, Ping

2016-10-01

Cell-based bioassays were effective method to assess the compound toxicity by cell viability, and the traditional label-based methods missed much information of cell growth due to endpoint detection, while the higher throughputs were demanded to obtain dynamic information. Cell-based biosensor methods can dynamically and continuously monitor with cell viability, however, the dynamic information was often ignored or seldom utilized in the toxin and drug assessment. Here, we reported a high-efficient and high-content cytotoxic recording method via dynamic and continuous cell-based impedance biosensor technology. The dynamic cell viability, inhibition ratio and growth rate were derived from the dynamic response curves from the cell-based impedance biosensor. The results showed that the biosensors has the dose-dependent manners to diarrhetic shellfish toxin, okadiac acid based on the analysis of the dynamic cell viability and cell growth status. Moreover, the throughputs of dynamic cytotoxicity were compared between cell-based biosensor methods and label-based endpoint methods. This cell-based impedance biosensor can provide a flexible, cost and label-efficient platform of cell viability assessment in the shellfish toxin screening fields.

Determination of morphological parameters of biological cells by analysis of scattered-light distributions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Burger, D.E.

1979-11-01

The extraction of morphological parameters from biological cells by analysis of light-scatter patterns is described. A light-scattering measurement system has been designed and constructed that allows one to visually examine and photographically record biological cells or cell models and measure the light-scatter pattern of an individual cell or cell model. Using a laser or conventional illumination, the imaging system consists of a modified microscope with a 35 mm camera attached to record the cell image or light-scatter pattern. Models of biological cells were fabricated. The dynamic range and angular distributions of light scattered from these models was compared to calculatedmore » distributions. Spectrum analysis techniques applied on the light-scatter data give the sought after morphological cell parameters. These results compared favorably to shape parameters of the fabricated cell models confirming the mathematical model procedure. For nucleated biological material, correct nuclear and cell eccentricity as well as the nuclear and cytoplasmic diameters were determined. A method for comparing the flow equivalent of nuclear and cytoplasmic size to the actual dimensions is shown. This light-scattering experiment provides baseline information for automated cytology. In its present application, it involves correlating average size as measured in flow cytology to the actual dimensions determined from this technique. (ERB)« less

Nemo regulates cell dynamics and represses the expression of miple, a midkine/pleiotrophin cytokine, during ommatidial rotation

PubMed Central

Muñoz-Soriano, Verónica; Ruiz, Carlos; Pérez-Alonso, Manuel; Mlodzik, Marek; Paricio, Nuria

2013-01-01

Ommatidial rotation is one of the most important events for correct patterning of the Drosophila eye. Although several signaling pathways are involved in this process, few genes have been shown to specifically affect it. One of them is nemo (nmo), which encodes a MAP-like protein kinase that regulates the rate of rotation throughout the entire process, and serves as a link between core planar cell polarity (PCP) factors and the E-cadherin–β-catenin complex. To determine more precisely the role of nmo in ommatidial rotation, live-imaging analyses in nmo mutant and wild-type early pupal eye discs were performed. We demonstrate that ommatidial rotation is not a continuous process, and that rotating and non-rotating interommatidial cells are very dynamic. Our in vivo analyses also show that nmo regulates the speed of rotation and is required in cone cells for correct ommatidial rotation, and that these cells as well as interommatidial cells are less dynamic in nmo mutants. Furthermore, microarray analyses of nmo and wild-type larval eye discs led us to identify new genes and signaling pathways related to nmo function during this process. One of them, miple, encodes the Drosophila ortholog of the midkine/pleiotrophin secreted cytokines that are involved in cell migration processes. miple is highly up-regulated in nmo mutant discs. Indeed, phenotypic analyses reveal that miple overexpression leads to ommatidial rotation defects. Genetic interaction assays suggest that miple is signaling through Ptp99A, the Drosophila ortholog of the vertebrate midkine/pleiotrophin PTPζ receptor. Accordingly, we propose that one of the roles of Nmo during ommatial rotation is to repress miple expression, which may in turn affect the dynamics in E-cadherin–β-catenin complexes. PMID:23428616

Motor Protein Myo1c Is a Podocyte Protein That Facilitates the Transport of Slit Diaphragm Protein Neph1 to the Podocyte Membrane ▿

PubMed Central

Arif, E.; Wagner, M. C.; Johnstone, D. B.; Wong, H. N.; George, B.; Pruthi, P. A.; Lazzara, M. J.; Nihalani, D.

2011-01-01

The podocyte proteins Neph1 and nephrin organize a signaling complex at the podocyte cell membrane that forms the structural framework for a functional glomerular filtration barrier. Mechanisms regulating the movement of these proteins to and from the membrane are currently unknown. This study identifies a novel interaction between Neph1 and the motor protein Myo1c, where Myo1c plays an active role in targeting Neph1 to the podocyte cell membrane. Using in vivo and in vitro experiments, we provide data supporting a direct interaction between Neph1 and Myo1c which is dynamic and actin dependent. Unlike wild-type Myo1c, the membrane localization of Neph1 was significantly reduced in podocytes expressing dominant negative Myo1c. In addition, Neph1 failed to localize at the podocyte cell membrane and cell junctions in Myo1c-depleted podocytes. We further demonstrate that similarly to Neph1, Myo1c also binds nephrin and reduces its localization at the podocyte cell membrane. A functional analysis of Myo1c knockdown cells showed defects in cell migration, as determined by a wound assay. In addition, the ability to form tight junctions was impaired in Myo1c knockdown cells, as determined by transepithelial electric resistance (TER) and bovine serum albumin (BSA) permeability assays. These results identify a novel Myo1c-dependent molecular mechanism that mediates the dynamic organization of Neph1 and nephrin at the slit diaphragm and is critical for podocyte function. PMID:21402783

Effects of geometry and cell-matrix interactions on the mechanics of 3D engineered microtissues

NASA Astrophysics Data System (ADS)

Bose, Prasenjit; Eyckmans, Jeroen; Chen, Christopher; Reich, Daniel

Approaches to measure and control cell-extracellular matrix (ECM) interactions in a dynamic mechanical environment are important both for studies of mechanobiology and for tissue design for bioengineering applications. We have developed a microtissue-based platform capable of controlling the ECM alignment of 3D engineered microtissues while simultaneously permitting measurement of cellular contractile forces and the tissues' mechanical properties. The tissues self-assemble from cell-laden collagen gels placed in micro-fabricated wells containing sets of flexible elastic pillars. Tissue geometry and ECM alignment are controlled by the pillars' number, shape and location. Optical tracking of the pillars provides readout of the tissues' contractile forces. Magnetic materials bound to selected pillars allow quasi-static or dynamic stretching of the tissue, and together with simultaneous measurements of the tissues' local dynamic strain field, enable characterization of the mechanical properties of the system, including their degree of anisotropy. Results on the effects of symmetry and degree of ECM alignment and organization on the role of cell-ECM interactions in determining tissue mechanical properties will be discussed. This work is supported by NSF CMMI-1463011 and CMMI-1462710.

Mathematical investigation of IP3-dependent calcium dynamics in astrocytes.

PubMed

Handy, Gregory; Taheri, Marsa; White, John A; Borisyuk, Alla

2017-06-01

We study evoked calcium dynamics in astrocytes, a major cell type in the mammalian brain. Experimental evidence has shown that such dynamics are highly variable between different trials, cells, and cell subcompartments. Here we present a qualitative analysis of a recent mathematical model of astrocyte calcium responses. We show how the major response types are generated in the model as a result of the underlying bifurcation structure. By varying key channel parameters, mimicking blockers used by experimentalists, we manipulate this underlying bifurcation structure and predict how the distributions of responses can change. We find that store-operated calcium channels, plasma membrane bound channels with little activity during calcium transients, have a surprisingly strong effect, underscoring the importance of considering these channels in both experiments and mathematical settings. Variation in the maximum flow in different calcium channels is also shown to determine the range of stable oscillations, as well as set the range of frequencies of the oscillations. Further, by conducting a randomized search through the parameter space and recording the resulting calcium responses, we create a database that can be used by experimentalists to help estimate the underlying channel distribution of their cells.

Culture of adult transgenic zebrafish retinal explants for live-cell imaging by multiphoton microscopy

PubMed Central

Lahne, Manuela; Gorsuch, Ryne A; Nelson, Craig M; Hyde, David R

2017-01-01

An endogenous regeneration program is initiated by Müller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The Müller glia re-enter the cell cycle and produce neuronal progenitor cells that undergo subsequent rounds of cell divisions and differentiate into the lost neuronal cell types. Both Müller glia and neuronal progenitor cell nuclei replicate their DNA and undergo mitosis in distinct locations of the retina i.e. they migrate between the basal inner nuclear layer (INL) and the outer nuclear layer (ONL), respectively, in a process described as interkinetic nuclear migration (INM). INM has predominantly been studied in the developing retina. To examine the dynamics of INM in the adult regenerating zebrafish retina in detail, live-cell imaging of fluorescently-labeled Müller glia/neuronal progenitor cells is required. Here, we provide the conditions to isolate and culture dorsal retinas from Tg[gfap:nGFP]mi2004 zebrafish that were exposed to constant intense light for 35 h. We also show that these retinal cultures are viable to perform live-cell imaging experiments, continuously acquiring z-stack images throughout the thickness of the retinal explant for up to 8 h using multiphoton microscopy to monitor the migratory behavior of gfap:nGFP-positive cells. In addition, we describe the details to perform post-imaging analysis to determine the velocity of apical and basal INM. To summarize, we established conditions to study the dynamics of INM in an adult model of neuronal regeneration. This will advance our understanding of this crucial cellular process and allow us to determine the mechanisms that control INM. PMID:28287581

Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy.

PubMed

Lahne, Manuela; Gorsuch, Ryne A; Nelson, Craig M; Hyde, David R

2017-02-24

An endogenous regeneration program is initiated by Müller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The Müller glia re-enter the cell cycle and produce neuronal progenitor cells that undergo subsequent rounds of cell divisions and differentiate into the lost neuronal cell types. Both Müller glia and neuronal progenitor cell nuclei replicate their DNA and undergo mitosis in distinct locations of the retina, i.e. they migrate between the basal Inner Nuclear Layer (INL) and the Outer Nuclear Layer (ONL), respectively, in a process described as Interkinetic Nuclear Migration (INM). INM has predominantly been studied in the developing retina. To examine the dynamics of INM in the adult regenerating zebrafish retina in detail, live-cell imaging of fluorescently-labeled Müller glia/neuronal progenitor cells is required. Here, we provide the conditions to isolate and culture dorsal retinas from Tg[gfap:nGFP] mi2004 zebrafish that were exposed to constant intense light for 35 h. We also show that these retinal cultures are viable to perform live-cell imaging experiments, continuously acquiring z-stack images throughout the thickness of the retinal explant for up to 8 h using multiphoton microscopy to monitor the migratory behavior of gfap:nGFP-positive cells. In addition, we describe the details to perform post-imaging analysis to determine the velocity of apical and basal INM. To summarize, we established conditions to study the dynamics of INM in an adult model of neuronal regeneration. This will advance our understanding of this crucial cellular process and allow us to determine the mechanisms that control INM.

Real-time Visualization of Tissue Dynamics during Embryonic Development and Malignant Transformation

NASA Astrophysics Data System (ADS)

Yamada, Kenneth

Tissues undergo dramatic changes in organization during embryonic development, as well as during cancer progression and invasion. Recent advances in microscopy now allow us to visualize and track directly the dynamic movements of tissues, their constituent cells, and cellular substructures. This behavior can now be visualized not only in regular tissue culture on flat surfaces (`2D' environments), but also in a variety of 3D environments that may provide physiological cues relevant to understanding dynamics within living organisms. Acquisition of imaging data using various microscopy modalities will provide rich opportunities for determining the roles of physical factors and for computational modeling of complex processes in living tissues. Direct visualization of real-time motility is providing insight into biology spanning multiple spatio-temporal scales. Many cells in our body are known to be in contact with connective tissue and other forms of extracellular matrix. They do so through microscopic cellular adhesions that bind to matrix proteins. In particular, fluorescence microscopy has revealed that cells dynamically probe and bend the matrix at the sites of cell adhesions, and that 3D matrix architecture, stiffness, and elasticity can each regulate migration of the cells. Conversely, cells remodel their local matrix as organs form or tumors invade. Cancer cells can invade tissues using microscopic protrusions that degrade the surrounding matrix; in this case, the local matrix protein concentration is more important for inducing the micro-invasive protrusions than stiffness. On the length scales of tissues, transiently high rates of individual cell movement appear to help establish organ architecture. In fact, isolated cells can self-organize to form tissue structures. In all of these cases, in-depth real-time visualization will ultimately provide the extensive data needed for computer modeling and for testing hypotheses in which physical forces interact closely with cell signaling to form organs or promote tumor invasion.

Impact of release dynamics of laser-irradiated polymer micropallets on the viability of selected adherent cells

PubMed Central

Ma, Huan; Mismar, Wael; Wang, Yuli; Small, Donald W.; Ras, Mat; Allbritton, Nancy L.; Sims, Christopher E.; Venugopalan, Vasan

2012-01-01

We use time-resolved interferometry, fluorescence assays and computational fluid dynamics (CFD) simulations to examine the viability of confluent adherent cell monolayers to selection via laser microbeam release of photoresist polymer micropallets. We demonstrate the importance of laser microbeam pulse energy and focal volume position relative to the glass–pallet interface in governing the threshold energies for pallet release as well as the pallet release dynamics. Measurements using time-resolved interferometry show that increases in laser pulse energy result in increasing pallet release velocities that can approach 10 m s−1 through aqueous media. CFD simulations reveal that the pallet motion results in cellular exposure to transient hydrodynamic shear stress amplitudes that can exceed 100 kPa on microsecond timescales, and which produces reduced cell viability. Moreover, CFD simulation results show that the maximum shear stress on the pallet surface varies spatially, with the largest shear stresses occurring on the pallet periphery. Cell viability of confluent cell monolayers on the pallet surface confirms that the use of larger pulse energies results in increased rates of necrosis for those cells situated away from the pallet centre, while cells situated at the pallet centre remain viable. Nevertheless, experiments that examine the viability of these cell monolayers following pallet release show that proper choices for laser microbeam pulse energy and focal volume position lead to the routine achievement of cell viability in excess of 90 per cent. These laser microbeam parameters result in maximum pallet release velocities below 6 m s−1 and cellular exposure of transient hydrodynamic shear stresses below 20 kPa. Collectively, these results provide a mechanistic understanding that relates pallet release dynamics and associated transient shear stresses with subsequent cellular viability. This provides a quantitative, mechanistic basis for determining optimal operating conditions for laser microbeam-based pallet release systems for the isolation and selection of adherent cells. PMID:22158840

Phenotypic Plasticity and Cell Fate Decisions in Cancer: Insights from Dynamical Systems Theory

PubMed Central

Kulkarni, Prakash; Levine, Herbert

2017-01-01

Waddington’s epigenetic landscape, a famous metaphor in developmental biology, depicts how a stem cell progresses from an undifferentiated phenotype to a differentiated one. The concept of “landscape” in the context of dynamical systems theory represents a high-dimensional space, in which each cell phenotype is considered as an “attractor” that is determined by interactions between multiple molecular players, and is buffered against environmental fluctuations. In addition, biological noise is thought to play an important role during these cell-fate decisions and in fact controls transitions between different phenotypes. Here, we discuss the phenotypic transitions in cancer from a dynamical systems perspective and invoke the concept of “cancer attractors”—hidden stable states of the underlying regulatory network that are not occupied by normal cells. Phenotypic transitions in cancer occur at varying levels depending on the context. Using epithelial-to-mesenchymal transition (EMT), cancer stem-like properties, metabolic reprogramming and the emergence of therapy resistance as examples, we illustrate how phenotypic plasticity in cancer cells enables them to acquire hybrid phenotypes (such as hybrid epithelial/mesenchymal and hybrid metabolic phenotypes) that tend to be more aggressive and notoriously resilient to therapies such as chemotherapy and androgen-deprivation therapy. Furthermore, we highlight multiple factors that may give rise to phenotypic plasticity in cancer cells, such as (a) multi-stability or oscillatory behaviors governed by underlying regulatory networks involved in cell-fate decisions in cancer cells, and (b) network rewiring due to conformational dynamics of intrinsically disordered proteins (IDPs) that are highly enriched in cancer cells. We conclude by discussing why a therapeutic approach that promotes “recanalization”, i.e., the exit from “cancer attractors” and re-entry into “normal attractors”, is more likely to succeed rather than a conventional approach that targets individual molecules/pathways. PMID:28640191

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)

PubMed Central

Samtleben, Samira; Jaepel, Juliane; Fecher, Caroline; Andreska, Thomas; Rehberg, Markus; Blum, Robert

2013-01-01

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca2+ indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca2+ indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca2+ indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca2+ indicator and a hydrophilic fluorescent dye/Ca2+ complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of ΔF/F0. PMID:23685703

Direct imaging of ER calcium with targeted-esterase induced dye loading (TED).

PubMed

Samtleben, Samira; Jaepel, Juliane; Fecher, Caroline; Andreska, Thomas; Rehberg, Markus; Blum, Robert

2013-05-07

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca(2+) indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca(2+) indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca(2+) indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca(2+) indicator and a hydrophilic fluorescent dye/Ca(2+) complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of ΔF/F0.

Impact of release dynamics of laser-irradiated polymer micropallets on the viability of selected adherent cells.

PubMed

Ma, Huan; Mismar, Wael; Wang, Yuli; Small, Donald W; Ras, Mat; Allbritton, Nancy L; Sims, Christopher E; Venugopalan, Vasan

2012-06-07

We use time-resolved interferometry, fluorescence assays and computational fluid dynamics (CFD) simulations to examine the viability of confluent adherent cell monolayers to selection via laser microbeam release of photoresist polymer micropallets. We demonstrate the importance of laser microbeam pulse energy and focal volume position relative to the glass-pallet interface in governing the threshold energies for pallet release as well as the pallet release dynamics. Measurements using time-resolved interferometry show that increases in laser pulse energy result in increasing pallet release velocities that can approach 10 m s(-1) through aqueous media. CFD simulations reveal that the pallet motion results in cellular exposure to transient hydrodynamic shear stress amplitudes that can exceed 100 kPa on microsecond timescales, and which produces reduced cell viability. Moreover, CFD simulation results show that the maximum shear stress on the pallet surface varies spatially, with the largest shear stresses occurring on the pallet periphery. Cell viability of confluent cell monolayers on the pallet surface confirms that the use of larger pulse energies results in increased rates of necrosis for those cells situated away from the pallet centre, while cells situated at the pallet centre remain viable. Nevertheless, experiments that examine the viability of these cell monolayers following pallet release show that proper choices for laser microbeam pulse energy and focal volume position lead to the routine achievement of cell viability in excess of 90 per cent. These laser microbeam parameters result in maximum pallet release velocities below 6 m s(-1) and cellular exposure of transient hydrodynamic shear stresses below 20 kPa. Collectively, these results provide a mechanistic understanding that relates pallet release dynamics and associated transient shear stresses with subsequent cellular viability. This provides a quantitative, mechanistic basis for determining optimal operating conditions for laser microbeam-based pallet release systems for the isolation and selection of adherent cells.

Determination of charge transfer resistance and capacitance of microbial fuel cell through a transient response analysis of cell voltage.

PubMed

Ha, Phuc Thi; Moon, Hyunsoo; Kim, Byung Hong; Ng, How Yong; Chang, In Seop

2010-03-15

An alternative method for determining the charge transfer resistance and double-layer capacitance of microbial fuel cells (MFCs), easily implemented without a potentiostat, was developed. A dynamic model with two parameters, the charge transfer resistance and double-layer capacitance of electrodes, was derived from a linear differential equation to depict the current generation with respect to activation overvoltage. This model was then used to fit the transient cell voltage response to the current step change during the continuous operation of a flat-plate type MFC fed with acetate. Variations of the charge transfer resistance and the capacitance value with respect to the MFC design conditions (biocatalyst existence and electrode area) and operating parameters (acetate concentration and buffer strength in the catholyte) were then determined to elucidate the validity of the proposed method. This model was able to describe the dynamic behavior of the MFC during current change in the activation loss region; having an R(2) value of over 0.99 in most tests. Variations of the charge transfer resistance value (thousands of Omega) according to the change of the design factors and operational factors were well-correlated with the corresponding MFC performances. However, though the capacitance values (approximately 0.02 F) reflected the expected trend according to the electrode area change and catalyst property, they did not show significant variation with changes in either the acetate concentration or buffer strength. (c) 2009 Elsevier B.V. All rights reserved.

Velocity measurements of heterogeneous RBC flow in capillary vessels using dynamic laser speckle signal.

PubMed

Li, Chenxi; Wang, Ruikang

2017-04-01

We propose an approach to measure heterogeneous velocities of red blood cells (RBCs) in capillary vessels using full-field time-varying dynamic speckle signals. The approach utilizes a low coherent laser speckle imaging system to record the instantaneous speckle pattern, followed by an eigen-decomposition-based filtering algorithm to extract dynamic speckle signal due to the moving RBCs. The velocity of heterogeneous RBC flows is determined by cross-correlating the temporal dynamic speckle signals obtained at adjacent locations. We verify the approach by imaging mouse pinna in vivo, demonstrating its capability for full-field RBC flow mapping and quantifying flow pattern with high resolution. It is expected to investigate the dynamic action of RBCs flow in capillaries under physiological changes.

Molecular Programs Underlying Asymmetric Stem Cell Division and Their Disruption in Malignancy.

PubMed

Mukherjee, Subhas; Brat, Daniel J

2017-01-01

Asymmetric division of stem cells is a highly conserved and tightly regulated process by which a single stem cell produces two unequal daughter cells. One retains its stem cell identity while the other becomes specialized through a differentiation program and loses stem cell properties. Coordinating these events requires control over numerous intra- and extracellular biological processes and signaling networks. In the initial stages, critical events include the compartmentalization of fate determining proteins within the mother cell and their subsequent passage to the appropriate daughter cell in order to direct their destiny. Disturbance of these events results in an altered dynamic of self-renewing and differentiation within the cell population, which is highly relevant to the growth and progression of cancer. Other critical events include proper asymmetric spindle assembly, extrinsic regulation through micro-environmental cues, and non-canonical signaling networks that impact cell division and fate determination. In this review, we discuss mechanisms that maintain the delicate balance of asymmetric cell division in normal tissues and describe the current understanding how some of these mechanisms are deregulated in cancer.

Temporal dynamic of wood formation in Pinus cembra along the alpine treeline ecotone and the effect of climate variables.

PubMed

Gruber, Andreas; Baumgartner, Daniel; Zimmermann, Jolanda; Oberhuber, Walter

2009-06-01

We determined the temporal dynamic of cambial activity and xylem development of stone pine (Pinus cembra L.) throughout the treeline ecotone. Repeated micro-sampling of the developing tree ring was carried out during the growing seasons 2006 and 2007 at the timberline (1950 m a.s.l.), treeline (2110 m a.s.l.) and within the krummholz belt (2180 m a.s.l.) and the influence of climate variables on intra-annual wood formation was determined.At the beginning of both growing seasons, highest numbers of cambial and enlarging cells were observed at the treeline. Soil temperatures at time of initiation of cambial activity were c. 1.5 °C higher at treeline (open canopy) compared to timberline (closed canopy), suggesting that a threshold root-zone temperature is involved in triggering onset of above ground stem growth.The rate of xylem cell production determined in two weekly intervals during June through August 2006-2007 was significantly correlated with air temperature (temperature sums expressed as degree-days and mean daily maximum temperature) at the timberline only. Lack of significant relationships between tracheid production and temperature variables at the treeline and within the krummholz belt support past dendroclimatological studies that more extreme environmental conditions (e.g., wind exposure, frost desiccation, late frost) increasingly control tree growth above timberline.Results of this study revealed that spatial and temporal (i.e. year-to-year) variability in timing and dynamic of wood formation of Pinus cembra is strongly influenced by local site factors within the treeline ecotone and the dynamics of seasonal temperature variation, respectively.

Atomistic molecular dynamics simulations of bioactive engrailed 1 interference peptides (EN1-iPeps).

PubMed

Gandhi, Neha S; Blancafort, Pilar; Mancera, Ricardo L

2018-04-27

The neural-specific transcription factor Engrailed 1 - is overexpressed in basal-like breast tumours. Synthetic interference peptides - comprising a cell-penetrating peptide/nuclear localisation sequence and the Engrailed 1-specific sequence from the N-terminus have been engineered to produce a strong apoptotic response in tumour cells overexpressing EN1, with no toxicity to normal or non Engrailed 1-expressing cells. Here scaled molecular dynamics simulations were used to study the conformational dynamics of these interference peptides in aqueous solution to characterise their structure and dynamics. Transitions from disordered to α-helical conformation, stabilised by hydrogen bonds and proline-aromatic interactions, were observed throughout the simulations. The backbone of the wild-type peptide folds to a similar conformation as that found in ternary complexes of anterior Hox proteins with conserved hexapeptide motifs important for recognition of pre-B-cell leukemia Homeobox 1, indicating that the motif may possess an intrinsic preference for helical structure. The predicted NMR chemical shifts of these peptides are consistent with the Hox hexapeptides in solution and Engrailed 2 NMR data. These findings highlight the importance of aromatic residues in determining the structure of Engrailed 1 interference peptides, shedding light on the rational design strategy of molecules that could be adopted to inhibit other transcription factors overexpressed in other cancer types, potentially including other transcription factor families that require highly conserved and cooperative protein-protein partnerships for biological activity.

Genetically Encoded Calcium Indicators For Studying Long-Term Calcium Dynamics During Apoptosis

PubMed Central

Garcia, M. Iveth; Chen, Jessica J.; Boehning, Darren

2017-01-01

Intracellular calcium release is essential for regulating almost all cellular functions. Specific spatio-temporal patterns of cytosolic calcium elevations are critical determinants of cell fate in response to pro-apoptotic cellular stressors. As the apoptotic program can take hours or days, measurement of long-term calcium dynamics are essential for understanding the mechanistic role of calcium in apoptotic cell death. Due to the technical limitations of using calcium-sensitive dyes to measure cytosolic calcium little is known about long-term calcium dynamics in living cells after treatment with apoptosis-inducing drugs. Genetically encoded calcium indicators could potentially overcome some of the limitations of calcium-sensitive dyes. Here, we compared the performance of the genetically encoded calcium indicators GCaMP6s and GCaMP6f with the ratiometric dye Fura-2. GCaMP6s performed as well or better than Fura-2 in detecting agonist-induced calcium transients. We then examined the utility of GCaMP6s for continuously measuring apoptotic calcium release over the course of ten hours after treatment with staurosporine. We found that GCaMP6s was suitable for measuring apoptotic calcium release over long time courses and revealed significant heterogeneity in calcium release dynamics in individual cells challenged with staurosporine. Our results suggest GCaMP6s is an excellent indicator for monitoring long-term changes cytosolic calcium during apoptosis. PMID:28073595

Using computer simulations to determine the limitations of dynamic clamp stimuli applied at the soma in mimicking distributed conductance sources.

PubMed

Lin, Risa J; Jaeger, Dieter

2011-05-01

In previous studies we used the technique of dynamic clamp to study how temporal modulation of inhibitory and excitatory inputs control the frequency and precise timing of spikes in neurons of the deep cerebellar nuclei (DCN). Although this technique is now widely used, it is limited to interpreting conductance inputs as being location independent; i.e., all inputs that are biologically distributed across the dendritic tree are applied to the soma. We used computer simulations of a morphologically realistic model of DCN neurons to compare the effects of purely somatic vs. distributed dendritic inputs in this cell type. We applied the same conductance stimuli used in our published experiments to the model. To simulate variability in neuronal responses to repeated stimuli, we added a somatic white current noise to reproduce subthreshold fluctuations in the membrane potential. We were able to replicate our dynamic clamp results with respect to spike rates and spike precision for different patterns of background synaptic activity. We found only minor differences in the spike pattern generation between focal or distributed input in this cell type even when strong inhibitory or excitatory bursts were applied. However, the location dependence of dynamic clamp stimuli is likely to be different for each cell type examined, and the simulation approach developed in the present study will allow a careful assessment of location dependence in all cell types.

Real-time analysis of Drosophila post-embryonic haemocyte behaviour.

PubMed

Sampson, Christopher J; Williams, Michael J

2012-01-01

The larval stage of the model organism Drosophila is frequently used to study host-pathogen interactions. During embryogenesis the cellular arm of the immune response, consisting of macrophage-like cells known as plasmatocytes, is extremely motile and functions to phagocytise pathogens and apoptotic bodies, as well as produce extracellular matrix. The cellular branch of the larval (post-embryonic) innate immune system consists of three cell types--plasmatocytes, crystal cells and lamellocytes--which are involved in the phagocytosis, encapsulation and melanisation of invading pathogens. Post-embryonic haemocyte motility is poorly understood thus further characterisation is required, for the purpose of standardisation. In order to examine post-embryonic haemocyte cytoskeletal dynamics or migration, the most commonly used system is in vitro cell lines. The current study employs an ex vivo system (an adaptation of in vitro cell incubation using primary cells), in which primary larval or pre-pupal haemocytes are isolated for short term analysis, in order to discover various aspects of their behaviour during events requiring cytoskeleton dynamics. The ex vivo method allows for real-time analysis and manipulation of primary post-embryonic haemocytes. This technique was used to characterise, and potentially standardised, larval and pre-pupal haemocyte cytoskeleton dynamics, assayed on different extracellular matrices. Using this method it was determined that, while larval haemocytes are unable to migrate, haemocytes recovered from pre-pupae are capable of migration.

Intrinsic islet heterogeneity and gap junction coupling determine spatiotemporal Ca²⁺ wave dynamics.

PubMed

Benninger, Richard K P; Hutchens, Troy; Head, W Steven; McCaughey, Michael J; Zhang, Min; Le Marchand, Sylvain J; Satin, Leslie S; Piston, David W

2014-12-02

Insulin is released from the islets of Langerhans in discrete pulses that are linked to synchronized oscillations of intracellular free calcium ([Ca(2+)]i). Associated with each synchronized oscillation is a propagating calcium wave mediated by Connexin36 (Cx36) gap junctions. A computational islet model predicted that waves emerge due to heterogeneity in β-cell function throughout the islet. To test this, we applied defined patterns of glucose stimulation across the islet using a microfluidic device and measured how these perturbations affect calcium wave propagation. We further investigated how gap junction coupling regulates spatiotemporal [Ca(2+)]i dynamics in the face of heterogeneous glucose stimulation. Calcium waves were found to originate in regions of the islet having elevated excitability, and this heterogeneity is an intrinsic property of islet β-cells. The extent of [Ca(2+)]i elevation across the islet in the presence of heterogeneity is gap-junction dependent, which reveals a glucose dependence of gap junction coupling. To better describe these observations, we had to modify the computational islet model to consider the electrochemical gradient between neighboring β-cells. These results reveal how the spatiotemporal [Ca(2+)]i dynamics of the islet depend on β-cell heterogeneity and cell-cell coupling, and are important for understanding the regulation of coordinated insulin release across the islet. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

A single-molecule view of gene regulation in cancer

NASA Astrophysics Data System (ADS)

Larson, Daniel

2013-03-01

Single-cell analysis has revealed that transcription is dynamic and stochastic, but tools are lacking that can determine the mechanism operating at a single gene. Here we utilize single-molecule observations of RNA in fixed and living cells to develop a single-cell model of steroid-receptor mediated gene activation. Steroid receptors coordinate a diverse range of responses in higher eukaryotes and are involved in a wide range of human diseases, including cancer. Steroid receptor response elements are present throughout the human genome and modulate chromatin remodeling and transcription in both a local and long-range fashion. As such, steroid receptor-mediated transcription is a paradigm of genetic control in the metazoan nucleus. Moreover, the ligand-dependent nature of these transcription factors makes them appealing targets for therapeutic intervention, necessitating a quantitative understanding of how receptors control output from target genes. We determine that steroids drive mRNA synthesis by frequency modulation of transcription. This digital behavior in single cells gives rise to the well-known analog dose response across the population. To test this model, we developed a light-activation technology to turn on a single gene and follow dynamic synthesis of RNA from the activated locus. The response delay is a measure of time required for chromatin remodeling at a single gene.

Dynamics of Adipocyte Turnover in Humans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Spalding, K; Arner, E; Westermark, P

2007-07-16

Obesity is increasing in an epidemic fashion in most countries and constitutes a public health problem by enhancing the risk for cardiovascular disease and metabolic disorders such as type 2 diabetes. Owing to the increase in obesity, life expectancy may start to decrease in developed countries for the first time in recent history. The factors determining fat mass in adult humans are not fully understood, but increased lipid storage in already developed fat cells is thought to be most important. We show that adipocyte number is a major determinant for the fat mass in adults. However, the number of fatmore » cells stays constant in adulthood in lean and obese and even under extreme conditions, indicating that the number of adipocytes is set during childhood and adolescence. To establish the dynamics within the stable population of adipocytes in adults, we have measured adipocyte turnover by analyzing the integration of {sup 14}C derived from nuclear bomb tests in genomic DNA. Approximately 10% of fat cells are renewed annually at all adult ages and levels of body mass index. Neither adipocyte death nor generation rate is altered in obesity, suggesting a tight regulation of fat cell number that is independent of metabolic profile in adulthood. The high turnover of adipocytes establishes a new therapeutic target for pharmacological intervention in obesity.« less

Nucleosome architecture throughout the cell cycle

PubMed Central

Deniz, Özgen; Flores, Oscar; Aldea, Martí; Soler-López, Montserrat; Orozco, Modesto

2016-01-01

Nucleosomes provide additional regulatory mechanisms to transcription and DNA replication by mediating the access of proteins to DNA. During the cell cycle chromatin undergoes several conformational changes, however the functional significance of these changes to cellular processes are largely unexplored. Here, we present the first comprehensive genome-wide study of nucleosome plasticity at single base-pair resolution along the cell cycle in Saccharomyces cerevisiae. We determined nucleosome organization with a specific focus on two regulatory regions: transcription start sites (TSSs) and replication origins (ORIs). During the cell cycle, nucleosomes around TSSs display rearrangements in a cyclic manner. In contrast to gap (G1 and G2) phases, nucleosomes have a fuzzier organization during S and M phases, Moreover, the choreography of nucleosome rearrangements correlate with changes in gene expression during the cell cycle, indicating a strong association between nucleosomes and cell cycle-dependent gene functionality. On the other hand, nucleosomes are more dynamic around ORIs along the cell cycle, albeit with tighter regulation in early firing origins, implying the functional role of nucleosomes on replication origins. Our study provides a dynamic picture of nucleosome organization throughout the cell cycle and highlights the subsequent impact on transcription and replication activity. PMID:26818620

Structural and conformational determinants of macrocycle cell permeability.

PubMed

Over, Björn; Matsson, Pär; Tyrchan, Christian; Artursson, Per; Doak, Bradley C; Foley, Michael A; Hilgendorf, Constanze; Johnston, Stephen E; Lee, Maurice D; Lewis, Richard J; McCarren, Patrick; Muncipinto, Giovanni; Norinder, Ulf; Perry, Matthew W D; Duvall, Jeremy R; Kihlberg, Jan

2016-12-01

Macrocycles are of increasing interest as chemical probes and drugs for intractable targets like protein-protein interactions, but the determinants of their cell permeability and oral absorption are poorly understood. To enable rational design of cell-permeable macrocycles, we generated an extensive data set under consistent experimental conditions for more than 200 non-peptidic, de novo-designed macrocycles from the Broad Institute's diversity-oriented screening collection. This revealed how specific functional groups, substituents and molecular properties impact cell permeability. Analysis of energy-minimized structures for stereo- and regioisomeric sets provided fundamental insight into how dynamic, intramolecular interactions in the 3D conformations of macrocycles may be linked to physicochemical properties and permeability. Combined use of quantitative structure-permeability modeling and the procedure for conformational analysis now, for the first time, provides chemists with a rational approach to design cell-permeable non-peptidic macrocycles with potential for oral absorption.

Lipid-linked cell wall precursors regulate membrane association of bacterial actin MreB

PubMed Central

Schirner, Kathrin; Eun, Ye-Jin; Dion, Mike; Luo, Yun; Helmann, John D.; Garner, Ethan C.; Walker, Suzanne

2014-01-01

Summary The bacterial actin homolog MreB, which is critical for rod shape determination, forms filaments that rotate around the cell width on the inner surface of the cytoplasmic membrane. What determines filament association with the membranes or with other cell wall elongation proteins is not known. Using specific chemical and genetic perturbations while following MreB filament motion, we find that MreB membrane association is an actively regulated process that depends on the presence of lipid-linked peptidoglycan precursors. When precursors are depleted, MreB filaments disassemble into the cytoplasm and peptidoglycan synthesis becomes disorganized. In cells that lack wall teichoic acids, but continue to make peptidoglycan, dynamic MreB filaments are observed, although their presence is not sufficient to establish a rod shape. We propose that the cell regulates MreB filament association with the membrane, allowing rapid and reversible inactivation of cell wall enzyme complexes in response to the inhibition of cell wall synthesis. PMID:25402772

Lipid-linked cell wall precursors regulate membrane association of bacterial actin MreB.

PubMed

Schirner, Kathrin; Eun, Ye-Jin; Dion, Mike; Luo, Yun; Helmann, John D; Garner, Ethan C; Walker, Suzanne

2015-01-01

The bacterial actin homolog MreB, which is crucial for rod shape determination, forms filaments that rotate around the cell width on the inner surface of the cytoplasmic membrane. What determines filament association with the membranes or with other cell wall elongation proteins is not known. Using specific chemical and genetic perturbations while following MreB filament motion, we find that MreB membrane association is an actively regulated process that depends on the presence of lipid-linked peptidoglycan precursors. When precursors are depleted, MreB filaments disassemble into the cytoplasm, and peptidoglycan synthesis becomes disorganized. In cells that lack wall teichoic acids but continue to make peptidoglycan, dynamic MreB filaments are observed, although their presence is not sufficient to establish a rod shape. We propose that the cell regulates MreB filament association with the membrane, allowing rapid and reversible inactivation of cell wall enzyme complexes in response to the inhibition of cell wall synthesis.

A viscoelastic-stochastic model of the effects of cytoskeleton remodelling on cell adhesion.

PubMed

Li, Long; Zhang, Wenyan; Wang, Jizeng

2016-10-01

Cells can adapt their mechanical properties through cytoskeleton remodelling in response to external stimuli when the cells adhere to the extracellular matrix (ECM). Many studies have investigated the effects of cell and ECM elasticity on cell adhesion. However, experiments determined that cells are viscoelastic and exhibiting stress relaxation, and the mechanism behind the effect of cellular viscoelasticity on the cell adhesion behaviour remains unclear. Therefore, we propose a theoretical model of a cluster of ligand-receptor bonds between two dissimilar viscoelastic media subjected to an applied tensile load. In this model, the distribution of interfacial traction is assumed to follow classical continuum viscoelastic equations, whereas the rupture and rebinding of individual molecular bonds are governed by stochastic equations. On the basis of this model, we determined that viscosity can significantly increase the lifetime, stability and dynamic strength of the adhesion cluster of molecular bonds, because deformation relaxation attributed to the viscoelastic property can increase the rebinding probability of each open bond and reduce the stress concentration in the adhesion area.

Using stroboscopic flow imaging to validate large-scale computational fluid dynamics simulations

NASA Astrophysics Data System (ADS)

Laurence, Ted A.; Ly, Sonny; Fong, Erika; Shusteff, Maxim; Randles, Amanda; Gounley, John; Draeger, Erik

2017-02-01

The utility and accuracy of computational modeling often requires direct validation against experimental measurements. The work presented here is motivated by taking a combined experimental and computational approach to determine the ability of large-scale computational fluid dynamics (CFD) simulations to understand and predict the dynamics of circulating tumor cells in clinically relevant environments. We use stroboscopic light sheet fluorescence imaging to track the paths and measure the velocities of fluorescent microspheres throughout a human aorta model. Performed over complex physiologicallyrealistic 3D geometries, large data sets are acquired with microscopic resolution over macroscopic distances.

Time-lapse electrical impedance spectroscopy for monitoring the cell cycle of single immobilized S. pombe cells.

PubMed

Zhu, Zhen; Frey, Olivier; Haandbaek, Niels; Franke, Felix; Rudolf, Fabian; Hierlemann, Andreas

2015-11-26

As a complement and alternative to optical methods, wide-band electrical impedance spectroscopy (EIS) enables multi-parameter, label-free and real-time detection of cellular and subcellular features. We report on a microfluidics-based system designed to reliably capture single rod-shaped Schizosaccharomyces pombe cells by applying suction through orifices in a channel wall. The system enables subsequent culturing of immobilized cells in an upright position, while dynamic changes in cell-cycle state and morphology were continuously monitored through EIS over a broad frequency range. Besides measuring cell growth, clear impedance signals for nuclear division have been obtained. The EIS system has been characterized with respect to sensitivity and detection limits. The spatial resolution in measuring cell length was 0.25 μm, which corresponds to approximately a 5-min interval of cell growth under standard conditions. The comprehensive impedance data sets were also used to determine the occurrence of nuclear division and cytokinesis. The obtained results have been validated through concurrent confocal imaging and plausibilized through comparison with finite-element modeling data. The possibility to monitor cellular and intracellular features of single S. pombe cells during the cell cycle at high spatiotemporal resolution renders the presented microfluidics-based EIS system a suitable tool for dynamic single-cell investigations.

Time-lapse electrical impedance spectroscopy for monitoring the cell cycle of single immobilized S. pombe cells

PubMed Central

Zhu, Zhen; Frey, Olivier; Haandbaek, Niels; Franke, Felix; Rudolf, Fabian; Hierlemann, Andreas

2015-01-01

As a complement and alternative to optical methods, wide-band electrical impedance spectroscopy (EIS) enables multi-parameter, label-free and real-time detection of cellular and subcellular features. We report on a microfluidics-based system designed to reliably capture single rod-shaped Schizosaccharomyces pombe cells by applying suction through orifices in a channel wall. The system enables subsequent culturing of immobilized cells in an upright position, while dynamic changes in cell-cycle state and morphology were continuously monitored through EIS over a broad frequency range. Besides measuring cell growth, clear impedance signals for nuclear division have been obtained. The EIS system has been characterized with respect to sensitivity and detection limits. The spatial resolution in measuring cell length was 0.25 μm, which corresponds to approximately a 5-min interval of cell growth under standard conditions. The comprehensive impedance data sets were also used to determine the occurrence of nuclear division and cytokinesis. The obtained results have been validated through concurrent confocal imaging and plausibilized through comparison with finite-element modeling data. The possibility to monitor cellular and intracellular features of single S. pombe cells during the cell cycle at high spatiotemporal resolution renders the presented microfluidics-based EIS system a suitable tool for dynamic single-cell investigations. PMID:26608589

Pea Border Cell Maturation and Release Involve Complex Cell Wall Structural Dynamics1[OPEN

PubMed Central

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 immunocarbohydrate microarray profiling, monosaccharide composition determination, Fourier-transformed infrared microspectroscopy, quantitative reverse transcription-PCR of cell wall biosynthetic genes, analysis of hydrolytic activities, transmission electron microscopy, and immunolocalization of cell wall components. Using this integrated glycobiology approach, we identified multiple novel modes of cell wall structural and compositional rearrangement during root cap growth and the release of border cells. Our findings provide a new level of detail about border cell maturation and enable us to develop a model of the separation process. We propose that loss of adhesion by the dissolution of homogalacturonan in the middle lamellae is augmented by an active biophysical process of cell curvature driven by the polarized distribution of xyloglucan and extensin epitopes. PMID:28400496

Concepts and tools for predictive modeling of microbial dynamics.

PubMed

Bernaerts, Kristel; Dens, Els; Vereecken, Karen; Geeraerd, Annemie H; Standaert, Arnout R; Devlieghere, Frank; Debevere, Johan; Van Impe, Jan F

2004-09-01

Description of microbial cell (population) behavior as influenced by dynamically changing environmental conditions intrinsically needs dynamic mathematical models. In the past, major effort has been put into the modeling of microbial growth and inactivation within a constant environment (static models). In the early 1990s, differential equation models (dynamic models) were introduced in the field of predictive microbiology. Here, we present a general dynamic model-building concept describing microbial evolution under dynamic conditions. Starting from an elementary model building block, the model structure can be gradually complexified to incorporate increasing numbers of influencing factors. Based on two case studies, the fundamentals of both macroscopic (population) and microscopic (individual) modeling approaches are revisited. These illustrations deal with the modeling of (i) microbial lag under variable temperature conditions and (ii) interspecies microbial interactions mediated by lactic acid production (product inhibition). Current and future research trends should address the need for (i) more specific measurements at the cell and/or population level, (ii) measurements under dynamic conditions, and (iii) more comprehensive (mechanistically inspired) model structures. In the context of quantitative microbial risk assessment, complexity of the mathematical model must be kept under control. An important challenge for the future is determination of a satisfactory trade-off between predictive power and manageability of predictive microbiology models.

Pattern Transitions in Bacterial Oscillating System under Nanofluidic Confinement

NASA Astrophysics Data System (ADS)

Shen, Jie-Pan; Chou, Chia-Fu

2011-03-01

Successful binary fission in E. coli relies on remarkable oscillatory behavior of the MinCDE protein system to determine the exact division site. The most favorable models to explain this fascinating spatiotemporal regulation on dynamic MinDE pattern formation in cells are based on reaction-diffusion scenario. Although not fully understood, geometric factors caused by bacterial morphology play a crucial role in MinDE dynamics. In the present study, bacteria were cultured, confined and reshaped in various micro/nanofluidic devices, to mimic either curvature changes of cell peripherals. Fluorescence imaging was utilized to detail the mode transitions in multiple MinDE patterns. The understanding of the physics in multiple pattern formations is further complemented via in silico modeling. The study synergizes the join merits of in vivo, in vitro and in silico approaches, to grasp the insight of stochastic dynamics inherited from the noisy mesoscopic biophysics. We acknowledge support from the Foresight Project, Academia Sinica.

Growth-mediated autochemotactic pattern formation in self-propelling bacteria

NASA Astrophysics Data System (ADS)

Mukherjee, Mrinmoy; Ghosh, Pushpita

2018-01-01

Bacteria, while developing a multicellular colony or biofilm, can undergo pattern formation by diverse intricate mechanisms. One such route is directional movement or chemotaxis toward or away from self-secreted or externally employed chemicals. In some bacteria, the self-produced signaling chemicals or autoinducers themselves act as chemoattractants or chemorepellents and thereby regulate the directional movements of the cells in the colony. In addition, bacteria follow a certain growth kinetics which is integrated in the process of colony development. Here, we study the interplay of bacterial growth dynamics, cell motility, and autochemotactic motion with respect to the self-secreted diffusive signaling chemicals in spatial pattern formation. Using a continuum model of motile bacteria, we show growth can act as a crucial tuning parameter in determining the spatiotemporal dynamics of a colony. In action of growth dynamics, while chemoattraction toward autoinducers creates arrested phase separation, pattern transitions and suppression can occur for a fixed chemorepulsive strength.

Spire, an actin nucleation factor, regulates cell division during Drosophila heart development.

PubMed

Xu, Peng; Johnson, Tamara L; Stoller-Conrad, Jessica R; Schulz, Robert A

2012-01-01

The Drosophila dorsal vessel is a beneficial model system for studying the regulation of early heart development. Spire (Spir), an actin-nucleation factor, regulates actin dynamics in many developmental processes, such as cell shape determination, intracellular transport, and locomotion. Through protein expression pattern analysis, we demonstrate that the absence of spir function affects cell division in Myocyte enhancer factor 2-, Tinman (Tin)-, Even-skipped- and Seven up (Svp)-positive heart cells. In addition, genetic interaction analysis shows that spir functionally interacts with Dorsocross, tin, and pannier to properly specify the cardiac fate. Furthermore, through visualization of double heterozygous embryos, we determines that spir cooperates with CycA for heart cell specification and division. Finally, when comparing the spir mutant phenotype with that of a CycA mutant, the results suggest that most Svp-positive progenitors in spir mutant embryos cannot undergo full cell division at cell cycle 15, and that Tin-positive progenitors are arrested at cell cycle 16 as double-nucleated cells. We conclude that Spir plays a crucial role in controlling dorsal vessel formation and has a function in cell division during heart tube morphogenesis.

Building a plant cell wall at a glance.

PubMed

Lampugnani, Edwin R; Khan, Ghazanfar Abbas; Somssich, Marc; Persson, Staffan

2018-01-29

Plant cells are surrounded by a strong polysaccharide-rich cell wall that aids in determining the overall form, growth and development of the plant body. Indeed, the unique shapes of the 40-odd cell types in plants are determined by their walls, as removal of the cell wall results in spherical protoplasts that are amorphic. Hence, assembly and remodeling of the wall is essential in plant development. Most plant cell walls are composed of a framework of cellulose microfibrils that are cross-linked to each other by heteropolysaccharides. The cell walls are highly dynamic and adapt to the changing requirements of the plant during growth. However, despite the importance of plant cell walls for plant growth and for applications that we use in our daily life such as food, feed and fuel, comparatively little is known about how they are synthesized and modified. In this Cell Science at a Glance article and accompanying poster, we aim to illustrate the underpinning cell biology of the synthesis of wall carbohydrates, and their incorporation into the wall, in the model plant Arabidopsis . © 2018. Published by The Company of Biologists Ltd.

