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Sample records for quantitative transcription dynamic

  1. Transcription Dynamics in Living Cells.

    PubMed

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

    2016-07-01

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

  2. In vivo Monitoring of Transcriptional Dynamics After Lower-Limb Muscle Injury Enables Quantitative Classification of Healing

    PubMed Central

    Aguilar, Carlos A.; Shcherbina, Anna; Ricke, Darrell O.; Pop, Ramona; Carrigan, Christopher T.; Gifford, Casey A.; Urso, Maria L.; Kottke, Melissa A.; Meissner, Alexander

    2015-01-01

    Traumatic lower-limb musculoskeletal injuries are pervasive amongst athletes and the military and typically an individual returns to activity prior to fully healing, increasing a predisposition for additional injuries and chronic pain. Monitoring healing progression after a musculoskeletal injury typically involves different types of imaging but these approaches suffer from several disadvantages. Isolating and profiling transcripts from the injured site would abrogate these shortcomings and provide enumerative insights into the regenerative potential of an individual’s muscle after injury. In this study, a traumatic injury was administered to a mouse model and healing progression was examined from 3 hours to 1 month using high-throughput RNA-Sequencing (RNA-Seq). Comprehensive dissection of the genome-wide datasets revealed the injured site to be a dynamic, heterogeneous environment composed of multiple cell types and thousands of genes undergoing significant expression changes in highly regulated networks. Four independent approaches were used to determine the set of genes, isoforms, and genetic pathways most characteristic of different time points post-injury and two novel approaches were developed to classify injured tissues at different time points. These results highlight the possibility to quantitatively track healing progression in situ via transcript profiling using high- throughput sequencing. PMID:26381351

  3. Transcriptional Interference: A quantitative approach to in vivo dynamics of RNAP on DNA.

    NASA Astrophysics Data System (ADS)

    Sneppen, Kim

    2007-03-01

    We present a mathematical model for transcriptional interference by RNA polymerase traffic in Escherichia coli. The model deals with the interference between the two promoters pA and pS. The RNAPs are injected onto the DNA through binding and formation of sitting duck complexes at the respective promoters, followed by subsequent formation of elongating complexes. Finally we discuss a combination of modeling and in vivo-experiments can be used to infer the interference-recruitment game that govern the core of the genetic switch in the temperate bacteriophages 186.K. Sneppen, I.B. Dodd, K.E. Shearwin, A.C. Palmer, R.A. Schubert, B.P. Callen, and J.B. Egan. J. Mol. Biol. 346:399 (2005)

  4. Quantitative regulation of FLC via coordinated transcriptional initiation and elongation

    PubMed Central

    Wu, Zhe; Ietswaart, Robert; Liu, Fuquan; Yang, Hongchun; Howard, Martin; Dean, Caroline

    2016-01-01

    The basis of quantitative regulation of gene expression is still poorly understood. In Arabidopsis thaliana, quantitative variation in expression of FLOWERING LOCUS C (FLC) influences the timing of flowering. In ambient temperatures, FLC expression is quantitatively modulated by a chromatin silencing mechanism involving alternative polyadenylation of antisense transcripts. Investigation of this mechanism unexpectedly showed that RNA polymerase II (Pol II) occupancy changes at FLC did not reflect RNA fold changes. Mathematical modeling of these transcriptional dynamics predicted a tight coordination of transcriptional initiation and elongation. This prediction was validated by detailed measurements of total and chromatin-bound FLC intronic RNA, a methodology appropriate for analyzing elongation rate changes in a range of organisms. Transcription initiation was found to vary ∼25-fold with elongation rate varying ∼8- to 12-fold. Premature sense transcript termination contributed very little to expression differences. This quantitative variation in transcription was coincident with variation in H3K36me3 and H3K4me2 over the FLC gene body. We propose different chromatin states coordinately influence transcriptional initiation and elongation rates and that this coordination is likely to be a general feature of quantitative gene regulation in a chromatin context. PMID:26699513

  5. Transcription Dynamics in Plant Immunity

    PubMed Central

    Moore, John W.; Loake, Gary J.; Spoel, Steven H.