The Water to Solute Permeability Ratio Governs the Osmotic Volume Dynamics in Beetroot Vacuoles.

PubMed

Vitali, Victoria; Sutka, Moira; Amodeo, Gabriela; Chara, Osvaldo; Ozu, Marcelo

2016-01-01

Plant cell vacuoles occupy up to 90% of the cell volume and, beyond their physiological function, are constantly subjected to water and solute exchange. The osmotic flow and vacuole volume dynamics relies on the vacuole membrane -the tonoplast- and its capacity to regulate its permeability to both water and solutes. The osmotic permeability coefficient ( P f ) is the parameter that better characterizes the water transport when submitted to an osmotic gradient. Usually, P f determinations are made in vitro from the initial rate of volume change, when a fast (almost instantaneous) osmolality change occurs. When aquaporins are present, it is accepted that initial volume changes are only due to water movements. However, in living cells osmotic changes are not necessarily abrupt but gradually imposed. Under these conditions, water flux might not be the only relevant driving force shaping the vacuole volume response. In this study, we quantitatively investigated volume dynamics of isolated Beta vulgaris root vacuoles under progressively applied osmotic gradients at different pH, a condition that modifies the tonoplast P f . We followed the vacuole volume changes while simultaneously determining the external osmolality time-courses and analyzing these data with mathematical modeling. Our findings indicate that vacuole volume changes, under progressively applied osmotic gradients, would not depend on the membrane elastic properties, nor on the non-osmotic volume of the vacuole, but on water and solute fluxes across the tonoplast. We found that the volume of the vacuole at the steady state is determined by the ratio of water to solute permeabilites ( P f / P s ), which in turn is ruled by pH. The dependence of the permeability ratio on pH can be interpreted in terms of the degree of aquaporin inhibition and the consequently solute transport modulation. This is relevant in many plant organs such as root, leaves, cotyledons, or stems that perform extensive rhythmic growth movements, which very likely involve considerable cell volume changes within seconds to hours.

The Water to Solute Permeability Ratio Governs the Osmotic Volume Dynamics in Beetroot Vacuoles

PubMed Central

Vitali, Victoria; Sutka, Moira; Amodeo, Gabriela; Chara, Osvaldo; Ozu, Marcelo

2016-01-01

Plant cell vacuoles occupy up to 90% of the cell volume and, beyond their physiological function, are constantly subjected to water and solute exchange. The osmotic flow and vacuole volume dynamics relies on the vacuole membrane -the tonoplast- and its capacity to regulate its permeability to both water and solutes. The osmotic permeability coefficient (Pf) is the parameter that better characterizes the water transport when submitted to an osmotic gradient. Usually, Pf determinations are made in vitro from the initial rate of volume change, when a fast (almost instantaneous) osmolality change occurs. When aquaporins are present, it is accepted that initial volume changes are only due to water movements. However, in living cells osmotic changes are not necessarily abrupt but gradually imposed. Under these conditions, water flux might not be the only relevant driving force shaping the vacuole volume response. In this study, we quantitatively investigated volume dynamics of isolated Beta vulgaris root vacuoles under progressively applied osmotic gradients at different pH, a condition that modifies the tonoplast Pf. We followed the vacuole volume changes while simultaneously determining the external osmolality time-courses and analyzing these data with mathematical modeling. Our findings indicate that vacuole volume changes, under progressively applied osmotic gradients, would not depend on the membrane elastic properties, nor on the non-osmotic volume of the vacuole, but on water and solute fluxes across the tonoplast. We found that the volume of the vacuole at the steady state is determined by the ratio of water to solute permeabilites (Pf/Ps), which in turn is ruled by pH. The dependence of the permeability ratio on pH can be interpreted in terms of the degree of aquaporin inhibition and the consequently solute transport modulation. This is relevant in many plant organs such as root, leaves, cotyledons, or stems that perform extensive rhythmic growth movements, which very likely involve considerable cell volume changes within seconds to hours. PMID:27695468

Modelling the host-pathogen interactions of macrophages and Candida albicans using Game Theory and dynamic optimization.

PubMed

Dühring, Sybille; Ewald, Jan; Germerodt, Sebastian; Kaleta, Christoph; Dandekar, Thomas; Schuster, Stefan

2017-07-01

The release of fungal cells following macrophage phagocytosis, called non-lytic expulsion, is reported for several fungal pathogens. On one hand, non-lytic expulsion may benefit the fungus in escaping the microbicidal environment of the phagosome. On the other hand, the macrophage could profit in terms of avoiding its own lysis and being able to undergo proliferation. To analyse the causes of non-lytic expulsion and the relevance of macrophage proliferation in the macrophage- Candida albicans interaction, we employ Evolutionary Game Theory and dynamic optimization in a sequential manner. We establish a game-theoretical model describing the different strategies of the two players after phagocytosis. Depending on the parameter values, we find four different Nash equilibria and determine the influence of the systems state of the host upon the game. As our Nash equilibria are a direct consequence of the model parameterization, we can depict several biological scenarios. A parameter region, where the host response is robust against the fungal infection, is determined. We further apply dynamic optimization to analyse whether macrophage mitosis is relevant in the host-pathogen interaction of macrophages and C. albicans For this, we study the population dynamics of the macrophage- C. albicans interactions and the corresponding optimal controls for the macrophages, indicating the best macrophage strategy of switching from proliferation to attacking fungal cells. © 2017 The Author(s).

Improving dynamic phytoplankton reserve-utilization models with an indirect proxy for internal nitrogen.

PubMed

Malerba, Martino E; Heimann, Kirsten; Connolly, Sean R

2016-09-07

Ecologists have often used indirect proxies to represent variables that are difficult or impossible to measure directly. In phytoplankton, the internal concentration of the most limiting nutrient in a cell determines its growth rate. However, directly measuring the concentration of nutrients within cells is inaccurate, expensive, destructive, and time-consuming, substantially impairing our ability to model growth rates in nutrient-limited phytoplankton populations. The red chlorophyll autofluorescence (hereafter "red fluorescence") signal emitted by a cell is highly correlated with nitrogen quota in nitrogen-limited phytoplankton species. The aim of this study was to evaluate the reliability of including flow cytometric red fluorescence as a proxy for internal nitrogen status to model phytoplankton growth rates. To this end, we used the classic Quota model and designed three approaches to calibrate its model parameters to data: where empirical observations on cell internal nitrogen quota were used to fit the model ("Nitrogen-Quota approach"), where quota dynamics were inferred only from changes in medium nutrient depletion and population density ("Virtual-Quota approach"), or where red fluorescence emission of a cell was used as an indirect proxy for its internal nitrogen quota ("Fluorescence-Quota approach"). Two separate analyses were carried out. In the first analysis, stochastic model simulations were parameterized from published empirical relationships and used to generate dynamics of phytoplankton communities reared under nitrogen-limited conditions. Quota models were fitted to the dynamics of each simulated species with the three different approaches and the performance of each model was compared. In the second analysis, we fit Quota models to laboratory time-series and we calculate the ability of each calibration approach to describe the observed trajectories of internal nitrogen quota in the culture. Results from both analyses concluded that the Fluorescence-Quota approach including per-cell red fluorescence as a proxy of internal nitrogen substantially improved the ability of Quota models to describe phytoplankton dynamics, while still accounting for the biologically important process of cell nitrogen storage. More broadly, many population models in ecology implicitly recognize the importance of accounting for storage mechanisms to describe the dynamics of individual organisms. Hence, the approach documented here with phytoplankton dynamics may also be useful for evaluating the potential of indirect proxies in other ecological systems. Copyright © 2016 Elsevier Ltd. All rights reserved.

Chemotaxis in densely populated tissue determines germinal center anatomy and cell motility: a new paradigm for the development of complex tissues.

PubMed

Hawkins, Jared B; Jones, Mark T; Plassmann, Paul E; Thorley-Lawson, David A

2011-01-01

Germinal centers (GCs) are complex dynamic structures that form within lymph nodes as an essential process in the humoral immune response. They represent a paradigm for studying the regulation of cell movement in the development of complex anatomical structures. We have developed a simulation of a modified cyclic re-entry model of GC dynamics which successfully employs chemotaxis to recapitulate the anatomy of the primary follicle and the development of a mature GC, including correctly structured mantle, dark and light zones. We then show that correct single cell movement dynamics (including persistent random walk and inter-zonal crossing) arise from this simulation as purely emergent properties. The major insight of our study is that chemotaxis can only achieve this when constrained by the known biological properties that cells are incompressible, exist in a densely packed environment, and must therefore compete for space. It is this interplay of chemotaxis and competition for limited space that generates all the complex and biologically accurate behaviors described here. Thus, from a single simple mechanism that is well documented in the biological literature, we can explain both higher level structure and single cell movement behaviors. To our knowledge this is the first GC model that is able to recapitulate both correctly detailed anatomy and single cell movement. This mechanism may have wide application for modeling other biological systems where cells undergo complex patterns of movement to produce defined anatomical structures with sharp tissue boundaries.

Extracellular calcium controls the expression of two different forms of ripple-like hippocampal oscillations.

PubMed

Aivar, Paloma; Valero, Manuel; Bellistri, Elisa; Menendez de la Prida, Liset

2014-02-19

Hippocampal high-frequency oscillations (HFOs) are prominent in physiological and pathological conditions. During physiological ripples (100-200 Hz), few pyramidal cells fire together coordinated by rhythmic inhibitory potentials. In the epileptic hippocampus, fast ripples (>200 Hz) reflect population spikes (PSs) from clusters of bursting cells, but HFOs in the ripple and the fast ripple range are vastly intermixed. What is the meaning of this frequency range? What determines the expression of different HFOs? Here, we used different concentrations of Ca(2+) in a physiological range (1-3 mM) to record local field potentials and single cells in hippocampal slices from normal rats. Surprisingly, we found that this sole manipulation results in the emergence of two forms of HFOs reminiscent of ripples and fast ripples recorded in vivo from normal and epileptic rats, respectively. We scrutinized the cellular correlates and mechanisms underlying the emergence of these two forms of HFOs by combining multisite, single-cell and paired-cell recordings in slices prepared from a rat reporter line that facilitates identification of GABAergic cells. We found a major effect of extracellular Ca(2+) in modulating intrinsic excitability and disynaptic inhibition, two critical factors shaping network dynamics. Moreover, locally modulating the extracellular Ca(2+) concentration in an in vivo environment had a similar effect on disynaptic inhibition, pyramidal cell excitability, and ripple dynamics. Therefore, the HFO frequency band reflects a range of firing dynamics of hippocampal networks.

Human Lysozyme Synergistically Enhances Bactericidal Dynamics and Lowers the Resistant Mutant Prevention Concentration for Metronidazole to Helicobacter pylori by Increasing Cell Permeability.

PubMed

Zhang, Xiaolin; Jiang, Anmin; Yu, Hao; Xiong, Youyi; Zhou, Guoliang; Qin, Meisong; Dou, Jinfeng; Wang, Jianfei

2016-10-28

Metronidazole (MNZ) is an effective agent that has been employed to eradicate Helicobacter pylori ( H. pylori ). The emergence of broad MNZ resistance in H. pylori has affected the efficacy of this therapeutic agent. The concentration of MNZ, especially the mutant prevention concentration (MPC), plays an important role in selecting or enriching resistant mutants and regulating therapeutic effects. A strategy to reduce the MPC that can not only effectively treat H. pylori but also prevent resistance mutations is needed. H. pylori is highly resistant to lysozyme. Lysozyme possesses a hydrolytic bacterial cell wall peptidoglycan and a cationic dependent mode. These effects can increase the permeability of bacterial cells and promote antibiotic absorption into bacterial cells. In this study, human lysozyme (hLYS) was used to probe its effects on the integrity of the H. pylori outer and inner membranes using as fluorescent probe hydrophobic 1- N -phenyl-naphthylamine (NPN) and the release of aspartate aminotransferase. Further studies using a propidium iodide staining method assessed whether hLYS could increase cell permeability and promote cell absorption. Finally, we determined the effects of hLYS on the bactericidal dynamics and MPC of MNZ in H. pylori . Our findings indicate that hLYS could dramatically increase cell permeability, reduce the MPC of MNZ for H. pylori , and enhance its bactericidal dynamic activity, demonstrating that hLYS could reduce the probability of MNZ inducing resistance mutations.

Biotechnology

NASA Image and Video Library

2003-05-05

Aboard the International Space Station (ISS), the Tissue Culture Medium (TCM) is the bioreactor vessel in which cell cultures are grown. With its two syringe ports, it is much like a bag used to administer intravenous fluid, except it allows gas exchange needed for life. The TCM contains cell culture medium, and when frozen cells are flown to the ISS, they are thawed and introduced to the TCM through the syringe ports. In the Cellular Biotechnology Operations Support System-Fluid Dynamics Investigation (CBOSS-FDI) experiment, several mixing procedures are being assessed to determine which method achieves the most uniform mixing of growing cells and culture medium.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Endele, Max; Etzrodt, Martin; Schroeder, Timm, E-mail: timm.schroeder@bsse.ethz.ch

Hematopoiesis is the cumulative consequence of finely tuned signaling pathways activated through extrinsic factors, such as local niche signals and systemic hematopoietic cytokines. Whether extrinsic factors actively instruct the lineage choice of hematopoietic stem and progenitor cells or are only selectively allowing survival and proliferation of already intrinsically lineage-committed cells has been debated over decades. Recent results demonstrated that cytokines can instruct lineage choice. However, the precise function of individual cytokine-triggered signaling molecules in inducing cellular events like proliferation, lineage choice, and differentiation remains largely elusive. Signal transduction pathways activated by different cytokine receptors are highly overlapping, but support themore » production of distinct hematopoietic lineages. Cellular context, signaling dynamics, and the crosstalk of different signaling pathways determine the cellular response of a given extrinsic signal. New tools to manipulate and continuously quantify signaling events at the single cell level are therefore required to thoroughly interrogate how dynamic signaling networks yield a specific cellular response. - Highlights: • Recent studies provided definite proof for lineage-instructive action of cytokines. • Signaling pathways involved in hematopoietic lineage instruction remain elusive. • New tools are emerging to quantitatively study dynamic signaling networks over time.« less

Living-Cell Microarrays

PubMed Central

Yarmush, Martin L.; King, Kevin R.

2011-01-01

Living cells are remarkably complex. To unravel this complexity, living-cell assays have been developed that allow delivery of experimental stimuli and measurement of the resulting cellular responses. High-throughput adaptations of these assays, known as living-cell microarrays, which are based on microtiter plates, high-density spotting, microfabrication, and microfluidics technologies, are being developed for two general applications: (a) to screen large-scale chemical and genomic libraries and (b) to systematically investigate the local cellular microenvironment. These emerging experimental platforms offer exciting opportunities to rapidly identify genetic determinants of disease, to discover modulators of cellular function, and to probe the complex and dynamic relationships between cells and their local environment. PMID:19413510

Long-Term Live Cell Imaging of Cell Migration: Effects of Pathogenic Fungi on Human Epithelial Cell Migration.

PubMed

Wöllert, Torsten; Langford, George M

2016-01-01

Long-term live cell imaging was used in this study to determine the responses of human epithelial cells to pathogenic biofilms formed by Candida albicans. Epithelial cells of the skin represent the front line of defense against invasive pathogens such as C. albicans but under certain circumstances, especially when the host's immune system is compromised, the skin barrier is breached. The mechanisms by which the fungal pathogen penetrates the skin and invade the deeper layers are not fully understood. In this study we used keratinocytes grown in culture as an in vitro model system to determine changes in host cell migration and the actin cytoskeleton in response to virulence factors produced by biofilms of pathogenic C. albicans. It is clear that changes in epithelial cell migration are part of the response to virulence factors secreted by biofilms of C. albicans and the actin cytoskeleton is the downstream effector that mediates cell migration. Our goal is to understand the mechanism by which virulence factors hijack the signaling pathways of the actin cytoskeleton to alter cell migration and thereby invade host tissues. To understand the dynamic changes of the actin cytoskeleton during infection, we used long-term live cell imaging to obtain spatial and temporal information of actin filament dynamics and to identify signal transduction pathways that regulate the actin cytoskeleton and its associated proteins. Long-term live cell imaging was achieved using a high resolution, multi-mode epifluorescence microscope equipped with specialized light sources, high-speed cameras with high sensitivity detectors, and specific biocompatible fluorescent markers. In addition to the multi-mode epifluorescence microscope, a spinning disk confocal long-term live cell imaging system (Olympus CV1000) equipped with a stage incubator to create a stable in vitro environment for long-term real-time and time-lapse microscopy was used. Detailed descriptions of these two long-term live cell imaging systems are provided.

Dynamic Analysis of Human Natural Killer Cell Response at Single-Cell Resolution in B-Cell Non-Hodgkin Lymphoma.

PubMed

Sarkar, Saheli; Sabhachandani, Pooja; Ravi, Dashnamoorthy; Potdar, Sayalee; Purvey, Sneha; Beheshti, Afshin; Evens, Andrew M; Konry, Tania

2017-01-01

Natural killer (NK) cells are phenotypically and functionally diverse lymphocytes that recognize and kill cancer cells. The susceptibility of target cancer cells to NK cell-mediated cytotoxicity depends on the strength and balance of regulatory (activating/inhibitory) ligands expressed on target cell surface. We performed gene expression arrays to determine patterns of NK cell ligands associated with B-cell non-Hodgkin lymphoma (b-NHL). Microarray analyses revealed significant upregulation of a multitude of NK-activating and costimulatory ligands across varied b-NHL cell lines and primary lymphoma cells, including ULBP1, CD72, CD48, and SLAMF6. To correlate genetic signatures with functional anti-lymphoma activity, we developed a dynamic and quantitative cytotoxicity assay in an integrated microfluidic droplet generation and docking array. Individual NK cells and target lymphoma cells were co-encapsulated in picoliter-volume droplets to facilitate monitoring of transient cellular interactions and NK cell effector outcomes at single-cell level. We identified significant variability in NK-lymphoma cell contact duration, frequency, and subsequent cytolysis. Death of lymphoma cells undergoing single contact with NK cells occurred faster than cells that made multiple short contacts. NK cells also killed target cells in droplets via contact-independent mechanisms that partially relied on calcium-dependent processes and perforin secretion, but not on cytokines (interferon-γ or tumor necrosis factor-α). We extended this technique to characterize functional heterogeneity in cytolysis of primary cells from b-NHL patients. Tumor cells from two diffuse large B-cell lymphoma patients showed similar contact durations with NK cells; primary Burkitt lymphoma cells made longer contacts and were lysed at later times. We also tested the cytotoxic efficacy of NK-92, a continuously growing NK cell line being investigated as an antitumor therapy, using our droplet-based bioassay. NK-92 cells were found to be more efficient in killing b-NHL cells compared with primary NK cells, requiring shorter contacts for faster killing activity. Taken together, our combined genetic and microfluidic analysis demonstrate b-NHL cell sensitivity to NK cell-based cytotoxicity, which was associated with significant heterogeneity in the dynamic interaction at single-cell level.

Viral immunogenicity determines epidemiological fitness in a cohort of DENV-1 infection in Brazil.

PubMed

Pinheiro, Tauyne Menegaldo; Mota, Mânlio Tasso de Oliveira; Watanabe, Aripuanã Sakurada Aranha; Biselli-Périco, Joice Matos; Drumond, Betânia Paiva; Ribeiro, Milene Rocha; Vedovello, Danila; Araújo, João Pessoa; Pimenta, Paulo Filemon Paolucci; Chaves, Bárbara Aparecida; Silva, Mayara Marques Carneiro da; Batista, Izabella Cristina Andrade; Papa, Michelle Premazzi; Meuren, Lana Monteiro; Lucas, Carolina Gonçalves de Oliveira; Matassoli, Flavio Lemos; Gil, Laura Helena Vega Gonzales; Bozzi, Adriana; Calzavara-Silva, Carlos Eduardo; Arruda, Luciana Barros de; Souza, Danielle da Glória de; Teixeira, Mauro Martins; Vasilakis, Nikos; Nogueira, Maurício Lacerda

2018-05-01

The dynamics of dengue virus (DENV) circulation depends on serotype, genotype and lineage replacement and turnover. In São José do Rio Preto, Brazil, we observed that the L6 lineage of DENV-1 (genotype V) remained the dominant circulating lineage even after the introduction of the L1 lineage. We investigated viral fitness and immunogenicity of the L1 and L6 lineages and which factors interfered with the dynamics of DENV epidemics. The results showed a more efficient replicative fitness of L1 over L6 in mosquitoes and in human and non-human primate cell lines. Infections by the L6 lineage were associated with reduced antigenicity, weak B and T cell stimulation and weak host immune system interactions, which were associated with higher viremia. Our data, therefore, demonstrate that reduced viral immunogenicity and consequent greater viremia determined the increased epidemiological fitness of DENV-1 L6 lineage in São José do Rio Preto.

Viral immunogenicity determines epidemiological fitness in a cohort of DENV-1 infection in Brazil

PubMed Central

Pinheiro, Tauyne Menegaldo; Mota, Mânlio Tasso de Oliveira; Biselli-Périco, Joice Matos; Ribeiro, Milene Rocha; Araújo, João Pessoa; Pimenta, Paulo Filemon Paolucci; da Silva, Mayara Marques Carneiro; Batista, Izabella Cristina Andrade; Meuren, Lana Monteiro; Lucas, Carolina Gonçalves de Oliveira; Matassoli, Flavio Lemos; Gil, Laura Helena Vega Gonzales; Bozzi, Adriana; Calzavara-Silva, Carlos Eduardo; de Souza, Danielle da Glória; Teixeira, Mauro Martins; Vasilakis, Nikos

2018-01-01

The dynamics of dengue virus (DENV) circulation depends on serotype, genotype and lineage replacement and turnover. In São José do Rio Preto, Brazil, we observed that the L6 lineage of DENV-1 (genotype V) remained the dominant circulating lineage even after the introduction of the L1 lineage. We investigated viral fitness and immunogenicity of the L1 and L6 lineages and which factors interfered with the dynamics of DENV epidemics. The results showed a more efficient replicative fitness of L1 over L6 in mosquitoes and in human and non-human primate cell lines. Infections by the L6 lineage were associated with reduced antigenicity, weak B and T cell stimulation and weak host immune system interactions, which were associated with higher viremia. Our data, therefore, demonstrate that reduced viral immunogenicity and consequent greater viremia determined the increased epidemiological fitness of DENV-1 L6 lineage in São José do Rio Preto. PMID:29813061

How to Study Thermal Applications of Open-Cell Metal Foam: Experiments and Computational Fluid Dynamics

PubMed Central

De Schampheleire, Sven; De Jaeger, Peter; De Kerpel, Kathleen; Ameel, Bernd; Huisseune, Henk; De Paepe, Michel

2016-01-01

This paper reviews the available methods to study thermal applications with open-cell metal foam. Both experimental and numerical work are discussed. For experimental research, the focus of this review is on the repeatability of the results. This is a major concern, as most studies only report the dependence of thermal properties on porosity and a number of pores per linear inch (PPI-value). A different approach, which is studied in this paper, is to characterize the foam using micro tomography scans with small voxel sizes. The results of these scans are compared to correlations from the open literature. Large differences are observed. For the numerical work, the focus is on studies using computational fluid dynamics. A novel way of determining the closure terms is proposed in this work. This is done through a numerical foam model based on micro tomography scan data. With this foam model, the closure terms are determined numerically. PMID:28787894

Do Memory CD4 T Cells Keep Their Cell-Type Programming: Plasticity versus Fate Commitment? Epigenome: A Dynamic Vehicle for Transmitting and Recording Cytokine Signaling.

PubMed

Johnson, John L; Vahedi, Golnaz

2018-03-01

CD4 + T cells are critical for the elimination of an immense array of microbial pathogens. Although there are aspects of helper T-cell differentiation that can be modeled as a classic cell-fate commitment, CD4 + T cells also maintain considerable flexibility in their transcriptional program. Here, we present an overview of chromatin biology during cellular reprogramming and, within this context, envision how the scope of cellular reprogramming may be expanded to further our understanding of the controversy surrounding CD4 + T lymphocyte plasticity or determinism. Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved.

FRAP and Photoconversion in Multiple Arbitrary Regions of Interest Using a Programmable Array Microscope (PAM)

PubMed Central

Hagen, Guy M.; Caarls, Wouter; Lidke, Keith A.; de Vries, Anthony H. B.; Fritsch, Cornelia; Barisas, B. George; Arndt-Jovin, Donna J.; Jovin, Thomas M.

2011-01-01

Photomanipulation (photobleaching, photoactivation, or photoconversion) is an essential tool in fluorescence microscopy. Fluorescence recovery after photobleaching (FRAP) is commonly used for the determination of lateral diffusion constants of membrane proteins, and can be conveniently implemented in confocal laser scanning microscopy (CLSM). Such determinations provide important information on molecular dynamics in live cells. However, the CLSM platform is inherently limited for FRAP because of its inflexible raster (spot) scanning format. We have implemented FRAP and photoactivation protocols using structured illumination and detection in a programmable array microscope (PAM). The patterns are arbitrary in number and shape, dynamic and adjustable to and by the sample characteristics. We have used multi-spot PAM-FRAP to measure the lateral diffusion of the erbB3 (HER3) receptor tyrosine kinase labeled by fusion with mCitrine on untreated cells and after treatment with reagents that perturb the cytoskeleton or plasma membrane or activate co-expressed erbB1 (HER1, the EGF receptor EGFR). We also show the versatility of the PAM for photoactivation in arbitrary regions of interest, in cells expressing erbB3 fused with the photoconvertible fluorescent protein dronpa. PMID:19208387

The Fluid Dynamics of Nascent Biofilms

NASA Astrophysics Data System (ADS)

Farthing, Nicola; Snow, Ben; Wilson, Laurence; Bees, Martin

2017-11-01

Many anti-biofilm approaches target mature biofilms with biochemical or physio-chemical interventions. We investigate the mechanics of interventions at an early stage that aim to inhibit biofilm maturation, focusing on hydrodynamics as cells transition from planktonic to surface-attached. Surface-attached cells generate flow fields that are relatively long-range compared with cells that are freely-swimming. We look at the effect of these flows on the biofilm formation. In particular, we use digital inline holographic microscopy to determine the three-dimensional flow due to a surface-attached cell and the effect this flow has on both tracers and other cells in the fluid. We compare experimental data with two models of cells on boundaries. The first approach utilizes slender body theory and captures many of the features of the experimental field. The second model develops a simple description in terms of singularity solutions of Stokes' flow, which produces qualitatively similar dynamics to both the experiments and more complex model but with significant computational savings. The range of validity of multiple cell arrangements is investigated. These two descriptions can be used to investigate the efficacy of actives developed by Unilever on nascent biofilms.

The dynamics of health in wild field vole populations: a haematological perspective

PubMed Central

Beldomenico, Pablo M.; Telfer, Sandra; Gebert, Stephanie; Lukomski, Lukasz; Bennett, Malcolm; Begon, Michael

2010-01-01

Summary Pathogens have been proposed as potentially important drivers of population dynamics, but while a few studies have investigated the impact of specific pathogens, the wealth of information provided by general indices of health has hardly been exploited. By evaluating haematological parameters in wild populations, our knowledge of the dynamics of health and infection may be better understood. Here, haematological dynamics in natural populations of field voles are investigated to determine environmental and host factors associated with indicators of inflammatory response (counts of monocytes and neutrophils) and of condition: measures of immunological investment (lymphocyte counts) and aerobic capacity (red blood cell counts). Individuals from three field vole populations were sampled monthly for 2 years. Comparisons with individuals kept under controlled conditions facilitated interpretation of field data. Mixed effects models were developed for each cell type to evaluate separately the effects of various factors on post-juvenile voles and mature breeding females. There were three well-characterized ‘physiological’ seasons. The immunological investment appeared lowest in winter (lowest lymphocyte counts), but red blood cells were at their highest levels and indices of inflammatory response at their lowest. Spring was characterized by a fall in red blood cell counts and peaks in indicators of inflammatory response. During the course of summer—autumn, red blood cell counts recovered, the immunological investment increased and the indicators of inflammatory response decreased. Poor body condition appeared to affect the inflammatory response (lower neutrophil and monocyte peaks) and the immunological investment (lower lymphocyte counts), providing evidence that the capacity to fight infection is dependent upon host condition. Breeding early in the year was most likely in females in better condition (high lymphocyte and red blood cell counts). All the haematological parameters were affected adversely by high population densities. PMID:18564292

F-BAR family proteins, emerging regulators for cell membrane dynamic changes-from structure to human diseases.

PubMed

Liu, Suxuan; Xiong, Xinyu; Zhao, Xianxian; Yang, Xiaofeng; Wang, Hong

2015-05-09

Eukaryotic cell membrane dynamics change in curvature during physiological and pathological processes. In the past ten years, a novel protein family, Fes/CIP4 homology-Bin/Amphiphysin/Rvs (F-BAR) domain proteins, has been identified to be the most important coordinators in membrane curvature regulation. The F-BAR domain family is a member of the Bin/Amphiphysin/Rvs (BAR) domain superfamily that is associated with dynamic changes in cell membrane. However, the molecular basis in membrane structure regulation and the biological functions of F-BAR protein are unclear. The pathophysiological role of F-BAR protein is unknown. This review summarizes the current understanding of structure and function in the BAR domain superfamily, classifies F-BAR family proteins into nine subfamilies based on domain structure, and characterizes F-BAR protein structure, domain interaction, and functional relevance. In general, F-BAR protein binds to cell membrane via F-BAR domain association with membrane phospholipids and initiates membrane curvature and scission via Src homology-3 (SH3) domain interaction with its partner proteins. This process causes membrane dynamic changes and leads to seven important cellular biological functions, which include endocytosis, phagocytosis, filopodium, lamellipodium, cytokinesis, adhesion, and podosome formation, via distinct signaling pathways determined by specific domain-binding partners. These cellular functions play important roles in many physiological and pathophysiological processes. We further summarize F-BAR protein expression and mutation changes observed in various diseases and developmental disorders. Considering the structure feature and functional implication of F-BAR proteins, we anticipate that F-BAR proteins modulate physiological and pathophysiological processes via transferring extracellular materials, regulating cell trafficking and mobility, presenting antigens, mediating extracellular matrix degradation, and transmitting signaling for cell proliferation.

An Analysis of Waves Underlying Grid Cell Firing in the Medial Enthorinal Cortex.

PubMed

Bonilla-Quintana, Mayte; Wedgwood, Kyle C A; O'Dea, Reuben D; Coombes, Stephen

2017-08-25

Layer II stellate cells in the medial enthorinal cortex (MEC) express hyperpolarisation-activated cyclic-nucleotide-gated (HCN) channels that allow for rebound spiking via an [Formula: see text] current in response to hyperpolarising synaptic input. A computational modelling study by Hasselmo (Philos. Trans. R. Soc. Lond. B, Biol. Sci. 369:20120523, 2013) showed that an inhibitory network of such cells can support periodic travelling waves with a period that is controlled by the dynamics of the [Formula: see text] current. Hasselmo has suggested that these waves can underlie the generation of grid cells, and that the known difference in [Formula: see text] resonance frequency along the dorsal to ventral axis can explain the observed size and spacing between grid cell firing fields. Here we develop a biophysical spiking model within a framework that allows for analytical tractability. We combine the simplicity of integrate-and-fire neurons with a piecewise linear caricature of the gating dynamics for HCN channels to develop a spiking neural field model of MEC. Using techniques primarily drawn from the field of nonsmooth dynamical systems we show how to construct periodic travelling waves, and in particular the dispersion curve that determines how wave speed varies as a function of period. This exhibits a wide range of long wavelength solutions, reinforcing the idea that rebound spiking is a candidate mechanism for generating grid cell firing patterns. Importantly we develop a wave stability analysis to show how the maximum allowed period is controlled by the dynamical properties of the [Formula: see text] current. Our theoretical work is validated by numerical simulations of the spiking model in both one and two dimensions.

Tumor evolution in space: the effects of competition colonization tradeoffs on tumor invasion dynamics.

PubMed

Orlando, Paul A; Gatenby, Robert A; Brown, Joel S

2013-01-01

We apply competition colonization tradeoff models to tumor growth and invasion dynamics to explore the hypothesis that varying selection forces will result in predictable phenotypic differences in cells at the tumor invasive front compared to those in the core. Spatially, ecologically, and evolutionarily explicit partial differential equation models of tumor growth confirm that spatial invasion produces selection pressure for motile phenotypes. The effects of the invasive phenotype on normal adjacent tissue determine the patterns of growth and phenotype distribution. If tumor cells do not destroy their environment, colonizer and competitive phenotypes coexist with the former localized at the invasion front and the latter, to the tumor interior. If tumors cells do destroy their environment, then cell motility is strongly selected resulting in accelerated invasion speed with time. Our results suggest that the widely observed genetic heterogeneity within cancers may not be the stochastic effect of random mutations. Rather, it may be the consequence of predictable variations in environmental selection forces and corresponding phenotypic adaptations.

Tumor Evolution in Space: The Effects of Competition Colonization Tradeoffs on Tumor Invasion Dynamics

PubMed Central

Orlando, Paul A.; Gatenby, Robert A.; Brown, Joel S.

2013-01-01

We apply competition colonization tradeoff models to tumor growth and invasion dynamics to explore the hypothesis that varying selection forces will result in predictable phenotypic differences in cells at the tumor invasive front compared to those in the core. Spatially, ecologically, and evolutionarily explicit partial differential equation models of tumor growth confirm that spatial invasion produces selection pressure for motile phenotypes. The effects of the invasive phenotype on normal adjacent tissue determine the patterns of growth and phenotype distribution. If tumor cells do not destroy their environment, colonizer and competitive phenotypes coexist with the former localized at the invasion front and the latter, to the tumor interior. If tumors cells do destroy their environment, then cell motility is strongly selected resulting in accelerated invasion speed with time. Our results suggest that the widely observed genetic heterogeneity within cancers may not be the stochastic effect of random mutations. Rather, it may be the consequence of predictable variations in environmental selection forces and corresponding phenotypic adaptations. PMID:23508890

Kinetic Monte Carlo and cellular particle dynamics simulations of multicellular systems

NASA Astrophysics Data System (ADS)

Flenner, Elijah; Janosi, Lorant; Barz, Bogdan; Neagu, Adrian; Forgacs, Gabor; Kosztin, Ioan

2012-03-01

Computer modeling of multicellular systems has been a valuable tool for interpreting and guiding in vitro experiments relevant to embryonic morphogenesis, tumor growth, angiogenesis and, lately, structure formation following the printing of cell aggregates as bioink particles. Here we formulate two computer simulation methods: (1) a kinetic Monte Carlo (KMC) and (2) a cellular particle dynamics (CPD) method, which are capable of describing and predicting the shape evolution in time of three-dimensional multicellular systems during their biomechanical relaxation. Our work is motivated by the need of developing quantitative methods for optimizing postprinting structure formation in bioprinting-assisted tissue engineering. The KMC and CPD model parameters are determined and calibrated by using an original computational-theoretical-experimental framework applied to the fusion of two spherical cell aggregates. The two methods are used to predict the (1) formation of a toroidal structure through fusion of spherical aggregates and (2) cell sorting within an aggregate formed by two types of cells with different adhesivities.

Charging and Transport Dynamics of a Flow-Through Electrode Capacitive Deionization System.

PubMed

Qu, Yatian; Campbell, Patrick G; Hemmatifar, Ali; Knipe, Jennifer M; Loeb, Colin K; Reidy, John J; Hubert, Mckenzie A; Stadermann, Michael; Santiago, Juan G

2018-01-11

We present a study of the interplay among electric charging rate, capacitance, salt removal, and mass transport in "flow-through electrode" capacitive deionization (CDI) systems. We develop two models describing coupled transport and electro-adsorption/desorption which capture salt removal dynamics. The first model is a simplified, unsteady zero-dimensional volume-averaged model which identifies dimensionless parameters and figures of merits associated with cell performance. The second model is a higher fidelity area-averaged model which captures both spatial and temporal responses of charging. We further conducted an experimental study of these dynamics and considered two salt transport regimes: (1) advection-limited regime and (2) dispersion-limited regime. We use these data to validate models. The study shows that, in the advection-limited regime, differential charge efficiency determines the salt adsorption at the early stage of the deionization process. Subsequently, charging transitions to a quasi-steady state where salt removal rate is proportional to applied current scaled by the inlet flow rate. In the dispersion-dominated regime, differential charge efficiency, cell volume, and diffusion rates govern adsorption dynamics and flow rate has little effect. In both regimes, the interplay among mass transport rate, differential charge efficiency, cell capacitance, and (electric) charging current governs salt removal in flow-through electrode CDI.

Mapping the dynamical organization of the cell nucleus through fluorescence correlation spectroscopy.

PubMed

Stortz, Martin; Angiolini, Juan; Mocskos, Esteban; Wolosiuk, Alejandro; Pecci, Adali; Levi, Valeria

2018-05-01

The hierarchical organization of the cell nucleus into specialized open reservoirs and the nucleoplasm overcrowding impose restrictions to the mobility of biomolecules and their interactions with nuclear targets. These properties determine that many nuclear functions such as transcription, replication, splicing or DNA repair are regulated by complex, dynamical processes that do not follow simple rules. Advanced fluorescence microscopy tools and, in particular, fluorescence correlation spectroscopy (FCS) provide complementary and exquisite information on the dynamics of fluorescent labeled molecules moving through the nuclear space and are helping us to comprehend the complexity of the nuclear structure. Here, we describe how FCS methods can be applied to reveal the dynamical organization of the nucleus in live cells. Specifically, we provide instructions for the preparation of cellular samples with fluorescent tagged proteins and detail how FCS can be easily instrumented in commercial confocal microscopes. In addition, we describe general rules to set the parameters for one and two-color experiments and the required controls for these experiments. Finally, we review the statistical analysis of the FCS data and summarize the use of numerical simulations as a complementary approach that helps us to understand the complex matrix of molecular interactions network within the nucleus. Copyright © 2017 Elsevier Inc. All rights reserved.

Drp1 levels constitutively regulate mitochondrial dynamics and cell survival in cortical neurons.

PubMed

Uo, Takuma; Dworzak, Jenny; Kinoshita, Chizuru; Inman, Denise M; Kinoshita, Yoshito; Horner, Philip J; Morrison, Richard S

2009-08-01

Mitochondria exist as dynamic networks that are constantly remodeled through the opposing actions of fusion and fission proteins. Changes in the expression of these proteins alter mitochondrial shape and size, and may promote or inhibit the propagation of apoptotic signals. Using mitochondrially targeted EGFP or DsRed2 to identify mitochondria, we observed a short, distinctly tubular mitochondrial morphology in postnatal cortical neurons in culture and in retinal ganglion cells in vivo, whereas longer, highly interconnected mitochondrial networks were detected in cortical astrocytes in vitro and non-neuronal cells in the retina in vivo. Differential expression patterns of fusion and fission proteins, in part, appear to determine these morphological differences as neurons expressed markedly high levels of Drp1 and OPA1 proteins compared to non-neuronal cells. This finding was corroborated using optic tissue samples. Moreover, cortical neurons expressed several splice variants of Drp1 including a neuron-specific isoform which incorporates exon 3. Knockdown or dominant-negative interference of endogenous Drp1 significantly increased mitochondrial length in both neurons and non-neuronal cells, but caused cell death only in cortical neurons. Conversely, depletion of the fusion protein, Mfn2, but not Mfn1, caused extensive mitochondrial fission and cell death. Thus, Drp1 and Mfn2 in normal cortical neurons not only regulate mitochondrial morphology, but are also required for cell survival. The present findings point to unique patterns of Drp1 expression and selective vulnerability to reduced levels of Drp1 expression/activity in neurons, and demonstrate that the regulation of mitochondrial dynamics must be tightly regulated in neurons.

Drp1 levels constitutively regulate mitochondrial dynamics and cell survival in cortical neurons

PubMed Central

Uo, Takuma; Dworzak, Jenny; Kinoshita, Chizuru; Inman, Denise M.; Kinoshita, Yoshito; Horner, Philip J.; Morrison, Richard S.

2009-01-01

Mitochondria exist as dynamic networks that are constantly remodeled through the opposing actions of fusion and fission proteins. Changes in the expression of these proteins alter mitochondrial shape and size, and may promote or inhibit the propagation of apoptotic signals. Using mitochondrially targeted EGFP or DsRed2 to identify mitochondria, we observed a short, distinctly tubular mitochondrial morphology in postnatal cortical neurons in culture and in retinal ganglion cells in vivo, whereas longer, highly interconnected mitochondrial networks were detected in cortical astrocytes in vitro and non-neuronal cells in the retina in vivo. Differential expression patterns of fusion and fission proteins, in part, appear to determine these morphological differences as neurons expressed markedly high levels of Drp1 and OPA1 proteins compared to non-neuronal cells. This finding was corroborated using optic tissue samples. Moreover, cortical neurons expressed several splice variants of Drp1 including a neuron-specific isoform which incorporates exon 3. Knockdown or dominant negative interference of endogenous Drp1 significantly increased mitochondrial length in both neurons and non-neuronal cells, but caused cell death only in cortical neurons. Conversely, depletion of the fusion protein, Mfn2, but not Mfn1, caused extensive mitochondrial fission and cell death. Thus, Drp1 and Mfn2 in normal cortical neurons not only regulate mitochondrial morphology, but are also required for cell survival. The present findings point to unique patterns of Drp1 expression and selective vulnerability to reduced levels of Drp1 expression/activity in neurons, and demonstrate that the regulation of mitochondrial dynamics must be tightly regulated in neurons. PMID:19445933

Dynamic O-linked N-acetylglucosamine modification of proteins affects stress responses and survival of mesothelial cells exposed to peritoneal dialysis fluids.

PubMed

Herzog, Rebecca; Bender, Thorsten O; Vychytil, Andreas; Bialas, Katarzyna; Aufricht, Christoph; Kratochwill, Klaus

2014-12-01

The ability of cells to respond and survive stressful conditions is determined, in part, by the attachment of O-linked N-acetylglucosamine (O-GlcNAc) to proteins (O-GlcNAcylation), a post-translational modification dependent on glucose and glutamine. This study investigates the role of dynamic O-GlcNAcylation of mesothelial cell proteins in cell survival during exposure to glucose-based peritoneal dialysis fluid (PDF). Immortalized human mesothelial cells and primary mesothelial cells, cultured from human omentum or clinical effluent of PD patients, were assessed for O-GlcNAcylation under normal conditions or after exposure to PDF. The dynamic status of O-GlcNAcylation and effects on cellular survival were investigated by chemical modulation with 6-diazo-5-oxo-L-norleucine (DON) to decrease or O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino N-phenyl carbamate (PUGNAc) to increase O-GlcNAc levels. Viability was decreased by reducing O-GlcNAc levels by DON, which also led to suppressed expression of the cytoprotective heat shock protein 72. In contrast, increasing O-GlcNAc levels by PUGNAc or alanyl-glutamine led to significantly improved cell survival paralleled by higher heat shock protein 72 levels during PDF treatment. Addition of alanyl-glutamine increased O-GlcNAcylation and partly counteracted its inhibition by DON, also leading to improved cell survival. Immunofluorescent analysis of clinical samples showed that the O-GlcNAc signal primarily originates from mesothelial cells. In conclusion, this study identified O-GlcNAcylation in mesothelial cells as a potentially important molecular mechanism after exposure to PDF. Modulating O-GlcNAc levels by clinically feasible interventions might evolve as a novel therapeutic target for the preservation of peritoneal membrane integrity in PD. Copyright © 2014 by the American Society of Nephrology.

Bioreactor validation and biocompatibility of Ag/poly(N-vinyl-2-pyrrolidone) hydrogel nanocomposites.

PubMed

Jovanović, Zeljka; Radosavljević, Aleksandra; Kačarević-Popović, Zorica; Stojkovska, Jasmina; Perić-Grujić, Aleksandra; Ristić, Mirjana; Matić, Ivana Z; Juranić, Zorica D; Obradovic, Bojana; Mišković-Stanković, Vesna

2013-05-01

Silver/poly(N-vinyl-2-pyrrolidone) (Ag/PVP) nanocomposites containing Ag nanoparticles at different concentrations were synthesized using γ-irradiation. Cytotoxicity of the obtained nanocomposites was determined by MTT assay in monolayer cultures of normal human immunocompetent peripheral blood mononuclear cells (PBMC) that were either non-stimulated or stimulated to proliferate by mitogen phytohemagglutinin (PHA), as well as in human cervix adenocarcinoma cell (HeLa) cultures. Silver release kinetics and mechanical properties of nanocomposites were investigated under bioreactor conditions in the simulated body fluid (SBF) at 37°C. The release of silver was monitored under static conditions, and in two types of bioreactors: perfusion bioreactors and a bioreactor with dynamic compression coupled with SBF perfusion simulating in vivo conditions in articular cartilage. Ag/PVP nanocomposites exhibited slight cytotoxic effects against PBMC at the estimated concentration of 0.4 μmol dm(-3), with negligible variations observed amongst different cell cultures investigated. Studies of the silver release kinetics indicated internal diffusion as the rate limiting step, determined by statistically comparable results obtained at all investigated conditions. However, silver release rate was slightly higher in the bioreactor with dynamic compression coupled with SBF perfusion as compared to the other two systems indicating the influence of dynamic compression. Modelling of silver release kinetics revealed potentials for optimization of Ag/PVP nanocomposites for particular applications as wound dressings or soft tissue implants. Copyright © 2013 Elsevier B.V. All rights reserved.

Assessment of Climate Driven Dynamics of Active Layer, Hydrological and Vegetation Status at the Qinghai-Tibet Plateau Using Dynamic Global Vegetation Model

NASA Astrophysics Data System (ADS)

Yang, Y.

2014-12-01

Extensive permafrost degradation starting from 1970s is observed at the Qinghai-Tibet Plateau , China. Degradation is attributed to an increase in mean annual ground temperature 0.1◦-0.5◦ C with mainly winter warming. The construction of Qinghai-Tibet Railway also influenced a state of permafrost in the area Permafrost degradation caused negative environmental consequences in the area. The areas covered by sand are expanding steadily making large concern of accelerating desertification. The general pathway of future joint dynamics of permafrost, vegetation and hydrological status at the Qinghai-Tibet Plateau is still poorly understood and foreseeable. Hydrology in the area is determined by heat-moisture dynamics of active layer. This dynamics is highly non-linear and depends as on external climatic variables temperature and precipitation, so on soil and rock properties (amount of sand against aeolian deposits in the Plateau) as well as vegetation cover, which determine thaw and freeze processes in the active layer and evaporation and run-off. SEVER DGVM was modified to include heat-moisture dynamics of active layer in the Qinghai-Tibet Plateau. SEVER DGVM imitates processes in 10 plant functional types at coarse resolution of 0.5 degrees. This model imitates behavior of average individual of each plant type in each grid cell through simulation years. Each of those grid cells processed independently. First, this model starts from "bare soil", placing a bit of each plant type and giving them some time to grow and achieve equilibrium. Then, including active layer thickness and soil moisture dynamics into this layer, it allows assessment of potential environmental dynamics in this area. Simulations demonstrate further degradation of pastureland and accelerating desertification processes in this vitally important water feed area for many Asian rivers. Negative environmental problems related to operation of Qinghai-Tibet are also assessed.

Velocity measurements of heterogeneous RBC flow in capillary vessels using dynamic laser speckle signal

PubMed Central

Li, Chenxi; Wang, Ruikang

2017-01-01

Abstract. We propose an approach to measure heterogeneous velocities of red blood cells (RBCs) in capillary vessels using full-field time-varying dynamic speckle signals. The approach utilizes a low coherent laser speckle imaging system to record the instantaneous speckle pattern, followed by an eigen-decomposition-based filtering algorithm to extract dynamic speckle signal due to the moving RBCs. The velocity of heterogeneous RBC flows is determined by cross-correlating the temporal dynamic speckle signals obtained at adjacent locations. We verify the approach by imaging mouse pinna in vivo, demonstrating its capability for full-field RBC flow mapping and quantifying flow pattern with high resolution. It is expected to investigate the dynamic action of RBCs flow in capillaries under physiological changes. PMID:28384709

Effects of Gravity on Cells, Tissues, and Organisms: Their Implications on Habitat and Human Support in Microgravity

NASA Technical Reports Server (NTRS)

Kizito, John

2004-01-01

This presentation will demonstrate that gravity plays a major role in advanced human life support in a closed habitat. The examples include, but are not limited to, control of purity in drinking water supplies (application of biocides), control of urine in space rodent habitats and operation of space septic tanks (waste management). Our goal is to understand and determine possible mechanisms that describe the process by which cells anchor to a substrate to form dynamic, vibrant communities of cells which influence human health in absence of gravity. The balance of all forces (mechanotransduction) acting on a cell will determine whether a cell thrives and multiplies or dies in a process called apoptosis and/or necrosis. The balance of forces are tightly coupled to the transport of nutrients and metabolic products (biochemotransduction) to and from the cell interface. We will highlight our effort to improve astronaut health by showing that microgravity life support systems have to be designed differently from those on Earth.

The dynamics of genome replication using deep sequencing

PubMed Central

Müller, Carolin A.; Hawkins, Michelle; Retkute, Renata; Malla, Sunir; Wilson, Ray; Blythe, Martin J.; Nakato, Ryuichiro; Komata, Makiko; Shirahige, Katsuhiko; de Moura, Alessandro P.S.; Nieduszynski, Conrad A.

2014-01-01

Eukaryotic genomes are replicated from multiple DNA replication origins. We present complementary deep sequencing approaches to measure origin location and activity in Saccharomyces cerevisiae. Measuring the increase in DNA copy number during a synchronous S-phase allowed the precise determination of genome replication. To map origin locations, replication forks were stalled close to their initiation sites; therefore, copy number enrichment was limited to origins. Replication timing profiles were generated from asynchronous cultures using fluorescence-activated cell sorting. Applying this technique we show that the replication profiles of haploid and diploid cells are indistinguishable, indicating that both cell types use the same cohort of origins with the same activities. Finally, increasing sequencing depth allowed the direct measure of replication dynamics from an exponentially growing culture. This is the first time this approach, called marker frequency analysis, has been successfully applied to a eukaryote. These data provide a high-resolution resource and methodological framework for studying genome biology. PMID:24089142

Extraction of Blebs in Human Embryonic Stem Cell Videos.

PubMed

Guan, Benjamin X; Bhanu, Bir; Talbot, Prue; Weng, Nikki Jo-Hao

2016-01-01

Blebbing is an important biological indicator in determining the health of human embryonic stem cells (hESC). Especially, areas of a bleb sequence in a video are often used to distinguish two cell blebbing behaviors in hESC: dynamic and apoptotic blebbings. This paper analyzes various segmentation methods for bleb extraction in hESC videos and introduces a bio-inspired score function to improve the performance in bleb extraction. Full bleb formation consists of bleb expansion and retraction. Blebs change their size and image properties dynamically in both processes and between frames. Therefore, adaptive parameters are needed for each segmentation method. A score function derived from the change of bleb area and orientation between consecutive frames is proposed which provides adaptive parameters for bleb extraction in videos. In comparison to manual analysis, the proposed method provides an automated fast and accurate approach for bleb sequence extraction.

Logical Modeling and Dynamical Analysis of Cellular Networks

PubMed Central

Abou-Jaoudé, Wassim; Traynard, Pauline; Monteiro, Pedro T.; Saez-Rodriguez, Julio; Helikar, Tomáš; Thieffry, Denis; Chaouiya, Claudine

2016-01-01

The logical (or logic) formalism is increasingly used to model regulatory and signaling networks. Complementing these applications, several groups contributed various methods and tools to support the definition and analysis of logical models. After an introduction to the logical modeling framework and to several of its variants, we review here a number of recent methodological advances to ease the analysis of large and intricate networks. In particular, we survey approaches to determine model attractors and their reachability properties, to assess the dynamical impact of variations of external signals, and to consistently reduce large models. To illustrate these developments, we further consider several published logical models for two important biological processes, namely the differentiation of T helper cells and the control of mammalian cell cycle. PMID:27303434

Cytokines Induce Faster Membrane Diffusion of MHC Class I and the Ly49A Receptor in a Subpopulation of Natural Killer Cells

PubMed Central

Bagawath-Singh, Sunitha; Staaf, Elina; Stoppelenburg, Arie Jan; Spielmann, Thiemo; Kambayashi, Taku; Widengren, Jerker; Johansson, Sofia

2016-01-01

Cytokines have the potential to drastically augment immune cell activity. Apart from altering the expression of a multitude of proteins, cytokines also affect immune cell dynamics. However, how cytokines affect the molecular dynamics within the cell membrane of immune cells has not been addressed previously. Molecular movement is a vital component of all biological processes, and the rate of motion is, thus, an inherent determining factor for the pace of such processes. Natural killer (NK) cells are cytotoxic lymphocytes, which belong to the innate immune system. By fluorescence correlation spectroscopy, we investigated the influence of cytokine stimulation on the membrane density and molecular dynamics of the inhibitory receptor Ly49A and its ligand, the major histocompatibility complex class I allele H-2Dd, in freshly isolated murine NK cells. H-2Dd was densely expressed and diffused slowly in resting NK cells. Ly49A was expressed at a lower density and diffused faster. The diffusion rate in resting cells was not altered by disrupting the actin cytoskeleton. A short-term stimulation with interleukin-2 or interferon-α + β did not change the surface density of moving H-2Dd or Ly49A, despite a slight upregulation at the cellular level of H-2Dd by interferon-α + β, and of Ly49A by IL-2. However, the molecular diffusion rates of both H-2Dd and Ly49A increased significantly. A multivariate analysis revealed that the increased diffusion was especially marked in a subpopulation of NK cells, where the diffusion rate was increased around fourfold compared to resting NK cells. After IL-2 stimulation, this subpopulation of NK cells also displayed lower density of Ly49A and higher brightness per entity, indicating that Ly49A may homo-cluster to a larger extent in these cells. A faster diffusion of inhibitory receptors could enable a faster accumulation of these molecules at the immune synapse with a target cell, eventually leading to a more efficient NK cell response. It has previously been assumed that cytokines regulate immune cells primarily via alterations of protein expression levels or posttranslational modifications. These findings suggest that cytokines may also modulate immune cell efficiency by increasing the molecular dynamics early on in the response. PMID:26870035

Model system for plant cell biology: GFP imaging in living onion epidermal cells

NASA Technical Reports Server (NTRS)

Scott, A.; Wyatt, S.; Tsou, P. L.; Robertson, D.; Allen, N. S.

1999-01-01

The ability to visualize organelle localization and dynamics is very useful in studying cellular physiological events. Until recently, this has been accomplished using a variety of staining methods. However, staining can give inaccurate information due to nonspecific staining, diffusion of the stain or through toxic effects. The ability to target green fluorescent protein (GFP) to various organelles allows for specific labeling of organelles in vivo. The disadvantages of GFP thus far have been the time and money involved in developing stable transformants or maintaining cell cultures for transient expression. In this paper, we present a rapid transient expression system using onion epidermal peels. We have localized GFP to various cellular compartments (including the cell wall) to illustrate the utility of this method and to visualize dynamics of these compartments. The onion epidermis has large, living, transparent cells in a monolayer, making them ideal for visualizing GFP. This method is easy and inexpensive, and it allows for testing of new GFP fusion proteins in a living tissue to determine deleterious effects and the ability to express before stable transformants are attempted.

Near-Membrane Refractometry Using Supercritical Angle Fluorescence.

PubMed

Brunstein, Maia; Roy, Lopamudra; Oheim, Martin

2017-05-09

Total internal reflection fluorescence (TIRF) microscopy and its variants are key technologies for visualizing the dynamics of single molecules or organelles in live cells. Yet truly quantitative TIRF remains problematic. One unknown hampering the interpretation of evanescent-wave excited fluorescence intensities is the undetermined cell refractive index (RI). Here, we use a combination of TIRF excitation and supercritical angle fluorescence emission detection to directly measure the average RI in the "footprint" region of the cell during image acquisition. Our RI measurement is based on the determination on a back-focal plane image of the critical angle separating evanescent and far-field fluorescence emission components. We validate our method by imaging mouse embryonic fibroblasts and BON cells. By targeting various dyes and fluorescent-protein chimeras to vesicles, the plasma membrane, as well as mitochondria and the endoplasmic reticulum, we demonstrate local RI measurements with subcellular resolution on a standard TIRF microscope, with a removable Bertrand lens as the only modification. Our technique has important applications for imaging axial vesicle dynamics and the mitochondrial energy state or detecting metabolically more active cancer cells. Copyright © 2017. Published by Elsevier Inc.

Processive movement of MreB-associated cell wall biosynthetic complexes in bacteria.

PubMed

Domínguez-Escobar, Julia; Chastanet, Arnaud; Crevenna, Alvaro H; Fromion, Vincent; Wedlich-Söldner, Roland; Carballido-López, Rut

2011-07-08

The peptidoglycan cell wall and the actin-like MreB cytoskeleton are major determinants of cell shape in rod-shaped bacteria. The prevailing model postulates that helical, membrane-associated MreB filaments organize elongation-specific peptidoglycan-synthesizing complexes along sidewalls. We used total internal reflection fluorescence microscopy to visualize the dynamic relation between MreB isoforms and cell wall synthesis in live Bacillus subtilis cells. During exponential growth, MreB proteins did not form helical structures. Instead, together with other morphogenetic factors, they assembled into discrete patches that moved processively along peripheral tracks perpendicular to the cell axis. Patch motility was largely powered by cell wall synthesis, and MreB polymers restricted diffusion of patch components in the membrane and oriented patch motion.

Dynamic mechanical measurement of the viscoelasticity of single adherent cells

NASA Astrophysics Data System (ADS)

Corbin, Elise A.; Adeniba, Olaoluwa O.; Ewoldt, Randy H.; Bashir, Rashid

2016-02-01

Many recent studies on the viscoelasticity of individual cells link mechanics with cellular function and health. Here, we introduce a measurement of the viscoelastic properties of individual human colon cancer cells (HT-29) using silicon pedestal microelectromechanical systems (MEMS) resonant sensors. We demonstrate that the viscoelastic properties of single adherent cells can be extracted by measuring a difference in vibrational amplitude of our resonant sensor platform. The magnitude of vibration of the pedestal sensor is measured using a laser Doppler vibrometer (LDV). A change in amplitude of the sensor, compared with the driving amplitude (amplitude ratio), is influenced by the mechanical properties of the adhered cells. The amplitude ratio of the fixed cells was greater than the live cells, with a p-value <0.0001. By combining the amplitude shift with the resonant frequency shift measure, we determined the elastic modulus and viscosity values of 100 Pa and 0.0031 Pa s, respectively. Our method using the change in amplitude of resonant MEMS devices can enable the determination of a refined solution space and could improve measuring the stiffness of cells.

Temporal dynamic of wood formation in Pinus cembra along the alpine treeline ecotone and the effect of climate variables

PubMed Central

Gruber, Andreas; Baumgartner, Daniel; Zimmermann, Jolanda; Oberhuber, Walter

2011-01-01

We determined the temporal dynamic of cambial activity and xylem development of stone pine (Pinus cembra L.) throughout the treeline ecotone. Repeated micro-sampling of the developing tree ring was carried out during the growing seasons 2006 and 2007 at the timberline (1950 m a.s.l.), treeline (2110 m a.s.l.) and within the krummholz belt (2180 m a.s.l.) and the influence of climate variables on intra-annual wood formation was determined. At the beginning of both growing seasons, highest numbers of cambial and enlarging cells were observed at the treeline. Soil temperatures at time of initiation of cambial activity were c. 1.5 °C higher at treeline (open canopy) compared to timberline (closed canopy), suggesting that a threshold root-zone temperature is involved in triggering onset of above ground stem growth. The rate of xylem cell production determined in two weekly intervals during June through August 2006-2007 was significantly correlated with air temperature (temperature sums expressed as degree-days and mean daily maximum temperature) at the timberline only. Lack of significant relationships between tracheid production and temperature variables at the treeline and within the krummholz belt support past dendroclimatological studies that more extreme environmental conditions (e.g., wind exposure, frost desiccation, late frost) increasingly control tree growth above timberline. Results of this study revealed that spatial and temporal (i.e. year-to-year) variability in timing and dynamic of wood formation of Pinus cembra is strongly influenced by local site factors within the treeline ecotone and the dynamics of seasonal temperature variation, respectively. PMID:21509148

Genetically encoded calcium indicators for studying long-term calcium dynamics during apoptosis.

PubMed

Garcia, M Iveth; Chen, Jessica J; Boehning, Darren

2017-01-01

Intracellular calcium release is essential for regulating almost all cellular functions. Specific spatio-temporal patterns of cytosolic calcium elevations are critical determinants of cell fate in response to pro-apoptotic cellular stressors. As the apoptotic program can take hours or days, measurement of long-term calcium dynamics are essential for understanding the mechanistic role of calcium in apoptotic cell death. Due to the technical limitations of using calcium-sensitive dyes to measure cytosolic calcium little is known about long-term calcium dynamics in living cells after treatment with apoptosis-inducing drugs. Genetically encoded calcium indicators could potentially overcome some of the limitations of calcium-sensitive dyes. Here, we compared the performance of the genetically encoded calcium indicators GCaMP6s and GCaMP6f with the ratiometric dye Fura-2. GCaMP6s performed as well or better than Fura-2 in detecting agonist-induced calcium transients. We then examined the utility of GCaMP6s for continuously measuring apoptotic calcium release over the course of ten hours after treatment with staurosporine. We found that GCaMP6s was suitable for measuring apoptotic calcium release over long time courses and revealed significant heterogeneity in calcium release dynamics in individual cells challenged with staurosporine. Our results suggest GCaMP6s is an excellent indicator for monitoring long-term changes cytosolic calcium during apoptosis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cell-free transmission of human adenovirus by passive mass transfer in cell culture simulated in a computer model.

PubMed

Yakimovich, Artur; Gumpert, Heidi; Burckhardt, Christoph J; Lütschg, Verena A; Jurgeit, Andreas; Sbalzarini, Ivo F; Greber, Urs F

2012-09-01

Viruses spread between cells, tissues, and organisms by cell-free and cell-cell transmissions. Both mechanisms enhance disease development, but it is difficult to distinguish between them. Here, we analyzed the transmission mode of human adenovirus (HAdV) in monolayers of epithelial cells by wet laboratory experimentation and a computer simulation. Using live-cell fluorescence microscopy and replication-competent HAdV2 expressing green fluorescent protein, we found that the spread of infection invariably occurred after cell lysis. It was affected by convection and blocked by neutralizing antibodies but was independent of second-round infections. If cells were overlaid with agarose, convection was blocked and round plaques developed around lytic infected cells. Infected cells that did not lyse did not give rise to plaques, highlighting the importance of cell-free transmission. Key parameters for cell-free virus transmission were the time from infection to lysis, the dose of free viruses determining infection probability, and the diffusion of single HAdV particles in aqueous medium. With these parameters, we developed an in silico model using multiscale hybrid dynamics, cellular automata, and particle strength exchange. This so-called white box model is based on experimentally determined parameters and reproduces viral infection spreading as a function of the local concentration of free viruses. These analyses imply that the extent of lytic infections can be determined by either direct plaque assays or can be predicted by calculations of virus diffusion constants and modeling.

The Integrated Role of Wnt/β-Catenin, N-Glycosylation, and E-Cadherin-Mediated Adhesion in Network Dynamics

PubMed Central

Vargas, Diego A.; Sun, Meng; Sadykov, Khikmet; Kukuruzinska, Maria A.; Zaman, Muhammad H.

2016-01-01

The cellular network composed of the evolutionarily conserved metabolic pathways of protein N-glycosylation, Wnt/β-catenin signaling pathway, and E-cadherin-mediated cell-cell adhesion plays pivotal roles in determining the balance between cell proliferation and intercellular adhesion during development and in maintaining homeostasis in differentiated tissues. These pathways share a highly conserved regulatory molecule, β-catenin, which functions as both a structural component of E-cadherin junctions and as a co-transcriptional activator of the Wnt/β-catenin signaling pathway, whose target is the N-glycosylation-regulating gene, DPAGT1. Whereas these pathways have been studied independently, little is known about the dynamics of their interaction. Here we present the first numerical model of this network in MDCK cells. Since the network comprises a large number of molecules with varying cell context and time-dependent levels of expression, it can give rise to a wide range of plausible cellular states that are difficult to track. Using known kinetic parameters for individual reactions in the component pathways, we have developed a theoretical framework and gained new insights into cellular regulation of the network. Specifically, we developed a mathematical model to quantify the fold-change in concentration of any molecule included in the mathematical representation of the network in response to a simulated activation of the Wnt/ β-catenin pathway with Wnt3a under different conditions. We quantified the importance of protein N-glycosylation and synthesis of the DPAGT1 encoded enzyme, GPT, in determining the abundance of cytoplasmic β-catenin. We confirmed the role of axin in β-catenin degradation. Finally, our data suggest that cell-cell adhesion is insensitive to E-cadherin recycling in the cell. We validate the model by inhibiting β-catenin-mediated activation of DPAGT1 expression and predicting changes in cytoplasmic β-catenin concentration and stability of E-cadherin junctions in response to DPAGT1 inhibition. We show the impact of pathway dysregulation through measurements of cell migration in scratch-wound assays. Collectively, our results highlight the importance of numerical analyses of cellular networks dynamics to gain insights into physiological processes and potential design of therapeutic strategies to prevent epithelial cell invasion in cancer. PMID:27427963

Ubx dynamically regulates Dpp signaling by repressing Dad expression during copper cell regeneration in the adult Drosophila midgut

PubMed Central

Li, Hongjie; Qi, Yanyan; Jasper, Heinrich

2016-01-01

The gastrointestinal (GI) tract of metazoans is lined by a series of regionally distinct epithelia. To maintain structure and function of the GI tract, regionally diversified differentiation of somatic stem cell (SC) lineages is critical. The adult Drosophila midgut provides an accessible model to study SC regulation and specification in a regionally defined manner. SCs of the posterior midgut (PM) have been studied extensively, but the control of SCs in the middle midgut (MM) is less well understood. The MM contains a stomach-like copper cell region (CCR) that is regenerated by gastric stem cells (GSSCs) and contains acid-secreting copper cells (CCs). Bmp-like Decapentaplegic (Dpp) signaling determines the identity of GSSCs, and is required for CC regeneration, yet the precise control of Dpp signaling activity in this lineage remains to be fully established. Here, we show that Dad, a negative feedback regulator of Dpp signaling, is dynamically regulated in the GSSC lineage to allow CC differentiation. Dad is highly expressed in GSSCs and their first daughter cells, the gastroblasts (GBs), but has to be repressed in differentiating CCs to allow Dpp-mediated differentiation into CCs. We find that the Hox gene ultrabithorax (Ubx) is required for this regulation. Loss of Ubx prevents Dad repression in the CCR, resulting in defective CC regeneration. Our study highlights the need for dynamic control of Dpp signaling activity in the differentiation of the GSSC lineage and identifies Ubx as a critical regulator of this process. PMID:27570230

Decoding the dynamics of cellular metabolism and the action of 3-bromopyruvate and 2-deoxyglucose using pulsed stable isotope-resolved metabolomics.

PubMed

Pietzke, Matthias; Zasada, Christin; Mudrich, Susann; Kempa, Stefan

2014-01-01

Cellular metabolism is highly dynamic and continuously adjusts to the physiological program of the cell. The regulation of metabolism appears at all biological levels: (post-) transcriptional, (post-) translational, and allosteric. This regulatory information is expressed in the metabolome, but in a complex manner. To decode such complex information, new methods are needed in order to facilitate dynamic metabolic characterization at high resolution. Here, we describe pulsed stable isotope-resolved metabolomics (pSIRM) as a tool for the dynamic metabolic characterization of cellular metabolism. We have adapted gas chromatography-coupled mass spectrometric methods for metabolomic profiling and stable isotope-resolved metabolomics. In addition, we have improved robustness and reproducibility and implemented a strategy for the absolute quantification of metabolites. By way of examples, we have applied this methodology to characterize central carbon metabolism of a panel of cancer cell lines and to determine the mode of metabolic inhibition of glycolytic inhibitors in times ranging from minutes to hours. Using pSIRM, we observed that 2-deoxyglucose is a metabolic inhibitor, but does not directly act on the glycolytic cascade.

Dynamics Determine Signaling in a Multicomponent System Associated with Rheumatoid Arthritis.

PubMed

Lindgren, Cecilia; Tyagi, Mohit; Viljanen, Johan; Toms, Johannes; Ge, Changrong; Zhang, Naru; Holmdahl, Rikard; Kihlberg, Jan; Linusson, Anna

2018-05-24

Strategies that target multiple components are usually required for treatment of diseases originating from complex biological systems. The multicomponent system consisting of the DR4 major histocompatibility complex type II molecule, the glycopeptide CII259-273 from type II collagen, and a T-cell receptor is associated with development of rheumatoid arthritis (RA). We introduced non-native amino acids and amide bond isosteres into CII259-273 and investigated the effect on binding to DR4 and the subsequent T-cell response. Molecular dynamics simulations revealed that complexes between DR4 and derivatives of CII259-273 were highly dynamic. Signaling in the overall multicomponent system was found to depend on formation of an appropriate number of dynamic intramolecular hydrogen bonds between DR4 and CII259-273, together with the positioning of the galactose moiety of CII259-273 in the DR4 binding groove. Interestingly, the system tolerated modifications at several positions in CII259-273, indicating opportunities to use analogues to increase our understanding of how rheumatoid arthritis develops and for evaluation as vaccines to treat RA.

A spatiotemporal characterization method for the dynamic cytoskeleton.

PubMed

Alhussein, Ghada; Shanti, Aya; Farhat, Ilyas A H; Timraz, Sara B H; Alwahab, Noaf S A; Pearson, Yanthe E; Martin, Matthew N; Christoforou, Nicolas; Teo, Jeremy C M

2016-05-01

The significant gap between quantitative and qualitative understanding of cytoskeletal function is a pressing problem; microscopy and labeling techniques have improved qualitative investigations of localized cytoskeleton behavior, whereas quantitative analyses of whole cell cytoskeleton networks remain challenging. Here we present a method that accurately quantifies cytoskeleton dynamics. Our approach digitally subdivides cytoskeleton images using interrogation windows, within which box-counting is used to infer a fractal dimension (Df ) to characterize spatial arrangement, and gray value intensity (GVI) to determine actin density. A partitioning algorithm further obtains cytoskeleton characteristics from the perinuclear, cytosolic, and periphery cellular regions. We validated our measurement approach on Cytochalasin-treated cells using transgenically modified dermal fibroblast cells expressing fluorescent actin cytoskeletons. This method differentiates between normal and chemically disrupted actin networks, and quantifies rates of cytoskeletal degradation. Furthermore, GVI distributions were found to be inversely proportional to Df , having several biophysical implications for cytoskeleton formation/degradation. We additionally demonstrated detection sensitivity of differences in Df and GVI for cells seeded on substrates with varying degrees of stiffness, and coated with different attachment proteins. This general approach can be further implemented to gain insights on dynamic growth, disruption, and structure of the cytoskeleton (and other complex biological morphology) due to biological, chemical, or physical stimuli. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

A spatiotemporal characterization method for the dynamic cytoskeleton

PubMed Central

Alhussein, Ghada; Shanti, Aya; Farhat, Ilyas A. H.; Timraz, Sara B. H.; Alwahab, Noaf S. A.; Pearson, Yanthe E.; Martin, Matthew N.; Christoforou, Nicolas

2016-01-01

The significant gap between quantitative and qualitative understanding of cytoskeletal function is a pressing problem; microscopy and labeling techniques have improved qualitative investigations of localized cytoskeleton behavior, whereas quantitative analyses of whole cell cytoskeleton networks remain challenging. Here we present a method that accurately quantifies cytoskeleton dynamics. Our approach digitally subdivides cytoskeleton images using interrogation windows, within which box‐counting is used to infer a fractal dimension (D f) to characterize spatial arrangement, and gray value intensity (GVI) to determine actin density. A partitioning algorithm further obtains cytoskeleton characteristics from the perinuclear, cytosolic, and periphery cellular regions. We validated our measurement approach on Cytochalasin‐treated cells using transgenically modified dermal fibroblast cells expressing fluorescent actin cytoskeletons. This method differentiates between normal and chemically disrupted actin networks, and quantifies rates of cytoskeletal degradation. Furthermore, GVI distributions were found to be inversely proportional to D f, having several biophysical implications for cytoskeleton formation/degradation. We additionally demonstrated detection sensitivity of differences in D f and GVI for cells seeded on substrates with varying degrees of stiffness, and coated with different attachment proteins. This general approach can be further implemented to gain insights on dynamic growth, disruption, and structure of the cytoskeleton (and other complex biological morphology) due to biological, chemical, or physical stimuli. © 2016 The Authors. Cytoskeleton Published by Wiley Periodicals, Inc. PMID:27015595

Statistical analysis of particle trajectories in living cells

NASA Astrophysics Data System (ADS)

Briane, Vincent; Kervrann, Charles; Vimond, Myriam

2018-06-01

Recent advances in molecular biology and fluorescence microscopy imaging have made possible the inference of the dynamics of molecules in living cells. Such inference allows us to understand and determine the organization and function of the cell. The trajectories of particles (e.g., biomolecules) in living cells, computed with the help of object tracking methods, can be modeled with diffusion processes. Three types of diffusion are considered: (i) free diffusion, (ii) subdiffusion, and (iii) superdiffusion. The mean-square displacement (MSD) is generally used to discriminate the three types of particle dynamics. We propose here a nonparametric three-decision test as an alternative to the MSD method. The rejection of the null hypothesis, i.e., free diffusion, is accompanied by claims of the direction of the alternative (subdiffusion or superdiffusion). We study the asymptotic behavior of the test statistic under the null hypothesis and under parametric alternatives which are currently considered in the biophysics literature. In addition, we adapt the multiple-testing procedure of Benjamini and Hochberg to fit with the three-decision-test setting, in order to apply the test procedure to a collection of independent trajectories. The performance of our procedure is much better than the MSD method as confirmed by Monte Carlo experiments. The method is demonstrated on real data sets corresponding to protein dynamics observed in fluorescence microscopy.

Dynamic model of a micro-tubular solid oxide fuel cell stack including an integrated cooling system

NASA Astrophysics Data System (ADS)

Hering, Martin; Brouwer, Jacob; Winkler, Wolfgang

2017-02-01

A novel dynamic micro-tubular solid oxide fuel cell (MT-SOFC) and stack model including an integrated cooling system is developed using a quasi three-dimensional, spatially resolved, transient thermodynamic, physical and electrochemical model that accounts for the complex geometrical relations between the cells and cooling-tubes. The modeling approach includes a simplified tubular geometry and stack design including an integrated cooling structure, detailed pressure drop and gas property calculations, the electrical and physical constraints of the stack design that determine the current, as well as control strategies for the temperature. Moreover, an advanced heat transfer balance with detailed radiative heat transfer between the cells and the integrated cooling-tubes, convective heat transfer between the gas flows and the surrounding structures and conductive heat transfer between the solid structures inside of the stack, is included. The detailed model can be used as a design basis for the novel MT-SOFC stack assembly including an integrated cooling system, as well as for the development of a dynamic system control strategy. The evaluated best-case design achieves very high electrical efficiency between around 75 and 55% in the entire power density range between 50 and 550 mW /cm2 due to the novel stack design comprising an integrated cooling structure.

Analysis of Video-Based Microscopic Particle Trajectories Using Kalman Filtering

PubMed Central

Wu, Pei-Hsun; Agarwal, Ashutosh; Hess, Henry; Khargonekar, Pramod P.; Tseng, Yiider

2010-01-01

Abstract The fidelity of the trajectories obtained from video-based particle tracking determines the success of a variety of biophysical techniques, including in situ single cell particle tracking and in vitro motility assays. However, the image acquisition process is complicated by system noise, which causes positioning error in the trajectories derived from image analysis. Here, we explore the possibility of reducing the positioning error by the application of a Kalman filter, a powerful algorithm to estimate the state of a linear dynamic system from noisy measurements. We show that the optimal Kalman filter parameters can be determined in an appropriate experimental setting, and that the Kalman filter can markedly reduce the positioning error while retaining the intrinsic fluctuations of the dynamic process. We believe the Kalman filter can potentially serve as a powerful tool to infer a trajectory of ultra-high fidelity from noisy images, revealing the details of dynamic cellular processes. PMID:20550894

A novel methodology for rapid digestion of rare earth element ores and determination by microwave plasma-atomic emission spectrometry and dynamic reaction cell-inductively coupled plasma-mass spectrometry.

PubMed

Helmeczi, Erick; Wang, Yong; Brindle, Ian D

2016-11-01

Short-wavelength infrared radiation has been successfully applied to accelerate the acid digestion of refractory rare-earth ore samples. Determinations were achieved with microwave plasma-atomic emission spectrometry (MP-AES) and dynamic reaction cell - inductively coupled plasma-mass spectrometry (DRC-ICP-MS). The digestion method developed was able to tackle high iron-oxide and silicate matrices using only phosphoric acid in a time frame of only 8min, and did not require perchloric or hydrofluoric acid. Additionally, excellent recoveries and reproducibilities of the rare earth elements, as well as uranium and thorium, were achieved. Digestions of the certified reference materials OREAS-465 and REE-1, with radically different mineralogies, delivered results that mirror those obtained by fusion processes. For the rare-earth CRM OKA-2, whose REE data are provisional, experimental data for the rare-earth elements were generally higher than the provisional values, often exceeding z-values of +2. Determined values for Th and U in this reference material, for which certified values are available, were in excellent agreement. Copyright © 2016 Elsevier B.V. All rights reserved.

Pea Border Cell Maturation and Release Involve Complex Cell Wall Structural Dynamics.

PubMed

Mravec, Jozef; Guo, Xiaoyuan; Hansen, Aleksander Riise; Schückel, Julia; Kračun, Stjepan Krešimir; Mikkelsen, Maria Dalgaard; Mouille, Grégory; Johansen, Ida Elisabeth; Ulvskov, Peter; Domozych, David S; Willats, William George Tycho

2017-06-01

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 immunocarbohydrate microarray profiling, monosaccharide composition determination, Fourier-transformed infrared microspectroscopy, quantitative reverse transcription-PCR of cell wall biosynthetic genes, analysis of hydrolytic activities, transmission electron microscopy, and immunolocalization of cell wall components. Using this integrated glycobiology approach, we identified multiple novel modes of cell wall structural and compositional rearrangement during root cap growth and the release of border cells. Our findings provide a new level of detail about border cell maturation and enable us to develop a model of the separation process. We propose that loss of adhesion by the dissolution of homogalacturonan in the middle lamellae is augmented by an active biophysical process of cell curvature driven by the polarized distribution of xyloglucan and extensin epitopes. © 2017 American Society of Plant Biologists. All Rights Reserved.

Organization and Dynamics of Receptor Proteins in a Plasma Membrane.

PubMed

Koldsø, Heidi; Sansom, Mark S P

2015-11-25

The interactions of membrane proteins are influenced by their lipid environment, with key lipid species able to regulate membrane protein function. Advances in high-resolution microscopy can reveal the organization and dynamics of proteins and lipids within living cells at resolutions <200 nm. Parallel advances in molecular simulations provide near-atomic-resolution models of the dynamics of the organization of membranes of in vivo-like complexity. We explore the dynamics of proteins and lipids in crowded and complex plasma membrane models, thereby closing the gap in length and complexity between computations and experiments. Our simulations provide insights into the mutual interplay between lipids and proteins in determining mesoscale (20-100 nm) fluctuations of the bilayer, and in enabling oligomerization and clustering of membrane proteins.

Dynamics of two-dimensional bubbles.

PubMed

Piedra, Saúl; Ramos, Eduardo; Herrera, J Ramón

2015-06-01

The dynamics of two-dimensional bubbles ascending under the influence of buoyant forces is numerically studied with a one-fluid model coupled with the front-tracking technique. The bubble dynamics are described by recording the position, shape, and orientation of the bubbles as functions of time. The qualitative properties of the bubbles and their terminal velocities are described in terms of the Eötvos (ratio of buoyancy to surface tension) and Archimedes numbers (ratio of buoyancy to viscous forces). The terminal Reynolds number result from the balance of buoyancy and drag forces and, consequently, is not an externally fixed parameter. In the cases that yield small Reynolds numbers, the bubbles follow straight paths and the wake is steady. A more interesting behavior is found at high Reynolds numbers where the bubbles follow an approximately periodic zigzag trajectory and an unstable wake with properties similar to the Von Karman vortex street is formed. The dynamical features of the motion of single bubbles are compared to experimental observations of air bubbles ascending in a water-filled Hele-Shaw cell. Although the comparison is not strictly valid in the sense that the effect of the lateral walls is not incorporated in the model, most of the dynamical properties observed are in good qualitative agreement with the numerical calculations. Hele-Shaw cells with different gaps have been used to determine the degree of approximation of the numerical calculation. It is found that for the relation between the terminal Reynolds number and the Archimedes number, the numerical calculations are closer to the observations of bubble dynamics in Hele-Shaw cells of larger gaps.

Exploratory Spatial Analysis of in vitro Respiratory Syncytial Virus Co-infections

PubMed Central

Simeonov, Ivan; Gong, Xiaoyan; Kim, Oekyung; Poss, Mary; Chiaromonte, Francesca; Fricks, John

2010-01-01

The cell response to virus infection and virus perturbation of that response is dynamic and is reflected by changes in cell susceptibility to infection. In this study, we evaluated the response of human epithelial cells to sequential infections with human respiratory syncytial virus strains A2 and B to determine if a primary infection with one strain will impact the ability of cells to be infected with the second as a function of virus strain and time elapsed between the two exposures. Infected cells were visualized with fluorescent markers, and location of all cells in the tissue culture well were identified using imaging software. We employed tools from spatial statistics to investigate the likelihood of a cell being infected given its proximity to a cell infected with either the homologous or heterologous virus. We used point processes, K-functions, and simulation procedures designed to account for specific features of our data when assessing spatial associations. Our results suggest that intrinsic cell properties increase susceptibility of cells to infection, more so for RSV-B than for RSV-A. Further, we provide evidence that the primary infection can decrease susceptibility of cells to the heterologous challenge virus but only at the 16 h time point evaluated in this study. Our research effort highlights the merits of integrating empirical and statistical approaches to gain greater insight on in vitro dynamics of virus-host interactions. PMID:21994640

Elements of the cellular metabolic structure

PubMed Central

De la Fuente, Ildefonso M.

2015-01-01

A large number of studies have demonstrated the existence of metabolic covalent modifications in different molecular structures, which are able to store biochemical information that is not encoded by DNA. Some of these covalent mark patterns can be transmitted across generations (epigenetic changes). Recently, the emergence of Hopfield-like attractor dynamics has been observed in self-organized enzymatic networks, which have the capacity to store functional catalytic patterns that can be correctly recovered by specific input stimuli. Hopfield-like metabolic dynamics are stable and can be maintained as a long-term biochemical memory. In addition, specific molecular information can be transferred from the functional dynamics of the metabolic networks to the enzymatic activity involved in covalent post-translational modulation, so that determined functional memory can be embedded in multiple stable molecular marks. The metabolic dynamics governed by Hopfield-type attractors (functional processes), as well as the enzymatic covalent modifications of specific molecules (structural dynamic processes) seem to represent the two stages of the dynamical memory of cellular metabolism (metabolic memory). Epigenetic processes appear to be the structural manifestation of this cellular metabolic memory. Here, a new framework for molecular information storage in the cell is presented, which is characterized by two functionally and molecularly interrelated systems: a dynamic, flexible and adaptive system (metabolic memory) and an essentially conservative system (genetic memory). The molecular information of both systems seems to coordinate the physiological development of the whole cell. PMID:25988183

Sodium-sulfur Cell Technology Flight Experiment (SSCT)

NASA Technical Reports Server (NTRS)

Halbach, Carl R.

1992-01-01

The sodium-sulfur battery is emerging as a prime high-temperature energy storage technology for space flight applications. A Na-S cell demonstration is planned for a 1995-96 NASA Space Shuttle flight which focuses on the microgravity effects on individual cells. The experiment is not optimized for battery performance as such. Rather, it maximizes the variety of operating conditions which the Na-S cell is capable of in a relatively short 5-day flight. The demonstration is designed to reveal the effects of microgravity by comparison with ground test control cells experiencing identical test conditions but with gravity. Specifically, limitations of transport dynamics and associated cell performance characteristics should be revealed. The Na-S Cell Technology Flight Experiment consists of three separate experiments designed to determine cell operating characteristics, detailed electrode kinetics and reactant distributions.

Tissue specific characterisation of Lim-kinase 1 expression during mouse embryogenesis

PubMed Central

Lindström, Nils O.; Neves, Carlos; McIntosh, Rebecca; Miedzybrodzka, Zosia; Vargesson, Neil; Collinson, J. Martin

2012-01-01

The Lim-kinase (LIMK) proteins are important for the regulation of the actin cytoskeleton, in particular the control of actin nucleation and depolymerisation via regulation of cofilin, and hence may control a large number of processes during development, including cell tensegrity, migration, cell cycling, and axon guidance. LIMK1/LIMK2 knockouts disrupt spinal cord morphogenesis and synapse formation but other tissues and developmental processes that require LIMK are yet to be fully determined. To identify tissues and cell-types that may require LIMK, we characterised the pattern of LIMK1 protein during mouse embryogenesis. We showed that LIMK1 displays an expression pattern that is temporally dynamic and tissue-specific. In several tissues LIMK1 is detected in cell-types that also express Wilms’ tumour protein 1 and that undergo transitions between epithelial and mesenchymal states, including the pleura, epicardium, kidney nephrons, and gonads. LIMK1 was also found in a subset of cells in the dorsal retina, and in mesenchymal cells surrounding the peripheral nerves. This detailed study of the spatial and temporal expression of LIMK1 shows that LIMK1 expression is more dynamic than previously reported, in particular at sites of tissue–tissue interactions guiding multiple developmental processes. PMID:21167960

Activation Loop Dynamics Determine the Different Catalytic Efficiencies of B Cell- and T Cell-Specific Tec Kinases

PubMed Central

Joseph, Raji E.; Kleino, Iivari; Wales, Thomas E.; Xie, Qian; Fulton, D. Bruce; Engen, John R.; Berg, Leslie J.; Andreotti, Amy H.

2014-01-01

Itk and Btk are nonreceptor tyrosine kinases of the Tec family that signal downstream of the T cell receptor (TCR) and B cell receptor (BCR), respectively. Despite their high sequence similarity and related signaling roles, Btk is a substantially more active kinase than Itk. We showed that substitution of six of the 619 amino acid residues of Itk with those of Btk was sufficient to completely switch the activities of Itk and Btk. The substitutions responsible for the swap in activity are all localized to the activation segment of the kinase domain. Nuclear magnetic resonance and hydrogen-deuterium exchange mass spectrometry analyses revealed that Itk and Btk had distinct protein dynamics in this region, which could explain the observed differences in catalytic efficiency between these kinases. Introducing Itk with enhanced activity into T cells led to enhanced and prolonged TCR signaling compared to that in cells with wild-type Itk. These findings imply that evolutionary pressures have led to Tec kinases having distinct enzymatic properties depending on the cellular context. We suggest that the weaker catalytic activities observed for T cell–specific kinases is one mechanism to regulate cellular activation and prevent aberrant immune responses. PMID:23982207

Eye on the B-ALL: B-cell receptor repertoires reveal persistence of numerous B-lymphoblastic leukemia subclones from diagnosis to relapse

PubMed Central

Bashford-Rogers, R J M; Nicolaou, K A; Bartram, J; Goulden, N J; Loizou, L; Koumas, L; Chi, J; Hubank, M; Kellam, P; Costeas, P A; Vassiliou, G S

2016-01-01

The strongest predictor of relapse in B-cell acute lymphoblastic leukemia (B-ALL) is the level of persistence of tumor cells after initial therapy. The high mutation rate of the B-cell receptor (BCR) locus allows high-resolution tracking of the architecture, evolution and clonal dynamics of B-ALL. Using longitudinal BCR repertoire sequencing, we find that the BCR undergoes an unexpectedly high level of clonal diversification in B-ALL cells through both somatic hypermutation and secondary rearrangements, which can be used for tracking the subclonal composition of the disease and detect minimal residual disease with unprecedented sensitivity. We go on to investigate clonal dynamics of B-ALL using BCR phylogenetic analyses of paired diagnosis-relapse samples and find that large numbers of small leukemic subclones present at diagnosis re-emerge at relapse alongside a dominant clone. Our findings suggest that in all informative relapsed patients, the survival of large numbers of clonogenic cells beyond initial chemotherapy is a surrogate for inherent partial chemoresistance or inadequate therapy, providing an increased opportunity for subsequent emergence of fully resistant clones. These results frame early cytoreduction as an important determinant of long-term outcome. PMID:27211266

Crystal structure and phase transition in (NH4)3WO2F5: from dynamic to static orientational disorder.

PubMed

Udovenko, Anatoly; Laptash, Natalia

2015-08-01

Single crystals of tungsten double salt (NH4)3WO2F5 = (NH4)3[WO2F4]F have been synthesized by solid-state reaction or from fluoride solution and its crystal structures at 296 and 193 K were determined by X-ray diffraction. At room temperature, the crystal structure of the compound is dynamically disordered with the ligand atoms statistically distributed on two positions (6e and 24m) of the Pm3m unit cell [a = 6.0298 (1) Å], and the tungsten atom dynamically disordered on 12 orientations forming a spatial cuboctahedron [W12] that enables the real geometry of cis-WO2F4 octahedron to be determined with two short W-O distances. On cooling, the compound undergoes a first-order phase transition with the symmetry change Pm3m → Pa3 and a doubling of the unit-cell parameter [a = 11.9635 (7) Å]. The ligand F(O) atoms statistically occupy two general 24d sites and form W1X6 and W2X6 octahedra, in which the O and F atoms are not crystallographically different that means a static orientational disorder of (NH4)3WO2F5.

Non-Kinetic Losses Caused by Electrochemical Carbon Corrosion in PEM Fuel Cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, Seh Kyu; Shao, Yuyan; Viswanathan, Vilayanur V.

2012-05-01

This paper presented non-kinetic losses in PEM fuel cells under an accelerated stress test of catalyst support. The cathode with carbon-supported Pt catalyst was prepared and characterized with potential hold at 1.2 V vs. SHE in PEM fuel cells. Irreversible losses caused by carbon corrosion were evaluated using a variety of electrochemical characterizations including cyclic voltammetry, linear sweep voltammetry, electrochemical impedance spectroscopy, and polarization technique. Ohmic losses at the cathode with potential hold were determined using its capacitive responses. Concentration losses in PEM fuel cells were analyzed in terms of Tafel behavior and thin film/flooded-agglomerate dynamics.

Collective and single cell behavior in epithelial contact inhibition.

PubMed

Puliafito, Alberto; Hufnagel, Lars; Neveu, Pierre; Streichan, Sebastian; Sigal, Alex; Fygenson, D Kuchnir; Shraiman, Boris I

2012-01-17

Control of cell proliferation is a fundamental aspect of tissue physiology central to morphogenesis, wound healing, and cancer. Although many of the molecular genetic factors are now known, the system level regulation of growth is still poorly understood. A simple form of inhibition of cell proliferation is encountered in vitro in normally differentiating epithelial cell cultures and is known as "contact inhibition." The study presented here provides a quantitative characterization of contact inhibition dynamics on tissue-wide and single cell levels. Using long-term tracking of cultured Madin-Darby canine kidney cells we demonstrate that inhibition of cell division in a confluent monolayer follows inhibition of cell motility and sets in when mechanical constraint on local expansion causes divisions to reduce cell area. We quantify cell motility and cell cycle statistics in the low density confluent regime and their change across the transition to epithelial morphology which occurs with increasing cell density. We then study the dynamics of cell area distribution arising through reductive division, determine the average mitotic rate as a function of cell size, and demonstrate that complete arrest of mitosis occurs when cell area falls below a critical value. We also present a simple computational model of growth mechanics which captures all aspects of the observed behavior. Our measurements and analysis show that contact inhibition is a consequence of mechanical interaction and constraint rather than interfacial contact alone, and define quantitative phenotypes that can guide future studies of molecular mechanisms underlying contact inhibition.

Stress and strain in the contractile and cytoskeletal filaments of airway smooth muscle.

PubMed

Deng, Linhong; Bosse, Ynuk; Brown, Nathan; Chin, Leslie Y M; Connolly, Sarah C; Fairbank, Nigel J; King, Greg G; Maksym, Geoffrey N; Paré, Peter D; Seow, Chun Y; Stephen, Newman L

2009-10-01

Stress and strain are omnipresent in the lung due to constant lung volume fluctuation associated with respiration, and they modulate the phenotype and function of all cells residing in the airways including the airway smooth muscle (ASM) cell. There is ample evidence that the ASM cell is very sensitive to its physical environment, and can alter its structure and/or function accordingly, resulting in either desired or undesired consequences. The forces that are either conferred to the ASM cell due to external stretching or generated inside the cell must be borne and transmitted inside the cytoskeleton (CSK). Thus, maintaining appropriate levels of stress and strain within the CSK is essential for maintaining normal function. Despite the importance, the mechanisms regulating/dysregulating ASM cytoskeletal filaments in response to stress and strain remained poorly understood until only recently. For example, it is now understood that ASM length and force are dynamically regulated, and both can adapt over a wide range of length, rendering ASM one of the most malleable living tissues. The malleability reflects the CSK's dynamic mechanical properties and plasticity, both of which strongly interact with the loading on the CSK, and all together ultimately determines airway narrowing in pathology. Here we review the latest advances in our understanding of stress and strain in ASM cells, including the organization of contractile and cytoskeletal filaments, range and adaptation of functional length, structural and functional changes of the cell in response to mechanical perturbation, ASM tone as a mediator of strain-induced responses, and the novel glassy dynamic behaviors of the CSK in relation to asthma pathophysiology.

Quantitative fluorescence correlation spectroscopy on DNA in living cells

NASA Astrophysics Data System (ADS)

Hodges, Cameron; Kafle, Rudra P.; Meiners, Jens-Christian

2017-02-01

FCS is a fluorescence technique conventionally used to study the kinetics of fluorescent molecules in a dilute solution. Being a non-invasive technique, it is now drawing increasing interest for the study of more complex systems like the dynamics of DNA or proteins in living cells. Unlike an ordinary dye solution, the dynamics of macromolecules like proteins or entangled DNA in crowded environments is often slow and subdiffusive in nature. This in turn leads to longer residence times of the attached fluorophores in the excitation volume of the microscope and artifacts from photobleaching abound that can easily obscure the signature of the molecular dynamics of interest and make quantitative analysis challenging.We discuss methods and procedures to make FCS applicable to quantitative studies of the dynamics of DNA in live prokaryotic and eukaryotic cells. The intensity autocorrelation is computed function from weighted arrival times of the photons on the detector that maximizes the information content while simultaneously correcting for the effect of photobleaching to yield an autocorrelation function that reflects only the underlying dynamics of the sample. This autocorrelation function in turn is used to calculate the mean square displacement of the fluorophores attached to DNA. The displacement data is more amenable to further quantitative analysis than the raw correlation functions. By using a suitable integral transform of the mean square displacement, we can then determine the viscoelastic moduli of the DNA in its cellular environment. The entire analysis procedure is extensively calibrated and validated using model systems and computational simulations.

Deciphering dynamics of clathrin-mediated endocytosis in a living organism

PubMed Central

Heidotting, Spencer P.; Huber, Scott D.

2016-01-01

Current understanding of clathrin-mediated endocytosis (CME) dynamics is based on detection and tracking of fluorescently tagged clathrin coat components within cultured cells. Because of technical limitations inherent to detection and tracking of single fluorescent particles, CME dynamics is not characterized in vivo, so the effects of mechanical cues generated during development of multicellular organisms on formation and dissolution of clathrin-coated structures (CCSs) have not been directly observed. Here, we use growth rates of fluorescence signals obtained from short CCS intensity trace fragments to assess CME dynamics. This methodology does not rely on determining the complete lifespan of individual endocytic assemblies. Therefore, it allows for real-time monitoring of spatiotemporal changes in CME dynamics and is less prone to errors associated with particle detection and tracking. We validate the applicability of this approach to in vivo systems by demonstrating the reduction of CME dynamics during dorsal closure of Drosophila melanogaster embryos. PMID:27458134

An assay to image neuronal microtubule dynamics in mice.

PubMed

Kleele, Tatjana; Marinković, Petar; Williams, Philip R; Stern, Sina; Weigand, Emily E; Engerer, Peter; Naumann, Ronald; Hartmann, Jana; Karl, Rosa M; Bradke, Frank; Bishop, Derron; Herms, Jochen; Konnerth, Arthur; Kerschensteiner, Martin; Godinho, Leanne; Misgeld, Thomas

2014-09-12

Microtubule dynamics in neurons play critical roles in physiology, injury and disease and determine microtubule orientation, the cell biological correlate of neurite polarization. Several microtubule binding proteins, including end-binding protein 3 (EB3), specifically bind to the growing plus tip of microtubules. In the past, fluorescently tagged end-binding proteins have revealed microtubule dynamics in vitro and in non-mammalian model organisms. Here, we devise an imaging assay based on transgenic mice expressing yellow fluorescent protein-tagged EB3 to study microtubules in intact mammalian neurites. Our approach allows measurement of microtubule dynamics in vivo and ex vivo in peripheral nervous system and central nervous system neurites under physiological conditions and after exposure to microtubule-modifying drugs. We find an increase in dynamic microtubules after injury and in neurodegenerative disease states, before axons show morphological indications of degeneration or regrowth. Thus increased microtubule dynamics might serve as a general indicator of neurite remodelling in health and disease.

[Establishment of fibroblast cell line and its biological characteristics in Matou goat].

PubMed

Li, Tianda; Liu, Chousheng; Wang, Zhigang; Zhang, Liping; Sun, Xiuzhu; Zhao, Junjin; Meng, Fei; Luo, Guihe; Zhu, Jinqing

2008-12-01

Taking Matou goat ear margin as the study material, we succeeded in established a fibroblast cell line by the method of explant culture directly. Observations on morphology, dynamic growth, determination of viability, analysis of karyotype, test of microorganism and other characteristics were detected. Results showed: Population Doubling Time (PDT) of cells was approximately 36 h; Cell viability was 96.7% after thawing; The status of cell After passage was constant; Analysis of chromosomal karyotyps indicated that diploid (2n=60) account for 98% in the cell line. Every index in the cell line met all the standard quality controls of ATCC in USA. The established of Matou goat ear fibroblast cell line has not only important genetic resources preserved at the cell level, but also valuable material for genome, postgenome and somatic cell nuclear transfer research.

Single-Cell and Single-Molecule Analysis of Gene Expression Regulation.

PubMed

Vera, Maria; Biswas, Jeetayu; Senecal, Adrien; Singer, Robert H; Park, Hye Yoon

2016-11-23

Recent advancements in single-cell and single-molecule imaging technologies have resolved biological processes in time and space that are fundamental to understanding the regulation of gene expression. Observations of single-molecule events in their cellular context have revealed highly dynamic aspects of transcriptional and post-transcriptional control in eukaryotic cells. This approach can relate transcription with mRNA abundance and lifetimes. Another key aspect of single-cell analysis is the cell-to-cell variability among populations of cells. Definition of heterogeneity has revealed stochastic processes, determined characteristics of under-represented cell types or transitional states, and integrated cellular behaviors in the context of multicellular organisms. In this review, we discuss novel aspects of gene expression of eukaryotic cells and multicellular organisms revealed by the latest advances in single-cell and single-molecule imaging technology.

Hepcidin as a possible marker in determination of malignancy degree and sensitivity of breast cancer cells to cytostatic drugs.

PubMed

Yalovenko, T M; Todor, I M; Lukianova, N Y; Chekhun, V F

2016-06-01

To investigate the role of hepcidin (Hepc) in the formation of cells malignant phenotype in vitro and its expression in the dyna-mics of growth of Walker-256 carcinosarcoma with different sensitivity to doxorubicin (Dox). The cell lines used in the analysis included T47D, MCF-7, MDA-MB-231, MDA-MB-468, MCF/CP, and MCF/Dox. Hepc expression was studied by immunocytochemical method. "Free" iron content was determined by EPR spectroscopy. Determination of Hepc expression in homogenates of tumor tissue and in blood serum of rats with Dox-sensitive and -resistant Walker-256 carcinosarcoma was performed. It was found that Hepc levels in breast cancer (BC) cells with high degree of malignancy (MDA-MB-231, MDA-MB-468) and drug-resistant phenotype (MCF/CP, MCF/Dox) were by 1.5-2 times higher (p < 0.05) in comparison with sensitive and less malignant BC cells. The development of drug-resistant phenotype in Walker-256 carcinosarcoma cells was accompanied by increasing of Hepc and "free" iron content (by 2.4 and 1.2 times, respectively). The data of in vitro and in vivo research evidenced on involvement of Hepc in formation of BC cells malignant phenotype and their resistance to Dox.

Piezo type mechanosensitive ion channel component 1 functions as a regulator of the cell fate determination of mesenchymal stem cells.

PubMed

Sugimoto, Asuna; Miyazaki, Aya; Kawarabayashi, Keita; Shono, Masayuki; Akazawa, Yuki; Hasegawa, Tomokazu; Ueda-Yamaguchi, Kimiko; Kitamura, Takamasa; Yoshizaki, Keigo; Fukumoto, Satoshi; Iwamoto, Tsutomu

2017-12-18

The extracellular environment regulates the dynamic behaviors of cells. However, the effects of hydrostatic pressure (HP) on cell fate determination of mesenchymal stem cells (MSCs) are not clearly understood. Here, we established a cell culture chamber to control HP. Using this system, we found that the promotion of osteogenic differentiation by HP is depend on bone morphogenetic protein 2 (BMP2) expression regulated by Piezo type mechanosensitive ion channel component 1 (PIEZO1) in MSCs. The PIEZO1 was expressed and induced after HP loading in primary MSCs and MSC lines, UE7T-13 and SDP11. HP and Yoda1, an activator of PIEZO1, promoted BMP2 expression and osteoblast differentiation, whereas inhibits adipocyte differentiation. Conversely, PIEZO1 inhibition reduced osteoblast differentiation and BMP2 expression. Furthermore, Blocking of BMP2 function by noggin inhibits HP induced osteogenic maker genes expression. In addition, in an in vivo model of medaka with HP loading, HP promoted caudal fin ray development whereas inhibition of piezo1 using GsMTx4 suppressed its development. Thus, our results suggested that PIEZO1 is responsible for HP and could functions as a factor for cell fate determination of MSCs by regulating BMP2 expression.

A viscoelastic–stochastic model of the effects of cytoskeleton remodelling on cell adhesion

PubMed Central

Li, Long; Zhang, Wenyan

2016-01-01

Cells can adapt their mechanical properties through cytoskeleton remodelling in response to external stimuli when the cells adhere to the extracellular matrix (ECM). Many studies have investigated the effects of cell and ECM elasticity on cell adhesion. However, experiments determined that cells are viscoelastic and exhibiting stress relaxation, and the mechanism behind the effect of cellular viscoelasticity on the cell adhesion behaviour remains unclear. Therefore, we propose a theoretical model of a cluster of ligand–receptor bonds between two dissimilar viscoelastic media subjected to an applied tensile load. In this model, the distribution of interfacial traction is assumed to follow classical continuum viscoelastic equations, whereas the rupture and rebinding of individual molecular bonds are governed by stochastic equations. On the basis of this model, we determined that viscosity can significantly increase the lifetime, stability and dynamic strength of the adhesion cluster of molecular bonds, because deformation relaxation attributed to the viscoelastic property can increase the rebinding probability of each open bond and reduce the stress concentration in the adhesion area. PMID:27853571

Biomechanical force in blood development: extrinsic physical cues drive pro-hematopoietic signaling

PubMed Central

Lee, Hyun Jung; Li, Nan; Evans, Siobahn M.; Diaz, Miguel F.; Wenzel, Pamela L.

2013-01-01

The hematopoietic system is dynamic during development and in adulthood, undergoing countless spatial and temporal transitions during the course of one’s life. Microenvironmental cues in the many unique hematopoietic niches differ, characterized by distinct soluble molecules, membrane-bound factors, and biophysical features that meet the changing needs of the blood system. Research from the last decade has revealed the importance of substrate elasticity and biomechanical force in determination of stem cell fate. Our understanding of the role of these factors in hematopoiesis is still relatively poor; however, the developmental origin of blood cells from the endothelium promts a model for comparison. Many endothelial mechanical sensors and second messenger systems may also determine hematopoietic stem cell fate, self renewal, and homing behaviors. Further, the intimate contact of hematopoietic cells with mechanosensitive cell types, including osteoblasts, endothelial cells, mesenchymal stem cells, and pericytes, places them in close proximity to paracrine signaling downstream of mechanical signals. The objective of this review is to present an overview of the sensors and intracellular signaling pathways activated by mechanical cues and highlight the role of mechanotransductive pathways in hematopoiesis. PMID:23850217

Multiscale modeling of the dynamics of multicellular systems

NASA Astrophysics Data System (ADS)

Kosztin, Ioan

2011-03-01

Describing the biomechanical properties of cellular systems, regarded as complex highly viscoelastic materials, is a difficult problem of great conceptual and practical value. Here we present a novel approach, referred to as the Cellular Particle Dynamics (CPD) method, for: (i) quantitatively relating biomechanical properties at the cell level to those at the multicellular and tissue level, and (ii) describing and predicting the time evolution of multicellular systems that undergo biomechanical relaxations. In CPD cells are modeled as an ensemble of cellular particles (CPs) that interact via short range contact interactions, characterized by an attractive (adhesive interaction) and a repulsive (excluded volume interaction) component. The time evolution of the spatial conformation of the multicellular system is determined by following the trajectories of all CPs through integration of their equations of motion. Cell and multicellular level biomechanical properties (e.g., viscosity, surface tension and shear modulus) are determined through the combined use of experiments and theory of continuum viscoelastic media. The same biomechanical properties are also ``measured'' computationally by employing the CPD method, the results being expressed in terms of CPD parameters. Once these parameters have been calibrated experimentally, the formalism provides a systematic framework to predict the time evolution of complex multicellular systems during shape-changing biomechanical transformations. By design, the CPD method is rather flexible and most suitable for multiscale modeling of multicellular system. The spatial level of detail of the system can be easily tuned by changing the number of CPs in a cell. Thus, CPD can be used equally well to describe both cell level processes (e.g., the adhesion of two cells) and tissue level processes (e.g., the formation of 3D constructs of millions of cells through bioprinting). Work supported by NSF [FIBR-0526854 and PHY-0957914]. Computer time provided by the University of Missouri Bioinformatics Consortium.

A nanobiosensor for dynamic single cell analysis during microvascular self-organization.

PubMed

Wang, S; Sun, J; Zhang, D D; Wong, P K

2016-10-14

The formation of microvascular networks plays essential roles in regenerative medicine and tissue engineering. Nevertheless, the self-organization mechanisms underlying the dynamic morphogenic process are poorly understood due to a paucity of effective tools for mapping the spatiotemporal dynamics of single cell behaviors. By establishing a single cell nanobiosensor along with live cell imaging, we perform dynamic single cell analysis of the morphology, displacement, and gene expression during microvascular self-organization. Dynamic single cell analysis reveals that endothelial cells self-organize into subpopulations with specialized phenotypes to form microvascular networks and identifies the involvement of Notch1-Dll4 signaling in regulating the cell subpopulations. The cell phenotype correlates with the initial Dll4 mRNA expression level and each subpopulation displays a unique dynamic Dll4 mRNA expression profile. Pharmacological perturbations and RNA interference of Notch1-Dll4 signaling modulate the cell subpopulations and modify the morphology of the microvascular network. Taken together, a nanobiosensor enables a dynamic single cell analysis approach underscoring the importance of Notch1-Dll4 signaling in microvascular self-organization.

Integration of actomyosin contractility with cell-cell adhesion during dorsal closure.

PubMed

Duque, Julia; Gorfinkiel, Nicole

2016-12-15

In this work, we combine genetic perturbation, time-lapse imaging and quantitative image analysis to investigate how pulsatile actomyosin contractility drives cell oscillations, apical cell contraction and tissue closure during morphogenesis of the amnioserosa, the main force-generating tissue during the dorsal closure in Drosophila We show that Myosin activity determines the oscillatory and contractile behaviour of amnioserosa cells. Reducing Myosin activity prevents cell shape oscillations and reduces cell contractility. By contrast, increasing Myosin activity increases the amplitude of cell shape oscillations and the time cells spend in the contracted phase relative to the expanded phase during an oscillatory cycle, promoting cell contractility and tissue closure. Furthermore, we show that in AS cells, Rok controls Myosin foci formation and Mbs regulates not only Myosin phosphorylation but also adhesion dynamics through control of Moesin phosphorylation, showing that Mbs coordinates actomyosin contractility with cell-cell adhesion during amnioserosa morphogenesis. © 2016. Published by The Company of Biologists Ltd.

Communication: On the origin of the non-Arrhenius behavior in water reorientation dynamics.

PubMed

Stirnemann, Guillaume; Laage, Damien

2012-07-21

We combine molecular dynamics simulations and analytic modeling to determine the origin of the non-Arrhenius temperature dependence of liquid water's reorientation and hydrogen-bond dynamics between 235 K and 350 K. We present a quantitative model connecting hydrogen-bond exchange dynamics to local structural fluctuations, measured by the asphericity of Voronoi cells associated with each water molecule. For a fixed local structure the regular Arrhenius behavior is recovered, and the global anomalous temperature dependence is demonstrated to essentially result from a continuous shift in the unimodal structure distribution upon cooling. The non-Arrhenius behavior can thus be explained without invoking an equilibrium between distinct structures. In addition, the large width of the homogeneous structural distribution is shown to cause a growing dynamical heterogeneity and a non-exponential relaxation at low temperature.

Dynamic epigenetic regulation of glioblastoma tumorigenicity through LSD1 modulation of MYC expression

PubMed Central

Kozono, David; Li, Jie; Nitta, Masayuki; Sampetrean, Oltea; Gonda, David; Kushwaha, Deepa S.; Merzon, Dmitry; Ramakrishnan, Valya; Zhu, Shan; Zhu, Kaya; Matsui, Hiroko; Harismendy, Olivier; Hua, Wei; Mao, Ying; Kwon, Chang-Hyuk; Saya, Hideyuki; Nakano, Ichiro; Pizzo, Donald P.; VandenBerg, Scott R.; Chen, Clark C.

2015-01-01

The available evidence suggests that the lethality of glioblastoma is driven by small subpopulations of cells that self-renew and exhibit tumorigenicity. It remains unclear whether tumorigenicity exists as a static property of a few cells or as a dynamically acquired property. We used tumor-sphere and xenograft formation as assays for tumorigenicity and examined subclones isolated from established and primary glioblastoma lines. Our results indicate that glioblastoma tumorigenicity is largely deterministic, yet the property can be acquired spontaneously at low frequencies. Further, these dynamic transitions are governed by epigenetic reprogramming through the lysine-specific demethylase 1 (LSD1). LSD depletion increases trimethylation of histone 3 lysine 4 at the avian myelocytomatosis viral oncogene homolog (MYC) locus, which elevates MYC expression. MYC, in turn, regulates oligodendrocyte lineage transcription factor 2 (OLIG2), SRY (sex determining region Y)-box 2 (SOX2), and POU class 3 homeobox 2 (POU3F2), a core set of transcription factors required for reprogramming glioblastoma cells into stem-like states. Our model suggests epigenetic regulation of key transcription factors governs transitions between tumorigenic states and provides a framework for glioblastoma therapeutic development. PMID:26159421

A Computational Approach for Modeling Neutron Scattering Data from Lipid Bilayers

DOE PAGES

Carrillo, Jan-Michael Y.; Katsaras, John; Sumpter, Bobby G.; ...

2017-01-12

Biological cell membranes are responsible for a range of structural and dynamical phenomena crucial to a cell's well-being and its associated functions. Due to the complexity of cell membranes, lipid bilayer systems are often used as biomimetic models. These systems have led to signficant insights into vital membrane phenomena such as domain formation, passive permeation and protein insertion. Experimental observations of membrane structure and dynamics are, however, limited in resolution, both spatially and temporally. Importantly, computer simulations are starting to play a more prominent role in interpreting experimental results, enabling a molecular under- standing of lipid membranes. Particularly, the synergymore » between scattering experiments and simulations offers opportunities for new discoveries in membrane physics, as the length and time scales probed by molecular dynamics (MD) simulations parallel those of experiments. We also describe a coarse-grained MD simulation approach that mimics neutron scattering data from large unilamellar lipid vesicles over a range of bilayer rigidity. Specfically, we simulate vesicle form factors and membrane thickness fluctuations determined from small angle neutron scattering (SANS) and neutron spin echo (NSE) experiments, respectively. Our simulations accurately reproduce trends from experiments and lay the groundwork for investigations of more complex membrane systems.« less

Transient Binding and Viscous Dissipation in Semi-flexible Polymer Networks

NASA Astrophysics Data System (ADS)

Lieleg, Oliver; Claessens, Mireille; Bausch, Andreas

2008-03-01

Nature specifically chooses from a myriad of actin binding proteins (ABPs) to tailor the cytoskeletal microstructure. Herein, cells rely on the dynamics of the cytoskeleton as its structural and mechanical adaptability is crucial to allow for dynamic processes. A molecular understanding of such biological complexity calls for an in vitro system with well-defined structural rearrangements and cross-linker dynamics to elucidate the physical origin of the unique viscoelastic properties of cells. As we present here, the frequency-dependent viscoelastic response of cross-linked in vitro actin networks is determined by the binding kinetics of cross-linking molecules. Independent from the particular network structure, the viscous dissipation (loss modulus) exhibits a pronounced minimum in an intermediate frequency which is dominated by elasticity. We show that in this frequency regime the molecular origin of the viscoelastic response is given by the non-static nature of actin/ABP bonds as they are subjugated to chemical on/off kinetics. The time scale of the resulting stress release is set by the lifetime distribution of the cross-linking molecule and therefore can be tuned independently from other relaxation mechanisms. We speculate that unbinding of distinct cross-links might be the molecular mechanism employed by cells for mechanosensing.

Dynamic changes in gene expression during human trophoblast differentiation.

PubMed

Handwerger, Stuart; Aronow, Bruce

2003-01-01

The genetic program that directs human placental differentiation is poorly understood. In a recent study, we used DNA microarray analyses to determine genes that are dynamically regulated during human placental development in an in vitro model system in which highly purified cytotrophoblast cells aggregate spontaneously and fuse to form a multinucleated syncytium that expresses placental lactogen, human chorionic gonadotropin, and other proteins normally expressed by fully differentiated syncytiotrophoblast cells. Of the 6918 genes present on the Incyte Human GEM V microarray that we analyzed over a 9-day period, 141 were induced and 256 were downregulated by more than 2-fold. The dynamically regulated genes fell into nine distinct kinetic patterns of induction or repression, as detected by the K-means algorithm. Classifying the genes according to functional characteristics, the regulated genes could be divided into six overall categories: cell and tissue structural dynamics, cell cycle and apoptosis, intercellular communication, metabolism, regulation of gene expression, and expressed sequence tags and function unknown. Gene expression changes within key functional categories were tightly coupled to the morphological changes that occurred during trophoblast differentiation. Within several key gene categories (e.g., cell and tissue structure), many genes were strongly activated, while others with related function were strongly repressed. These findings suggest that trophoblast differentiation is augmented by "categorical reprogramming" in which the ability of induced genes to function is enhanced by diminished synthesis of other genes within the same category. We also observed categorical reprogramming in human decidual fibroblasts decidualized in vitro in response to progesterone, estradiol, and cyclic AMP. While there was little overlap between genes that are dynamically regulated during trophoblast differentiation versus decidualization, many of the categories in which genes were strongly activated also contained genes whose expression was strongly diminished. Taken together, these findings point to a fundamental role for simultaneous induction and repression of mRNAs that encode functionally related proteins during the differentiation process.

Electrical properties of a light-addressable microelectrode chip with high electrode density for extracellular stimulation and recording of excitable cells.

PubMed

Bucher, V; Brunner, B; Leibrock, C; Schubert, M; Nisch, W

2001-05-01

A light-addressable microelectrode chip with 3600 TiN electrodes was fabricated. Amorphous silicon (a-Si:H) serves as a photo conductor. The electrodes on the chip are addressed by a laser spot and electrical properties of the system are determined. DC measurements show a dark to bright dynamic of 10(6)-10(7). The AC impedance dynamic @ 1 kHz/100 mV and thus the signal-to-noise-ratio is determined to 60. This value is quite sufficient for electrophysiological measurements. For the first time, recordings from cardiac myocytes are reported using the principle of light-addressing. Measurements were done with a standard laser scan microscope (Zeiss LSM 410).

Cell cycle tracking for irradiated and unirradiated bystander cells in a single colony with exposure to a soft X-ray microbeam.

PubMed

Kaminaga, Kiichi; Noguchi, Miho; Narita, Ayumi; Hattori, Yuya; Usami, Noriko; Yokoya, Akinari

2016-11-01

To establish a new experimental technique to explore the photoelectric and subsequent Auger effects on the cell cycles of soft X-ray microbeam-irradiated cells and unirradiated bystander cells in a single colony. Several cells located in the center of a microcolony of HeLa-Fucci cells consisting of 20-80 cells were irradiated with soft X-ray (5.35 keV) microbeam using synchrotron radiation as a light source. All cells in the colony were tracked for 72 h by time-lapse microscopy imaging. Cell cycle progression, division, and death of each cell in the movies obtained were analyzed by pedigree assay. The number of cell divisions in the microcolony was also determined. The fates of these cells were clarified by tracking both irradiated and unirradiated bystander cells. Irradiated cells showed significant cell cycle retardation, explosive cell death, or cell fusion after a few divisions. These serious effects were also observed in 15 and 26% of the bystander cells for 10 and 20 Gy irradiation, respectively, and frequently appeared in at least two daughter or granddaughter cells from a single-parent cell. We successfully tracked the fates of microbeam-irradiated cells and unirradiated bystander cells with live cell recordings, which have revealed the dynamics of soft X-ray irradiated and unirradiated bystander cells for the first time. Notably, cell deaths or cell cycle arrests frequently arose in closely related cells. These details would not have been revealed by a conventional immunostaining imaging method. Our approach promises to reveal the dynamic cellular effects of soft X-ray microbeam irradiation and subsequent Auger processes from various endpoints in future studies.

Cell Electrosensitization Exists Only in Certain Electroporation Buffers.

PubMed

Dermol, Janja; Pakhomova, Olga N; Pakhomov, Andrei G; Miklavčič, Damijan

2016-01-01

Electroporation-induced cell sensitization was described as the occurrence of a delayed hypersensitivity to electric pulses caused by pretreating cells with electric pulses. It was achieved by increasing the duration of the electroporation treatment at the same cumulative energy input. It could be exploited in electroporation-based treatments such as electrochemotherapy and tissue ablation with irreversible electroporation. The mechanisms responsible for cell sensitization, however, have not yet been identified. We investigated cell sensitization dynamics in five different electroporation buffers. We split a pulse train into two trains varying the delay between them and measured the propidium uptake by fluorescence microscopy. By fitting the first-order model to the experimental results, we determined the uptake due to each train (i.e. the first and the second) and the corresponding resealing constant. Cell sensitization was observed in the growth medium but not in other tested buffers. The effect of pulse repetition frequency, cell size change, cytoskeleton disruption and calcium influx do not adequately explain cell sensitization. Based on our results, we can conclude that cell sensitization is a sum of several processes and is buffer dependent. Further research is needed to determine its generality and to identify underlying mechanisms.

Cell Electrosensitization Exists Only in Certain Electroporation Buffers

PubMed Central

Dermol, Janja; Pakhomova, Olga N.; Pakhomov, Andrei G.; Miklavčič, Damijan

2016-01-01

Electroporation-induced cell sensitization was described as the occurrence of a delayed hypersensitivity to electric pulses caused by pretreating cells with electric pulses. It was achieved by increasing the duration of the electroporation treatment at the same cumulative energy input. It could be exploited in electroporation-based treatments such as electrochemotherapy and tissue ablation with irreversible electroporation. The mechanisms responsible for cell sensitization, however, have not yet been identified. We investigated cell sensitization dynamics in five different electroporation buffers. We split a pulse train into two trains varying the delay between them and measured the propidium uptake by fluorescence microscopy. By fitting the first-order model to the experimental results, we determined the uptake due to each train (i.e. the first and the second) and the corresponding resealing constant. Cell sensitization was observed in the growth medium but not in other tested buffers. The effect of pulse repetition frequency, cell size change, cytoskeleton disruption and calcium influx do not adequately explain cell sensitization. Based on our results, we can conclude that cell sensitization is a sum of several processes and is buffer dependent. Further research is needed to determine its generality and to identify underlying mechanisms. PMID:27454174

Morphology and Performance of Polymer Solar Cell Characterized by DPD Simulation and Graph Theory.

PubMed

Du, Chunmiao; Ji, Yujin; Xue, Junwei; Hou, Tingjun; Tang, Jianxin; Lee, Shuit-Tong; Li, Youyong

2015-11-19

The morphology of active layers in the bulk heterojunction (BHJ) solar cells is critical to the performance of organic photovoltaics (OPV). Currently, there is limited information for the morphology from transmission electron microscopy (TEM) techniques. Meanwhile, there are limited approaches to predict the morphology /efficiency of OPV. Here we use Dissipative Particle Dynamics (DPD) to determine 3D morphology of BHJ solar cells and show DPD to be an efficient approach to predict the 3D morphology. Based on the 3D morphology, we estimate the performance indicator of BHJ solar cells by using graph theory. Specifically, we study poly (3-hexylthiophene)/[6, 6]-phenyl-C61butyric acid methyl ester (P3HT/PCBM) BHJ solar cells. We find that, when the volume fraction of PCBM is in the region 0.4 ∼ 0.5, P3HT/PCBM will show bi-continuous morphology and optimum performance, consistent with experimental results. Further, the optimum temperature (413 K) for the morphology and performance of P3HT/PCBM is in accord with annealing results. We find that solvent additive plays a critical role in the desolvation process of P3HT/PCBM BHJ solar cell. Our approach provides a direct method to predict dynamic 3D morphology and performance indicator for BHJ solar cells.

Relationship between intracellular pH, metabolic co-factors and caspase-3 activation in cancer cells during apoptosis.

PubMed

Sergeeva, Tatiana F; Shirmanova, Marina V; Zlobovskaya, Olga A; Gavrina, Alena I; Dudenkova, Varvara V; Lukina, Maria M; Lukyanov, Konstantin A; Zagaynova, Elena V

2017-03-01

A complex cascade of molecular events occurs in apoptotic cells but cell-to-cell variability significantly complicates determination of the order and interconnections between different processes. For better understanding of the mechanisms of programmed cell death, dynamic simultaneous registration of several parameters is required. In this paper we used multiparameter fluorescence microscopy to analyze energy metabolism, intracellular pH and caspase-3 activation in living cancer cells in vitro during staurosporine-induced apoptosis. We performed metabolic imaging of two co-factors, NAD(P)H and FAD, and used the genetically encoded pH-indicator SypHer1 and the FRET-based sensor for caspase-3 activity, mKate2-DEVD-iRFP, to visualize these parameters by confocal fluorescence microscopy and two-photon fluorescence lifetime imaging microscopy. The correlation between energy metabolism, intracellular pH and caspase-3 activation and their dynamic changes were studied in CT26 cancer cells during apoptosis. Induction of apoptosis was accompanied by a switch to oxidative phosphorylation, cytosol acidification and caspase-3 activation. We showed that alterations in cytosolic pH and the activation of oxidative phosphorylation are relatively early events associated with the induction of apoptosis. Copyright © 2017 Elsevier B.V. All rights reserved.

SPECT Imaging for in vivo tracking of NIS containing stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Zhenghong

2013-04-02

The proposed study contains two groups of imaging experiments: 1) human mesenchymal stem cells supporting in vivo survival of unrelated donor hematopoietic stem cells; 2) gene transduction and selection of mutant MGMT genes on human hematopoietic stem cells conferring resistance to BC+BCNU. There is increasing evidence that adult human tissues harbor stem and progenitor cells that can be used for therapeutic purposes. We had focused on the Mesenchymal Stem Cells (MSCs) found in human bone marrow and investigated these cells in the context of autologous and allogeneic hematopoietic stem cell transplantation to a) facilitate rapid hematopoietic engraftment in cancer patientsmore » receiving high dose chemotherapy and b) to modulate the graft-versus-host disease (GVHD). We have demonstrated that culture-expanded autologous and allogeneic MSCs can be safely infused into humans and the preliminary results showed that MSCs facilitate hematopoietic engraftment and reduce GVHD. On the other hand, studies of gene transfer with drug resistant selection suggest major perturbations to the process of hematopoietic reconstitution and the confounding issue of organ toxicity and recovery that takes place in the host. We have found that limiting numbers of hematopoietic stem cells transduced with MGMT repopulate the bone marrow of primary and secondary recipient mice. We are also particularly interested in the dynamics of engraftment and selection in regions of bones, liver, spleen and lung, where we have previously seen marked evidence of engraftment. All the measurements have required animal sacrifice and single point determinations of engraftment in individual and cohorts of mice. Heretofore it has not been possible to study the dynamics of engraftment and enrichment. In the upcoming application, we propose to develop an imaging method to track intravenously infused stem cells in vivo at preset time points to understand their homing and proliferation. Specifically, we propose to use Na+/I- symporter (NIS) gene as a reporter gene (imagene) for non-invasive imaging of infused stem cells distribution and persistence in vivo on small animal models. NIS is an intrinsic membrane glycoprotein that mediates active iodide (I-) uptake into normal thyroid follicular cells and other cells. The advantages of using NIS for non-invasive and repeated scintigraphic imaging in this application are: a) NIS is not a foreign gene and thus eliminate the immunoresponse problem; b) radiotracer or substrate for NIS is simply radioiodide (I-125, I- 123, I-124, and I-124) or [Tc-99m]-pertechnetate, no radiosynthesis is needed. It has been shown that NIS gene transfer can induce radioactive iodide uptake in a variety of cells and that xenografts expressing exogenous NIS could be imaged by non-invasive scintigraphic imaging. The specific aims are: 1.Determine the feasibility, stability and physiological effects of human NIS gene expression on human HSCs and MSCs in vitro. 2.Determine the engraftment of human HSC and MSC co-infused in NOD-SCID mice. 3.Transduce both a drug resistance gene and an imagene into bone marrow stem cells, and follow the dynamics of engraftment after selection in real time.« less

Engineering challenges of BioNEMS: the integration of microfluidics, micro- and nanodevices, models and external control for systems biology.

PubMed

Wikswo, J P; Prokop, A; Baudenbacher, F; Cliffel, D; Csukas, B; Velkovsky, M

2006-08-01

Systems biology, i.e. quantitative, postgenomic, postproteomic, dynamic, multiscale physiology, addresses in an integrative, quantitative manner the shockwave of genetic and proteomic information using computer models that may eventually have 10(6) dynamic variables with non-linear interactions. Historically, single biological measurements are made over minutes, suggesting the challenge of specifying 10(6) model parameters. Except for fluorescence and micro-electrode recordings, most cellular measurements have inadequate bandwidth to discern the time course of critical intracellular biochemical events. Micro-array expression profiles of thousands of genes cannot determine quantitative dynamic cellular signalling and metabolic variables. Major gaps must be bridged between the computational vision and experimental reality. The analysis of cellular signalling dynamics and control requires, first, micro- and nano-instruments that measure simultaneously multiple extracellular and intracellular variables with sufficient bandwidth; secondly, the ability to open existing internal control and signalling loops; thirdly, external BioMEMS micro-actuators that provide high bandwidth feedback and externally addressable intracellular nano-actuators; and, fourthly, real-time, closed-loop, single-cell control algorithms. The unravelling of the nested and coupled nature of cellular control loops requires simultaneous recording of multiple single-cell signatures. Externally controlled nano-actuators, needed to effect changes in the biochemical, mechanical and electrical environment both outside and inside the cell, will provide a major impetus for nanoscience.

Dynamics and control of the ERK signaling pathway: Sensitivity, bistability, and oscillations.

PubMed

Arkun, Yaman; Yasemi, Mohammadreza

2018-01-01

Cell signaling is the process by which extracellular information is transmitted into the cell to perform useful biological functions. The ERK (extracellular-signal-regulated kinase) signaling controls several cellular processes such as cell growth, proliferation, differentiation and apoptosis. The ERK signaling pathway considered in this work starts with an extracellular stimulus and ends with activated (double phosphorylated) ERK which gets translocated into the nucleus. We model and analyze this complex pathway by decomposing it into three functional subsystems. The first subsystem spans the initial part of the pathway from the extracellular growth factor to the formation of the SOS complex, ShC-Grb2-SOS. The second subsystem includes the activation of Ras which is mediated by the SOS complex. This is followed by the MAPK subsystem (or the Raf-MEK-ERK pathway) which produces the double phosphorylated ERK upon being activated by Ras. Although separate models exist in the literature at the subsystems level, a comprehensive model for the complete system including the important regulatory feedback loops is missing. Our dynamic model combines the existing subsystem models and studies their steady-state and dynamic interactions under feedback. We establish conditions under which bistability and oscillations exist for this important pathway. In particular, we show how the negative and positive feedback loops affect the dynamic characteristics that determine the cellular outcome.

Live-cell imaging combined with immunofluorescence, RNA, or DNA FISH to study the nuclear dynamics and expression of the X-inactivation center.

PubMed

Pollex, Tim; Piolot, Tristan; Heard, Edith

2013-01-01

Differentiation of embryonic stem cells is accompanied by changes of gene expression and chromatin and chromosome dynamics. One of the most impressive examples for these changes is inactivation of one of the two X chromosomes occurring upon differentiation of mouse female embryonic stem cells. With a few exceptions, these events have been mainly studied in fixed cells. In order to better understand the dynamics, kinetics, and order of events during differentiation, one needs to employ live-cell imaging techniques. Here, we describe a combination of live-cell imaging with techniques that can be used in fixed cells (e.g., RNA FISH) to correlate locus dynamics or subnuclear localization with, e.g., gene expression. To study locus dynamics in female ES cells, we generated cell lines containing TetO arrays in the X-inactivation center, the locus on the X chromosome regulating X-inactivation, which can be visualized upon expression of TetR fused to fluorescent proteins. We will use this system to elaborate on how to generate ES cell lines for live-cell imaging of locus dynamics, how to culture ES cells prior to live-cell imaging, and to describe typical live-cell imaging conditions for ES cells using different microscopes. Furthermore, we will explain how RNA, DNA FISH, or immunofluorescence can be applied following live-cell imaging to correlate gene expression with locus dynamics.

Limiting Energy Dissipation Induces Glassy Kinetics in Single-Cell High-Precision Responses

PubMed Central

Das, Jayajit

2016-01-01

Single cells often generate precise responses by involving dissipative out-of-thermodynamic-equilibrium processes in signaling networks. The available free energy to fuel these processes could become limited depending on the metabolic state of an individual cell. How does limiting dissipation affect the kinetics of high-precision responses in single cells? I address this question in the context of a kinetic proofreading scheme used in a simple model of early-time T cell signaling. Using exact analytical calculations and numerical simulations, I show that limiting dissipation qualitatively changes the kinetics in single cells marked by emergence of slow kinetics, large cell-to-cell variations of copy numbers, temporally correlated stochastic events (dynamic facilitation), and ergodicity breaking. Thus, constraints in energy dissipation, in addition to negatively affecting ligand discrimination in T cells, can create a fundamental difficulty in determining single-cell kinetics from cell-population results. PMID:26958894

Matrix Remodeling in Pulmonary Fibrosis and Emphysema

PubMed Central

O’Reilly, Philip; Antony, Veena B.; Gaggar, Amit

2016-01-01

Pulmonary fibrosis and emphysema are chronic lung diseases characterized by a progressive decline in lung function, resulting in significant morbidity and mortality. A hallmark of these diseases is recurrent or persistent alveolar epithelial injury, typically caused by common environmental exposures such as cigarette smoke. We propose that critical determinants of the outcome of the injury-repair processes that result in fibrosis versus emphysema are mesenchymal cell fate and associated extracellular matrix dynamics. In this review, we explore the concept that regulation of mesenchymal cells under the influence of soluble factors, in particular transforming growth factor-β1, and the extracellular matrix determine the divergent tissue remodeling responses seen in pulmonary fibrosis and emphysema. PMID:26741177

A New Approach for Determining Phase Response Curves Reveals that Purkinje Cells Can Act as Perfect Integrators

PubMed Central

Roth, Arnd; Häusser, Michael

2010-01-01

Cerebellar Purkinje cells display complex intrinsic dynamics. They fire spontaneously, exhibit bistability, and via mutual network interactions are involved in the generation of high frequency oscillations and travelling waves of activity. To probe the dynamical properties of Purkinje cells we measured their phase response curves (PRCs). PRCs quantify the change in spike phase caused by a stimulus as a function of its temporal position within the interspike interval, and are widely used to predict neuronal responses to more complex stimulus patterns. Significant variability in the interspike interval during spontaneous firing can lead to PRCs with a low signal-to-noise ratio, requiring averaging over thousands of trials. We show using electrophysiological experiments and simulations that the PRC calculated in the traditional way by sampling the interspike interval with brief current pulses is biased. We introduce a corrected approach for calculating PRCs which eliminates this bias. Using our new approach, we show that Purkinje cell PRCs change qualitatively depending on the firing frequency of the cell. At high firing rates, Purkinje cells exhibit single-peaked, or monophasic PRCs. Surprisingly, at low firing rates, Purkinje cell PRCs are largely independent of phase, resembling PRCs of ideal non-leaky integrate-and-fire neurons. These results indicate that Purkinje cells can act as perfect integrators at low firing rates, and that the integration mode of Purkinje cells depends on their firing rate. PMID:20442875

Genetic and epigenetic effects in sex determination.

PubMed

Gunes, Sezgin Ozgur; Metin Mahmutoglu, Asli; Agarwal, Ashok

2016-12-01

Sex determination is a complex and dynamic process with multiple genetic and environmental causes, in which germ and somatic cells receive various sex-specific features. During the fifth week of fetal life, the bipotential embryonic gonad starts to develop in humans. In the bipotential gonadal tissue, certain cell groups start to differentiate to form the ovaries or testes. Despite considerable efforts and advances in identifying the mechanisms playing a role in sex determination and differentiation, the underlying mechanisms of the exact functions of many genes, gene-gene interactions, and epigenetic modifications that are involved in different stages of this cascade are not completely understood. This review aims at discussing current data on the genetic effects via genes and epigenetic mechanisms that affect the regulation of sex determination. Birth Defects Research (Part C) 108:321-336, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Telomere length dynamics in human memory T cells specific for viruses causing acute or latent infections

PubMed Central

2013-01-01

Background Declining telomere length (TL) is associated with T cell senescence. While TL in naïve and memory T cells declines with increasing age, there is limited data on TL dynamics in virus-specific memory CD4+ T cells in healthy adults. We combined BrdU-labeling of virus-stimulated T cells followed with flow cytometry-fluorescent in situ hybridization for TL determination. We analyzed TL in T cells specific for several virus infections: non-recurring acute (vaccinia virus, VACV), recurring-acute (influenza A virus, IAV), and reactivating viruses (varicella-zoster virus, VZV, and cytomegalovirus, CMV) in 10 healthy subjects. Additionally, five subjects provided multiple blood samples separated by up to 10 years. Results VACV- and CMV-specific T cells had longer average TL than IAV-specific CD4+ T cells. Although most virus-specific cells were CD45RA-, we observed a minor population of BrdU+ CD45RA+ T cells characterized by long telomeres. Longitudinal analysis demonstrated a slow decline in average TL in virus-specific T cells. However, in one subject, VZV reactivation led to an increase in average TL in VZV-specific memory T cells, suggesting a conversion of longer TL cells from the naïve T cell repertoire. Conclusions TLs in memory CD4+ T cells in otherwise healthy adults are heterogeneous and follow distinct virus-specific kinetics. These findings suggests that the distribution of TL and the creation and maintenance of long TL memory T cells could be important for the persistence of long-lived T cell memory. PMID:23971624

Telomere length dynamics in human memory T cells specific for viruses causing acute or latent infections.

PubMed

O'Bryan, Joel M; Woda, Marcia; Co, Mary; Mathew, Anuja; Rothman, Alan L

2013-08-26

Declining telomere length (TL) is associated with T cell senescence. While TL in naïve and memory T cells declines with increasing age, there is limited data on TL dynamics in virus-specific memory CD4+ T cells in healthy adults. We combined BrdU-labeling of virus-stimulated T cells followed with flow cytometry-fluorescent in situ hybridization for TL determination. We analyzed TL in T cells specific for several virus infections: non-recurring acute (vaccinia virus, VACV), recurring-acute (influenza A virus, IAV), and reactivating viruses (varicella-zoster virus, VZV, and cytomegalovirus, CMV) in 10 healthy subjects. Additionally, five subjects provided multiple blood samples separated by up to 10 years. VACV- and CMV-specific T cells had longer average TL than IAV-specific CD4+ T cells. Although most virus-specific cells were CD45RA-, we observed a minor population of BrdU+ CD45RA+ T cells characterized by long telomeres. Longitudinal analysis demonstrated a slow decline in average TL in virus-specific T cells. However, in one subject, VZV reactivation led to an increase in average TL in VZV-specific memory T cells, suggesting a conversion of longer TL cells from the naïve T cell repertoire. TLs in memory CD4+ T cells in otherwise healthy adults are heterogeneous and follow distinct virus-specific kinetics. These findings suggests that the distribution of TL and the creation and maintenance of long TL memory T cells could be important for the persistence of long-lived T cell memory.

Dynamized Preparations in Cell Culture

PubMed Central

Sunila, Ellanzhiyil Surendran; Preethi, Korengath Chandran; Kuttan, Girija

2009-01-01

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. PMID:18955237

Lattice dynamics of the rare-earth element samarium

NASA Astrophysics Data System (ADS)

Bauder, Olga; Piekarz, Przemysław; Barla, Alessandro; Sergueev, Ilya; Rüffer, Rudolf; ŁaŻewski, Jan; Baumbach, Tilo; Parlinski, Krzysztof; Stankov, Svetoslav

2013-12-01

The lattice dynamics of samarium is determined by in situ low-temperature nuclear inelastic scattering on a single crystalline (0001)Sm film, a polycrystalline Sm foil, and by first-principles theory. The ab initio calculated phonon dispersion relations and phonon density of states for the Sm-type structure and the double hexagonal-close-packed (dhcp) lattice, characteristic for light lanthanides, are compared. The dhcp unit cell, which is a factor of 2.24 smaller in height, exhibits more pronounced vibrational anisotropy in comparison to the Sm-type structure. The analysis reveals a minor influence of the spin-orbit coupling in the Sm atom on the lattice dynamics. A broadening of the longitudinal peak, not found in the calculations, suggests the influence of electron correlations on lattice dynamics in metallic samarium.

Oscillatory Protein Expression Dynamics Endows Stem Cells with Robust Differentiation Potential

PubMed Central

Kaneko, Kunihiko

2011-01-01

The lack of understanding of stem cell differentiation and proliferation is a fundamental problem in developmental biology. Although gene regulatory networks (GRNs) for stem cell differentiation have been partially identified, the nature of differentiation dynamics and their regulation leading to robust development remain unclear. Herein, using a dynamical system modeling cell approach, we performed simulations of the developmental process using all possible GRNs with a few genes, and screened GRNs that could generate cell type diversity through cell-cell interactions. We found that model stem cells that both proliferated and differentiated always exhibited oscillatory expression dynamics, and the differentiation frequency of such stem cells was regulated, resulting in a robust number distribution. Moreover, we uncovered the common regulatory motifs for stem cell differentiation, in which a combination of regulatory motifs that generated oscillatory expression dynamics and stabilized distinct cellular states played an essential role. These findings may explain the recently observed heterogeneity and dynamic equilibrium in cellular states of stem cells, and can be used to predict regulatory networks responsible for differentiation in stem cell systems. PMID:22073296

A Hybrid Cellular Automaton Model of Clonal Evolution in Cancer: The Emergence of the Glycolytic Phenotype

PubMed Central

Gerlee, P.; Anderson, A.R.A.

2009-01-01

We present a cellular automaton model of clonal evolution in cancer aimed at investigating the emergence of the glycolytic phenotype. In the model each cell is equipped with a micro-environment response network that determines the behaviour or phenotype of the cell based on the local environment. The response network is modelled using a feed-forward neural network, which is subject to mutations when the cells divide. This implies that cells might react differently to the environment and when space and nutrients are limited only the fittest cells will survive. With this model we have investigated the impact of the environment on the growth dynamics of the tumour. In particular we have analysed the influence of the tissue oxygen concentration and extra-cellular matrix density on the dynamics of the model. We found that the environment influences both the growth and evolutionary dynamics of the tumour. For low oxygen concentration we observe tumours with a fingered morphology, while increasing the matrix density gives rise to more compact tumours with wider fingers. The distribution of phenotypes in the tumour is also affected, and we observe that the glycolytic phenotype is most likely to emerge in a poorly oxygenated tissue with a high matrix density. Our results suggest that it is the combined effect of the oxygen concentration and matrix density that creates an environment where the glycolytic phenotype has a growth advantage and consequently is most likely to appear. PMID:18068192

The relationship between node degree and dissipation rate in networks of diffusively coupled oscillators and its significance for pancreatic beta cells.

PubMed

Gosak, Marko; Stožer, Andraž; Markovič, Rene; Dolenšek, Jurij; Marhl, Marko; Rupnik, Marjan Slak; Perc, Matjaž

2015-07-01

Self-sustained oscillatory dynamics is a motion along a stable limit cycle in the phase space, and it arises in a wide variety of mechanical, electrical, and biological systems. Typically, oscillations are due to a balance between energy dissipation and generation. Their stability depends on the properties of the attractor, in particular, its dissipative characteristics, which in turn determine the flexibility of a given dynamical system. In a network of oscillators, the coupling additionally contributes to the dissipation, and hence affects the robustness of the oscillatory solution. Here, we therefore investigate how a heterogeneous network structure affects the dissipation rate of individual oscillators. First, we show that in a network of diffusively coupled oscillators, the dissipation is a linearly decreasing function of the node degree, and we demonstrate this numerically by calculating the average divergence of coupled Hopf oscillators. Subsequently, we use recordings of intracellular calcium dynamics in pancreatic beta cells in mouse acute tissue slices and the corresponding functional connectivity networks for an experimental verification of the presented theory. We use methods of nonlinear time series analysis to reconstruct the phase space and calculate the sum of Lyapunov exponents. Our analysis reveals a clear tendency of cells with a higher degree, that is, more interconnected cells, having more negative values of divergence, thus confirming our theoretical predictions. We discuss these findings in the context of energetic aspects of signaling in beta cells and potential risks for pathological changes in the tissue.

3D Spatially Resolved Models of the Intracellular Dynamics of the Hepatitis C Genome Replication Cycle

PubMed Central

Reiter, Sebastian; Grillo, Alfio; Herrmann, Eva; Wittum, Gabriel

2017-01-01

Mathematical models of virus dynamics have not previously acknowledged spatial resolution at the intracellular level despite substantial arguments that favor the consideration of intracellular spatial dependence. The replication of the hepatitis C virus (HCV) viral RNA (vRNA) occurs within special replication complexes formed from membranes derived from endoplasmatic reticulum (ER). These regions, termed membranous webs, are generated primarily through specific interactions between nonstructural virus-encoded proteins (NSPs) and host cellular factors. The NSPs are responsible for the replication of the vRNA and their movement is restricted to the ER surface. Therefore, in this study we developed fully spatio-temporal resolved models of the vRNA replication cycle of HCV. Our simulations are performed upon realistic reconstructed cell structures—namely the ER surface and the membranous webs—based on data derived from immunostained cells replicating HCV vRNA. We visualized 3D simulations that reproduced dynamics resulting from interplay of the different components of our models (vRNA, NSPs, and a host factor), and we present an evaluation of the concentrations for the components within different regions of the cell. Thus far, our model is restricted to an internal portion of a hepatocyte and is qualitative more than quantitative. For a quantitative adaption to complete cells, various additional parameters will have to be determined through further in vitro cell biology experiments, which can be stimulated by the results described in the present study. PMID:28973992

Particle Size/ Grain Size Correlation and Mechanical Properties of Spark Plasma Sintered 8Y-ZrO2, MgAl2O4, and Al2O3 Based Composites

NASA Astrophysics Data System (ADS)

Karandikar, Keyur Kashinath

Solution-processed nanomaterials such as lead sulfide (PbS) colloidal quantum dots (CQDs) combine various manufacturing benefits and facile spectral tunability. However, the low mobility of CQD films limits its power conversion efficiency in photovoltaic cells. Here, I employ a novel femtosecond transient absorption (fs-TA) technique to determine the mobility of PbS CQD films that have undergone state of the art surface treatments. A significant mobility increase from 3 x 10-2 to 5 x 10 -1 cm2 V-1 s-1 was determined for iodide passivated and novel perovskite-shelled PbS CQDs, respectively. I performed, for the first time, temperature-dependent ultrafast carrier dynamics in perovskite-shelled CQDs using fs-TA, and determined an activation energy of 14 meV required for carrier hopping. Complementary studies that used time-of-flight measurements to determine the mobility in solar cell configuration corroborated the fs-TA method. Taken together, these results indicate a promising avenue toward improved CQD solar cells.

ZapE Is a Novel Cell Division Protein Interacting with FtsZ and Modulating the Z-Ring Dynamics

PubMed Central

Marteyn, Benoit S.; Karimova, Gouzel; Fenton, Andrew K.; Gazi, Anastasia D.; West, Nicholas; Touqui, Lhousseine; Prevost, Marie-Christine; Betton, Jean-Michel; Poyraz, Oemer; Ladant, Daniel; Gerdes, Kenn; Sansonetti, Philippe J.; Tang, Christoph M.

2014-01-01

ABSTRACT Bacterial cell division requires the formation of a mature divisome complex positioned at the midcell. The localization of the divisome complex is determined by the correct positioning, assembly, and constriction of the FtsZ ring (Z-ring). Z-ring constriction control remains poorly understood and (to some extent) controversial, probably due to the fact that this phenomenon is transient and controlled by numerous factors. Here, we characterize ZapE, a novel ATPase found in Gram-negative bacteria, which is required for growth under conditions of low oxygen, while loss of zapE results in temperature-dependent elongation of cell shape. We found that ZapE is recruited to the Z-ring during late stages of the cell division process and correlates with constriction of the Z-ring. Overexpression or inactivation of zapE leads to elongation of Escherichia coli and affects the dynamics of the Z-ring during division. In vitro, ZapE destabilizes FtsZ polymers in an ATP-dependent manner. PMID:24595368

Dynamic Finite Element Predictions for Mars Sample Return Cellular Impact Test #4

NASA Technical Reports Server (NTRS)

Fasanella, Edwin L.; Billings, Marcus D.

2001-01-01

The nonlinear, transient dynamic finite element code, MSC.Dytran, was used to simulate an impact test of an energy absorbing Earth Entry Vehicle (EEV) that will impact without a parachute. EEVOs are designed to return materials from asteroids, comets, or planets for laboratory analysis on Earth. The EEV concept uses an energy absorbing cellular structure designed to contain and limit the acceleration of space exploration samples during Earth impact. The spherical shaped cellular structure is composed of solid hexagonal and pentagonal foam-filled cells with hybrid graphite-epoxy/Kevlar cell walls. Space samples fit inside a smaller sphere at the center of the EEVOs cellular structure. Pre-test analytical predictions were compared with the test results from a bungee accelerator. The model used to represent the foam and the proper failure criteria for the cell walls were critical in predicting the impact loads of the cellular structure. It was determined that a FOAM1 model for the foam and a 20% failure strain criteria for the cell walls gave an accurate prediction of the acceleration pulse for cellular impact.

Ultrafast Electron Dynamics in Solar Energy Conversion.

PubMed

Ponseca, Carlito S; Chábera, Pavel; Uhlig, Jens; Persson, Petter; Sundström, Villy

2017-08-23

Electrons are the workhorses of solar energy conversion. Conversion of the energy of light to electricity in photovoltaics, or to energy-rich molecules (solar fuel) through photocatalytic processes, invariably starts with photoinduced generation of energy-rich electrons. The harvesting of these electrons in practical devices rests on a series of electron transfer processes whose dynamics and efficiencies determine the function of materials and devices. To capture the energy of a photogenerated electron-hole pair in a solar cell material, charges of opposite sign have to be separated against electrostatic attractions, prevented from recombining and being transported through the active material to electrodes where they can be extracted. In photocatalytic solar fuel production, these electron processes are coupled to chemical reactions leading to storage of the energy of light in chemical bonds. With the focus on the ultrafast time scale, we here discuss the light-induced electron processes underlying the function of several molecular and hybrid materials currently under development for solar energy applications in dye or quantum dot-sensitized solar cells, polymer-fullerene polymer solar cells, organometal halide perovskite solar cells, and finally some photocatalytic systems.

Neural dynamics of feedforward and feedback processing in figure-ground segregation

PubMed Central

Layton, Oliver W.; Mingolla, Ennio; Yazdanbakhsh, Arash

2014-01-01

Determining whether a region belongs to the interior or exterior of a shape (figure-ground segregation) is a core competency of the primate brain, yet the underlying mechanisms are not well understood. Many models assume that figure-ground segregation occurs by assembling progressively more complex representations through feedforward connections, with feedback playing only a modulatory role. We present a dynamical model of figure-ground segregation in the primate ventral stream wherein feedback plays a crucial role in disambiguating a figure's interior and exterior. We introduce a processing strategy whereby jitter in RF center locations and variation in RF sizes is exploited to enhance and suppress neural activity inside and outside of figures, respectively. Feedforward projections emanate from units that model cells in V4 known to respond to the curvature of boundary contours (curved contour cells), and feedback projections from units predicted to exist in IT that strategically group neurons with different RF sizes and RF center locations (teardrop cells). Neurons (convex cells) that preferentially respond when centered on a figure dynamically balance feedforward (bottom-up) information and feedback from higher visual areas. The activation is enhanced when an interior portion of a figure is in the RF via feedback from units that detect closure in the boundary contours of a figure. Our model produces maximal activity along the medial axis of well-known figures with and without concavities, and inside algorithmically generated shapes. Our results suggest that the dynamic balancing of feedforward signals with the specific feedback mechanisms proposed by the model is crucial for figure-ground segregation. PMID:25346703

Standardized extract of Bacopa monnieri (CDRI-08): Effect on germ cell dynamics and possible mechanisms of its beneficial action on spermatogenesis and sperm quality in male mice.

PubMed

Patel, Shishir Kumar; Singh, Shilpi; Singh, Shio Kumar

2017-12-09

Bacopa monnieri (BM) is used in traditional medicine as nerve tonic. We have recently shown that CDRI-08, a standardized extract of BM, improves testicular functions and epididymal sperm quality in Parkes (P) mice. The aim of the present study was to investigate the effect of CDRI-08 on germ cell dynamics and mechanisms of its action on spermatogenesis and sperm quality in P mice, and to determine the chemical profile of the extract. CDRI-08 (40 and 80 mg/kg body weight) was orally administered to male mice for 28 days. Germ cell dynamics, oxidative stress parameters in testis and sperm, and expressions of nuclear factor-erythroid-2-related factor-2 (Nrf2), phosphorylated protein kinase B (p-Akt) and upstream kinases in mitogen-activated protein kinase (MAPK) pathway namely MAP2K1, MAP2K2 and MKK4 in the testis were evaluated. The treatment potentiated germ cell dynamics and improved sperm quality by enhancing antioxidant enzymes activities. The beneficial effects of CDRI-08 in the testis involve p-Akt-mediated activation of Nrf2, thereby enhancing antioxidant enzymes activities; upregulation of MAP2K1 and MAP2K2 and suppression of MKK4 are also implicated in this action. A total of 26 phytocomponents were identified in CDRI-08 by GC-MS. The results suggest that CDRI-08 also may prove useful in improving reproductive health in males. Copyright © 2017 Elsevier Inc. All rights reserved.

Neural dynamics of feedforward and feedback processing in figure-ground segregation.

PubMed

Layton, Oliver W; Mingolla, Ennio; Yazdanbakhsh, Arash

2014-01-01

Determining whether a region belongs to the interior or exterior of a shape (figure-ground segregation) is a core competency of the primate brain, yet the underlying mechanisms are not well understood. Many models assume that figure-ground segregation occurs by assembling progressively more complex representations through feedforward connections, with feedback playing only a modulatory role. We present a dynamical model of figure-ground segregation in the primate ventral stream wherein feedback plays a crucial role in disambiguating a figure's interior and exterior. We introduce a processing strategy whereby jitter in RF center locations and variation in RF sizes is exploited to enhance and suppress neural activity inside and outside of figures, respectively. Feedforward projections emanate from units that model cells in V4 known to respond to the curvature of boundary contours (curved contour cells), and feedback projections from units predicted to exist in IT that strategically group neurons with different RF sizes and RF center locations (teardrop cells). Neurons (convex cells) that preferentially respond when centered on a figure dynamically balance feedforward (bottom-up) information and feedback from higher visual areas. The activation is enhanced when an interior portion of a figure is in the RF via feedback from units that detect closure in the boundary contours of a figure. Our model produces maximal activity along the medial axis of well-known figures with and without concavities, and inside algorithmically generated shapes. Our results suggest that the dynamic balancing of feedforward signals with the specific feedback mechanisms proposed by the model is crucial for figure-ground segregation.

Targeting Paclitaxel-Loaded Nanoparticles to Ovarian Cancer

DTIC Science & Technology

2011-05-01

with each other causes the polymer to collapse to form a nanoparticle of ~20 nm in aqueous solutions as determined by dynamic light scattering (2, 8...molecular target in tumor cells and tumor stroma. Cancer Res. 2008;68:7210-8. 19. von Maltzahn G, Ren Y, Park JH, Min DH, Kotamraju VR, Jayakumar J, et

Mechanism of intermediate filament recognition by plakin repeat domains revealed by envoplakin targeting of vimentin

NASA Astrophysics Data System (ADS)

Fogl, Claudia; Mohammed, Fiyaz; Al-Jassar, Caezar; Jeeves, Mark; Knowles, Timothy J.; Rodriguez-Zamora, Penelope; White, Scott A.; Odintsova, Elena; Overduin, Michael; Chidgey, Martyn

2016-03-01

Plakin proteins form critical connections between cell junctions and the cytoskeleton; their disruption within epithelial and cardiac muscle cells cause skin-blistering diseases and cardiomyopathies. Envoplakin has a single plakin repeat domain (PRD) which recognizes intermediate filaments through an unresolved mechanism. Herein we report the crystal structure of envoplakin's complete PRD fold, revealing binding determinants within its electropositive binding groove. Four of its five internal repeats recognize negatively charged patches within vimentin via five basic determinants that are identified by nuclear magnetic resonance spectroscopy. Mutations of the Lys1901 or Arg1914 binding determinants delocalize heterodimeric envoplakin from intracellular vimentin and keratin filaments in cultured cells. Recognition of vimentin is abolished when its residues Asp112 or Asp119 are mutated. The latter slot intermediate filament rods into basic PRD domain grooves through electrosteric complementarity in a widely applicable mechanism. Together this reveals how plakin family members form dynamic linkages with cytoskeletal frameworks.

Dynamic phenotypic restructuring of the CD4 and CD8 T-cell subsets with age in healthy humans: a compartmental model analysis.

PubMed

Jackola, D R; Hallgren, H M

1998-11-16

In healthy humans, phenotypic restructuring occurs with age within the CD3+ T-lymphocyte complement. This is characterized by a non-linear decrease of the percentage of 'naive' (CD45RA+) cells and a corresponding non-linear increase of the percentage of 'memory' (CD45R0+) cells among both the CD4+ and CD8+ T-cell subsets. We devised a simple compartmental model to study the age-dependent kinetics of phenotypic restructuring. We also derived differential equations whose parameters determined yearly gains minus losses of the percentage and absolute numbers of circulating naive cells, yearly gains minus losses of the percentage and absolute numbers of circulating memory cells, and the yearly rate of conversion of naive to memory cells. Solutions of these evaluative differential equations demonstrate the following: (1) the memory cell complement 'resides' within its compartment for a longer time than the naive cell complement within its compartment for both CD4 and CD8 cells; (2) the average, annual 'turnover rate' is the same for CD4 and CD8 naive cells. In contrast, the average, annual 'turnover rate' for memory CD8 cells is 1.5 times that of memory CD4 cells; (3) the average, annual conversion rate of CD4 naive cells to memory cells is twice that of the CD8 conversion rate; (4) a transition in dynamic restructuring occurs during the third decade of life that is due to these differences in turnover and conversion rates, between and from naive to memory cells.

Transcription closed and open complex dynamics studies reveal balance between genetic determinants and co-factors

NASA Astrophysics Data System (ADS)

Sala, Adrien; Shoaib, Muhammad; Anufrieva, Olga; Mutharasu, Gnanavel; Jahan Hoque, Rawnak; Yli-Harja, Olli; Kandhavelu, Meenakshisundaram

2015-05-01

In E. coli, promoter closed and open complexes are key steps in transcription initiation, where magnesium-dependent RNA polymerase catalyzes RNA synthesis. However, the exact mechanism of initiation remains to be fully elucidated. Here, using single mRNA detection and dual reporter studies, we show that increased intracellular magnesium concentration affects Plac initiation complex formation resulting in a highly dynamic process over the cell growth phases. Mg2+ regulates transcription transition, which modulates bimodality of mRNA distribution in the exponential phase. We reveal that Mg2+ regulates the size and frequency of the mRNA burst by changing the open complex duration. Moreover, increasing magnesium concentration leads to higher intrinsic and extrinsic noise in the exponential phase. RNAP-Mg2+ interaction simulation reveals critical movements creating a shorter contact distance between aspartic acid residues and Nucleotide Triphosphate residues and increasing electrostatic charges in the active site. Our findings provide unique biophysical insights into the balanced mechanism of genetic determinants and magnesium ion in transcription initiation regulation during cell growth.

Dynamic cell culture on porous biopolymer microcarriers in a spinner flask for bone tissue engineering: a feasibility study.

PubMed

Jin, Guang-Zhen; Park, Jeong-Hui; Seo, Seog-Jin; Kim, Hae-Won

2014-07-01

Porous microspherical carriers have great promise for cell culture and tissue engineering. Dynamic cultures enable more uniform cell population and effective differentiation than static cultures. Here we applied dynamic spinner flask culture for the loading and multiplication of cells onto porous biopolymer microcarriers. The abilities of the microcarriers to populate cells and to induce osteogenic differentiation were examined and the feasibility of in vivo delivery of the constructs was addressed. Over time, the porous microcarriers enabled cell adhesion and expansion under proper dynamic culture conditions. Osteogenic markers were substantially expressed by the dynamic cell cultures. The cell-cultured microcarriers implanted in the mouse subcutaneous tissue for 4 weeks showed excellent tissue compatibility, with minimal inflammatory signs and significant induction of bone tissues. This first report on dynamic culture of porous biopolymer microcarriers providing an effective tool for bone tissue engineering.

Impaired Bioenergetics in Mutant Mitochondrial DNA Determines Cell Fate During Seizure-Like Activity.

PubMed

Kovac, Stjepana; Preza, Elisavet; Houlden, Henry; Walker, Matthew C; Abramov, Andrey Y

2018-04-27

Mutations in genes affecting mitochondrial proteins are increasingly recognised in patients with epilepsy, but the factors determining cell fate during seizure activity in these mutations remain unknown. Fluorescent dye imaging techniques were applied to fibroblast cell lines from patients suffering from common mitochondrial mutations and to age-matched controls. Using live cell imaging techniques in fibroblasts, we show that fibroblasts with mutations in the mitochondrial genome had reduced mitochondrial membrane potential and NADH pools and higher redox indices, indicative of respiratory chain dysfunction. Increasing concentrations of ferutinin, a Ca 2+ ionophore, led to oscillatory Ca 2+ signals in fibroblasts resembling dynamic Ca 2+ changes that occur during seizure-like activity. Co-monitoring of mitochondrial membrane potential (ΔΨ m ) changes induced by ferutinin showed accelerated membrane depolarisation and cell collapse in fibroblasts with mutations in the mitochondrial genome when compared to controls. Ca 2+ flash photolysis using caged Ca 2+ confirmed impaired Ca 2+ handling in fibroblasts with mitochondrial mutations. Findings indicate that intracellular Ca 2+ levels cannot be compensated during periods of hyperexcitability, leading to Ca 2+ overload and subsequent cell death in mitochondrial diseases.

Analysis of B Cell Repertoire Dynamics Following Hepatitis B Vaccination in Humans, and Enrichment of Vaccine-specific Antibody Sequences.

PubMed

Galson, Jacob D; Trück, Johannes; Fowler, Anna; Clutterbuck, Elizabeth A; Münz, Márton; Cerundolo, Vincenzo; Reinhard, Claudia; van der Most, Robbert; Pollard, Andrew J; Lunter, Gerton; Kelly, Dominic F

2015-12-01

Generating a diverse B cell immunoglobulin repertoire is essential for protection against infection. The repertoire in humans can now be comprehensively measured by high-throughput sequencing. Using hepatitis B vaccination as a model, we determined how the total immunoglobulin sequence repertoire changes following antigen exposure in humans, and compared this to sequences from vaccine-specific sorted cells. Clonal sequence expansions were seen 7 days after vaccination, which correlated with vaccine-specific plasma cell numbers. These expansions caused an increase in mutation, and a decrease in diversity and complementarity-determining region 3 sequence length in the repertoire. We also saw an increase in sequence convergence between participants 14 and 21 days after vaccination, coinciding with an increase of vaccine-specific memory cells. These features allowed development of a model for in silico enrichment of vaccine-specific sequences from the total repertoire. Identifying antigen-specific sequences from total repertoire data could aid our understanding B cell driven immunity, and be used for disease diagnostics and vaccine evaluation.

Stoichiometry of Nck-dependent actin polymerization in living cells

PubMed Central

Ditlev, Jonathon A.; Michalski, Paul J.; Huber, Greg; Rivera, Gonzalo M.; Mohler, William A.

2012-01-01

Regulation of actin dynamics through the Nck/N-WASp (neural Wiskott–Aldrich syndrome protein)/Arp2/3 pathway is essential for organogenesis, cell invasiveness, and pathogen infection. Although many of the proteins involved in this pathway are known, the detailed mechanism by which it functions remains undetermined. To examine the signaling mechanism, we used a two-pronged strategy involving computational modeling and quantitative experimentation. We developed predictions for Nck-dependent actin polymerization using the Virtual Cell software system. In addition, we used antibody-induced aggregation of membrane-targeted Nck SH3 domains to test these predictions and to determine how the number of molecules in Nck aggregates and the density of aggregates affected localized actin polymerization in living cells. Our results indicate that the density of Nck molecules in aggregates is a critical determinant of actin polymerization. Furthermore, results from both computational simulations and experimentation support a model in which the Nck/N-WASp/Arp2/3 stoichiometry is 4:2:1. These results provide new insight into activities involving localized actin polymerization, including tumor cell invasion, microbial pathogenesis, and T cell activation. PMID:22613834

Assembly kinetics determine the architecture of α-actinin crosslinked F-actin networks.

PubMed

Falzone, Tobias T; Lenz, Martin; Kovar, David R; Gardel, Margaret L

2012-05-29

The actin cytoskeleton is organized into diverse meshworks and bundles that support many aspects of cell physiology. Understanding the self-assembly of these actin-based structures is essential for developing predictive models of cytoskeletal organization. Here we show that the competing kinetics of bundle formation with the onset of dynamic arrest arising from filament entanglements and crosslinking determine the architecture of reconstituted actin networks formed with α-actinin crosslinks. Crosslink-mediated bundle formation only occurs in dilute solutions of highly mobile actin filaments. As actin polymerization proceeds, filament mobility and bundle formation are arrested concomitantly. By controlling the onset of dynamic arrest, perturbations to actin assembly kinetics dramatically alter the architecture of biochemically identical samples. Thus, the morphology of reconstituted F-actin networks is a kinetically determined structure similar to those formed by physical gels and glasses. These results establish mechanisms controlling the structure and mechanics in diverse semiflexible biopolymer networks.

Altered Liver Proteoglycan/Glycosaminoglycan Structure as a Manifestation of Extracellular Matrix Remodeling upon BCG-induced Granulomatosis in Mice.

PubMed

Kim, L B; Shkurupy, V A; Putyatina, A N

2017-01-01

Experimental BCG-induced granulomatosis in mice was used to study changes in the dynamics of individual liver proteoglycan components reflecting phasic extracellular matrix remodeling, determined by the host-parasite interaction and associated with granuloma development. In the early BCG-granulomatosis period, the increase in individual proteoglycan components promotes granuloma formation, providing conditions for mycobacteria adhesion to host cells, migration of phagocytic cells from circulation, and cell-cell interaction leading to granuloma development and fibrosis. Later, reduced reserve capacity of the extracellular matrix, development of interstitial fibrosis and granuloma fibrosis can lead to trophic shortage for cells within the granulomas, migration of macrophages out of them, and development of spontaneous necrosis and apoptosis typical of tuberculosis.

Accounting for Space—Quantification of Cell-To-Cell Transmission Kinetics Using Virus Dynamics Models.

PubMed

Kumberger, Peter; Durso-Cain, Karina; Uprichard, Susan L; Dahari, Harel; Graw, Frederik

2018-04-17

Mathematical models based on ordinary differential equations (ODE) that describe the population dynamics of viruses and infected cells have been an essential tool to characterize and quantify viral infection dynamics. Although an important aspect of viral infection is the dynamics of viral spread, which includes transmission by cell-free virions and direct cell-to-cell transmission, models used so far ignored cell-to-cell transmission completely, or accounted for this process by simple mass-action kinetics between infected and uninfected cells. In this study, we show that the simple mass-action approach falls short when describing viral spread in a spatially-defined environment. Using simulated data, we present a model extension that allows correct quantification of cell-to-cell transmission dynamics within a monolayer of cells. By considering the decreasing proportion of cells that can contribute to cell-to-cell spread with progressing infection, our extension accounts for the transmission dynamics on a single cell level while still remaining applicable to standard population-based experimental measurements. While the ability to infer the proportion of cells infected by either of the transmission modes depends on the viral diffusion rate, the improved estimates obtained using our novel approach emphasize the need to correctly account for spatial aspects when analyzing viral spread.

Separation of red blood cells in deep deterministic lateral displacement devices

NASA Astrophysics Data System (ADS)

Kabacaoglu, Gokberk; Biros, George

2017-11-01

Microfluidic cell separation techniques are of great interest since they help rapid medical diagnoses and tests. Deterministic lateral displacement (DLD) is one of them. A DLD device consists of arrays of pillars. Main flow and alignment of the pillars define two different directions. Size-based separation of rigid spherical particles is possible as they follow one of these directions depending on their sizes. However, the separation of non-spherical deformable particles such as red blood cells (RBCs) is more complicated than that due to their intricate dynamics. We study the separation of RBCs in DLD using an in-house integral equation solver. We systematically investigate the effects of the interior fluid viscosity and the membrane elasticity of an RBC on its behavior. These mechanical properties of a cell determine its deformability, which can be altered by several diseases. We particularly consider deep devices in which an RBC can show rich dynamics such as tank-treading and tumbling. It turns out that strong hydrodynamic lift force moves the tank-treading cells along the pillars and downward force leads the tumbling ones to move with the flow. Thereby, deformability-based separation of RBCs is possible.

Protein adsorption in microengraving immunoassays.

PubMed

Song, Qing

2015-10-16

Microengraving is a novel immunoassay for characterizing multiple protein secretions from single cells. During the immunoassay, characteristic diffusion and kinetic time scales  and  determine the time for molecular diffusion of proteins secreted from the activated single lymphocytes and subsequent binding onto the glass slide surface respectively. Our results demonstrate that molecular diffusion plays important roles in the early stage of protein adsorption dynamics which shifts to a kinetic controlled mechanism in the later stage. Similar dynamic pathways are observed for protein adsorption with significantly fast rates and rapid shifts in transport mechanisms when  is increased a hundred times from 0.313 to 31.3. Theoretical adsorption isotherms follow the trend of experimentally obtained data. Adsorption isotherms indicate that amount of proteins secreted from individual cells and subsequently captured on a clean glass slide surface increases monotonically with time. Our study directly validates that protein secretion rates can be quantified by the microengraving immunoassay. This will enable us to apply microengraving immunoassays to quantify secretion rates from 10⁴-10⁵ single cells in parallel, screen antigen-specific cells with the highest secretion rate for clonal expansion and quantitatively reveal cellular heterogeneity within a small cell sample.

Protein Adsorption in Microengraving Immunoassays

PubMed Central

Song, Qing

2015-01-01

Microengraving is a novel immunoassay forcharacterizing multiple protein secretions from single cells. During the immunoassay, characteristic diffusion and kinetic time scales τD and τK determine the time for molecular diffusion of proteins secreted from the activated single lymphocytes and subsequent binding onto the glass slide surface respectively. Our results demonstrate that molecular diffusion plays important roles in the early stage of protein adsorption dynamics which shifts to a kinetic controlled mechanism in the later stage. Similar dynamic pathways are observed for protein adsorption with significantly fast rates and rapid shifts in transport mechanisms when C0* is increased a hundred times from 0.313 to 31.3. Theoretical adsorption isotherms follow the trend of experimentally obtained data. Adsorption isotherms indicate that amount of proteins secreted from individual cells and subsequently captured on a clean glass slide surface increases monotonically with time. Our study directly validates that protein secretion rates can be quantified by the microengraving immunoassay. This will enable us to apply microengraving immunoassays to quantify secretion rates from 104–105 single cells in parallel, screen antigen-specific cells with the highest secretion rate for clonal expansion and quantitatively reveal cellular heterogeneity within a small cell sample. PMID:26501282

Cooperation among cancer cells as public goods games on Voronoi networks.

PubMed

Archetti, Marco

2016-05-07

Cancer cells produce growth factors that diffuse and sustain tumour proliferation, a form of cooperation that can be studied using mathematical models of public goods in the framework of evolutionary game theory. Cell populations, however, form heterogeneous networks that cannot be described by regular lattices or scale-free networks, the types of graphs generally used in the study of cooperation. To describe the dynamics of growth factor production in populations of cancer cells, I study public goods games on Voronoi networks, using a range of non-linear benefits that account for the known properties of growth factors, and different types of diffusion gradients. The results are surprisingly similar to those obtained on regular graphs and different from results on scale-free networks, revealing that network heterogeneity per se does not promote cooperation when public goods diffuse beyond one-step neighbours. The exact shape of the diffusion gradient is not crucial, however, whereas the type of non-linear benefit is an essential determinant of the dynamics. Public goods games on Voronoi networks can shed light on intra-tumour heterogeneity, the evolution of resistance to therapies that target growth factors, and new types of cell therapy. Copyright © 2016 Elsevier Ltd. All rights reserved.

GSK3 as a Sensor Determining Cell Fate in the Brain.

PubMed

Cole, Adam R

2012-01-01

Glycogen synthase kinase 3 (GSK3) is an unusual serine/threonine kinase that controls many neuronal functions, including neurite outgrowth, synapse formation, neurotransmission, and neurogenesis. It mediates these functions by phosphorylating a wide range of substrates involved in gene transcription, metabolism, apoptosis, cytoskeletal dynamics, signal transduction, lipid membrane dynamics, and trafficking, amongst others. This complicated list of diverse substrates generally follow a more simple pattern: substrates negatively regulated by GSK3-mediated phosphorylation favor a proliferative/survival state, while substrates positively regulated by GSK3 favor a more differentiated/functional state. Accordingly, GSK3 activity is higher in differentiated cells than undifferentiated cells and physiological (Wnt, growth factors) and pharmacological inhibitors of GSK3 promote the proliferative capacity of embryonic stem cells. In the brain, the level of GSK3 activity influences neural progenitor cell proliferation/differentiation in neuroplasticity and repair, as well as efficient neurotransmission in differentiated adult neurons. While defects in GSK3 activity are unlikely to be the primary cause of neurodegenerative diseases, therapeutic regulation of its activity to promote a proliferative/survival versus differentiated/mature functional environment in the brain could be a powerful strategy for treatment of neurodegenerative and other mental disorders.

GSK3 as a Sensor Determining Cell Fate in the Brain

PubMed Central

Cole, Adam R.

2012-01-01

Glycogen synthase kinase 3 (GSK3) is an unusual serine/threonine kinase that controls many neuronal functions, including neurite outgrowth, synapse formation, neurotransmission, and neurogenesis. It mediates these functions by phosphorylating a wide range of substrates involved in gene transcription, metabolism, apoptosis, cytoskeletal dynamics, signal transduction, lipid membrane dynamics, and trafficking, amongst others. This complicated list of diverse substrates generally follow a more simple pattern: substrates negatively regulated by GSK3-mediated phosphorylation favor a proliferative/survival state, while substrates positively regulated by GSK3 favor a more differentiated/functional state. Accordingly, GSK3 activity is higher in differentiated cells than undifferentiated cells and physiological (Wnt, growth factors) and pharmacological inhibitors of GSK3 promote the proliferative capacity of embryonic stem cells. In the brain, the level of GSK3 activity influences neural progenitor cell proliferation/differentiation in neuroplasticity and repair, as well as efficient neurotransmission in differentiated adult neurons. While defects in GSK3 activity are unlikely to be the primary cause of neurodegenerative diseases, therapeutic regulation of its activity to promote a proliferative/survival versus differentiated/mature functional environment in the brain could be a powerful strategy for treatment of neurodegenerative and other mental disorders. PMID:22363258

Modeling population dynamics of mitochondria in mammalian cells

NASA Astrophysics Data System (ADS)

Kornick, Kellianne; Das, Moumita

Mitochondria are organelles located inside eukaryotic cells and are essential for several key cellular processes such as energy (ATP) production, cell signaling, differentiation, and apoptosis. All organisms are believed to have low levels of variation in mitochondrial DNA (mtDNA), and alterations in mtDNA are connected to a range of human health conditions, including epilepsy, heart failure, Parkinsons disease, diabetes, and multiple sclerosis. Therefore, understanding how changes in mtDNA accumulate over time and are correlated to changes in mitochondrial function and cell properties can have a profound impact on our understanding of cell physiology and the origins of some diseases. Motivated by this, we develop and study a mathematical model to determine which cellular parameters have the largest impact on mtDNA population dynamics. The model consists of coupled ODEs to describe subpopulations of healthy and dysfunctional mitochondria subject to mitochondrial fission, fusion, autophagy, and mutation. We study the time evolution and stability of each sub-population under specific selection biases and pressures by tuning specific terms in our model. Our results may provide insights into how sub-populations of mitochondria survive and evolve under different selection pressures. This work was supported by a Grant from the Moore Foundation.

Mitochondrial Dysfunction in Cancer

PubMed Central

Boland, Michelle L.; Chourasia, Aparajita H.; Macleod, Kay F.

2013-01-01

A mechanistic understanding of how mitochondrial dysfunction contributes to cell growth and tumorigenesis is emerging beyond Warburg as an area of research that is under-explored in terms of its significance for clinical management of cancer. Work discussed in this review focuses less on the Warburg effect and more on mitochondria and how dysfunctional mitochondria modulate cell cycle, gene expression, metabolism, cell viability, and other established aspects of cell growth and stress responses. There is increasing evidence that key oncogenes and tumor suppressors modulate mitochondrial dynamics through important signaling pathways and that mitochondrial mass and function vary between tumors and individuals but the significance of these events for cancer are not fully appreciated. We explore the interplay between key molecules involved in mitochondrial fission and fusion and in apoptosis, as well as in mitophagy, biogenesis, and spatial dynamics of mitochondria and consider how these distinct mechanisms are coordinated in response to physiological stresses such as hypoxia and nutrient deprivation. Importantly, we examine how deregulation of these processes in cancer has knock on effects for cell proliferation and growth. We define major forms of mitochondrial dysfunction and address the extent to which the functional consequences of such dysfunction can be determined and exploited for cancer diagnosis and treatment. PMID:24350057

Mechanism of dynamic reorientation of cortical microtubules due to mechanical stress.

PubMed

Muratov, Alexander; Baulin, Vladimir A

2015-12-01

Directional growth caused by gravitropism and corresponding bending of plant cells has been explored since 19th century, however, many aspects of mechanisms underlying the perception of gravity at the molecular level are still not well known. Perception of gravity in root and shoot gravitropisms is usually attributed to gravisensitive cells, called statocytes, which exploit sedimentation of macroscopic and heavy organelles, amyloplasts, to sense the direction of gravity. Gravity stimulus is then transduced into distal elongation zone, which is several mm far from statocytes, where it causes stretching. It is suggested that gravity stimulus is conveyed by gradients in auxin flux. We propose a theoretical model that may explain how concentration gradients and/or stretching may indirectly affect the global orientation of cortical microtubules, attached to the cell membrane and induce their dynamic reorientation perpendicular to the gradients. In turn, oriented microtubule arrays direct the growth and orientation of cellulose microfibrils, forming part of the cell external skeleton and determine the shape of the cell. Reorientation of microtubules is also observed in reaction to light in phototropism and mechanical bending, thus suggesting universality of the proposed mechanism. Copyright © 2015 Elsevier B.V. All rights reserved.

Ligand-Binding Properties and Conformational Dynamics of Autolysin Repeat Domains in Staphylococcal Cell Wall Recognition

PubMed Central

Zoll, Sebastian; Schlag, Martin; Shkumatov, Alexander V.; Rautenberg, Maren; Svergun, Dmitri I.; Götz, Friedrich

2012-01-01

The bifunctional major autolysin Atl plays a key role in staphylococcal cell separation. Processing of Atl yields catalytically active amidase (AM) and glucosaminidase (GL) domains that are each fused to repeating units. The two repeats of AM (R1 and R2) target the enzyme to the septum, where it cleaves murein between dividing cells. We have determined the crystal structure of R2, which reveals that each repeat folds into two half-open β-barrel subunits. We further demonstrate that lipoteichoic acid serves as a receptor for the repeats and that this interaction depends on conserved surfaces in each subunit. Small-angle X-ray scattering of the mature amidase reveals the presence of flexible linkers separating the AM, R1, and R2 units. Different levels of flexibility for each linker provide mechanistic insights into the conformational dynamics of the full-length protein and the roles of its components in cell wall association and catalysis. Our analysis supports a model in which the repeats direct the catalytic AM domain to the septum, where it can optimally perform the final step of cell division. PMID:22609916

Dynamics from Seconds to Hours in Hodgkin-Huxley Model with Time-Dependent Ion Concentrations and Buffer Reservoirs

PubMed Central

Hübel, Niklas; Dahlem, Markus A.

2014-01-01

The classical Hodgkin-Huxley (HH) model neglects the time-dependence of ion concentrations in spiking dynamics. The dynamics is therefore limited to a time scale of milliseconds, which is determined by the membrane capacitance multiplied by the resistance of the ion channels, and by the gating time constants. We study slow dynamics in an extended HH framework that includes time-dependent ion concentrations, pumps, and buffers. Fluxes across the neuronal membrane change intra- and extracellular ion concentrations, whereby the latter can also change through contact to reservoirs in the surroundings. Ion gain and loss of the system is identified as a bifurcation parameter whose essential importance was not realized in earlier studies. Our systematic study of the bifurcation structure and thus the phase space structure helps to understand activation and inhibition of a new excitability in ion homeostasis which emerges in such extended models. Also modulatory mechanisms that regulate the spiking rate can be explained by bifurcations. The dynamics on three distinct slow times scales is determined by the cell volume-to-surface-area ratio and the membrane permeability (seconds), the buffer time constants (tens of seconds), and the slower backward buffering (minutes to hours). The modulatory dynamics and the newly emerging excitable dynamics corresponds to pathological conditions observed in epileptiform burst activity, and spreading depression in migraine aura and stroke, respectively. PMID:25474648

Single-cell dynamics of mast cell-CD4+ CD25+ regulatory T cell interactions.

PubMed

Frossi, Barbara; D'Incà, Federica; Crivellato, Enrico; Sibilano, Riccardo; Gri, Giorgia; Mongillo, Marco; Danelli, Luca; Maggi, Laura; Pucillo, Carlo E

2011-07-01

The biological behavior of immune cells is determined by their intrinsic properties and interactions with other cell populations within their microenvironment. Several studies have confirmed the existence of tight spatial interactions between mast cells (MCs) and Tregs in different settings. For instance, we have recently identified the functional cross-talk between MCs and Tregs, through the OX40L-OX40 axis, as a new mechanism of reciprocal influence. However, there is scant information regarding the single-cell dynamics of this process. In this study, time-lapse video microscopy revealed direct interactions between Tregs and MCs in both murine and human cell co-cultures, resulting in the inhibition of the MC degranulation response. MCs incubated with WT, but not OX40-deficient, Tregs mediated numerous and long-lasting interactions and displayed different morphological features lacking the classical signs of exocytosis. MC degranulation and Ca2+ mobilization upon activation were inhibited by Tregs on a single-cell basis, without affecting overall cytokine secretion. Transmission electron microscopy showed ultrastructural evidence of vesicle-mediated secretion reconcilable with the morphological pattern of piecemeal degranulation. Our results suggest that MC morphological and functional changes following MC-Treg interactions can be ascribed to cell-cell contact and represent a transversal, non-species-specific mechanism of immune response regulation. Further research, looking at the molecular composition of this interaction will broaden our understanding of its contribution to immunity. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Heterogeneity for IGF-II production maintained by public goods dynamics in neuroendocrine pancreatic cancer.

PubMed

Archetti, Marco; Ferraro, Daniela A; Christofori, Gerhard

2015-02-10

The extensive intratumor heterogeneity revealed by sequencing cancer genomes is an essential determinant of tumor progression, diagnosis, and treatment. What maintains heterogeneity remains an open question because competition within a tumor leads to a strong selection for the fittest subclone. Cancer cells also cooperate by sharing molecules with paracrine effects, such as growth factors, and heterogeneity can be maintained if subclones depend on each other for survival. Without strict interdependence between subclones, however, nonproducer cells can free-ride on the growth factors produced by neighboring producer cells, a collective action problem known in game theory as the "tragedy of the commons," which has been observed in microbial cell populations. Here, we report that similar dynamics occur in cancer cell populations. Neuroendocrine pancreatic cancer (insulinoma) cells that do not produce insulin-like growth factor II (IGF-II) grow slowly in pure cultures but have a proliferation advantage in mixed cultures, where they can use the IGF-II provided by producer cells. We show that, as predicted by evolutionary game theory, producer cells do not go extinct because IGF-II acts as a nonlinear public good, creating negative frequency-dependent selection that leads to a stable coexistence of the two cell types. Intratumor cell heterogeneity can therefore be maintained even without strict interdependence between cell subclones. Reducing the amount of growth factors available within a tumor may lead to a reduction in growth followed by a new equilibrium, which may explain relapse in therapies that target growth factors.

Studies on fluid dynamics of the flow field and gas transfer in orbitally shaken tubes.

PubMed

Zhu, Li-Kuan; Song, Bo-Yan; Wang, Zhen-Long; Monteil, Dominique T; Shen, Xiao; Hacker, David L; De Jesus, Maria; Wurm, Florian M

2017-01-01

Orbitally shaken cylindrical bioreactors [OrbShake bioreactors (OSRs)] without an impeller or sparger are increasingly being used for the suspension cultivation of mammalian cells. Among small volume OSRs, 50-mL tubes with a ventilated cap (OSR50), originally derived from standard laboratory centrifuge tubes with a conical bottom, have found many applications including high-throughput screening for the optimization of cell cultivation conditions. To better understand the fluid dynamics and gas transfer rates at the liquid surface in OSR50, we established a three-dimensional simulation model of the unsteady liquid forms (waves) in this vessel. The studies verified that the operating conditions have a large effect on the interfacial surface. The volumetric mass transfer coefficient (k L a) was determined experimentally and from simulations under various working conditions. We also determined the liquid-phase mass transfer coefficient (k L ) and the specific interfacial area (a) under different conditions to demonstrate that the value of a affected the gas transfer rate more than did the value of k L . High oxygen transfer rates, sufficient for supporting the high-density culture of mammalian cells, were found. Finally, the average axial velocity of the liquid was identified to be an important parameter for maintaining cells in suspension. Overall these studies provide valuable insights into the preferable operating conditions for the OSR50, such as those needed for cell cultures requiring high oxygen levels. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:192-200, 2017. © 2016 American Institute of Chemical Engineers.

Intestinal stem cells: no longer immortal but ever so clever....

PubMed

Edgar, Bruce A

2012-05-30

To maintain tissue homeostasis, stem cells must balance self-renewal with differentiation. In some stem cell lineages this process is 'hard-wired' by the asymmetric partitioning of determinants at division, such that one stem cell daughter always remains pluripotent and other differentiates. But in a dynamic tissue like the intestinal epithelium, which might need to repair itself following an infection or expand to digest the fall harvest, this balancing act requires more flexibility. Recent studies of intestinal stem cell (ISC) lineages in the fruit fly and mouse provide new insights into how this plasticity is achieved. The mechanisms in these two homologous but rather different organs have remarkable similarities, and so are likely relevant to how stem cell pools are controlled in organs other than the intestine.

Early transcriptional and epigenetic regulation of CD8+ T cell differentiation revealed by single-cell RNA-seq

PubMed Central

Kakaradov, Boyko; Arsenio, Janilyn; Widjaja, Christella E.; He, Zhaoren; Aigner, Stefan; Metz, Patrick J.; Yu, Bingfei; Wehrens, Ellen J.; Lopez, Justine; Kim, Stephanie H.; Zuniga, Elina I.; Goldrath, Ananda W.; Chang, John T.; Yeo, Gene W.

2017-01-01

SUMMARY During microbial infection, responding CD8+ T lymphocytes differentiate into heterogeneous subsets that together provide immediate and durable protection. To elucidate the dynamic transcriptional changes that underlie this process, we applied a single-cell RNA sequencing approach and analyzed individual CD8+ T lymphocytes sequentially throughout the course of a viral infection in vivo. Our analyses revealed a striking transcriptional divergence among cells that had undergone their first division and identified previously unknown molecular determinants controlling CD8+ T lymphocyte fate specification. These findings suggest a model of terminal effector cell differentiation initiated by an early burst of transcriptional activity and subsequently refined by epigenetic silencing of transcripts associated with memory lymphocytes, highlighting the power and necessity of single-cell approaches. PMID:28218746

The diet-derived short chain fatty acid propionate improves beta-cell function in humans and stimulates insulin secretion from human islets in vitro.

PubMed

Pingitore, Attilio; Chambers, Edward S; Hill, Thomas; Maldonado, Inmaculada Ruz; Liu, Bo; Bewick, Gavin; Morrison, Douglas J; Preston, Tom; Wallis, Gareth A; Tedford, Catriona; Castañera González, Ramón; Huang, Guo C; Choudhary, Pratik; Frost, Gary; Persaud, Shanta J

2017-02-01

Diet-derived short chain fatty acids (SCFAs) improve glucose homeostasis in vivo, but the role of individual SCFAs and their mechanisms of action have not been defined. This study evaluated the effects of increasing colonic delivery of the SCFA propionate on β-cell function in humans and the direct effects of propionate on isolated human islets in vitro. For 24 weeks human subjects ingested an inulin-propionate ester that delivers propionate to the colon. Acute insulin, GLP-1 and non-esterified fatty acid (NEFA) levels were quantified pre- and post-supplementation in response to a mixed meal test. Expression of the SCFA receptor FFAR2 in human islets was determined by western blotting and immunohistochemistry. Dynamic insulin secretion from perifused human islets was quantified by radioimmunoassay and islet apoptosis was determined by quantification of caspase 3/7 activities. Colonic propionate delivery in vivo was associated with improved β-cell function with increased insulin secretion that was independent of changes in GLP-1 levels. Human islet β-cells expressed FFAR2 and propionate potentiated dynamic glucose-stimulated insulin secretion in vitro, an effect that was dependent on signalling via protein kinase C. Propionate also protected human islets from apoptosis induced by the NEFA sodium palmitate and inflammatory cytokines. Our results indicate that propionate has beneficial effects on β-cell function in vivo, and in vitro analyses demonstrated that it has direct effects to potentiate glucose-stimulated insulin release and maintain β-cell mass through inhibition of apoptosis. These observations support ingestion of propiogenic dietary fibres to maintain healthy glucose homeostasis. © 2016 John Wiley & Sons Ltd.

Two-photon Photoactivation to Measure Histone Exchange Dynamics in Plant Root Cells.

PubMed

Rosa, Stefanie; Shaw, Peter

2015-10-20

Chromatin-binding proteins play a crucial role in chromatin structure and gene expression. Direct binding of chromatin proteins both maintains and regulates transcriptional states. It is therefore important to study the binding properties of these proteins in vivo within the natural environment of the nucleus. Photobleaching, photoactivation and photoconversion (photoswitching) can provide a non-invasive experimental approach to study dynamic properties of living cells and organisms. We used photoactivation to determine exchange dynamics of histone H2B in plant stem cells of the root (Rosa et al. , 2014). The stem cells of the root are located in the middle of the tissue, which made it impossible to carry out photoactivation of sufficiently small and well-defined sub-cellular regions with conventional laser illumination in the confocal microscope, mainly because scattering and refraction effects within the root tissue dispersed the focal spot and caused photoactivation of too large a region. We therefore used 2-photon activation, which has much better inherent resolution of the illuminated region. This is because the activation depends on simultaneous absorption of two or more photons, which in turns depends on the square (or higher power) of the intensity-a much sharper peak. In this protocol we will describe the experimental procedure to perform two-photon photoactivation experiments and the corresponding image analysis. This protocol can be used for nuclear proteins tagged with photoactivable GFP (PA-GFP) expressed in root tissues.

Lifespan differences in nonlinear dynamics during rest and auditory oddball performance.

PubMed

Müller, Viktor; Lindenberger, Ulman

2012-07-01

Electroencephalographic recordings (EEG) were used to assess age-associated differences in nonlinear brain dynamics during both rest and auditory oddball performance in children aged 9.0-12.8 years, younger adults, and older adults. We computed nonlinear coupling dynamics and dimensional complexity, and also determined spectral alpha power as an indicator of cortical reactivity. During rest, both nonlinear coupling and spectral alpha power decreased with age, whereas dimensional complexity increased. In contrast, when attending to the deviant stimulus, nonlinear coupling increased with age, and complexity decreased. Correlational analyses showed that nonlinear measures assessed during auditory oddball performance were reliably related to an independently assessed measure of perceptual speed. We conclude that cortical dynamics during rest and stimulus processing undergo substantial reorganization from childhood to old age, and propose that lifespan age differences in nonlinear dynamics during stimulus processing reflect lifespan changes in the functional organization of neuronal cell assemblies. © 2012 Blackwell Publishing Ltd.

Local and global influences on protein turnover in neurons and glia

PubMed Central

Dörrbaum, Aline R; Kochen, Lisa

2018-01-01

Regulation of protein turnover allows cells to react to their environment and maintain homeostasis. Proteins can show different turnover rates in different tissue, but little is known about protein turnover in different brain cell types. We used dynamic SILAC to determine half-lives of over 5100 proteins in rat primary hippocampal cultures as well as in neuron-enriched and glia-enriched cultures ranging from <1 to >20 days. In contrast to synaptic proteins, membrane proteins were relatively shorter-lived and mitochondrial proteins were longer-lived compared to the population. Half-lives also correlate with protein functions and the dynamics of the complexes they are incorporated in. Proteins in glia possessed shorter half-lives than the same proteins in neurons. The presence of glia sped up or slowed down the turnover of neuronal proteins. Our results demonstrate that both the cell-type of origin as well as the nature of the extracellular environment have potent influences on protein turnover. PMID:29914620

Dynamic metabolic exchange governs a marine algal-bacterial interaction.

PubMed

Segev, Einat; Wyche, Thomas P; Kim, Ki Hyun; Petersen, Jörn; Ellebrandt, Claire; Vlamakis, Hera; Barteneva, Natasha; Paulson, Joseph N; Chai, Liraz; Clardy, Jon; Kolter, Roberto

2016-11-18

Emiliania huxleyi is a model coccolithophore micro-alga that generates vast blooms in the ocean. Bacteria are not considered among the major factors influencing coccolithophore physiology. Here we show through a laboratory model system that the bacterium Phaeobacter inhibens , a well-studied member of the Roseobacter group, intimately interacts with E. huxleyi. While attached to the algal cell, bacteria initially promote algal growth but ultimately kill their algal host. Both algal growth enhancement and algal death are driven by the bacterially-produced phytohormone indole-3-acetic acid. Bacterial production of indole-3-acetic acid and attachment to algae are significantly increased by tryptophan, which is exuded from the algal cell. Algal death triggered by bacteria involves activation of pathways unique to oxidative stress response and programmed cell death. Our observations suggest that bacteria greatly influence the physiology and metabolism of E. huxleyi. Coccolithophore-bacteria interactions should be further studied in the environment to determine whether they impact micro-algal population dynamics on a global scale.

Predict the glass transition temperature of glycerol-water binary cryoprotectant by molecular dynamic simulation.

PubMed

Li, Dai-Xi; Liu, Bao-Lin; Liu, Yi-shu; Chen, Cheng-lung

2008-04-01

Vitrification is proposed to be the best way for the cryopreservation of organs. The glass transition temperature (T(g)) of vitrification solutions is a critical parameter of fundamental importance for cryopreservation by vitrification. The instruments that can detect the thermodynamic, mechanical and dielectric changes of a substance may be used to determine the glass transition temperature. T(g) is usually measured by using differential scanning calorimetry (DSC). In this study, the T(g) of the glycerol-aqueous solution (60%, wt/%) was determined by isothermal-isobaric molecular dynamic simulation (NPT-MD). The software package Discover in Material Studio with the Polymer Consortium Force Field (PCFF) was used for the simulation. The state parameters of heat capacity at constant pressure (C(p)), density (rho), amorphous cell volume (V(cell)) and specific volume (V(specific)) and radial distribution function (rdf) were obtained by NPT-MD in the temperature range of 90-270K. These parameters showed a discontinuity at a specific temperature in the plot of state parameter versus temperature. The temperature at the discontinuity is taken as the simulated T(g) value for glycerol-water binary solution. The T(g) values determined by simulation method were compared with the values in the literatures. The simulation values of T(g) (160.06-167.51K) agree well with the DSC results (163.60-167.10K) and the DMA results (159.00K). We drew the conclusion that molecular dynamic simulation (MDS) is a potential method for investigating the glass transition temperature (T(g)) of glycerol-water binary cryoprotectants and may be used for other vitrification solutions.

Dynamic morphology applied to human and animal leukemia cells.

PubMed

Haemmerli, G; Felix, H; Sträuli, P

1979-08-01

Dynamic morphology, which describes the shape and surface architecture of fixed cells in terms related to their behavior in the living state, is based on the concurrent use of two methods: scanning electron microscopy and microcinematography. This combination has both advantages and disadvantages. In this study on leukemic cells, we were able to draw the following conclusions about the usefulness of dynamic morphology. It confirms that white blood cells do not flatten on a glass substrate; they stay spherical and are either round or polarized. Round cells of similar size, whatever their origin, cannot be classified by dynamic morphology. Polarized cells can be classified as blasts, promyelocytes, myelocytes, granulocytes and lymphocytes, although polarized blast cells of different origins cannot be differentiated. Dynamic morphology cannot classify the same cell type as benign or malignant.

A novel grid-based mesoscopic model for evacuation dynamics

NASA Astrophysics Data System (ADS)

Shi, Meng; Lee, Eric Wai Ming; Ma, Yi

2018-05-01

This study presents a novel grid-based mesoscopic model for evacuation dynamics. In this model, the evacuation space is discretised into larger cells than those used in microscopic models. This approach directly computes the dynamic changes crowd densities in cells over the course of an evacuation. The density flow is driven by the density-speed correlation. The computation is faster than in traditional cellular automata evacuation models which determine density by computing the movements of each pedestrian. To demonstrate the feasibility of this model, we apply it to a series of practical scenarios and conduct a parameter sensitivity study of the effect of changes in time step δ. The simulation results show that within the valid range of δ, changing δ has only a minor impact on the simulation. The model also makes it possible to directly acquire key information such as bottleneck areas from a time-varied dynamic density map, even when a relatively large time step is adopted. We use the commercial software AnyLogic to evaluate the model. The result shows that the mesoscopic model is more efficient than the microscopic model and provides more in-situ details (e.g., pedestrian movement pattern) than the macroscopic models.

Simultaneous quantification of actin monomer and filament dynamics with modeling-assisted analysis of photoactivation

PubMed Central

Kapustina, Maryna; Read, Tracy-Ann

2016-01-01

ABSTRACT Photoactivation allows one to pulse-label molecules and obtain quantitative data about their behavior. We have devised a new modeling-based analysis for photoactivatable actin experiments that simultaneously measures properties of monomeric and filamentous actin in a three-dimensional cellular environment. We use this method to determine differences in the dynamic behavior of β- and γ-actin isoforms, showing that both inhabit filaments that depolymerize at equal rates but that β-actin exists in a higher monomer-to-filament ratio. We also demonstrate that cofilin (cofilin 1) equally accelerates depolymerization of filaments made from both isoforms, but is only required to maintain the β-actin monomer pool. Finally, we used modeling-based analysis to assess actin dynamics in axon-like projections of differentiating neuroblastoma cells, showing that the actin monomer concentration is significantly depleted as the axon develops. Importantly, these results would not have been obtained using traditional half-time analysis. Given that parameters of the publicly available modeling platform can be adjusted to suit the experimental system of the user, this method can easily be used to quantify actin dynamics in many different cell types and subcellular compartments. PMID:27831495

Microscale Mechanics of Actin Networks During Dynamic Assembly and Dissociation

NASA Astrophysics Data System (ADS)

Gurmessa, Bekele; Robertson-Anderson, Rae; Ross, Jennifer; Nguyen, Dan; Saleh, Omar

Actin is one of the key components of the cytoskeleton, enabling cells to move and divide while maintaining shape by dynamic polymerization, dissociation and crosslinking. Actin polymerization and network formation is driven by ATP hydrolysis and varies depending on the concentrations of actin monomers and crosslinking proteins. The viscoelastic properties of steady-state actin networks have been well-characterized, yet the mechanical properties of these non-equilibrium systems during dynamic assembly and disassembly remain to be understood. We use semipermeable microfluidic devices to induce in situ dissolution and re-polymerization of entangled and crosslinked actin networks, by varying ATP concentrations in real-time, while measuring the mechanical properties during disassembly and re-assembly. We use optical tweezers to sinusoidally oscillate embedded microspheres and measure the resulting force at set time-intervals and in different regions of the network during cyclic assembly/disassembly. We determine the time-dependent viscoelastic properties of non-equilibrium network intermediates and the reproducibility and homogeneity of network formation and dissolution. Results inform the role that cytoskeleton reorganization plays in the dynamic multifunctional mechanics of cells. NSF CAREER Award (DMR-1255446) and a Scialog Collaborative Innovation Award funded by Research Corporation for Scientific Advancement (Grant No. 24192).

Monitoring intra-annual dynamics of wood formation with microcores and dendrometers in Picea abies at two different altitudes.

PubMed

Cocozza, Claudia; Palombo, Caterina; Tognetti, Roberto; La Porta, Nicola; Anichini, Monica; Giovannelli, Alessio; Emiliani, Giovanni

2016-07-01

Seasonal analyses of cambial cell production and day-by-day stem radial increment can help to elucidate how climate modulates wood formation in conifers. Intra-annual dynamics of wood formation were determined with microcores and dendrometers and related to climatic signals in Norway spruce (Picea abies (L.) Karst.). The seasonal dynamics of these processes were observed at two sites of different altitude, Savignano (650 m a.s.l.) and Lavazè (1800 m a.s.l.) in the Italian Alps. Seasonal dynamics of cambial activity were found to be site specific, indicating that the phenology of cambial cell production is highly variable and plastic with altitude. There was a site-specific trend in the number of cells in the wall thickening phase, with the maximum cell production in early July (DOY 186) at Savignano and in mid-July (DOY 200) at Lavazè. The formation of mature cells showed similar trends at the two sites, although different numbers of cells and timing of cell differentiation were visible in the model shapes; at the end of ring formation in 2010, the number of cells was four times higher at Savignano (106.5 cells) than at Lavazè (26.5 cells). At low altitudes, microcores and dendrometers described the radial growth patterns comparably, though the dendrometer function underlined the higher upper asymptote of maximum growth in comparison with the cell production function. In contrast, at high altitude, these functions exhibited different trends. The best model was obtained by fitting functions of the Gompertz model to the experimental data. By combining radial growth and cambial activity indices we defined a model system able to synchronize these processes. Processes of adaptation of the pattern of xylogenesis occurred, enabling P. abies to occupy sites with contrasting climatic conditions. The use of daily climatic variables in combination with plant functional traits obtained by sensors and/or destructive sampling could provide a suitable tool to better investigate the effect of disturbances on response strategies in trees and, consequently, contribute to improving our prediction of tree growth and species resilience based on climate scenarios. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Dynamic adsorption properties of n-alkyl glucopyranosides determine their ability to inhibit cytolysis mediated by acoustic cavitation.

PubMed

Sostaric, Joe Z; Miyoshi, Norio; Cheng, Jason Y; Riesz, Peter

2008-10-09

Suspensions of human leukemia (HL-60) cells readily undergo cytolysis when exposed to ultrasound above the acoustic cavitation threshold. However, n-alkyl glucopyranosides (hexyl, heptyl, and octyl) completely inhibit ultrasound-induced (1057 kHz) cytolysis (Sostaric, et al. Free Radical Biol. Med. 2005, 39, 1539-1548). The efficacy of protection from ultrasound-induced cytolysis was determined by the n-alkyl chain length of the glucopyranosides, indicating that protection efficacy depended on adsorption of n-alkyl glucopyranosides to the gas/solution interface of cavitation bubbles and/or the lipid membrane of cells. The current study tests the hypothesis that "sonoprotection" (i.e., protection of cells from ultrasound-induced cytolysis) in vitro depends on the adsorption of glucopyranosides at the gas/solution interface of cavitation bubbles. To test this hypothesis, the effect of ultrasound frequency (from 42 kHz to 1 MHz) on the ability of a homologous series of n-alkyl glucopyranosides to protect cells from ultrasound-induced cytolysis was investigated. It is expected that ultrasound frequency will affect sonoprotection ability since the nature of the cavitation bubble field will change. This will affect the relative importance of the possible mechanisms for ultrasound-induced cytolysis. Additionally, ultrasound frequency will affect the lifetime and rate of change of the surface area of cavitation bubbles, hence the dynamically controlled adsorption of glucopyranosides to their surface. The data support the hypothesis that sonoprotection efficiency depends on the ability of glucopyranosides to adsorb at the gas/solution interface of cavitation bubbles.

Abstraction and Idealization in Biomedicine: The Nonautonomous Theory of Acute Cell Injury.

PubMed

DeGracia, Donald J; Taha, Doaa; Tri Anggraini, Fika; Sutariya, Shreya; Rababeh, Gabriel; Huang, Zhi-Feng

2018-02-27

Neuroprotection seeks to halt cell death after brain ischemia and has been shown to be possible in laboratory studies. However, neuroprotection has not been successfully translated into clinical practice, despite voluminous research and controlled clinical trials. We suggested these failures may be due, at least in part, to the lack of a general theory of cell injury to guide research into specific injuries. The nonlinear dynamical theory of acute cell injury was introduced to ameliorate this situation. Here we present a revised nonautonomous nonlinear theory of acute cell injury and show how to interpret its solutions in terms of acute biomedical injuries. The theory solutions demonstrate the complexity of possible outcomes following an idealized acute injury and indicate that a "one size fits all" therapy is unlikely to be successful. This conclusion is offset by the fact that the theory can (1) determine if a cell has the possibility to survive given a specific acute injury, and (2) calculate the degree of therapy needed to cause survival. To appreciate these conclusions, it is necessary to idealize and abstract complex physical systems to identify the fundamental mechanism governing the injury dynamics. The path of abstraction and idealization in biomedical research opens the possibility for medical treatments that may achieve engineering levels of precision.

Abstraction and Idealization in Biomedicine: The Nonautonomous Theory of Acute Cell Injury

PubMed Central

DeGracia, Donald J.; Taha, Doaa; Tri Anggraini, Fika; Sutariya, Shreya; Rababeh, Gabriel; Huang, Zhi-Feng

2018-01-01

Neuroprotection seeks to halt cell death after brain ischemia and has been shown to be possible in laboratory studies. However, neuroprotection has not been successfully translated into clinical practice, despite voluminous research and controlled clinical trials. We suggested these failures may be due, at least in part, to the lack of a general theory of cell injury to guide research into specific injuries. The nonlinear dynamical theory of acute cell injury was introduced to ameliorate this situation. Here we present a revised nonautonomous nonlinear theory of acute cell injury and show how to interpret its solutions in terms of acute biomedical injuries. The theory solutions demonstrate the complexity of possible outcomes following an idealized acute injury and indicate that a “one size fits all” therapy is unlikely to be successful. This conclusion is offset by the fact that the theory can (1) determine if a cell has the possibility to survive given a specific acute injury, and (2) calculate the degree of therapy needed to cause survival. To appreciate these conclusions, it is necessary to idealize and abstract complex physical systems to identify the fundamental mechanism governing the injury dynamics. The path of abstraction and idealization in biomedical research opens the possibility for medical treatments that may achieve engineering levels of precision. PMID:29495539

Magnetic Particle Spectroscopy Reveals Dynamic Changes in the Magnetic Behavior of Very Small Superparamagnetic Iron Oxide Nanoparticles During Cellular Uptake and Enables Determination of Cell-Labeling Efficacy.

PubMed

Poller, Wolfram C; Löwa, Norbert; Wiekhorst, Frank; Taupitz, Matthias; Wagner, Susanne; Möller, Konstantin; Baumann, Gert; Stangl, Verena; Trahms, Lutz; Ludwig, Antje

2016-02-01

In vivo tracking of nanoparticle-labeled cells by magnetic resonance imaging (MRI) crucially depends on accurate determination of cell-labeling efficacy prior to transplantation. Here, we analyzed the feasibility and accuracy of magnetic particle spectroscopy (MPS) for estimation of cell-labeling efficacy in living THP-1 cells incubated with very small superparamagnetic iron oxide nanoparticles (VSOP). Cell viability and proliferation capacity were not affected by the MPS measurement procedure. In VSOP samples without cell contact, MPS enabled highly accurate quantification. In contrast, MPS constantly overestimated the amount of cell associated and internalized VSOP. Analyses of the MPS spectrum shape expressed as harmonic ratio A₅/A₃ revealed distinct changes in the magnetic behavior of VSOP in response to cellular uptake. These changes were proportional to the deviation between MPS and actual iron amount, therefore allowing for adjusted iron quantification. Transmission electron microscopy provided visual evidence that changes in the magnetic properties correlated with cell surface interaction of VSOP as well as with alterations of particle structure and arrangement during the phagocytic process. Altogether, A₅/A₃-adjusted MPS enables highly accurate, cell-preserving VSOP quantification and furthermore provides information on the magnetic characteristics of internalized VSOP.

Dynamic changes in quality of life after three first-line therapies for EGFR mutation-positive advanced non-small-cell lung cancer.

PubMed

Yang, Szu-Chun; Lin, Chien-Chung; Lai, Wu-Wei; Chang, Sheng-Mao; Hwang, Jing-Shiang; Su, Wu-Chou; Wang, Jung-Der

2018-01-01

Three different tyrosine kinase inhibitors have been approved as first-line therapies for epidermal growth factor receptor (EGFR) mutation-positive advanced non-small-cell lung cancer with similar overall survival. This study determined dynamic changes in quality of life (QoL) for patients using these therapies after controlling for potential confounders. From 2011 to 2016, we prospectively assessed the utility values and QoL scores of patients using the EuroQol five-dimension and World Health Organization Quality-of-Life - Brief questionnaires. QoL functions after initiation of treatment were estimated using a kernel-smoothing method. Dynamic changes in major determinants were repeatedly assessed for constructing mixed models. A total of 344 patients were enrolled, with 934 repeated assessments. After controlling for performance status, disease progression, EGFR mutation subtype and other confounders, the mixed models showed significantly lower QoL scores for afatinib versus gefitinib in the physical, psychological and social domains, and 10 facets. The differences seemed to appear 10 months after initiation of treatment. In contrast, there was no significant difference between erlotinib and gefitinib in the scores of all domains and facets. QoL in patients receiving afatinib seemed to be lower than in those receiving gefitinib. Since the sample sizes in this study were relatively small, more studies are warranted to corroborate these results.

Application of speckle dynamics for studying metabolic activity of cell cultures with herpes virus

NASA Astrophysics Data System (ADS)

Vladimirov, A. P.; Bakharev, A. A.; Malygin, A. S.; Mikhaylova, J. A.; Borodin, E. M.; Poryvayeva, A. P.; Glinskikh, N. P.

2014-05-01

The report considers the results of the experiments in which digital values of light intensity I and the image area correlation index η values were recorded on a real-time basis for one or two days. Three cell cultures with viruses along with intact cultures were investigated. High correlation of dependence of η values on time t values was demonstrated for three cultures. The η=η(t) and I=I(t) dependences for cells with and without viruses differ considerably. It was shown that the presence of viruses could be determined as early as ten minutes after measurements were started.

Visualizing Molecular Diffusion through Passive Permeability Barriers in Cells: Conventional and Novel Approaches

PubMed Central

Lin, Yu-Chun; Phua, Siew Cheng; Lin, Benjamin; Inoue, Takanari

2013-01-01

Diffusion barriers are universal solutions for cells to achieve distinct organizations, compositions, and activities within a limited space. The influence of diffusion barriers on the spatiotemporal dynamics of signaling molecules often determines cellular physiology and functions. Over the years, the passive permeability barriers in various subcellular locales have been characterized using elaborate analytical techniques. In this review, we will summarize the current state of knowledge on the various passive permeability barriers present in mammalian cells. We will conclude with a description of several conventional techniques and one new approach based on chemically-inducible diffusion trap (C-IDT) for probing permeable barriers. PMID:23731778

Model-Based Analysis of Cell Cycle Responses to Dynamically Changing Environments

PubMed Central

Seaton, Daniel D; Krishnan, J

2016-01-01

Cell cycle progression is carefully coordinated with a cell’s intra- and extracellular environment. While some pathways have been identified that communicate information from the environment to the cell cycle, a systematic understanding of how this information is dynamically processed is lacking. We address this by performing dynamic sensitivity analysis of three mathematical models of the cell cycle in Saccharomyces cerevisiae. We demonstrate that these models make broadly consistent qualitative predictions about cell cycle progression under dynamically changing conditions. For example, it is shown that the models predict anticorrelated changes in cell size and cell cycle duration under different environments independently of the growth rate. This prediction is validated by comparison to available literature data. Other consistent patterns emerge, such as widespread nonmonotonic changes in cell size down generations in response to parameter changes. We extend our analysis by investigating glucose signalling to the cell cycle, showing that known regulation of Cln3 translation and Cln1,2 transcription by glucose is sufficient to explain the experimentally observed changes in cell cycle dynamics at different glucose concentrations. Together, these results provide a framework for understanding the complex responses the cell cycle is capable of producing in response to dynamic environments. PMID:26741131

Dynamic contact guidance of migrating cells

NASA Astrophysics Data System (ADS)

Losert, Wolfgang; Sun, Xiaoyu; Guven, Can; Driscoll, Meghan; Fourkas, John

2014-03-01

We investigate the effects of nanotopographical surfaces on the cell migration and cell shape dynamics of the amoeba Dictyostelium discoideum. Amoeboid motion exhibits significant contact guidance along surfaces with nanoscale ridges or grooves. We show quantitatively that nanoridges spaced 1.5 μm apart exhibit the greatest contact guidance efficiency. Using principal component analysis, we characterize the dynamics of the cell shape modulated by the coupling between the cell membrane and ridges. We show that motion parallel to the ridges is enhanced, while the turning, at the largest spatial scales, is suppressed. Since protrusion dynamics are principally governed by actin dynamics, we imaged the actin polymerization of cells on ridges. We found that actin polymerization occurs preferentially along nanoridges in a ``monorail'' like fashion. The ridges then provide us with a tool to study actin dynamics in an effectively reduced dimensional system.

Evaluating Cell Processes, Quality, and Biomarkers in Pluripotent Stem Cells Using Video Bioinformatics

PubMed Central

Lin, Sabrina C.; Bays, Brett C.; Omaiye, Esther; Bhanu, Bir; Talbot, Prue

2016-01-01

There is a foundational need for quality control tools in stem cell laboratories engaged in basic research, regenerative therapies, and toxicological studies. These tools require automated methods for evaluating cell processes and quality during in vitro passaging, expansion, maintenance, and differentiation. In this paper, an unbiased, automated high-content profiling toolkit, StemCellQC, is presented that non-invasively extracts information on cell quality and cellular processes from time-lapse phase-contrast videos. Twenty four (24) morphological and dynamic features were analyzed in healthy, unhealthy, and dying human embryonic stem cell (hESC) colonies to identify those features that were affected in each group. Multiple features differed in the healthy versus unhealthy/dying groups, and these features were linked to growth, motility, and death. Biomarkers were discovered that predicted cell processes before they were detectable by manual observation. StemCellQC distinguished healthy and unhealthy/dying hESC colonies with 96% accuracy by non-invasively measuring and tracking dynamic and morphological features over 48 hours. Changes in cellular processes can be monitored by StemCellQC and predictions can be made about the quality of pluripotent stem cell colonies. This toolkit reduced the time and resources required to track multiple pluripotent stem cell colonies and eliminated handling errors and false classifications due to human bias. StemCellQC provided both user-specified and classifier-determined analysis in cases where the affected features are not intuitive or anticipated. Video analysis algorithms allowed assessment of biological phenomena using automatic detection analysis, which can aid facilities where maintaining stem cell quality and/or monitoring changes in cellular processes are essential. In the future StemCellQC can be expanded to include other features, cell types, treatments, and differentiating cells. PMID:26848582

T-Cell Artificial Focal Triggering Tools: Linking Surface Interactions with Cell Response

PubMed Central

Carpentier, Benoît; Pierobon, Paolo; Hivroz, Claire; Henry, Nelly

2009-01-01

T-cell activation is a key event in the immune system, involving the interaction of several receptor ligand pairs in a complex intercellular contact that forms between T-cell and antigen-presenting cells. Molecular components implicated in contact formation have been identified, but the mechanism of activation and the link between molecular interactions and cell response remain poorly understood due to the complexity and dynamics exhibited by whole cell-cell conjugates. Here we demonstrate that simplified model colloids grafted so as to target appropriate cell receptors can be efficiently used to explore the relationship of receptor engagement to the T-cell response. Using immortalized Jurkat T cells, we monitored both binding and activation events, as seen by changes in the intracellular calcium concentration. Our experimental strategy used flow cytometry analysis to follow the short time scale cell response in populations of thousands of cells. We targeted both T-cell receptor CD3 (TCR/CD3) and leukocyte-function-associated antigen (LFA-1) alone or in combination. We showed that specific engagement of TCR/CD3 with a single particle induced a transient calcium signal, confirming previous results and validating our approach. By decreasing anti-CD3 particle density, we showed that contact nucleation was the most crucial and determining step in the cell-particle interaction under dynamic conditions, due to shear stress produced by hydrodynamic flow. Introduction of LFA-1 adhesion molecule ligands at the surface of the particle overcame this limitation and elucidated the low TCR/CD3 ligand density regime. Despite their simplicity, model colloids induced relevant biological responses which consistently echoed whole cell behavior. We thus concluded that this biophysical approach provides useful tools for investigating initial events in T-cell activation, and should enable the design of intelligent artificial systems for adoptive immunotherapy. PMID:19274104

Evaluating Cell Processes, Quality, and Biomarkers in Pluripotent Stem Cells Using Video Bioinformatics.

PubMed

Zahedi, Atena; On, Vincent; Lin, Sabrina C; Bays, Brett C; Omaiye, Esther; Bhanu, Bir; Talbot, Prue

2016-01-01

There is a foundational need for quality control tools in stem cell laboratories engaged in basic research, regenerative therapies, and toxicological studies. These tools require automated methods for evaluating cell processes and quality during in vitro passaging, expansion, maintenance, and differentiation. In this paper, an unbiased, automated high-content profiling toolkit, StemCellQC, is presented that non-invasively extracts information on cell quality and cellular processes from time-lapse phase-contrast videos. Twenty four (24) morphological and dynamic features were analyzed in healthy, unhealthy, and dying human embryonic stem cell (hESC) colonies to identify those features that were affected in each group. Multiple features differed in the healthy versus unhealthy/dying groups, and these features were linked to growth, motility, and death. Biomarkers were discovered that predicted cell processes before they were detectable by manual observation. StemCellQC distinguished healthy and unhealthy/dying hESC colonies with 96% accuracy by non-invasively measuring and tracking dynamic and morphological features over 48 hours. Changes in cellular processes can be monitored by StemCellQC and predictions can be made about the quality of pluripotent stem cell colonies. This toolkit reduced the time and resources required to track multiple pluripotent stem cell colonies and eliminated handling errors and false classifications due to human bias. StemCellQC provided both user-specified and classifier-determined analysis in cases where the affected features are not intuitive or anticipated. Video analysis algorithms allowed assessment of biological phenomena using automatic detection analysis, which can aid facilities where maintaining stem cell quality and/or monitoring changes in cellular processes are essential. In the future StemCellQC can be expanded to include other features, cell types, treatments, and differentiating cells.

The mechanical response of talin

NASA Astrophysics Data System (ADS)

Yao, Mingxi; Goult, Benjamin T.; Klapholz, Benjamin; Hu, Xian; Toseland, Christopher P.; Guo, Yingjian; Cong, Peiwen; Sheetz, Michael P.; Yan, Jie

2016-07-01

Talin, a force-bearing cytoplasmic adapter essential for integrin-mediated cell adhesion, links the actin cytoskeleton to integrin-based cell-extracellular matrix adhesions at the plasma membrane. Its C-terminal rod domain, which contains 13 helical bundles, plays important roles in mechanosensing during cell adhesion and spreading. However, how the structural stability and transition kinetics of the 13 helical bundles of talin are utilized in the diverse talin-dependent mechanosensing processes remains poorly understood. Here we report the force-dependent unfolding and refolding kinetics of all talin rod domains. Using experimentally determined kinetics parameters, we determined the dynamics of force fluctuation during stretching of talin under physiologically relevant pulling speeds and experimentally measured extension fluctuation trajectories. Our results reveal that force-dependent stochastic unfolding and refolding of talin rod domains make talin a very effective force buffer that sets a physiological force range of only a few pNs in the talin-mediated force transmission pathway.

Determinants of Mammalian Nucleolar Architecture

PubMed Central

Farley, Katherine I.; Surovtseva, Yulia; Merkel, Janie; Baserga, Susan J.

2015-01-01

The nucleolus is responsible for the production of ribosomes, essential machines which synthesize all proteins needed by the cell. The structure of human nucleoli is highly dynamic and is directly related to its functions in ribosome biogenesis. Despite the importance of this organelle, the intricate relationship between nucleolar structure and function remains largely unexplored. How do cells control nucleolar formation and function? What are the minimal requirements for making a functional nucleolus? Here we review what is currently known regarding mammalian nucleolar formation at nucleolar organizer regions (NORs), which can be studied by observing the dissolution and reformation of the nucleolus during each cell division. Additionally, the nucleolus can be examined by analyzing how alterations in nucleolar function manifest in differences in nucleolar architecture. Furthermore, changes in nucleolar structure and function are correlated with cancer, highlighting the importance of studying the determinants of nucleolar formation. PMID:25670395

Stability of a general delayed virus dynamics model with humoral immunity and cellular infection

NASA Astrophysics Data System (ADS)

Elaiw, A. M.; Raezah, A. A.; Alofi, A. S.

2017-06-01

In this paper, we investigate the dynamical behavior of a general nonlinear model for virus dynamics with virus-target and infected-target incidences. The model incorporates humoral immune response and distributed time delays. The model is a four dimensional system of delay differential equations where the production and removal rates of the virus and cells are given by general nonlinear functions. We derive the basic reproduction parameter R˜0 G and the humoral immune response activation number R˜1 G and establish a set of conditions on the general functions which are sufficient to determine the global dynamics of the models. We use suitable Lyapunov functionals and apply LaSalle's invariance principle to prove the global asymptotic stability of the all equilibria of the model. We confirm the theoretical results by numerical simulations.

Modal analysis and dynamic stresses for acoustically excited shuttle insulation tiles

NASA Technical Reports Server (NTRS)

Ojalvo, I. U.; Ogilvie, P. L.

1975-01-01

Improvements and extensions to the RESIST computer program developed for determining the normalized modal stress response of shuttle insulation tiles are described. The new version of RESIST can accommodate primary structure panels with closed-cell stringers, in addition to the capability for treating open-cell stringers. In addition, the present version of RESIST numerically solves vibration problems several times faster than its predecessor. A new digital computer program, titled ARREST (Acoustic Response of Reusable Shuttle Tiles) is also described. Starting with modal information contained on output tapes from RESIST computer runs, ARREST determines RMS stresses, deflections and accelerations of shuttle panels with reusable surface insulation tiles. Both programs are applicable to stringer stiffened structural panels with or without reusable surface insulation titles.

Preface of the "Symposium on Mathematical Models and Methods to investigate Heterogeneity in Cell and Cell Population Biology"

NASA Astrophysics Data System (ADS)

Clairambault, Jean

2016-06-01

This session investigates hot topics related to mathematical representations of cell and cell population dynamics in biology and medicine, in particular, but not only, with applications to cancer. Methods in mathematical modelling and analysis, and in statistical inference using single-cell and cell population data, should contribute to focus this session on heterogeneity in cell populations. Among other methods are proposed: a) Intracellular protein dynamics and gene regulatory networks using ordinary/partial/delay differential equations (ODEs, PDEs, DDEs); b) Representation of cell population dynamics using agent-based models (ABMs) and/or PDEs; c) Hybrid models and multiscale models to integrate single-cell dynamics into cell population behaviour; d) Structured cell population dynamics and asymptotic evolution w.r.t. relevant traits; e) Heterogeneity in cancer cell populations: origin, evolution, phylogeny and methods of reconstruction; f) Drug resistance as an evolutionary phenotype: predicting and overcoming it in therapeutics; g) Theoretical therapeutic optimisation of combined drug treatments in cancer cell populations and in populations of other organisms, such as bacteria.

Self-Organizing Global Gene Expression Regulated through Criticality: Mechanism of the Cell-Fate Change

PubMed Central

Tsuchiya, Masa; Giuliani, Alessandro; Hashimoto, Midori; Erenpreisa, Jekaterina; Yoshikawa, Kenichi

2016-01-01

Background A fundamental issue in bioscience is to understand the mechanism that underlies the dynamic control of genome-wide expression through the complex temporal-spatial self-organization of the genome to regulate the change in cell fate. We address this issue by elucidating a physically motivated mechanism of self-organization. Principal Findings Building upon transcriptome experimental data for seven distinct cell fates, including early embryonic development, we demonstrate that self-organized criticality (SOC) plays an essential role in the dynamic control of global gene expression regulation at both the population and single-cell levels. The novel findings are as follows: i) Mechanism of cell-fate changes: A sandpile-type critical transition self-organizes overall expression into a few transcription response domains (critical states). A cell-fate change occurs by means of a dissipative pulse-like global perturbation in self-organization through the erasure of initial-state critical behaviors (criticality). Most notably, the reprogramming of early embryo cells destroys the zygote SOC control to initiate self-organization in the new embryonal genome, which passes through a stochastic overall expression pattern. ii) Mechanism of perturbation of SOC controls: Global perturbations in self-organization involve the temporal regulation of critical states. Quantitative evaluation of this perturbation in terminal cell fates reveals that dynamic interactions between critical states determine the critical-state coherent regulation. The occurrence of a temporal change in criticality perturbs this between-states interaction, which directly affects the entire genomic system. Surprisingly, a sub-critical state, corresponding to an ensemble of genes that shows only marginal changes in expression and consequently are considered to be devoid of any interest, plays an essential role in generating a global perturbation in self-organization directed toward the cell-fate change. Conclusion and Significance ‘Whole-genome’ regulation of gene expression through self-regulatory SOC control complements gene-by-gene fine tuning and represents a still largely unexplored non-equilibrium statistical mechanism that is responsible for the massive reprogramming of genome expression. PMID:27997556

Fluid Dynamic Modeling to Support the Development of Flow-Based Hepatocyte Culture Systems for Metabolism Studies

PubMed Central

Pedersen, Jenny M.; Shim, Yoo-Sik; Hans, Vaibhav; Phillips, Martin B.; Macdonald, Jeffrey M.; Walker, Glenn; Andersen, Melvin E.; Clewell, Harvey J.; Yoon, Miyoung

2016-01-01

Accurate prediction of metabolism is a significant outstanding challenge in toxicology. The best predictions are based on experimental data from in vitro systems using primary hepatocytes. The predictivity of the primary hepatocyte-based culture systems, however, is still limited due to well-known phenotypic instability and rapid decline of metabolic competence within a few hours. Dynamic flow bioreactors for three-dimensional cell cultures are thought to be better at recapitulating tissue microenvironments and show potential to improve in vivo extrapolations of chemical or drug toxicity based on in vitro test results. These more physiologically relevant culture systems hold potential for extending metabolic competence of primary hepatocyte cultures as well. In this investigation, we used computational fluid dynamics to determine the optimal design of a flow-based hepatocyte culture system for evaluating chemical metabolism in vitro. The main design goals were (1) minimization of shear stress experienced by the cells to maximize viability, (2) rapid establishment of a uniform distribution of test compound in the chamber, and (3) delivery of sufficient oxygen to cells to support aerobic respiration. Two commercially available flow devices – RealBio® and QuasiVivo® (QV) – and a custom developed fluidized bed bioreactor were simulated, and turbulence, flow characteristics, test compound distribution, oxygen distribution, and cellular oxygen consumption were analyzed. Experimental results from the bioreactors were used to validate the simulation results. Our results indicate that maintaining adequate oxygen supply is the most important factor to the long-term viability of liver bioreactor cultures. Cell density and system flow patterns were the major determinants of local oxygen concentrations. The experimental results closely corresponded to the in silico predictions. Of the three bioreactors examined in this study, we were able to optimize the experimental conditions for long-term hepatocyte cell culture using the QV bioreactor. This system facilitated the use of low system volumes coupled with higher flow rates. This design supports cellular respiration by increasing oxygen concentrations in the vicinity of the cells and facilitates long-term kinetic studies of low clearance test compounds. These two goals were achieved while simultaneously keeping the shear stress experienced by the cells within acceptable limits. PMID:27747210

Differential roles of two delayed rectifier potassium currents in regulation of ventricular action potential duration and arrhythmia susceptibility.

PubMed

Devenyi, Ryan A; Ortega, Francis A; Groenendaal, Willemijn; Krogh-Madsen, Trine; Christini, David J; Sobie, Eric A

2017-04-01

Arrhythmias result from disruptions to cardiac electrical activity, although the factors that control cellular action potentials are incompletely understood. We combined mathematical modelling with experiments in heart cells from guinea pigs to determine how cellular electrical activity is regulated. A mismatch between modelling predictions and the experimental results allowed us to construct an improved, more predictive mathematical model. The balance between two particular potassium currents dictates how heart cells respond to perturbations and their susceptibility to arrhythmias. Imbalances of ionic currents can destabilize the cardiac action potential and potentially trigger lethal cardiac arrhythmias. In the present study, we combined mathematical modelling with information-rich dynamic clamp experiments to determine the regulation of action potential morphology in guinea pig ventricular myocytes. Parameter sensitivity analysis was used to predict how changes in ionic currents alter action potential duration, and these were tested experimentally using dynamic clamp, a technique that allows for multiple perturbations to be tested in each cell. Surprisingly, we found that a leading mathematical model, developed with traditional approaches, systematically underestimated experimental responses to dynamic clamp perturbations. We then re-parameterized the model using a genetic algorithm, which allowed us to estimate ionic current levels in each of the cells studied. This unbiased model adjustment consistently predicted an increase in the rapid delayed rectifier K + current and a drastic decrease in the slow delayed rectifier K + current, and this prediction was validated experimentally. Subsequent simulations with the adjusted model generated the clinically relevant prediction that the slow delayed rectifier is better able to stabilize the action potential and suppress pro-arrhythmic events than the rapid delayed rectifier. In summary, iterative coupling of simulations and experiments enabled novel insight into how the balance between cardiac K + currents influences ventricular arrhythmia susceptibility. © 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.

Arabidopsis Regenerating Protoplast: A Powerful Model System for Combining the Proteomics of Cell Wall Proteins and the Visualization of Cell Wall Dynamics

PubMed Central

Yokoyama, Ryusuke; Kuki, Hiroaki; Kuroha, Takeshi; Nishitani, Kazuhiko

2016-01-01

The development of a range of sub-proteomic approaches to the plant cell wall has identified many of the cell wall proteins. However, it remains difficult to elucidate the precise biological role of each protein and the cell wall dynamics driven by their actions. The plant protoplast provides an excellent means not only for characterizing cell wall proteins, but also for visualizing the dynamics of cell wall regeneration, during which cell wall proteins are secreted. It therefore offers a unique opportunity to investigate the de novo construction process of the cell wall. This review deals with sub-proteomic approaches to the plant cell wall through the use of protoplasts, a methodology that will provide the basis for further exploration of cell wall proteins and cell wall dynamics. PMID:28248244

Non-invasive In-cell Determination of Free Cytosolic [NAD+]/[NADH] Ratios Using Hyperpolarized Glucose Show Large Variations in Metabolic Phenotypes*

PubMed Central

Christensen, Caspar Elo; Karlsson, Magnus; Winther, Jakob R.; Jensen, Pernille Rose; Lerche, Mathilde H.

2014-01-01

Accumulating evidence suggest that the pyridine nucleotide NAD has far wider biological functions than its classical role in energy metabolism. NAD is used by hundreds of enzymes that catalyze substrate oxidation and, as such, it plays a key role in various biological processes such as aging, cell death, and oxidative stress. It has been suggested that changes in the ratio of free cytosolic [NAD+]/[NADH] reflects metabolic alterations leading to, or correlating with, pathological states. We have designed an isotopically labeled metabolic bioprobe of free cytosolic [NAD+]/[NADH] by combining a magnetic enhancement technique (hyperpolarization) with cellular glycolytic activity. The bioprobe reports free cytosolic [NAD+]/[NADH] ratios based on dynamically measured in-cell [pyruvate]/[lactate] ratios. We demonstrate its utility in breast and prostate cancer cells. The free cytosolic [NAD+]/[NADH] ratio determined in prostate cancer cells was 4 times higher than in breast cancer cells. This higher ratio reflects a distinct metabolic phenotype of prostate cancer cells consistent with previously reported alterations in the energy metabolism of these cells. As a reporter on free cytosolic [NAD+]/[NADH] ratio, the bioprobe will enable better understanding of the origin of diverse pathological states of the cell as well as monitor cellular consequences of diseases and/or treatments. PMID:24302737

Identification of viscous droplets' physical properties that determine droplet behaviors in inertial microfluidics

NASA Astrophysics Data System (ADS)

Hur, Soojung Claire

2013-11-01

Inertial effects in microfluidic systems have recently recognized as a robust and passive way of focusing and ordering microscale particles and cells continuously. Moreover, theoretical analysis has shown that there exists a force away from channel walls in Poiseuille flow that locates deformable particles closer to the channel center than rigid counterparts. Then, the particle deformability can be extrapolated from the positions of particles with known sizes in the channel. Here, behaviors of various viscous droplets in inertial flow were investigated to identify critical properties determining their dynamic lateral position. Fluorinated oil solutions (μ = 1.7 mPas and 5 mPas) containing droplets (1mPas< μ<1.3Pas) were injected into a microfluidic channel with a syringe pump (8 < Rc < 50). Interfacial tension between aqueous and oil phases were varied by adding controlled amount of a surfactant. The diameter, a, deformability, Def, and dynamic lateral position, Xeq, were determined using high-speed microscopy. Xeq, was found to correlate with the particle Capillary Number, CaP, regardless of droplet viscosities when CaP <0.02 or CaP >0.2, suggesting that the viscous drag from the continuous phase and the interfacial tension were competing factors determining Xeq. Experimental results suggested that (i) interplay among droplet's viscosity, interfacial tension and inertia of carrier fluid determines dynamic lateral position of droplets and (ii) the dominant property varies at a different regime.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vassilevska, Tanya

This is the first code, designed to run on a desktop, which models the intracellular replication and the cell-to-cell infection and demonstrates virus evolution at the molecular level. This code simulates the infection of a population of "idealized biological cells" (represented as objects that do not divide or have metabolism) with "virus" (represented by its genetic sequence), the replication and simultaneous mutation of the virus which leads to evolution of the population of genetically diverse viruses. The code is built to simulate single-stranded RNA viruses. The input for the code is 1. the number of biological cells in the culture,more » 2. the initial composition of the virus population, 3. the reference genome of the RNA virus, 4. the coordinates of the genome regions and their significance and, 5. parameters determining the dynamics of virus replication, such as the mutation rate. The simulation ends when all cells have been infected or when no more infections occurs after a given number of attempts. The code has the ability to simulate the evolution of the virus in serial passage of cell "cultures", i.e. after the end of a simulation, a new one is immediately scheduled with a new culture of infected cells. The code outputs characteristics of the resulting virus population dynamics and genetic composition of the virus population, such as the top dominant genomes, percentage of a genome with specific characteristics.« less

Unusual spiral wave dynamics in the Kessler-Levine model of an excitable medium.

PubMed

Oikawa, N; Bodenschatz, E; Zykov, V S

2015-05-01

The Kessler-Levine model is a two-component reaction-diffusion system that describes spatiotemporal dynamics of the messenger molecules in a cell-to-cell signaling process during the aggregation of social amoeba cells. An excitation wave arising in the model has a phase wave at the wave back, which simply follows the wave front after a fixed time interval with the same propagation velocity. Generally speaking, the medium excitability and the refractoriness are two important factors which determine the spiral wave dynamics in any excitable media. The model allows us to separate these two factors relatively easily since the medium refractoriness can be changed independently of the medium excitability. For rigidly rotating waves, the universal relationship has been established by using a modified free-boundary approach, which assumes that the front and the back of a propagating wave are thin in comparison to the wave plateau. By taking a finite thickness of the domain boundary into consideration, the validity of the proposed excitability measure has been essentially improved. A novel method of numerical simulation to suppress the spiral wave instabilities is introduced. The trajectories of the spiral tip observed for a long refractory period have been investigated under a systematic variation of the medium refractoriness.

Filopodial retraction force is generated by cortical actin dynamics and controlled by reversible tethering at the tip

PubMed Central

Bornschlögl, Thomas; Romero, Stéphane; Vestergaard, Christian L.; Joanny, Jean-François; Van Nhieu, Guy Tran; Bassereau, Patricia

2013-01-01

Filopodia are dynamic, finger-like plasma membrane protrusions that sense the mechanical and chemical surroundings of the cell. Here, we show in epithelial cells that the dynamics of filopodial extension and retraction are determined by the difference between the actin polymerization rate at the tip and the retrograde flow at the base of the filopodium. Adhesion of a bead to the filopodial tip locally reduces actin polymerization and leads to retraction via retrograde flow, reminiscent of a process used by pathogens to invade cells. Using optical tweezers, we show that filopodial retraction occurs at a constant speed against counteracting forces up to 50 pN. Our measurements point toward retrograde flow in the cortex together with frictional coupling between the filopodial and cortical actin networks as the main retraction-force generator for filopodia. The force exerted by filopodial retraction, however, is limited by the connection between filopodial actin filaments and the membrane at the tip. Upon mechanical rupture of the tip connection, filopodia exert a passive retraction force of 15 pN via their plasma membrane. Transient reconnection at the tip allows filopodia to continuously probe their surroundings in a load-and-fail manner within a well-defined force range. PMID:24198333

Automatic assessment of dynamic contrast-enhanced MRI in an ischemic rat hindlimb model: an exploratory study of transplanted multipotent progenitor cells.

PubMed

Hsu, Li-Yueh; Wragg, Andrew; Anderson, Stasia A; Balaban, Robert S; Boehm, Manfred; Arai, Andrew E

2008-02-01

This study presents computerized automatic image analysis for quantitatively evaluating dynamic contrast-enhanced MRI in an ischemic rat hindlimb model. MRI at 7 T was performed on animals in a blinded placebo-controlled experiment comparing multipotent adult progenitor cell-derived progenitor cell (MDPC)-treated, phosphate buffered saline (PBS)-injected, and sham-operated rats. Ischemic and non-ischemic limb regions of interest were automatically segmented from time-series images for detecting changes in perfusion and late enhancement. In correlation analysis of the time-signal intensity histograms, the MDPC-treated limbs correlated well with their corresponding non-ischemic limbs. However, the correlation coefficient of the PBS control group was significantly lower than that of the MDPC-treated and sham-operated groups. In semi-quantitative parametric maps of contrast enhancement, there was no significant difference in hypo-enhanced area between the MDPC and PBS groups at early perfusion-dependent time frames. However, the late-enhancement area was significantly larger in the PBS than the MDPC group. The results of this exploratory study show that MDPC-treated rats could be objectively distinguished from PBS controls. The differences were primarily determined by late contrast enhancement of PBS-treated limbs. These computerized methods appear promising for assessing perfusion and late enhancement in dynamic contrast-enhanced MRI.

Manganese induces mitochondrial dynamics impairment and apoptotic cell death: a study in human Gli36 cells.

PubMed

Alaimo, Agustina; Gorojod, Roxana M; Miglietta, Esteban A; Villarreal, Alejandro; Ramos, Alberto J; Kotler, Mónica L

2013-10-25

Manganese (Mn) is an essential trace element due to its participation in many physiological processes. However, overexposure to this metal leads to a neurological disorder known as Manganism whose clinical manifestations and molecular mechanisms resemble Parkinson's disease. Several lines of evidence implicate astrocytes as an early target of Mn neurotoxicity being the mitochondria the most affected organelles. The aim of this study was to investigate the possible mitochondrial dynamics alterations in Mn-exposed human astrocytes. Therefore, we employed Gli36 cells which express the astrocytic markers GFAP and S100B. We demonstrated that Mn triggers the mitochondrial apoptotic pathway revealed by increased Bax/Bcl-2 ratio, by the loss of mitochondrial membrane potential and by caspase-9 activation. This apoptotic program may be in turn responsible of caspase-3/7 activation, PARP-1 cleavage, chromatin condensation and fragmentation. In addition, we determined that Mn induces deregulation in mitochondria-shaping proteins (Opa-1, Mfn-2 and Drp-1) expression levels in parallel with the disruption of the mitochondrial network toward to an exacerbated fragmentation. Since mitochondrial dynamics is altered in several neurodegenerative diseases, these proteins could become future targets to be considered in Manganism treatment. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Unusual spiral wave dynamics in the Kessler-Levine model of an excitable medium

NASA Astrophysics Data System (ADS)

Oikawa, N.; Bodenschatz, E.; Zykov, V. S.

2015-05-01

The Kessler-Levine model is a two-component reaction-diffusion system that describes spatiotemporal dynamics of the messenger molecules in a cell-to-cell signaling process during the aggregation of social amoeba cells. An excitation wave arising in the model has a phase wave at the wave back, which simply follows the wave front after a fixed time interval with the same propagation velocity. Generally speaking, the medium excitability and the refractoriness are two important factors which determine the spiral wave dynamics in any excitable media. The model allows us to separate these two factors relatively easily since the medium refractoriness can be changed independently of the medium excitability. For rigidly rotating waves, the universal relationship has been established by using a modified free-boundary approach, which assumes that the front and the back of a propagating wave are thin in comparison to the wave plateau. By taking a finite thickness of the domain boundary into consideration, the validity of the proposed excitability measure has been essentially improved. A novel method of numerical simulation to suppress the spiral wave instabilities is introduced. The trajectories of the spiral tip observed for a long refractory period have been investigated under a systematic variation of the medium refractoriness.

Golgi Positioning

PubMed Central

Yadav, Smita; Linstedt, Adam D.

2011-01-01

The Golgi apparatus in mammalian cells is positioned near the centrosome-based microtubule-organizing center (Fig. 1). Secretory cargo moves inward in membrane carriers for delivery to Golgi membranes in which it is processed and packaged for transport outward to the plasma membrane. Cytoplasmic dynein motor proteins (herein termed dynein) primarily mediate inward cargo carrier movement and Golgi positioning. These motors move along microtubules toward microtubule minus-ends embedded in centrosomes. Centripetal motility is controlled by a host of regulators whose precise functions remain to be determined. Significantly, a specific Golgi receptor for dynein has not been identified. This has impaired progress toward elucidation of membrane-motor-microtubule attachment in the periphery and, after inward movement, recycling of the motor for another round. Pericentrosomal positioning of the Golgi apparatus is dynamic. It is regulated during critical cellular processes such as mitosis, differentiation, cell polarization, and cell migration. Positioning is also important as it aligns the Golgi along an axis of cell polarity. In certain cell types, this promotes secretion directed to the proximal plasma membrane domain thereby maintaining specializations critical for diverse processes including wound healing, immunological synapse formation, and axon determination. PMID:21504874

Characterization of tissue biomechanics and mechanical signaling in uterine leiomyoma☆

PubMed Central

Norian, John M.; Owen, Carter M.; Taboas, Juan; Korecki, Casey; Tuan, Rocky; Malik, Minnie; Catherino, William H.; Segars, James H.

2012-01-01

Leiomyoma are common tumors arising within the uterus that feature excessive deposition of a stiff, disordered extracellular matrix (ECM). Mechanical stress is a critical determinant of excessive ECM deposition and increased mechanical stress has been shown to be involved in tumorigenesis. Here we tested the viscoelastic properties of leiomyoma and characterized dynamic and static mechanical signaling in leiomyoma cells using three approaches, including measurement of active RhoA. We found that the peak strain and pseudo-dynamic modulus of leiomyoma tissue was significantly increased relative to matched myometrium. In addition, leiomyoma cells demonstrated an attenuated response to applied cyclic uniaxial strain and to variation in substrate stiffness, relative to myometrial cells. However, on a flexible pronectin-coated silicone substrate, basal levels and lysophosphatidic acid-stimulated levels of activated RhoA were similar between leiomyoma and myometrial cells. In contrast, leiomyoma cells plated on a rigid polystyrene substrate had elevated levels of active RhoA, compared to myometrial cells. The results indicate that viscoelastic properties of the ECM of leiomyoma contribute significantly to the tumor’s inherent stiffness and that leiomyoma cells have an attenuated sensitivity to mechanical cues. The findings suggest there may be a fundamental alteration in the communication between the external mechanical environment (extracellular forces) and reorganization of the actin cytoskeleton mediated by RhoA in leiomyoma cells. Additional research will be needed to elucidate the mechanism(s) responsible for the attenuated mechanical signaling in leiomyoma cells. PMID:21983114

Involvements of γδT Lymphocytes in Acute and Chronic Skin Wound Repair.

PubMed

Xu, Peng; Fu, Xiujun; Xiao, Nin; Guo, Yuanyuan; Pei, Qing; Peng, Yinbo; Zhang, Yifan; Yao, Min

2017-08-01

Wound healing involves three stages including inflammation, proliferation, and tissue remodeling. The underlying mechanisms remain to be further elucidated. The inflammation is characterized by spatially and temporally changing patterns of various leukocyte subsets. It is regarded as the most crucial stage since the inflammatory response is instrumental to supplying various factors and cytokines that orchestrate healing events. As a subtype of T lymphocytes, γδ T cells play an important role in skin homeostasis, tumor immunosurveillance, and wound repair. However, either the dynamics of γδ T cells in healing process or the anticipated association of γδ T cells with chronic or refractory wounds were not well understood. In this study, we determine the dynamics of γδ T cells and γδ T cell-produced effectors during acute and chronic wound repair by establishing a third-degree burn model in mice skin or human skin from diabetic patients. Our data show that the involvement of γδ T cells in acute and chronic skin wound healing. The protein levels and mRNA expressions of γδ T cell-produced effectors were increased in acute healing model, whereas those effectors were decreased in chronic repair, suggesting γδ T cells are essential for wound repair. This study probes into the significant relevance of γδ T cells with effective wound repair and provides new enlightenments for the mechanisms of the formation of chronic and/or refractory wounds.

DNA-Binding Kinetics Determines the Mechanism of Noise-Induced Switching in Gene Networks

PubMed Central

Tse, Margaret J.; Chu, Brian K.; Roy, Mahua; Read, Elizabeth L.

2015-01-01

Gene regulatory networks are multistable dynamical systems in which attractor states represent cell phenotypes. Spontaneous, noise-induced transitions between these states are thought to underlie critical cellular processes, including cell developmental fate decisions, phenotypic plasticity in fluctuating environments, and carcinogenesis. As such, there is increasing interest in the development of theoretical and computational approaches that can shed light on the dynamics of these stochastic state transitions in multistable gene networks. We applied a numerical rare-event sampling algorithm to study transition paths of spontaneous noise-induced switching for a ubiquitous gene regulatory network motif, the bistable toggle switch, in which two mutually repressive genes compete for dominant expression. We find that the method can efficiently uncover detailed switching mechanisms that involve fluctuations both in occupancies of DNA regulatory sites and copy numbers of protein products. In addition, we show that the rate parameters governing binding and unbinding of regulatory proteins to DNA strongly influence the switching mechanism. In a regime of slow DNA-binding/unbinding kinetics, spontaneous switching occurs relatively frequently and is driven primarily by fluctuations in DNA-site occupancies. In contrast, in a regime of fast DNA-binding/unbinding kinetics, switching occurs rarely and is driven by fluctuations in levels of expressed protein. Our results demonstrate how spontaneous cell phenotype transitions involve collective behavior of both regulatory proteins and DNA. Computational approaches capable of simulating dynamics over many system variables are thus well suited to exploring dynamic mechanisms in gene networks. PMID:26488666

Dynamical System Modeling of Immune Reconstitution after Allogeneic Stem Cell Transplantation Identifies Patients at Risk for Adverse Outcomes

PubMed Central

Toor, Amir A.; Sabo, Roy T.; Roberts, Catherine H.; Moore, Bonny L.; Salman, Salman R.; Scalora, Allison F.; Aziz, May T.; Shubar Ali, Ali S.; Hall, Charles E.; Meier, Jeremy; Thorn, Radhika M.; Wang, Elaine; Song, Shiyu; Miller, Kristin; Rizzo, Kathryn; Clark, William B.; McCarty, John M.; Chung, Harold M.; Manjili, Masoud H.; Neale, Michael C.

2016-01-01

Systems that evolve over time and follow mathematical laws as they evolve are called dynamical systems. Lymphocyte recovery and clinical outcomes in 41 allograft recipients conditioned using antithymocyte globulin (ATG) and 4.5-Gy total body irradiation were studied to determine if immune reconstitution could be described as a dynamical system. Survival, relapse, and graft-versus-host disease (GVHD) were not significantly different in 2 cohorts of patients receiving different doses of ATG. However, donor-derived CD3+ cell reconstitution was superior in the lower ATG dose cohort, and there were fewer instances of donor lymphocyte infusion (DLI). Lymphoid recovery was plotted in each individual over time and demonstrated 1 of 3 sigmoid growth patterns: Pattern A (n = 15) had rapid growth with high lymphocyte counts, pattern B (n = 14) had slower growth with intermediate recovery, and pattern C (n = 10) had poor lymphocyte reconstitution. There was a significant association between lymphocyte recovery patterns and both the rate of change of donor-derived CD3+ at day 30 after stem cell transplantation (SCT) and clinical outcomes. GVHD was observed more frequently with pattern A, relapse and DLI more so with pattern C, with a consequent survival advantage in patients with patterns A and B. We conclude that evaluating immune reconstitution after SCT as a dynamical system may differentiate patients at risk of adverse outcomes and allow early intervention to modulate that risk. PMID:25849208

Electrochemical energy storage subsystems study, volume 1

NASA Technical Reports Server (NTRS)

Miller, F. Q.; Richardson, P. W.; Graff, C. L.; Jordan, M. V.; Patterson, V. L.

1981-01-01

The effects on life cycle costs (LCC) of major design and performance technology parameters for multi kW LEO and GEO energy storage subsystems using NiCd and NiH2 batteries and fuel cell/electrolysis cell devices were examined. Design, performance and LCC dynamic models are developed based on mission and system/subsystem requirements and existing or derived physical and cost data relationships. The models define baseline designs and costs. The major design and performance parameters are each varied to determine their influence on LCC around the baseline values.

Haemoglobin content modulated deformation dynamics of red blood cells on a compact disc.

PubMed

Kar, Shantimoy; Ghosh, Uddipta; Maiti, Tapas Kumar; Chakraborty, Suman

2015-12-21

We investigate the deformation characteristics of red blood cells (RBCs) on a rotating compact disc platform. Our study brings out the interplay between haemoglobin content and RBC deformability in a centrifugally actuated microfluidic environment. We reveal that RBC deformations follow the similar trend of principal stress distributed throughout the radial direction, rendering an insight into the mechano-physical processes involved. This study can be used as a diagnostic marker to determine haematological disorders in diseased blood samples tested on compact disc based microfluidic platforms.

Electrochemical Energy Storage Subsystems Study, Volume 2

NASA Technical Reports Server (NTRS)

Miller, F. Q.; Richardson, P. W.; Graff, C. L.; Jordan, M. V.; Patterson, V. L.

1981-01-01

The effects on life cycle costs (LCC) of major design and performance technology parameters for multi kW LEO and GEO energy storage subsystems using NiCd and NiH2 batteries and fuel cell/electrolysis cell devices were examined. Design, performance and LCC dynamic models are developed based on mission and system/subsystem requirements and existing or derived physical and cost data relationships. The models are exercised to define baseline designs and costs. Then the major design and performance parameters are each varied to determine their influence on LCC around the baseline values.

A technique to measure rotordynamic coefficients in hydrostatic bearings

NASA Technical Reports Server (NTRS)

Capaldi, Russell J.

1993-01-01

An experimental technique is described for measuring the rotordynamic coefficients of fluid film journal bearings. The bearing tester incorporates a double-spool shaft assembly that permits independent control over the journal spin speed and the frequency of an adjustable-magnitude circular orbit. This configuration yields data that enables determination of the full linear anisotropic rotordynamic coefficient matrices. The dynamic force measurements were made simultaneously with two independent systems, one with piezoelectric load cells and the other with strain gage load cells. Some results are presented for a four-recess, oil-fed hydrostatic journal bearing.

Automated single cell microbioreactor for monitoring intracellular dynamics and cell growth in free solution†

PubMed Central

Johnson-Chavarria, Eric M.; Agrawal, Utsav; Tanyeri, Melikhan; Kuhlman, Thomas E.

2014-01-01

We report an automated microfluidic-based platform for single cell analysis that allows for cell culture in free solution with the ability to control the cell growth environment. Using this approach, cells are confined by the sole action of gentle fluid flow, thereby enabling non-perturbative analysis of cell growth away from solid boundaries. In addition, the single cell microbioreactor allows for precise and time-dependent control over cell culture media, with the combined ability to observe the dynamics of non-adherent cells over long time scales. As a proof-of-principle demonstration, we used the platform to observe dynamic cell growth, gene expression, and intracellular diffusion of repressor proteins while precisely tuning the cell growth environment. Overall, this microfluidic approach enables the direct observation of cellular dynamics with exquisite control over environmental conditions, which will be useful for quantifying the behaviour of single cells in well-defined media. PMID:24836754

Exploring Instructive Physiological Signaling with the Bioelectric Tissue Simulation Engine

PubMed Central

Pietak, Alexis; Levin, Michael

2016-01-01

Bioelectric cell properties have been revealed as powerful targets for modulating stem cell function, regenerative response, developmental patterning, and tumor reprograming. Spatio-temporal distributions of endogenous resting potential, ion flows, and electric fields are influenced not only by the genome and external signals but also by their own intrinsic dynamics. Ion channels and electrical synapses (gap junctions) both determine, and are themselves gated by, cellular resting potential. Thus, the origin and progression of bioelectric patterns in multicellular tissues is complex, which hampers the rational control of voltage distributions for biomedical interventions. To improve understanding of these dynamics and facilitate the development of bioelectric pattern control strategies, we developed the BioElectric Tissue Simulation Engine (BETSE), a finite volume method multiphysics simulator, which predicts bioelectric patterns and their spatio-temporal dynamics by modeling ion channel and gap junction activity and tracking changes to the fundamental property of ion concentration. We validate performance of the simulator by matching experimentally obtained data on membrane permeability, ion concentration and resting potential to simulated values, and by demonstrating the expected outcomes for a range of well-known cases, such as predicting the correct transmembrane voltage changes for perturbation of single cell membrane states and environmental ion concentrations, in addition to the development of realistic transepithelial potentials and bioelectric wounding signals. In silico experiments reveal factors influencing transmembrane potential are significantly different in gap junction-networked cell clusters with tight junctions, and identify non-linear feedback mechanisms capable of generating strong, emergent, cluster-wide resting potential gradients. The BETSE platform will enable a deep understanding of local and long-range bioelectrical dynamics in tissues, and assist the development of specific interventions to achieve greater control of pattern during morphogenesis and remodeling. PMID:27458581

Molecular Determinants and Dynamics of Hepatitis C Virus Secretion

PubMed Central

Coller, Kelly E.; Heaton, Nicholas S.; Berger, Kristi L.; Cooper, Jacob D.; Saunders, Jessica L.; Randall, Glenn

2012-01-01

The current model of hepatitis C virus (HCV) production involves the assembly of virions on or near the surface of lipid droplets, envelopment at the ER in association with components of VLDL synthesis, and egress via the secretory pathway. However, the cellular requirements for and a mechanistic understanding of HCV secretion are incomplete at best. We combined an RNA interference (RNAi) analysis of host factors for infectious HCV secretion with the development of live cell imaging of HCV core trafficking to gain a detailed understanding of HCV egress. RNAi studies identified multiple components of the secretory pathway, including ER to Golgi trafficking, lipid and protein kinases that regulate budding from the trans-Golgi network (TGN), VAMP1 vesicles and adaptor proteins, and the recycling endosome. Our results support a model wherein HCV is infectious upon envelopment at the ER and exits the cell via the secretory pathway. We next constructed infectious HCV with a tetracysteine (TC) tag insertion in core (TC-core) to monitor the dynamics of HCV core trafficking in association with its cellular cofactors. In order to isolate core protein movements associated with infectious HCV secretion, only trafficking events that required the essential HCV assembly factor NS2 were quantified. TC-core traffics to the cell periphery along microtubules and this movement can be inhibited by nocodazole. Sub-populations of TC-core localize to the Golgi and co-traffic with components of the recycling endosome. Silencing of the recycling endosome component Rab11a results in the accumulation of HCV core at the Golgi. The majority of dynamic core traffics in association with apolipoprotein E (ApoE) and VAMP1 vesicles. This study identifies many new host cofactors of HCV egress, while presenting dynamic studies of HCV core trafficking in infected cells. PMID:22241992

Alpha-actinin binding kinetics modulate cellular dynamics and force generation

PubMed Central

Ehrlicher, Allen J.; Krishnan, Ramaswamy; Guo, Ming; Bidan, Cécile M.; Weitz, David A.; Pollak, Martin R.

2015-01-01

The actin cytoskeleton is a key element of cell structure and movement whose properties are determined by a host of accessory proteins. Actin cross-linking proteins create a connected network from individual actin filaments, and though the mechanical effects of cross-linker binding affinity on actin networks have been investigated in reconstituted systems, their impact on cellular forces is unknown. Here we show that the binding affinity of the actin cross-linker α-actinin 4 (ACTN4) in cells modulates cytoplasmic mobility, cellular movement, and traction forces. Using fluorescence recovery after photobleaching, we show that an ACTN4 mutation that causes human kidney disease roughly triples the wild-type binding affinity of ACTN4 to F-actin in cells, increasing the dissociation time from 29 ± 13 to 86 ± 29 s. This increased affinity creates a less dynamic cytoplasm, as demonstrated by reduced intracellular microsphere movement, and an approximate halving of cell speed. Surprisingly, these less motile cells generate larger forces. Using traction force microscopy, we show that increased binding affinity of ACTN4 increases the average contractile stress (from 1.8 ± 0.7 to 4.7 ± 0.5 kPa), and the average strain energy (0.4 ± 0.2 to 2.1 ± 0.4 pJ). We speculate that these changes may be explained by an increased solid-like nature of the cytoskeleton, where myosin activity is more partitioned into tension and less is dissipated through filament sliding. These findings demonstrate the impact of cross-linker point mutations on cell dynamics and forces, and suggest mechanisms by which such physical defects lead to human disease. PMID:25918384

The cell wall of Arabidopsis thaliana influences actin network dynamics.

PubMed

Tolmie, Frances; Poulet, Axel; McKenna, Joseph; Sassmann, Stefan; Graumann, Katja; Deeks, Michael; Runions, John

2017-07-20

In plant cells, molecular connections link the cell wall-plasma membrane-actin cytoskeleton to form a continuum. It is hypothesized that the cell wall provides stable anchor points around which the actin cytoskeleton remodels. Here we use live cell imaging of fluorescently labelled marker proteins to quantify the organization and dynamics of the actin cytoskeleton and to determine the impact of disrupting connections within the continuum. Labelling of the actin cytoskeleton with green fluorescent protein (GFP)-fimbrin actin-binding domain 2 (FABD2) resulted in a network composed of fine filaments and thicker bundles that appeared as a highly dynamic remodelling meshwork. This differed substantially from the GFP-Lifeact-labelled network that appeared much more sparse with thick bundles that underwent 'simple movement', in which the bundles slightly change position, but in such a manner that the structure of the network was not substantially altered during the time of observation. Label-dependent differences in actin network morphology and remodelling necessitated development of two new image analysis techniques. The first of these, 'pairwise image subtraction', was applied to measurement of the more rapidly remodelling actin network labelled with GFP-FABD2, while the second, 'cumulative fluorescence intensity', was used to measure bulk remodelling of the actin cytoskeleton when labelled with GFP-Lifeact. In each case, these analysis techniques show that the actin cytoskeleton has a decreased rate of bulk remodelling when the cell wall-plasma membrane-actin continuum is disrupted either by plasmolysis or with isoxaben, a drug that specifically inhibits cellulose deposition. Changes in the rate of actin remodelling also affect its functionality, as observed by alteration in Golgi body motility. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Structure-Functional Basis of Ion Transport in Sodium–Calcium Exchanger (NCX) Proteins

PubMed Central

Giladi, Moshe; Shor, Reut; Lisnyansky, Michal; Khananshvili, Daniel

2016-01-01

The membrane-bound sodium–calcium exchanger (NCX) proteins shape Ca2+ homeostasis in many cell types, thus participating in a wide range of physiological and pathological processes. Determination of the crystal structure of an archaeal NCX (NCX_Mj) paved the way for a thorough and systematic investigation of ion transport mechanisms in NCX proteins. Here, we review the data gathered from the X-ray crystallography, molecular dynamics simulations, hydrogen–deuterium exchange mass-spectrometry (HDX-MS), and ion-flux analyses of mutants. Strikingly, the apo NCX_Mj protein exhibits characteristic patterns in the local backbone dynamics at particular helix segments, thereby possessing characteristic HDX profiles, suggesting structure-dynamic preorganization (geometric arrangements of catalytic residues before the transition state) of conserved α1 and α2 repeats at ion-coordinating residues involved in transport activities. Moreover, dynamic preorganization of local structural entities in the apo protein predefines the status of ion-occlusion and transition states, even though Na+ or Ca2+ binding modifies the preceding backbone dynamics nearby functionally important residues. Future challenges include resolving the structural-dynamic determinants governing the ion selectivity, functional asymmetry and ion-induced alternating access. Taking into account the structural similarities of NCX_Mj with the other proteins belonging to the Ca2+/cation exchanger superfamily, the recent findings can significantly improve our understanding of ion transport mechanisms in NCX and similar proteins. PMID:27879668

Structure-Functional Basis of Ion Transport in Sodium-Calcium Exchanger (NCX) Proteins.

PubMed

Giladi, Moshe; Shor, Reut; Lisnyansky, Michal; Khananshvili, Daniel

2016-11-22

The membrane-bound sodium-calcium exchanger (NCX) proteins shape Ca 2+ homeostasis in many cell types, thus participating in a wide range of physiological and pathological processes. Determination of the crystal structure of an archaeal NCX (NCX_Mj) paved the way for a thorough and systematic investigation of ion transport mechanisms in NCX proteins. Here, we review the data gathered from the X-ray crystallography, molecular dynamics simulations, hydrogen-deuterium exchange mass-spectrometry (HDX-MS), and ion-flux analyses of mutants. Strikingly, the apo NCX_Mj protein exhibits characteristic patterns in the local backbone dynamics at particular helix segments, thereby possessing characteristic HDX profiles, suggesting structure-dynamic preorganization (geometric arrangements of catalytic residues before the transition state) of conserved α₁ and α₂ repeats at ion-coordinating residues involved in transport activities. Moreover, dynamic preorganization of local structural entities in the apo protein predefines the status of ion-occlusion and transition states, even though Na⁺ or Ca 2+ binding modifies the preceding backbone dynamics nearby functionally important residues. Future challenges include resolving the structural-dynamic determinants governing the ion selectivity, functional asymmetry and ion-induced alternating access. Taking into account the structural similarities of NCX_Mj with the other proteins belonging to the Ca 2+ /cation exchanger superfamily, the recent findings can significantly improve our understanding of ion transport mechanisms in NCX and similar proteins.

Fundamental limits on dynamic inference from single-cell snapshots

PubMed Central

Weinreb, Caleb; Tusi, Betsabeh K.; Socolovsky, Merav

2018-01-01

Single-cell expression profiling reveals the molecular states of individual cells with unprecedented detail. Because these methods destroy cells in the process of analysis, they cannot measure how gene expression changes over time. However, some information on dynamics is present in the data: the continuum of molecular states in the population can reflect the trajectory of a typical cell. Many methods for extracting single-cell dynamics from population data have been proposed. However, all such attempts face a common limitation: for any measured distribution of cell states, there are multiple dynamics that could give rise to it, and by extension, multiple possibilities for underlying mechanisms of gene regulation. Here, we describe the aspects of gene expression dynamics that cannot be inferred from a static snapshot alone and identify assumptions necessary to constrain a unique solution for cell dynamics from static snapshots. We translate these constraints into a practical algorithmic approach, population balance analysis (PBA), which makes use of a method from spectral graph theory to solve a class of high-dimensional differential equations. We use simulations to show the strengths and limitations of PBA, and then apply it to single-cell profiles of hematopoietic progenitor cells (HPCs). Cell state predictions from this analysis agree with HPC fate assays reported in several papers over the past two decades. By highlighting the fundamental limits on dynamic inference faced by any method, our framework provides a rigorous basis for dynamic interpretation of a gene expression continuum and clarifies best experimental designs for trajectory reconstruction from static snapshot measurements. PMID:29463712

Chondrocyte response to cyclic hydrostatic pressure in alginate versus pellet culture.

PubMed

Elder, Steven H; Sanders, Shawn W; McCulley, William R; Marr, Misti L; Shim, Joon W; Hasty, Karen A

2006-04-01

Cells are often cultured at high density (e.g., confluent monolayer and as pellets) to promote chondrogenic differentiation and to maintain the chondrocyte phenotype. They are also frequently suspended in hydrogels such as agarose or alginate for the same purposes. These culture techniques differ markedly with respect to frequency of direct contact between cells and overall intercellular spacing. Because these factors may significantly affect mechanotransduction, the purpose of this study was to determine if the response of articular chondrocytes to cyclic hydrostatic pressure would depend on the culture condition. Primary articular chondrocytes from young and mature pigs were cultured either as pellets or suspended in alginate beads. Both groups were exposed to dynamic hydrostatic pressure (4 MPa, 1 Hz, 5400 cycles per day) for 7 days. Cell proliferation was unaffected by pressure, but pressurized chondrocytes in pellet culture had significantly greater sGAG content and incorporated [3H]proline at a higher rate than nonpressurized controls. Electron microscopy revealed a fibrous extracellular matrix (ECM) surrounding pellets, but not cells in alginate. In addition, expression of Connexin 43 (Cx43) mRNA was slightly lower in alginate than in pellet cultures and was not significantly altered by loading. Thus, metabolic response of chondrocytes to dynamic hydrostatic pressure was affected by culture technique; chondrocytes cultured as pellets exhibited the classical anabolic response to dynamic hydrostatic pressure, but those in alginate did not. Although cell-ECM interaction could be important, the differential response is not likely attributable to differential expression of Cx43 mRNA. Copyright 2006 Orthopaedic Research Society

Isotropic actomyosin dynamics promote organization of the apical cell cortex in epithelial cells.

PubMed

Klingner, Christoph; Cherian, Anoop V; Fels, Johannes; Diesinger, Philipp M; Aufschnaiter, Roland; Maghelli, Nicola; Keil, Thomas; Beck, Gisela; Tolić-Nørrelykke, Iva M; Bathe, Mark; Wedlich-Soldner, Roland

2014-10-13

Although cortical actin plays an important role in cellular mechanics and morphogenesis, there is surprisingly little information on cortex organization at the apical surface of cells. In this paper, we characterize organization and dynamics of microvilli (MV) and a previously unappreciated actomyosin network at the apical surface of Madin-Darby canine kidney cells. In contrast to short and static MV in confluent cells, the apical surfaces of nonconfluent epithelial cells (ECs) form highly dynamic protrusions, which are often oriented along the plane of the membrane. These dynamic MV exhibit complex and spatially correlated reorganization, which is dependent on myosin II activity. Surprisingly, myosin II is organized into an extensive network of filaments spanning the entire apical membrane in nonconfluent ECs. Dynamic MV, myosin filaments, and their associated actin filaments form an interconnected, prestressed network. Interestingly, this network regulates lateral mobility of apical membrane probes such as integrins or epidermal growth factor receptors, suggesting that coordinated actomyosin dynamics contributes to apical cell membrane organization. © 2014 Klingner et al.

Coordinated dynamic encoding in the retina using opposing forms of plasticity

PubMed Central

Kastner, David B.; Baccus, Stephen A.

2011-01-01

The range of natural inputs encoded by a neuron often exceeds its dynamic range. To overcome this limitation, neural populations divide their inputs among different cell classes, as with rod and cone photoreceptors, and adapt by shifting their dynamic range. We report that the dynamic behavior of retinal ganglion cells in salamanders, mice, and rabbits is divided into two opposing forms of short-term plasticity in different cell classes. One population of cells exhibited sensitization—a persistent elevated sensitivity following a strong stimulus. This novel dynamic behavior compensates for the information loss caused by the known process of adaptation occurring in a separate cell population. The two populations divide the dynamic range of inputs, with sensitizing cells encoding weak signals, and adapting cells encoding strong signals. In the two populations, the linear, threshold and adaptive properties are linked to preserve responsiveness when stimulus statistics change, with one population maintaining the ability to respond when the other fails. PMID:21909086

Assessing the dynamic biofilm removal of sulfonated phenolics using CP-OCT

NASA Astrophysics Data System (ADS)

Englund, K.; Nikrad, J.; Jones, R.

2017-02-01

Examining the physical mechanisms related to biofilm removal of sulfonated phenolics (SP) is difficult using conventional microscopy techniques. A custom flow cell system integrated with a real time cross polarization optical coherence tomography system investigated the dynamic speed of biofilm removal when oral multi-species biofilms are exposed to SP under sheer stress. The Near infrared 1310-nm CP-OCT system non-destructively imaged fluid immersed oral biofilms at nearly 30 frames/s. This dynamic imaging was able to determine the cohesive and adhesion related disruption of SP on oral biofilms adhering to tooth like surfaces. For multi-species biofilms that are initially grown without the presence of sucrose, the disruption of biofilms on saliva coated hydroxyapatite (HA) is dominated as a adhesive failure at the HA-biofilm interface. For multi-species biofilms that are grown in the presence of sucrose, the disruption is dominated by cohesive disruption followed by adhesive failure. This novel CP-OCT flow cell assay has the potential to examine rapid interactions between anti-biofilm agents and tooth like surfaces.

Assembly of the MHC I peptide-loading complex determined by a conserved ionic lock-switch

PubMed Central

Blees, Andreas; Reichel, Katrin; Trowitzsch, Simon; Fisette, Olivier; Bock, Christoph; Abele, Rupert; Hummer, Gerhard; Schäfer, Lars V.; Tampé, Robert

2015-01-01

Salt bridges in lipid bilayers play a decisive role in the dynamic assembly and downstream signaling of the natural killer and T-cell receptors. Here, we describe the identification of an inter-subunit salt bridge in the membrane within yet another key component of the immune system, the peptide-loading complex (PLC). The PLC regulates cell surface presentation of self-antigens and antigenic peptides via molecules of the major histocompatibility complex class I. We demonstrate that a single salt bridge in the membrane between the transporter associated with antigen processing TAP and the MHC I-specific chaperone tapasin is essential for the assembly of the PLC and for efficient MHC I antigen presentation. Molecular modeling and all-atom molecular dynamics simulations suggest an ionic lock-switch mechanism for the binding of TAP to tapasin, in which an unfavorable uncompensated charge in the ER-membrane is prevented through complex formation. Our findings not only deepen the understanding of the interaction network within the PLC, but also provide evidence for a general interaction principle of dynamic multiprotein membrane complexes in immunity. PMID:26611325

Characterizing active cytoskeletal dynamics with magnetic microposts

NASA Astrophysics Data System (ADS)

Shi, Yu; Henry, Steven; Crocker, John; Reich, Daniel

Characterization of an active matter system such as the cellular cytoskeleton requires knowledge of three frequency dependent quantities: the dynamic shear modulus, G*(ω) describing its viscoelasticity, the Fourier power spectrum of forces in the material due to internal force generators f (ω) , and the spectrum of the material's active strain fluctuations x(ω) . Via use of PDMS micropost arrays with magnetic nanowires embedded in selected posts, we measure the local complex modulus of cells through mechanical actuation of the magnetic microposts. The micrometer scale microposts are also used as passive probes to measure simultaneously the frequency dependent strain fluctuations. We present data on 3T3 fibroblasts, where we find power law behavior for both the frequency dependence of cells' modulus | G (ω) | ω 0 . 27 and the power spectrum of strain fluctuations |x(ω) | ω-2 . Results for the power spectrum of active cytoskeletal stresses determined from these two measurements, and implications of this mesoscale characterization of cytoskeletal dynamics for cellular biophysics will also be discussed. Supported in part by NIH Grant 1R01HL127087.

Quantitative analysis of cellular proteome alterations in human influenza A virus-infected mammalian cell lines.

PubMed

Vester, Diana; Rapp, Erdmann; Gade, Dörte; Genzel, Yvonne; Reichl, Udo

2009-06-01

Over the last years virus-host cell interactions were investigated in numerous studies. Viral strategies for evasion of innate immune response, inhibition of cellular protein synthesis and permission of viral RNA and protein production were disclosed. With quantitative proteome technology, comprehensive studies concerning the impact of viruses on the cellular machinery of their host cells at protein level are possible. Therefore, 2-D DIGE and nanoHPLC-nanoESI-MS/MS analysis were used to qualitatively and quantitatively determine the dynamic cellular proteome responses of two mammalian cell lines to human influenza A virus infection. A cell line used for vaccine production (MDCK) was compared with a human lung carcinoma cell line (A549) as a reference model. Analyzing 2-D gels of the proteomes of uninfected and influenza-infected host cells, 16 quantitatively altered protein spots (at least +/-1.7-fold change in relative abundance, p<0.001) were identified for both cell lines. Most significant changes were found for keratins, major components of the cytoskeleton system, and for Mx proteins, interferon-induced key components of the host cell defense. Time series analysis of infection processes allowed the identification of further proteins that are described to be involved in protein synthesis, signal transduction and apoptosis events. Most likely, these proteins are required for supporting functions during influenza viral life cycle or host cell stress response. Quantitative proteome-wide profiling of virus infection can provide insights into complexity and dynamics of virus-host cell interactions and may accelerate antiviral research and support optimization of vaccine manufacturing processes.

Cell type-specific response to high intracellular loading of polyacrylic acid-coated magnetic nanoparticles

PubMed Central

Lojk, Jasna; Bregar, Vladimir B; Rajh, Maruša; Miš, Katarina; Kreft, Mateja Erdani; Pirkmajer, Sergej; Veranič, Peter; Pavlin, Mojca

2015-01-01

Magnetic nanoparticles (NPs) are a special type of NP with a ferromagnetic, electron-dense core that enables several applications such as cell tracking, hyperthermia, and magnetic separation, as well as multimodality. So far, superparamagnetic iron oxide NPs (SPIONs) are the only clinically approved type of metal oxide NPs, but cobalt ferrite NPs have properties suitable for biomedical applications as well. In this study, we analyzed the cellular responses to magnetic cobalt ferrite NPs coated with polyacrylic acid (PAA) in three cell types: Chinese Hamster Ovary (CHO), mouse melanoma (B16) cell line, and primary human myoblasts (MYO). We compared the internalization pathway, intracellular trafficking, and intracellular fate of our NPs using fluorescence and transmission electron microscopy (TEM) as well as quantified NP uptake and analyzed uptake dynamics. We determined cell viability after 24 or 96 hours’ exposure to increasing concentrations of NPs, and quantified the generation of reactive oxygen species (ROS) upon 24 and 48 hours’ exposure. Our NPs have been shown to readily enter and accumulate in cells in high quantities using the same two endocytic pathways; mostly by macropinocytosis and partially by clathrin-mediated endocytosis. The cell types differed in their uptake rate, the dynamics of intracellular trafficking, and the uptake capacity, as well as in their response to higher concentrations of internalized NPs. The observed differences in cell responses stress the importance of evaluation of NP–cell interactions on several different cell types for better prediction of possible toxic effects on different cell and tissue types in vivo. PMID:25733835

Molecular basis of cell integrity and morphogenesis in Saccharomyces cerevisiae.

PubMed Central

Cid, V J; Durán, A; del Rey, F; Snyder, M P; Nombela, C; Sánchez, M

1995-01-01

In fungi and many other organisms, a thick outer cell wall is responsible for determining the shape of the cell and for maintaining its integrity. The budding yeast Saccharomyces cerevisiae has been a useful model organism for the study of cell wall synthesis, and over the past few decades, many aspects of the composition, structure, and enzymology of the cell wall have been elucidated. The cell wall of budding yeasts is a complex and dynamic structure; its arrangement alters as the cell grows, and its composition changes in response to different environmental conditions and at different times during the yeast life cycle. In the past few years, we have witnessed a profilic genetic and molecular characterization of some key aspects of cell wall polymer synthesis and hydrolysis in the budding yeast. Furthermore, this organism has been the target of numerous recent studies on the topic of morphogenesis, which have had an enormous impact on our understanding of the intracellular events that participate in directed cell wall synthesis. A number of components that direct polarized secretion, including those involved in assembly and organization of the actin cytoskeleton, secretory pathways, and a series of novel signal transduction systems and regulatory components have been identified. Analysis of these different components has suggested pathways by which polarized secretion is directed and controlled. Our aim is to offer an overall view of the current understanding of cell wall dynamics and of the complex network that controls polarized growth at particular stages of the budding yeast cell cycle and life cycle. PMID:7565410

Transposable element dynamics and PIWI regulation impacts lncRNA and gene expression diversity in Drosophila ovarian cell cultures.

PubMed

Sytnikova, Yuliya A; Rahman, Reazur; Chirn, Gung-Wei; Clark, Josef P; Lau, Nelson C

2014-12-01

Piwi proteins and Piwi-interacting RNAs (piRNAs) repress transposable elements (TEs) from mobilizing in gonadal cells. To determine the spectrum of piRNA-regulated targets that may extend beyond TEs, we conducted a genome-wide survey for transcripts associated with PIWI and for transcripts affected by PIWI knockdown in Drosophila ovarian somatic sheet (OSS) cells, a follicle cell line expressing the Piwi pathway. Despite the immense sequence diversity among OSS cell piRNAs, our analysis indicates that TE transcripts are the major transcripts associated with and directly regulated by PIWI. However, several coding genes were indirectly regulated by PIWI via an adjacent de novo TE insertion that generated a nascent TE transcript. Interestingly, we noticed that PIWI-regulated genes in OSS cells greatly differed from genes affected in a related follicle cell culture, ovarian somatic cells (OSCs). Therefore, we characterized the distinct genomic TE insertions across four OSS and OSC lines and discovered dynamic TE landscapes in gonadal cultures that were defined by a subset of active TEs. Particular de novo TEs appeared to stimulate the expression of novel candidate long noncoding RNAs (lncRNAs) in a cell lineage-specific manner, and some of these TE-associated lncRNAs were associated with PIWI and overlapped PIWI-regulated genes. Our analyses of OSCs and OSS cells demonstrate that despite having a Piwi pathway to suppress endogenous mobile elements, gonadal cell TE landscapes can still dramatically change and create transcriptome diversity. © 2014 Sytnikova et al.; Published by Cold Spring Harbor Laboratory Press.

Membrane potential dynamics of grid cells

PubMed Central

Domnisoru, Cristina; Kinkhabwala, Amina A.; Tank, David W.

2014-01-01

During navigation, grid cells increase their spike rates in firing fields arranged on a strikingly regular triangular lattice, while their spike timing is often modulated by theta oscillations. Oscillatory interference models of grid cells predict theta amplitude modulations of membrane potential during firing field traversals, while competing attractor network models predict slow depolarizing ramps. Here, using in-vivo whole-cell recordings, we tested these models by directly measuring grid cell intracellular potentials in mice running along linear tracks in virtual reality. Grid cells had large and reproducible ramps of membrane potential depolarization that were the characteristic signature tightly correlated with firing fields. Grid cells also exhibited intracellular theta oscillations that influenced their spike timing. However, the properties of theta amplitude modulations were not consistent with the view that they determine firing field locations. Our results support cellular and network mechanisms in which grid fields are produced by slow ramps, as in attractor models, while theta oscillations control spike timing. PMID:23395984

Four-point bending protocols to study the effects of dynamic strain in osteoblastic cells in vitro.

PubMed

Galea, Gabriel L; Price, Joanna S

2015-01-01

Strain engendered within bone tissue by mechanical loading of the skeleton is a major influence on the processes of bone modeling and remodeling and so a critical determinant of bone mass and architecture. The cells best placed to respond to strain in bone tissue are the resident osteocytes and osteoblasts. To address the mechanisms of strain-related responses in osteoblast-like cells, our group uses both in vivo and in vitro approaches, including a system of four-point bending of the substrate on which cells are cultured. A range of cell lines can be studied using this system but we routinely compare their responses to those in primary cultures of osteoblast-like cells derived from explants of mouse long bones. These cells show a range of well-characterized responses to physiological levels of strain, including increased proliferation, which in vivo is a feature of the osteogenic response.

Time scale of diffusion in molecular and cellular biology

NASA Astrophysics Data System (ADS)

Holcman, D.; Schuss, Z.

2014-05-01

Diffusion is the driver of critical biological processes in cellular and molecular biology. The diverse temporal scales of cellular function are determined by vastly diverse spatial scales in most biophysical processes. The latter are due, among others, to small binding sites inside or on the cell membrane or to narrow passages between large cellular compartments. The great disparity in scales is at the root of the difficulty in quantifying cell function from molecular dynamics and from simulations. The coarse-grained time scale of cellular function is determined from molecular diffusion by the mean first passage time of molecular Brownian motion to a small targets or through narrow passages. The narrow escape theory (NET) concerns this issue. The NET is ubiquitous in molecular and cellular biology and is manifested, among others, in chemical reactions, in the calculation of the effective diffusion coefficient of receptors diffusing on a neuronal cell membrane strewn with obstacles, in the quantification of the early steps of viral trafficking, in the regulation of diffusion between the mother and daughter cells during cell division, and many other cases. Brownian trajectories can represent the motion of a molecule, a protein, an ion in solution, a receptor in a cell or on its membrane, and many other biochemical processes. The small target can represent a binding site or an ionic channel, a hidden active site embedded in a complex protein structure, a receptor for a neurotransmitter on the membrane of a neuron, and so on. The mean time to attach to a receptor or activator determines diffusion fluxes that are key regulators of cell function. This review describes physical models of various subcellular microdomains, in which the NET coarse-grains the molecular scale to a higher cellular-level, thus clarifying the role of cell geometry in determining subcellular function.

Directing stem cell fate on hydrogel substrates by controlling cell geometry, matrix mechanics and adhesion ligand composition.

PubMed

Lee, Junmin; Abdeen, Amr A; Zhang, Douglas; Kilian, Kristopher A

2013-11-01

There is a dynamic relationship between physical and biochemical signals presented in the stem cell microenvironment to guide cell fate determination. Model systems that modulate cell geometry, substrate stiffness or matrix composition have proved useful in exploring how these signals influence stem cell fate. However, the interplay between these physical and biochemical cues during differentiation remains unclear. Here, we demonstrate a microengineering strategy to vary single cell geometry and the composition of adhesion ligands - on substrates that approximate the mechanical properties of soft tissues - to study adipogenesis and neurogenesis in adherent mesenchymal stem cells. Cells cultured in small circular islands show elevated expression of adipogenesis markers while cells that spread in anisotropic geometries tend to express elevated neurogenic markers. Arraying different combinations of matrix protein in a myriad of 2D and pseudo-3D geometries reveals optimal microenvironments for controlling the differentiation of stem cells to these "soft" lineages without the use of media supplements. © 2013 Elsevier Ltd. All rights reserved.

Limiting Energy Dissipation Induces Glassy Kinetics in Single-Cell High-Precision Responses.

PubMed

Das, Jayajit

2016-03-08

Single cells often generate precise responses by involving dissipative out-of-thermodynamic-equilibrium processes in signaling networks. The available free energy to fuel these processes could become limited depending on the metabolic state of an individual cell. How does limiting dissipation affect the kinetics of high-precision responses in single cells? I address this question in the context of a kinetic proofreading scheme used in a simple model of early-time T cell signaling. Using exact analytical calculations and numerical simulations, I show that limiting dissipation qualitatively changes the kinetics in single cells marked by emergence of slow kinetics, large cell-to-cell variations of copy numbers, temporally correlated stochastic events (dynamic facilitation), and ergodicity breaking. Thus, constraints in energy dissipation, in addition to negatively affecting ligand discrimination in T cells, can create a fundamental difficulty in determining single-cell kinetics from cell-population results. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Intestinal Stem Cell Dynamics: A Story of Mice and Humans.

PubMed

Hodder, Michael C; Flanagan, Dustin J; Sansom, Owen J

2018-06-01

Stem cell dynamics define the probability of accumulating mutations within the intestinal epithelium. In this issue of Cell Stem Cell, Nicholson et al. (2018) report that human intestinal stem cell dynamics differ significantly from those of mice and establish that oncogenic mutations are more likely to expand; therefore, "normal" epithelium may carry multiple mutations. Copyright © 2018 Elsevier Inc. All rights reserved.

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

NASA Astrophysics Data System (ADS)

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.

Construction of mutant TKGFP for real-time imaging of temporal dynamics of HIF-1 signal transduction activity mediated by hypoxia and reoxygenation in tumors in living mice.

PubMed

Hsieh, Chia-Hung; Kuo, Jung-Wen; Lee, Yi-Jang; Chang, Chi-Wei; Gelovani, Juri G; Liu, Ren-Shyan

2009-12-01

The herpes simplex virus type 1 thymidine kinase (HSV1-tk)/green fluorescent protein (TKGFP) dual-reporter gene and a multimodality imaging approach play a critical role in monitoring therapeutic gene expression, immune cell trafficking, and protein-protein interactions in translational molecular-genetic imaging. However, the cytotoxicity and low temporal resolution of TKGFP limits its application in studies that require a rapid turnover of the reporter. The purpose of this study was to construct a novel mutant TKGFP fusion reporter gene with low cytotoxicity and high temporal resolution for use in the real-time monitoring of temporal dynamics and spatial heterogeneity of hypoxia-inducible factor 1 (HIF-1) signal transduction activity mediated by hypoxia and reoxygenation in vitro and in vivo. Destabilized TKGFP was produced by inserting the nuclear export signal (NES) sequence at the N terminus and fusing the degradation domain of mouse ornithine decarboxylase (dMODC) at the C terminus. The stability of TKGFP in living NG4TL4 cells was determined by Western blot analysis, HSV1-tk enzyme activity assay, and flow cytometric analysis. The suitability of NESTKGFP:dMODC as a transcription reporter was investigated by linking it to a promoter consisting of 8 copies of hypoxia-responsive elements, whose activities depend on HIF-1. The dynamic transcriptional events mediated by hypoxia and reoxygenation were monitored by NESTKGFP:dMODC or TKGFP and determined by optical imaging and PET. Unlike TKGFP, NESTKGFP:dMODC was unstable in the presence of cycloheximide and showed a short half-life of protein and enzyme activity. Rapid turnover of NESTKGFP:dMODC occurred in a 26S proteasome-dependent manner. Furthermore, NESTKGFP:dMODC showed an upregulated expression and low cytotoxicity in living cells. Studies of hypoxia-responsive TKGFP and NESTKGFP:dMODC expression showed that NESTKGFP:dMODC as a reporter gene had better temporal resolution than did TKGFP for monitoring the dynamic transcriptional events mediated by hypoxia and reoxygenation; the TKGFP expression level was not optimal for the purpose of monitoring. In translational molecular-genetic imaging, NESTKGFP:dMODC as a reporter gene, together with optical imaging and PET, allows the direct monitoring of transcription induction and easy determination of its association with other biochemical changes.

Microfocus diffraction from different regions of a protein crystal: structural variations and unit-cell polymorphism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thompson, Michael C.; Cascio, Duilio; Yeates, Todd O.

Real macromolecular crystals can be non-ideal in a myriad of ways. This often creates challenges for structure determination, while also offering opportunities for greater insight into the crystalline state and the dynamic behavior of macromolecules. To evaluate whether different parts of a single crystal of a dynamic protein, EutL, might be informative about crystal and protein polymorphism, a microfocus X-ray synchrotron beam was used to collect a series of 18 separate data sets from non-overlapping regions of the same crystal specimen. A principal component analysis (PCA) approach was employed to compare the structure factors and unit cells across the datamore » sets, and it was found that the 18 data sets separated into two distinct groups, with largeRvalues (in the 40% range) and significant unit-cell variations between the members of the two groups. This categorization mapped the different data-set types to distinct regions of the crystal specimen. Atomic models of EutL were then refined against two different data sets obtained by separately merging data from the two distinct groups. A comparison of the two resulting models revealed minor but discernable differences in certain segments of the protein structure, and regions of higher deviation were found to correlate with regions where larger dynamic motions were predicted to occur by normal-mode molecular-dynamics simulations. The findings emphasize that large spatially dependent variations may be present across individual macromolecular crystals. This information can be uncovered by simultaneous analysis of multiple partial data sets and can be exploited to reveal new insights about protein dynamics, while also improving the accuracy of the structure-factor data ultimately obtained in X-ray diffraction experiments.« less

On the origins of the universal dynamics of endogenous granules in mammalian cells.

PubMed

Vanapalli, Siva A; Li, Yixuan; Mugele, Frieder; Duits, Michel H G

2009-12-01

Endogenous granules (EGs) that consist of lipid droplets and mitochondria have been commonly used to assess intracellular mechanical properties via multiple particle tracking microrheology (MPTM). Despite their widespread use, the nature of interaction of EGs with the cytoskeletal network and the type of forces driving their dynamics--both of which are crucial for the interpretation of the results from MPTM technique--are yet to be resolved. In this report, we study the dynamics of endogenous granules in mammalian cells using particle tracking methods. We find that the ensemble dynamics of EGs is diffusive in three types of mammalian cells (endothelial cells, smooth muscle cells and fibroblasts), thereby suggesting an apparent universality in their dynamical behavior. Moreover, in a given cell, the amplitude of the mean-squared displacement for EGs is an order of magnitude larger than that of injected particles. This observation along with results from ATP depletion and temperature intervention studies suggests that cytoskeletal active forces drive the dynamics of EGs. To elucidate the dynamical origin of the diffusive-like nonthermal motion, we consider three active force generation mechanisms--molecular motor transport, actomyosin contractility and microtubule polymerization forces. We test these mechanisms using pharmacological interventions. Experimental evidence and model calculations suggest that EGs are intimately linked to microtubules and that microtubule polymerization forces drive their dynamics. Thus, endogenous granules could serve as non-invasive probes for microtubule network dynamics in mammalian cells.

Tcf21 regulates the specification and maturation of proepicardial cells

PubMed Central

Tandon, Panna; Miteva, Yana V.; Kuchenbrod, Lauren M.; Cristea, Ileana M.; Conlon, Frank L.

2013-01-01

The epicardium is a mesothelial cell layer essential for vertebrate heart development and pertinent for cardiac repair post-injury in the adult. The epicardium initially forms from a dynamic precursor structure, the proepicardial organ, from which cells migrate onto the heart surface. During the initial stage of epicardial development crucial epicardial-derived cell lineages are thought to be determined. Here, we define an essential requirement for transcription factor Tcf21 during early stages of epicardial development in Xenopus, and show that depletion of Tcf21 results in a disruption in proepicardial cell specification and failure to form a mature epithelial epicardium. Using a mass spectrometry-based approach we defined Tcf21 interactions and established its association with proteins that function as transcriptional co-repressors. Furthermore, using an in vivo systems-based approach, we identified a panel of previously unreported proepicardial precursor genes that are persistently expressed in the epicardial layer upon Tcf21 depletion, thereby confirming a primary role for Tcf21 in the correct determination of the proepicardial lineage. Collectively, these studies lead us to propose that Tcf21 functions as a transcriptional repressor to regulate proepicardial cell specification and the correct formation of a mature epithelial epicardium. PMID:23637334

An RNA tool kit to study the status of mouse ES cells: sex determination and stemness.

PubMed

Jay, F; Ciaudo, C

2013-09-01

Mouse embryonic stem cells (mESCs) are pluripotent stem cells derived from the inner cell mass of the blastocyst. They can be maintained under controlled culture conditions in a pluripotent state, or be induced to differentiate into all derivatives of the three primary germ layers: ectoderm, endoderm and mesoderm. Several studies have characterised the coding and non-coding (nc) RNA repertoires of mESCs, uncovering highly dynamic variations during the process of differentiation, but also qualitative differences pertaining to sex. For example, up-regulation of the long non-coding RNA Xist on the X chromosome induces gene silencing and X inactivation exclusively during female mESC differentiation. In contrast, specific small RNAs have been shown to be up-regulated during male mESC differentiation. Here, we illustrate how a small set of key coding and ncRNAs can be exploited as dynamic and sensitive markers of the stemness and/or the differentiation status of male or female mESC lines. We describe adapted techniques for the extended characterization and analysis of mESCs from as little material as that cultured in a single 75cm(2) flask. Copyright © 2013 Elsevier Inc. All rights reserved.

Single molecule super-resolution imaging of proteins in living Salmonella enterica using self-labelling enzymes

PubMed Central

Barlag, Britta; Beutel, Oliver; Janning, Dennis; Czarniak, Frederik; Richter, Christian P.; Kommnick, Carina; Göser, Vera; Kurre, Rainer; Fabiani, Florian; Erhardt, Marc; Piehler, Jacob; Hensel, Michael

2016-01-01

The investigation of the subcellular localization, dynamics and interaction of proteins and protein complexes in prokaryotes is complicated by the small size of the cells. Super-resolution microscopy (SRM) comprise various new techniques that allow light microscopy with a resolution that can be up to ten-fold higher than conventional light microscopy. Application of SRM techniques to living prokaryotes demands the introduction of suitable fluorescent probes, usually by fusion of proteins of interest to fluorescent proteins with properties compatible to SRM. Here we describe an approach that is based on the genetically encoded self-labelling enzymes HaloTag and SNAP-tag. Proteins of interest are fused to HaloTag or SNAP-tag and cell permeable substrates can be labelled with various SRM-compatible fluorochromes. Fusions of the enzyme tags to subunits of a type I secretion system (T1SS), a T3SS, the flagellar rotor and a transcription factor were generated and analysed in living Salmonella enterica. The new approach is versatile in tagging proteins of interest in bacterial cells and allows to determine the number, relative subcellular localization and dynamics of protein complexes in living cells. PMID:27534893

Drug-induced cellular death dynamics monitored by a highly sensitive organic electrochemical system.

PubMed

Romeo, Agostino; Tarabella, Giuseppe; D'Angelo, Pasquale; Caffarra, Cristina; Cretella, Daniele; Alfieri, Roberta; Petronini, Pier Giorgio; Iannotta, Salvatore

2015-06-15

We propose and demonstrate a sensitive diagnostic device based on an Organic Electrochemical Transistor (OECT) for direct in-vitro monitoring cell death. The system efficiently monitors cell death dynamics, being able to detect signals related to specific death mechanisms, namely necrosis or early/late apoptosis, demonstrating a reproducible correlation between the OECT electrical response and the trends of standard cell death assays. The innovative design of the Twell-OECT system has been modeled to better correlate electrical signals with cell death dynamics. To qualify the device, we used a human lung adenocarcinoma cell line (A549) that was cultivated on the micro-porous membrane of a Transwell (Twell) support, and exposed to the anticancer drug doxorubicin. Time-dependent and dose-dependent dynamics of A549 cells exposed to doxorubicin are evaluated by monitoring cell death upon exposure to a range of doses and times that fully covers the protocols used in cancer treatment. The demonstrated ability to directly monitor cell stress and death dynamics upon drug exposure using simple electronic devices and, possibly, achieving selectivity to different cell dynamics is of great interest for several application fields, including toxicology, pharmacology, and therapeutics. Copyright © 2015 Elsevier B.V. All rights reserved.

Dynamic organization of myristoylated Src in the live cell plasma membrane

DOE Office of Scientific and Technical Information (OSTI.GOV)

Smith, Adam W.; Huang, Hector H.; Endres, Nicholas F.

The spatial organization of lipid-anchored proteins in the plasma membrane directly influences cell signaling, but measuring such organization in situ is experimentally challenging. The canonical oncogene, c-Src, is a lipid anchored protein that plays a key role in integrin-mediated signal transduction within focal adhesions and cell–cell junctions. Because of its activity in specific plasma membrane regions, structural motifs within the protein have been hypothesized to play an important role in its subcellular localization. This study used a combination of time-resolved fluorescence fluctuation spectroscopy and super-resolution microscopy to quantify the dynamic organization of c-Src in live cell membranes. Pulsed-interleaved excitation fluorescencemore » cross-correlation spectroscopy (PIE–FCCS) showed that a small fraction of c-Src transiently sorts into membrane clusters that are several times larger than the monomers. Photoactivated localization microscopy (PALM) confirmed that c-Src partitions into clusters with low probability and showed that the characteristic size of the clusters is 10–80 nm. Finally, time-resolved fluorescence anisotropy measurements were used to quantify the rotational mobility of c-Src to determine how it interacts with its local environment. Altogether, these results build a quantitative description of the mobility and clustering behavior of the c-Src nonreceptor tyrosine kinase in the live cell plasma membrane.« less

Dynamic organization of myristoylated Src in the live cell plasma membrane

DOE PAGES

Smith, Adam W.; Huang, Hector H.; Endres, Nicholas F.; ...

2016-01-15

The spatial organization of lipid-anchored proteins in the plasma membrane directly influences cell signaling, but measuring such organization in situ is experimentally challenging. The canonical oncogene, c-Src, is a lipid anchored protein that plays a key role in integrin-mediated signal transduction within focal adhesions and cell–cell junctions. Because of its activity in specific plasma membrane regions, structural motifs within the protein have been hypothesized to play an important role in its subcellular localization. This study used a combination of time-resolved fluorescence fluctuation spectroscopy and super-resolution microscopy to quantify the dynamic organization of c-Src in live cell membranes. Pulsed-interleaved excitation fluorescencemore » cross-correlation spectroscopy (PIE–FCCS) showed that a small fraction of c-Src transiently sorts into membrane clusters that are several times larger than the monomers. Photoactivated localization microscopy (PALM) confirmed that c-Src partitions into clusters with low probability and showed that the characteristic size of the clusters is 10–80 nm. Finally, time-resolved fluorescence anisotropy measurements were used to quantify the rotational mobility of c-Src to determine how it interacts with its local environment. Altogether, these results build a quantitative description of the mobility and clustering behavior of the c-Src nonreceptor tyrosine kinase in the live cell plasma membrane.« less

Exploring the context of the lung proteome within the airway mucosa following allergen challenge.

PubMed

Fehniger, Thomas E; Sato-Folatre, José-Gabriel; Malmström, Johan; Berglund, Magnus; Lindberg, Claes; Brange, Charlotte; Lindberg, Henrik; Marko-Varga, György

2004-01-01

The lung proteome is a dynamic collection of specialized proteins related to pulmonary function. Many cells of different derivations, activation states, and levels of maturity contribute to the changing environment, which produces the lung proteome. Inflammatory cells reacting to environmental challenge, for example from allergens, produce and secrete proteins which have profound effects on both resident and nonresident cells located in airways, alveoli, and the vascular tree which provides blood cells to the parenchyma alveolar bed for gas exchange. In an experimental model of allergic airway inflammation, we have compared control and allergen challenged lung compartments to determine global protein expression patterns using 2D-gel electrophoresis and subsequent spot identification by MS/MS mass spectrometry. We have then specifically isolated the epithelial mucosal layer, which lines conducting airways, from control and allergen challenged lungs, using laser capture technology and performed proteome identification on these selected cell samples. A central component of our investigations has been to contextually relate the histological features of the dynamic pulmonary environment to the changes in protein expression observed following challenge. Our results provide new information of the complexity of the submucosa/epithelium interface and the mechanisms behind the transformation of airway epithelium from normal steady states to functionally activated states.

Modeling the effects of vorinostat in vivo reveals both transient and delayed HIV transcriptional activation and minimal killing of latently infected cells

DOE PAGES

Ke, Ruian; Lewin, Sharon R.; Elliott, Julian H.; ...

2015-10-23

Recent efforts to cure human immunodeficiency virus type-1 (HIV-1) infection have focused on developing latency reversing agents as a first step to eradicate the latent reservoir. The histone deacetylase inhibitor, vorinostat, has been shown to activate HIV RNA transcription in CD4+ T-cells and alter host cell gene transcription in HIV-infected individuals on antiretroviral therapy. In order to understand how latently infected cells respond dynamically to vorinostat treatment and determine the impact of vorinostat on reservoir size in vivo, we have constructed viral dynamic models of latency that incorporate vorinostat treatment. We fitted these models to data collected from a recentmore » clinical trial in which vorinostat was administered daily for 14 days to HIV-infected individuals on suppressive ART. The results show that HIV transcription is increased transiently during the first few hours or days of treatment and that there is a delay before a sustained increase of HIV transcription, whose duration varies among study participants and may depend on the long term impact of vorinostat on host gene expression. Parameter estimation suggests that in latently infected cells, HIV transcription induced by vorinostat occurs at lower levels than in productively infected cells. Lastly, the estimated loss rate of transcriptionally induced cells remains close to baseline in most study participants, suggesting vorinostat treatment does not induce latently infected cell killing and thus reduce the latent reservoir in vivo.« less

Modeling the effects of vorinostat in vivo reveals both transient and delayed HIV transcriptional activation and minimal killing of latently infected cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ke, Ruian; Lewin, Sharon R.; Elliott, Julian H.

Recent efforts to cure human immunodeficiency virus type-1 (HIV-1) infection have focused on developing latency reversing agents as a first step to eradicate the latent reservoir. The histone deacetylase inhibitor, vorinostat, has been shown to activate HIV RNA transcription in CD4+ T-cells and alter host cell gene transcription in HIV-infected individuals on antiretroviral therapy. In order to understand how latently infected cells respond dynamically to vorinostat treatment and determine the impact of vorinostat on reservoir size in vivo, we have constructed viral dynamic models of latency that incorporate vorinostat treatment. We fitted these models to data collected from a recentmore » clinical trial in which vorinostat was administered daily for 14 days to HIV-infected individuals on suppressive ART. The results show that HIV transcription is increased transiently during the first few hours or days of treatment and that there is a delay before a sustained increase of HIV transcription, whose duration varies among study participants and may depend on the long term impact of vorinostat on host gene expression. Parameter estimation suggests that in latently infected cells, HIV transcription induced by vorinostat occurs at lower levels than in productively infected cells. Lastly, the estimated loss rate of transcriptionally induced cells remains close to baseline in most study participants, suggesting vorinostat treatment does not induce latently infected cell killing and thus reduce the latent reservoir in vivo.« less

Simultaneous quantification of 17 trace elements in blood by dynamic reaction cell inductively coupled plasma mass spectrometry (DRC-ICP-MS) equipped with a high-efficiency sample introduction system.

PubMed

D'Ilio, S; Violante, N; Di Gregorio, M; Senofonte, O; Petrucci, F

2006-10-10

A quadrupole inductively coupled plasma mass spectrometer (Q-ICP-MS) equipped with a dynamic reaction cell (DRC) and coupled with a desolvating nebulization system (APEX-IR) was employed to determine 17 elements (Al, As, Ba, Cd, Co, Cr, Li, Mn, Mo, Ni, Pb, Sb, Se, Sn, Sr, V, and Zr) in blood samples. Ammonia (for Al, Cr, Mn, and V) and O2 (for As and Se) were used as reacting gases. Selection of the best flow rate of the gases and optimization of the quadrupole dynamic bandpass tuning parameter (RPq) were carried out, using digested blood diluted 1+9 with deionized water and spiked with 1 microg L(-1) of Al, Cr, Mn, V and 5 microgL(-1) of As and Se. Detection limits were determined in digested blood using the 3sigma criterion. The desolvating system allowed a sufficient sensitivity to be achieved to determine elements at levels of ng L(-1) without detriment of signal stability. The accuracy of the method was tested with the whole blood certified reference material (CRM), certified for Al, As, Cd, Co, Cr, Mn, Mo, Ni, Pb, Sb, Se, and V, and with indicative values for Ba, Li, Sn, Sr, and Zr. The addition calibration approach was chosen for analysis. In order to confirm the DRC data, samples were also analyzed by means of sector field inductively coupled plasma mass spectrometry (SF-ICP-MS), operating in medium (m/Deltam=4000) and high (m/Deltam=10,000) resolution mode and achieving a good agreement between the two techniques.

Fluid Mechanics of Blood Clot Formation.

PubMed

Fogelson, Aaron L; Neeves, Keith B

2015-01-01

Intravascular blood clots form in an environment in which hydrodynamic forces dominate and in which fluid-mediated transport is the primary means of moving material. The clotting system has evolved to exploit fluid dynamic mechanisms and to overcome fluid dynamic challenges to ensure that clots that preserve vascular integrity can form over the wide range of flow conditions found in the circulation. Fluid-mediated interactions between the many large deformable red blood cells and the few small rigid platelets lead to high platelet concentrations near vessel walls where platelets contribute to clotting. Receptor-ligand pairs with diverse kinetic and mechanical characteristics work synergistically to arrest rapidly flowing cells on an injured vessel. Variations in hydrodynamic stresses switch on and off the function of key clotting polymers. Protein transport to, from, and within a developing clot determines whether and how fast it grows. We review ongoing experimental and modeling research to understand these and related phenomena.

Three-Dimensional Simulation of Liquid Drop Dynamics Within Unsaturated Vertical Hele-Shaw Cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hai Huang; Paul Meakin

A three-dimensional, multiphase fluid flow model with volume of fluid-interface tracking was developed and applied to study the multiphase dynamics of moving liquid drops of different sizes within vertical Hele-Shaw cells. The simulated moving velocities are significantly different from those obtained from a first-order analytical approximation, based on simple force-balance concepts. The simulation results also indicate that the moving drops can exhibit a variety of shapes and that the transition among these different shapes is largely determined by the moving velocities. More important, there is a transition from a linear moving regime at small capillary numbers, in which the capillarymore » number scales linearly with the Bond number, to a nonlinear moving regime at large capillary numbers, in which the moving drop releases a train of droplets from its trailing edge. The train of droplets forms a variety of patterns at different moving velocities.« less

Fluid Mechanics of Blood Clot Formation

NASA Astrophysics Data System (ADS)

Fogelson, Aaron L.; Neeves, Keith B.

2015-01-01

Intravascular blood clots form in an environment in which hydrodynamic forces dominate and in which fluid-mediated transport is the primary means of moving material. The clotting system has evolved to exploit fluid dynamic mechanisms and to overcome fluid dynamic challenges to ensure that clots that preserve vascular integrity can form over the wide range of flow conditions found in the circulation. Fluid-mediated interactions between the many large deformable red blood cells and the few small rigid platelets lead to high platelet concentrations near vessel walls where platelets contribute to clotting. Receptor-ligand pairs with diverse kinetic and mechanical characteristics work synergistically to arrest rapidly flowing cells on an injured vessel. Variations in hydrodynamic stresses switch on and off the function of key clotting polymers. Protein transport to, from, and within a developing clot determines whether and how fast it grows. We review ongoing experimental and modeling research to understand these and related phenomena.

Vertebrate homologues of Frodo are dynamically expressed during embryonic development in tissues undergoing extensive morphogenetic movements.

PubMed

Hunter, Nina L; Hikasa, Hiroki; Dymecki, Susan M; Sokol, Sergei Y

2006-01-01

Frodo has been identified as a protein interacting with Dishevelled, an essential mediator of the Wnt signaling pathway, critical for the determination of cell fate and polarity in embryonic development. In this study, we use specific gene probes to characterize stage- and tissue-specific expression patterns of the mouse Frodo homologue and compare them with Frodo expression patterns in Xenopus embryos. In situ hybridization analysis of mouse Frodo transcripts demonstrates that, similar to Xenopus Frodo, mouse Frodo is expressed in primitive streak mesoderm, neuroectoderm, neural crest, presomitic mesoderm, and somites. In many cases, Frodo expression is confined to tissues undergoing extensive morphogenesis, suggesting that Frodo may be involved in the regulation of cell shape and motility. Highly conserved dynamic expression patterns of Frodo homologues indicate a similar function for these proteins in different vertebrates. 2005 Wiley-Liss, Inc.

Large-scale inference of protein tissue origin in gram-positive sepsis plasma using quantitative targeted proteomics

PubMed Central

Malmström, Erik; Kilsgård, Ola; Hauri, Simon; Smeds, Emanuel; Herwald, Heiko; Malmström, Lars; Malmström, Johan

2016-01-01

The plasma proteome is highly dynamic and variable, composed of proteins derived from surrounding tissues and cells. To investigate the complex processes that control the composition of the plasma proteome, we developed a mass spectrometry-based proteomics strategy to infer the origin of proteins detected in murine plasma. The strategy relies on the construction of a comprehensive protein tissue atlas from cells and highly vascularized organs using shotgun mass spectrometry. The protein tissue atlas was transformed to a spectral library for highly reproducible quantification of tissue-specific proteins directly in plasma using SWATH-like data-independent mass spectrometry analysis. We show that the method can determine drastic changes of tissue-specific protein profiles in blood plasma from mouse animal models with sepsis. The strategy can be extended to several other species advancing our understanding of the complex processes that contribute to the plasma proteome dynamics. PMID:26732734

Jagged–Delta asymmetry in Notch signaling can give rise to a Sender/Receiver hybrid phenotype

PubMed Central

Boareto, Marcelo; Jolly, Mohit Kumar; Lu, Mingyang; Onuchic, José N.; Clementi, Cecilia; Ben-Jacob, Eshel

2015-01-01

Notch signaling pathway mediates cell-fate determination during embryonic development, wound healing, and tumorigenesis. This pathway is activated when the ligand Delta or the ligand Jagged of one cell interacts with the Notch receptor of its neighboring cell, releasing the Notch Intracellular Domain (NICD) that activates many downstream target genes. NICD affects ligand production asymmetrically––it represses Delta, but activates Jagged. Although the dynamical role of Notch–Jagged signaling remains elusive, it is widely recognized that Notch–Delta signaling behaves as an intercellular toggle switch, giving rise to two distinct fates that neighboring cells adopt––Sender (high ligand, low receptor) and Receiver (low ligand, high receptor). Here, we devise a specific theoretical framework that incorporates both Delta and Jagged in Notch signaling circuit to explore the functional role of Jagged in cell-fate determination. We find that the asymmetric effect of NICD renders the circuit to behave as a three-way switch, giving rise to an additional state––a hybrid Sender/Receiver (medium ligand, medium receptor). This phenotype allows neighboring cells to both send and receive signals, thereby attaining similar fates. We also show that due to the asymmetric effect of the glycosyltransferase Fringe, different outcomes are generated depending on which ligand is dominant: Delta-mediated signaling drives neighboring cells to have an opposite fate; Jagged-mediated signaling drives the cell to maintain a similar fate to that of its neighbor. We elucidate the role of Jagged in cell-fate determination and discuss its possible implications in understanding tumor–stroma cross-talk, which frequently entails Notch–Jagged communication. PMID:25605936

During running in place, grid cells integrate elapsed time and distance run

PubMed Central

Kraus, Benjamin J.; Brandon, Mark P.; Robinson, Robert J.; Connerney, Michael A.; Hasselmo, Michael E.; Eichenbaum, Howard

2015-01-01

Summary The spatial scale of grid cells may be provided by self-generated motion information or by external sensory information from environmental cues. To determine whether grid cell activity reflects distance traveled or elapsed time independent of external information, we recorded grid cells as animals ran in place on a treadmill. Grid cell activity was only weakly influenced by location but most grid cells and other neurons recorded from the same electrodes strongly signaled a combination of distance and time, with some signaling only distance or time. Grid cells were more sharply tuned to time and distance than non-grid cells. Many grid cells exhibited multiple firing fields during treadmill running, parallel to the periodic firing fields observed in open fields, suggesting a common mode of information processing. These observations indicate that, in the absence of external dynamic cues, grid cells integrate self-generated distance and time information to encode a representation of experience. PMID:26539893

Whole-genome fingerprint of the DNA methylome during human B cell differentiation.

PubMed

Kulis, Marta; Merkel, Angelika; Heath, Simon; Queirós, Ana C; Schuyler, Ronald P; Castellano, Giancarlo; Beekman, Renée; Raineri, Emanuele; Esteve, Anna; Clot, Guillem; Verdaguer-Dot, Néria; Duran-Ferrer, Martí; Russiñol, Nuria; Vilarrasa-Blasi, Roser; Ecker, Simone; Pancaldi, Vera; Rico, Daniel; Agueda, Lidia; Blanc, Julie; Richardson, David; Clarke, Laura; Datta, Avik; Pascual, Marien; Agirre, Xabier; Prosper, Felipe; Alignani, Diego; Paiva, Bruno; Caron, Gersende; Fest, Thierry; Muench, Marcus O; Fomin, Marina E; Lee, Seung-Tae; Wiemels, Joseph L; Valencia, Alfonso; Gut, Marta; Flicek, Paul; Stunnenberg, Hendrik G; Siebert, Reiner; Küppers, Ralf; Gut, Ivo G; Campo, Elías; Martín-Subero, José I

2015-07-01

We analyzed the DNA methylome of ten subpopulations spanning the entire B cell differentiation program by whole-genome bisulfite sequencing and high-density microarrays. We observed that non-CpG methylation disappeared upon B cell commitment, whereas CpG methylation changed extensively during B cell maturation, showing an accumulative pattern and affecting around 30% of all measured CpG sites. Early differentiation stages mainly displayed enhancer demethylation, which was associated with upregulation of key B cell transcription factors and affected multiple genes involved in B cell biology. Late differentiation stages, in contrast, showed extensive demethylation of heterochromatin and methylation gain at Polycomb-repressed areas, and genes with apparent functional impact in B cells were not affected. This signature, which has previously been linked to aging and cancer, was particularly widespread in mature cells with an extended lifespan. Comparing B cell neoplasms with their normal counterparts, we determined that they frequently acquire methylation changes in regions already undergoing dynamic methylation during normal B cell differentiation.

Active tissue stiffness modulation controls valve interstitial cell phenotype and osteogenic potential in 3D culture.

PubMed

Duan, Bin; Yin, Ziying; Hockaday Kang, Laura; Magin, Richard L; Butcher, Jonathan T

2016-05-01

Calcific aortic valve disease (CAVD) progression is a highly dynamic process whereby normally fibroblastic valve interstitial cells (VIC) undergo osteogenic differentiation, maladaptive extracellular matrix (ECM) composition, structural remodeling, and tissue matrix stiffening. However, how VIC with different phenotypes dynamically affect matrix properties and how the altered matrix further affects VIC phenotypes in response to physiological and pathological conditions have not yet been determined. In this study, we develop 3D hydrogels with tunable matrix stiffness to investigate the dynamic interplay between VIC phenotypes and matrix biomechanics. We find that VIC populated within hydrogels with valve leaflet like stiffness differentiate towards myofibroblasts in osteogenic media, but surprisingly undergo osteogenic differentiation when cultured within lower initial stiffness hydrogels. VIC differentiation progressively stiffens the hydrogel microenvironment, which further upregulates both early and late osteogenic markers. These findings identify a dynamic positive feedback loop that governs acceleration of VIC calcification. Temporal stiffening of pathologically lower stiffness matrix back to normal level, or blocking the mechanosensitive RhoA/ROCK signaling pathway, delays the osteogenic differentiation process. Therefore, direct ECM biomechanical modulation can affect VIC phenotypes towards and against osteogenic differentiation in 3D culture. These findings highlight the importance of the homeostatic maintenance of matrix stiffness to restrict pathological VIC differentiation. We implement 3D hydrogels with tunable matrix stiffness to investigate the dynamic interaction between valve interstitial cells (VIC, major cell population in heart valve) and matrix biomechanics. This work focuses on how human VIC responses to changing 3D culture environments. Our findings identify a dynamic positive feedback loop that governs acceleration of VIC calcification, which is the hallmark of calcific aortic valve disease. Temporal stiffening of pathologically lower stiffness matrix back to normal level, or blocking the mechanosensitive signaling pathway, delays VIC osteogenic differentiation. Our findings provide an improved understanding of VIC-matrix interactions to aid in interpretation of VIC calcification studies in vitro and suggest that ECM disruption resulting in local tissue stiffness decreases may promote calcific aortic valve disease. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

An analysis of cross-coupling of a multicomponent jet engine test stand using finite element modeling techniques

NASA Technical Reports Server (NTRS)

Schweikhard, W. G.; Singnoi, W. N.

1985-01-01

A two axis thrust measuring system was analyzed by using a finite a element computer program to determine the sensitivities of the thrust vectoring nozzle system to misalignment of the load cells and applied loads, and the stiffness of the structural members. Three models were evaluated: (1) the basic measuring element and its internal calibration load cells; (2) the basic measuring element and its external load calibration equipment; and (3) the basic measuring element, external calibration load frame and the altitude facility support structure. Alignment of calibration loads was the greatest source of error for multiaxis thrust measuring systems. Uniform increases or decreases in stiffness of the members, which might be caused by the selection of the materials, have little effect on the accuracy of the measurements. It is found that the POLO-FINITE program is a viable tool for designing and analyzing multiaxis thrust measurement systems. The response of the test stand to step inputs that might be encountered with thrust vectoring tests was determined. The dynamic analysis show a potential problem for measuring the dynamic response characteristics of thrust vectoring systems because of the inherently light damping of the test stand.

ICP-MS determination of trace elements in aerosols using a dynamic reaction cell: first results in PM10 comparing road and aerial traffic in Nice area (France).

PubMed

Fabretti, Jean-François; Sauret, Nathalie; Gal, Jean-François; Maria, Pierre-Charles; Schärer, Urs

2007-09-01

An analytical methodology was developed for the determination of 21 trace elements in suspended particulate matter (PM) using a microwave digestion procedure associated with an inductively coupled plasma mass spectrometry (ICP-MS). The dynamic reaction cell (DRC) of the instrument was carefully optimized to eliminate polyatomic species causing spectral interferences for three specified elements (Cr, Fe, Mn). With this method, the detection limits based on the analysis of seven quartz fibre filters (QFF) considering a one-week sample (250 m3) varied between 0.2 and 650 pg m(-3) for trace elements and between 2.1 and 5.6 ng m(-3) for major elements (Fe, Ti, Zn). The recovery of the analytes was tested with NIST SRM 1648 urban dust within 10% of the certified values only for 3-4 mg of material. The first results were discussed for a field campaign which was carried out simultaneously in the heaviest traffic road tunnel of the centre of Nice and near the landing-taking-off runways in the international airport of Nice Côte d'Azur. The behaviour of some combustion tracers was especially studied.

Design and evaluation of a microfluidic system for inhibition studies of yeast cell signaling

NASA Astrophysics Data System (ADS)

Hamngren, Charlotte; Dinér, Peter; Grøtli, Morten; Goksör, Mattias; Adiels, Caroline B.

2012-10-01

In cell signaling, different perturbations lead to different responses and using traditional biological techniques that result in averaged data may obscure important cell-to-cell variations. The aim of this study was to develop and evaluate a four-inlet microfluidic system that enables single-cell analysis by investigating the effect on Hog1 localization post a selective Hog1 inhibitor treatment during osmotic stress. Optical tweezers was used to position yeast cells in an array of desired size and density inside the microfluidic system. By changing the flow rates through the inlet channels, controlled and rapid introduction of two different perturbations over the cell array was enabled. The placement of the cells was determined by diffusion rates flow simulations. The system was evaluated by monitoring the subcellular localization of a fluorescently tagged kinase of the yeast "High Osmolarity Glycerol" (HOG) pathway, Hog1-GFP. By sequential treatment of the yeast cells with a selective Hog1 kinase inhibitor and sorbitol, the subcellular localization of Hog1-GFP was analysed on a single-cell level. The results showed impaired Hog1-GFP nuclear localization, providing evidence of a congenial design. The setup made it possible to remove and add an agent within 2 seconds, which is valuable for investigating the dynamic signal transduction pathways and cannot be done using traditional methods. We are confident that the features of the four-inlet microfluidic system will be a valuable tool and hence contribute significantly to unravel the mechanisms of the HOG pathway and similar dynamic signal transduction pathways.

Static and dynamic strain energy release rates in toughened thermosetting composite laminates

NASA Technical Reports Server (NTRS)

Cairns, Douglas S.

1992-01-01

In this work, the static and dynamic fracture properties of several thermosetting resin based composite laminates are presented. Two classes of materials are explored. These are homogeneous, thermosetting resins and toughened, multi-phase, thermosetting resin systems. Multi-phase resin materials have shown enhancement over homogenous materials with respect to damage resistance. The development of new dynamic tests are presented for composite laminates based on Width Tapered Double Cantilevered Beam (WTDCB) for Mode 1 fracture and the End Notched Flexure (ENF) specimen. The WTDCB sample was loaded via a low inertia, pneumatic cylinder to produce rapid cross-head displacements. A high rate, piezo-electric load cell and an accelerometer were mounted on the specimen. A digital oscilloscope was used for data acquisition. Typical static and dynamic load versus displacement plots are presented. The ENF specimen was impacted in three point bending with an instrumented impact tower. Fracture initiation and propagation energies under static and dynamic conditions were determined analytically and experimentally. The test results for Mode 1 fracture are relatively insensitive to strain rate effects for the laminates tested in this study. The test results from Mode 2 fracture indicate that the toughened systems provide superior fracture initiation and higher resistance to propagation under dynamic conditions. While the static fracture properties of the homogeneous systems may be relatively high, the apparent Mode 2 dynamic critical strain energy release rate drops significantly. The results indicate that static Mode 2 fracture testing is inadequate for determining the fracture performance of composite structures subjected to conditions such as low velocity impact. A good correlation between the basic Mode 2 dynamic fracture properties and the performance is a combined material/structural Compression After Impact (CAI) test is found. These results underscore the importance of examining rate-dependent behavior for determining the longevity of structures manufactured from composite materials.

Microenvironmental Stiffness of 3D Polymeric Structures to Study Invasive Rates of Cancer Cells.

PubMed

Lemma, Enrico Domenico; Spagnolo, Barbara; Rizzi, Francesco; Corvaglia, Stefania; Pisanello, Marco; De Vittorio, Massimo; Pisanello, Ferruccio

2017-11-01

Cells are highly dynamic elements, continuously interacting with the extracellular environment. Mechanical forces sensed and applied by cells are responsible for cellular adhesion, motility, and deformation, and are heavily involved in determining cancer spreading and metastasis formation. Cell/extracellular matrix interactions are commonly analyzed with the use of hydrogels and 3D microfabricated scaffolds. However, currently available techniques have a limited control over the stiffness of microscaffolds and do not allow for separating environmental properties from biological processes in driving cell mechanical behavior, including nuclear deformability and cell invasiveness. Herein, a new approach is presented to study tumor cell invasiveness by exploiting an innovative class of polymeric scaffolds based on two-photon lithography to control the stiffness of deterministic microenvironments in 3D. This is obtained by fine-tuning of the laser power during the lithography, thus locally modifying both structural and mechanical properties in the same fabrication process. Cage-like structures and cylindric stent-like microscaffolds are fabricated with different Young's modulus and stiffness gradients, allowing obtaining new insights on the mechanical interplay between tumor cells and the surrounding environments. In particular, cell invasion is mostly driven by softer architectures, and the introduction of 3D stiffness "weak spots" is shown to boost the rate at which cancer cells invade the scaffolds. The possibility to modulate structural compliance also allowed estimating the force distribution exerted by a single cell on the scaffold, revealing that both pushing and pulling forces are involved in the cell-structure interaction. Overall, exploiting this method to obtain a wide range of 3D architectures with locally engineered stiffness can pave the way for unique applications to study tumor cell dynamics. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

NASA Astrophysics Data System (ADS)

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.

Dynamic Finite Element Predictions for Mars Sample Return Cellular Impact Test #4

NASA Technical Reports Server (NTRS)

Fasanella, Edwin L.; Billings, Marcus D.

2001-01-01

The nonlinear finite element program MSC.Dytran was used to predict the impact pulse for (he drop test of an energy absorbing cellular structure. This pre-test simulation was performed to aid in the design of an energy absorbing concept for a highly reliable passive Earth Entry Vehicle (EEV) that will directly impact the Earth without a parachute. In addition, a goal of the simulation was to bound the acceleration pulse produced and delivered to the simulated space cargo container. EEV's are designed to return materials from asteroids, comets, or planets for laboratory analysis on Earth. The EEV concept uses an energy absorbing cellular structure designed to contain and limit the acceleration of space exploration samples during Earth impact. The spherical shaped cellular structure is composed of solid hexagonal and pentagonal foam-filled cells with hybrid graphite-epoxy/Kevlar cell walls. Space samples fit inside a smaller sphere at the enter of the EEV's cellular structure. The material models and failure criteria were varied to determine their effect on the resulting acceleration pulse. Pre-test analytical predictions using MSC.Dytran were compared with the test results obtained from impact test #4 using bungee accelerator located at the NASA Langley Research Center Impact Dynamics Research Facility. The material model used to represent the foam and the proper failure criteria for the cell walls were critical in predicting the impact loads of the cellular structure. It was determined that a FOAMI model for the foam and a 20% failure strain criteria for the cell walls gave an accurate prediction of the acceleration pulse for drop test #4.

Molecular modeling of the conformational dynamics of the cellular prion protein

NASA Astrophysics Data System (ADS)

Nguyen, Charles; Colling, Ian; Bartz, Jason; Soto, Patricia

2014-03-01

Prions are infectious agents responsible for transmissible spongiform encephalopathies (TSEs), a type of fatal neurodegenerative disease in mammals. Prions propagate biological information by conversion of the non-pathological version of the prion protein to the infectious conformation, PrPSc. A wealth of knowledge has shed light on the nature and mechanism of prion protein conversion. In spite of the significance of this problem, we are far from fully understanding the conformational dynamics of the cellular isoform. To remedy this situation we employ multiple biomolecular modeling techniques such as docking and molecular dynamics simulations to map the free energy landscape and determine what specific regions of the prion protein are most conductive to binding. The overall goal is to characterize the conformational dynamics of the cell form of the prion protein, PrPc, to gain insight into inhibition pathways against misfolding. NE EPSCoR FIRST Award to Patricia Soto.

Chemical imaging of molecular changes in a hydrated single cell by dynamic secondary ion mass spectrometry and super-resolution microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hua, Xin; Szymanski, Craig; Wang, Zhaoying

2016-01-01

Chemical imaging of single cells is important in capturing biological dynamics. Single cell correlative imaging is realized between structured illumination microscopy (SIM) and time-of-flight secondary ion mass spectrometry (ToF-SIMS) using System for Analysis at the Liquid Vacuum Interface (SALVI), a multimodal microreactor. SIM characterized cells and guided subsequent ToF-SIMS analysis. Dynamic ToF-SIMS provided time- and space-resolved cell molecular mapping. Lipid fragments were identified in the hydrated cell membrane. Principal component analysis was used to elucidate chemical component differences among mouse lung cells that uptake zinc oxide nanoparticles. Our results provided submicron chemical spatial mapping for investigations of cell dynamics atmore » the molecular level.« less

Quantifying Cell Fate Decisions for Differentiation and Reprogramming of a Human Stem Cell Network: Landscape and Biological Paths

PubMed Central

Li, Chunhe; Wang, Jin

2013-01-01

Cellular reprogramming has been recently intensively studied experimentally. We developed a global potential landscape and kinetic path framework to explore a human stem cell developmental network composed of 52 genes. We uncovered the underlying landscape for the stem cell network with two basins of attractions representing stem and differentiated cell states, quantified and exhibited the high dimensional biological paths for the differentiation and reprogramming process, connecting the stem cell state and differentiated cell state. Both the landscape and non-equilibrium curl flux determine the dynamics of cell differentiation jointly. Flux leads the kinetic paths to be deviated from the steepest descent gradient path, and the corresponding differentiation and reprogramming paths are irreversible. Quantification of paths allows us to find out how the differentiation and reprogramming occur and which important states they go through. We show the developmental process proceeds as moving from the stem cell basin of attraction to the differentiation basin of attraction. The landscape topography characterized by the barrier heights and transition rates quantitatively determine the global stability and kinetic speed of cell fate decision process for development. Through the global sensitivity analysis, we provided some specific predictions for the effects of key genes and regulation connections on the cellular differentiation or reprogramming process. Key links from sensitivity analysis and biological paths can be used to guide the differentiation designs or reprogramming tactics. PMID:23935477

Cellular fluid mechanics.

PubMed

Kamm, Roger D

2002-01-01

The coupling of fluid dynamics and biology at the level of the cell is an intensive area of investigation because of its critical role in normal physiology and disease. Microcirculatory flow has been a focus for years, owing to the complexity of cell-cell or cell-glycocalyx interactions. Noncirculating cells, particularly those that comprise the walls of the circulatory system, experience and respond biologically to fluid dynamic stresses. In this article, we review the more recent studies of circulating cells, with an emphasis on the role of the glycocalyx on red-cell motion in small capillaries and on the deformation of leukocytes passing through the microcirculation. We also discuss flows in the vicinity of noncirculating cells, the influence of fluid dynamic shear stress on cell biology, and diffusion in the lipid bi-layer, all in the context of the important fluid-dynamic phenomena.

ODE constrained mixture modelling: a method for unraveling subpopulation structures and dynamics.

PubMed

Hasenauer, Jan; Hasenauer, Christine; Hucho, Tim; Theis, Fabian J

2014-07-01

Functional cell-to-cell variability is ubiquitous in multicellular organisms as well as bacterial populations. Even genetically identical cells of the same cell type can respond differently to identical stimuli. Methods have been developed to analyse heterogeneous populations, e.g., mixture models and stochastic population models. The available methods are, however, either incapable of simultaneously analysing different experimental conditions or are computationally demanding and difficult to apply. Furthermore, they do not account for biological information available in the literature. To overcome disadvantages of existing methods, we combine mixture models and ordinary differential equation (ODE) models. The ODE models provide a mechanistic description of the underlying processes while mixture models provide an easy way to capture variability. In a simulation study, we show that the class of ODE constrained mixture models can unravel the subpopulation structure and determine the sources of cell-to-cell variability. In addition, the method provides reliable estimates for kinetic rates and subpopulation characteristics. We use ODE constrained mixture modelling to study NGF-induced Erk1/2 phosphorylation in primary sensory neurones, a process relevant in inflammatory and neuropathic pain. We propose a mechanistic pathway model for this process and reconstructed static and dynamical subpopulation characteristics across experimental conditions. We validate the model predictions experimentally, which verifies the capabilities of ODE constrained mixture models. These results illustrate that ODE constrained mixture models can reveal novel mechanistic insights and possess a high sensitivity.

Dynamics of heat shock protein 60 in endothelial cells exposed to cigarette smoke extract

PubMed Central

Kreutmayer, Simone Barbara; Messner, Barbara; Knoflach, Michael; Henderson, Blair; Niederegger, Harald; Böck, Günther; Van der Zee, Ruurd; Wick, Georg; Bernhard, David

2011-01-01

Heat shock protein 60 (HSP60), expressed on the surface of endothelial cells (ECs) stressed by e.g. oxidized LDL or mechanical shear, was shown to function as an auto-antigen and thus as a pro-atherosclerotic molecule. The aim of this study was to determine whether cigarette smoke chemicals can lead to the activation of the “HSP60 pathway.” It was also our aim to elucidate the dynamics of HSP60 from gene expression to endothelial surface expression and secretion. Here we show for the first time that the exposure of human umbilical vein endothelial cells (HUVECs) to cigarette smoke extract (CSE) results in an up-regulation of HSP60 mRNA. Live cell imaging analysis of a HSP60-EYFP fusion protein construct transfected into ECs revealed that mitochondrial structures collapse in response to CSE exposure. As a result, HSP60 is released from the mitochondria, transported to the cell surface, and released into the cell culture supernatant. Analysis of HSP60 in the sera of healthy young individuals exposed to secondhand smoke revealed significantly elevated levels of HSP60. Cigarette smoking is one of the most relevant risk factors for atherosclerosis. Herein, we provide evidence that cigarette smoke may initiate atherosclerosis in the sense of the “auto-immune hypothesis of atherosclerosis.” PMID:21798264

Dynamics of heat shock protein 60 in endothelial cells exposed to cigarette smoke extract.

PubMed

Kreutmayer, Simone Barbara; Messner, Barbara; Knoflach, Michael; Henderson, Blair; Niederegger, Harald; Böck, Günther; Van der Zee, Ruurd; Wick, Georg; Bernhard, David

2011-11-01

Heat shock protein 60 (HSP60), expressed on the surface of endothelial cells (ECs) stressed by e.g. oxidized LDL or mechanical shear, was shown to function as an auto-antigen and thus as a pro-atherosclerotic molecule. The aim of this study was to determine whether cigarette smoke chemicals can lead to the activation of the "HSP60 pathway." It was also our aim to elucidate the dynamics of HSP60 from gene expression to endothelial surface expression and secretion. Here we show for the first time that the exposure of human umbilical vein endothelial cells (HUVECs) to cigarette smoke extract (CSE) results in an up-regulation of HSP60 mRNA. Live cell imaging analysis of a HSP60-EYFP fusion protein construct transfected into ECs revealed that mitochondrial structures collapse in response to CSE exposure. As a result, HSP60 is released from the mitochondria, transported to the cell surface, and released into the cell culture supernatant. Analysis of HSP60 in the sera of healthy young individuals exposed to secondhand smoke revealed significantly elevated levels of HSP60. Cigarette smoking is one of the most relevant risk factors for atherosclerosis. Herein, we provide evidence that cigarette smoke may initiate atherosclerosis in the sense of the "auto-immune hypothesis of atherosclerosis." Copyright © 2011 Elsevier Ltd. All rights reserved.

Auditory hair cell centrioles undergo confined Brownian motion throughout the developmental migration of the kinocilium.

PubMed

Lepelletier, Léa; de Monvel, Jacques Boutet; Buisson, Johanna; Desdouets, Chantal; Petit, Christine

2013-07-02

Planar polarization of the forming hair bundle, the mechanosensory antenna of auditory hair cells, depends on the poorly characterized center-to-edge displacement of a primary cilium, the kinocilium, at their apical surface. Taking advantage of the gradient of hair cell differentiation along the cochlea, we reconstituted a map of the kinocilia displacements in the mouse embryonic cochlea. We then developed a cochlear organotypic culture and video-microscopy approach to monitor the movements of the kinocilium basal body (mother centriole) and its daughter centriole, which we analyzed using particle tracking and modeling. We found that both hair cell centrioles undergo confined Brownian movements around their equilibrium positions, under the apparent constraint of a radial restoring force of ∼0.1 pN. This magnitude depended little on centriole position, suggesting nonlinear interactions with constraining, presumably cytoskeletal elements. The only dynamic change observed during the period of kinocilium migration was a doubling of the centrioles' confinement area taking place early in the process. It emerges from these static and dynamic observations that kinocilia migrate gradually in parallel with the organization of hair cells into rows during cochlear neuroepithelium extension. Analysis of the confined motion of hair cell centrioles under normal and pathological conditions should help determine which structures contribute to the restoring force exerting on them. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Probing GFP-actin diffusion in living cells using fluorescence correlation spectroscopy.

PubMed

Engelke, Hanna; Heinrich, Doris; Rädler, Joachim O

2010-12-22

The cytoskeleton of eukaryotic cells is continuously remodeled by polymerization and depolymerization of actin. Consequently, the relative content of polymerized filamentous actin (F-actin) and monomeric globular actin (G-actin) is subject to temporal and spatial fluctuations. Since fluorescence correlation spectroscopy (FCS) can measure the diffusion of fluorescently labeled actin it seems likely that FCS allows us to determine the dynamics and hence indirectly the structural properties of the cytoskeleton components with high spatial resolution. To this end we investigate the FCS signal of GFP-actin in living Dictyostelium discoideum cells and explore the inherent spatial and temporal signatures of the actin cytoskeleton. Using the free green fluorescent protein (GFP) as a reference, we find that actin diffusion inside cells is dominated by G-actin and slower than diffusion in diluted cell extract. The FCS signal in the dense cortical F-actin network near the cell membrane is probed using the cytoskeleton protein LIM and is found to be slower than cytosolic G-actin diffusion. Furthermore, we show that polymerization of the cytoskeleton induced by Jasplakinolide leads to a substantial decrease of G-actin diffusion. Pronounced fluctuations in the distribution of the FCS correlation curves can be induced by latrunculin, which is known to induce actin waves. Our work suggests that the FCS signal of GFP-actin in combination with scanning or spatial correlation techniques yield valuable information about the local dynamics and concomitant cytoskeletal properties.

Auditory Hair Cell Centrioles Undergo Confined Brownian Motion Throughout the Developmental Migration of the Kinocilium

PubMed Central

Lepelletier, Léa; de Monvel, Jacques Boutet; Buisson, Johanna; Desdouets, Chantal; Petit, Christine

2013-01-01

Planar polarization of the forming hair bundle, the mechanosensory antenna of auditory hair cells, depends on the poorly characterized center-to-edge displacement of a primary cilium, the kinocilium, at their apical surface. Taking advantage of the gradient of hair cell differentiation along the cochlea, we reconstituted a map of the kinocilia displacements in the mouse embryonic cochlea. We then developed a cochlear organotypic culture and video-microscopy approach to monitor the movements of the kinocilium basal body (mother centriole) and its daughter centriole, which we analyzed using particle tracking and modeling. We found that both hair cell centrioles undergo confined Brownian movements around their equilibrium positions, under the apparent constraint of a radial restoring force of ∼0.1 pN. This magnitude depended little on centriole position, suggesting nonlinear interactions with constraining, presumably cytoskeletal elements. The only dynamic change observed during the period of kinocilium migration was a doubling of the centrioles’ confinement area taking place early in the process. It emerges from these static and dynamic observations that kinocilia migrate gradually in parallel with the organization of hair cells into rows during cochlear neuroepithelium extension. Analysis of the confined motion of hair cell centrioles under normal and pathological conditions should help determine which structures contribute to the restoring force exerting on them. PMID:23823223

A multicolour graph as a complete topological invariant for \\Omega-stable flows without periodic trajectories on surfaces

NASA Astrophysics Data System (ADS)

Kruglov, V. E.; Malyshev, D. S.; Pochinka, O. V.

2018-01-01

Studying the dynamics of a flow on surfaces by partitioning the phase space into cells with the same limit behaviour of trajectories within a cell goes back to the classical papers of Andronov, Pontryagin, Leontovich and Maier. The types of cells (the number of which is finite) and how the cells adjoin one another completely determine the topological equivalence class of a flow with finitely many special trajectories. If one trajectory is chosen in every cell of a rough flow without periodic orbits, then the cells are partitioned into so-called triangular regions of the same type. A combinatorial description of such a partition gives rise to the three-colour Oshemkov-Sharko graph, the vertices of which correspond to the triangular regions, and the edges to separatrices connecting them. Oshemkov and Sharko proved that such flows are topologically equivalent if and only if the three-colour graphs of the flows are isomorphic, and described an algorithm of distinguishing three-colour graphs. But their algorithm is not efficient with respect to graph theory. In the present paper, we describe the dynamics of Ω-stable flows without periodic trajectories on surfaces in the language of four-colour graphs, present an efficient algorithm for distinguishing such graphs, and develop a realization of a flow from some abstract graph. Bibliography: 17 titles.

Feasibility study of solid oxide fuel cell engines integrated with sprinter gas turbines: Modeling, design and control

NASA Astrophysics Data System (ADS)

Jia, Zhenzhong; Sun, Jing; Dobbs, Herb; King, Joel

2015-02-01

Conventional recuperating solid oxide fuel cell (SOFC)/gas turbine (GT) system suffers from its poor dynamic capability and load following performance. To meet the fast, safe and efficient load following requirements for mobile applications, a sprinter SOFC/GT system concept is proposed in this paper. In the proposed system, an SOFC stack operating at fairly constant temperature provides the baseline power with high efficiency while the fast dynamic capability of the GT-generator is fully explored for fast dynamic load following. System design and control studies have been conducted by using an SOFC/GT system model consisting of experimentally-verified component models. In particular, through analysis of the steady-state simulation results, an SOFC operation strategy is proposed to maintain fairly constant SOFC power (less than 2% power variation) and temperature (less than 2 K temperature variation) over the entire load range. A system design procedure well-suited to the proposed system has also been developed to help determining component sizes and the reference steady-state operation line. In addition, control analysis has been studied for both steady-state and transient operations. Simulation results suggest that the proposed system holds the promise to achieve fast and safe transient operations by taking full advantage of the fast dynamics of the GT-generator.

Patient-specific modeling and analysis of dynamic behavior of individual sickle red blood cells under hypoxic conditions

NASA Astrophysics Data System (ADS)

Li, Xuejin; Du, E.; Li, Zhen; Tang, Yu-Hang; Lu, Lu; Dao, Ming; Karniadakis, George

2015-11-01

Sickle cell anemia is an inherited blood disorder exhibiting heterogeneous morphology and abnormal dynamics under hypoxic conditions. We developed a time-dependent cell model that is able to simulate the dynamic processes of repeated sickling and unsickling of red blood cells (RBCs) under physiological conditions. By using the kinetic cell model with parameters derived from patient-specific data, we present a mesoscopic computational study of the dynamic behavior of individual sickle RBCs flowing in a microfluidic channel with multiple microgates. We investigate how individual sickle RBCs behave differently from healthy ones in channel flow, and analyze the alteration of cellular behavior and response to single-cell capillary obstruction induced by cell rheologic rigidification and morphological change due to cell sickling under hypoxic conditions. We also simulate the flow dynamics of sickle RBCs treated with hydroxyurea (HU) and quantify the relative enhancement of hemodynamic performance of HU. This work was supported by the National Institutes of Health (NIH) Grant U01HL114476.

Mars power system concept definition study. Volume 1: Study results

NASA Technical Reports Server (NTRS)

Littman, Franklin D.

1994-01-01

A preliminary top level study was completed to define power system concepts applicable to Mars surface applications. This effort included definition of power system requirements and selection of power systems with the potential for high commonality. These power systems included dynamic isotope, Proton Exchange Membrane (PEM) regenerative fuel cell, sodium sulfur battery, photovoltaic, and reactor concepts. Design influencing factors were identified. Characterization studies were then done for each concept to determine system performance, size/volume, and mass. Operations studies were done to determine emplacement/deployment maintenance/servicing, and startup/shutdown requirements. Technology development roadmaps were written for each candidate power system (included in Volume 2). Example power system architectures were defined and compared on a mass basis. The dynamic isotope power system and nuclear reactor power system architectures had significantly lower total masses than the photovoltaic system architectures. Integrated development and deployment time phasing plans were completed for an example DIPS and reactor architecture option to determine the development strategies required to meet the mission scenario requirements.

Nanoparticle Tracking Analysis for Determination of Hydrodynamic Diameter, Concentration, and Zeta-Potential of Polyplex Nanoparticles.

PubMed

Wilson, David R; Green, Jordan J

2017-01-01

Nanoparticle tracking analysis (NTA) is a recently developed nanoparticle characterization technique that offers certain advantages over dynamic light scattering for characterizing polyplex nanoparticles in particular. Dynamic light scattering results in intensity-weighted average measurements of nanoparticle characteristics. In contrast, NTA directly tracks individual particles, enabling concentration measurements as well as the direct determination of number-weighted particle size and zeta-potential. A direct number-weighted assessment of nanoparticle characteristics is particularly useful for polydisperse samples of particles, including many varieties of gene delivery particles that can be prone to aggregation. Here, we describe the synthesis of poly(beta-amino ester)/deoxyribonucleic acid (PBAE/DNA) polyplex nanoparticles and their characterization using NTA to determine hydrodynamic diameter, zeta-potential, and concentration. Additionally, we detail methods of labeling nucleic acids with fluorophores to assess only those polyplex nanoparticles containing plasmids via NTA. Polymeric gene delivery of exogenous plasmid DNA has great potential for treating a wide variety of diseases by inducing cells to express a gene of interest.

Assembly Kinetics Determine the Architecture of α-actinin Crosslinked F-actin Networks

PubMed Central

Falzone, Tobias T.; Lenz, Martin; Kovar, David R.; Gardel, Margaret L.

2013-01-01

The actin cytoskeleton is organized into diverse meshworks and bundles that support many aspects of cell physiology. Understanding the self-assembly of these actin-based structures is essential for developing predictive models of cytoskeletal organization. Here we show that the competing kinetics of bundle formation with the onset of dynamic arrest arising from filament entanglements and cross-linking determine the architecture of reconstituted actin networks formed with α-actinin cross-links. Cross-link mediated bundle formation only occurs in dilute solutions of highly mobile actin filaments. As actin polymerization proceeds, filament mobility and bundle formation are arrested concomitantly. By controlling the onset of dynamic arrest, perturbations to actin assembly kinetics dramatically alter the architecture of biochemically identical samples. Thus, the morphology of reconstituted F-actin networks is a kinetically determined structure similar to those formed by physical gels and glasses. These results establish mechanisms controlling the structure and mechanics in diverse semi-flexible biopolymer networks. PMID:22643888