    2011-01-01

    Plant cells maintain sophisticated gene transcription programs to regulate their development, communication, and response to the environment. Environmental stress cues, such as pathogen encounter, lead to dramatic reprogramming of transcription to favor stress responses over normal cellular functions. Transcription reprogramming is conferred by the concerted action of myriad transcription (co)factors that function directly or indirectly to recruit or release RNA Polymerase II. To establish an effective defense response, cells require transcription (co)factors to deploy their activity rapidly, transiently, spatially, and hierarchically. Recent findings suggest that in plant immunity these requirements are met by posttranslational modifications that accurately regulate transcription (co)factor activity as well as by sequential pulse activation of specific gene transcription programs that provide feedback and feedforward properties to the defense gene network. Here, we integrate these recent findings from plant defense studies into the emerging field of transcription dynamics in eukaryotes. PMID:21841124

  6. Workshop on quantitative dynamic stratigraphy

    SciTech Connect

    Cross, T.A.

    1988-04-01

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

  7. Eukaryotic transcriptional dynamics: from single molecules to cell populations

    PubMed Central

    Coulon, Antoine; Chow, Carson C.; Singer, Robert H.; Larson, Daniel R.

    2013-01-01

    Transcriptional regulation is achieved through combinatorial interactions between regulatory elements in the human genome and a vast range of factors that modulate the recruitment and activity of RNA polymerase. Experimental approaches for studying transcription in vivo now extend from single-molecule techniques to genome-wide measurements. Parallel to these developments is the need for testable quantitative and predictive models for understanding gene regulation. These conceptual models must also provide insight into the dynamics of transcription and the variability that is observed at the single-cell level. In this Review, we discuss recent results on transcriptional regulation and also the models those results engender. We show how a non-equilibrium description informs our view of transcription by explicitly considering time-and energy-dependence at the molecular level. PMID:23835438

  8. Quantitative imaging of transcription in living Drosophila embryos links polymerase activity to patterning.

    PubMed

    Garcia, Hernan G; Tikhonov, Mikhail; Lin, Albert; Gregor, Thomas

    2013-11-01

    Spatiotemporal patterns of gene expression are fundamental to every developmental program. The resulting macroscopic domains have been mainly characterized by their levels of gene products. However, the establishment of such patterns results from differences in the dynamics of microscopic events in individual cells such as transcription. It is unclear how these microscopic decisions lead to macroscopic patterns, as measurements in fixed tissue cannot access the underlying transcriptional dynamics. In vivo transcriptional dynamics have long been approached in single-celled organisms, but never in a multicellular developmental context. Here, we directly address how boundaries of gene expression emerge in the Drosophila embryo by measuring the absolute number of actively transcribing polymerases in real time in individual nuclei. Specifically, we show that the formation of a boundary cannot be quantitatively explained by the rate of mRNA production in each cell, but instead requires amplification of the dynamic range of the expression boundary. This amplification is accomplished by nuclei randomly adopting active or inactive states of transcription, leading to a collective effect where the fraction of active nuclei is modulated in space. Thus, developmental patterns are not just the consequence of reproducible transcriptional dynamics in individual nuclei, but are the result of averaging expression over space and time.

  9. Deciphering Transcriptional Dynamics In Vivo by Counting Nascent RNA Molecules

    PubMed Central

    Choubey, Sandeep; Kondev, Jane; Sanchez, Alvaro

    2015-01-01

    Abstract Deciphering how the regulatory DNA sequence of a gene dictates its expression in response to intra and extracellular cues is one of the leading challenges in modern genomics. The development of novel single-cell sequencing and imaging techniques, as well as a better exploitation of currently available single-molecule imaging techniques, provides an avenue to interrogate the process of transcription and its dynamics in cells by quantifying the number of RNA polymerases engaged in the transcription of a gene (or equivalently the number of nascent RNAs) at a given moment in time. In this paper, we propose that measurements of the cell-to-cell variability in the number of nascent RNAs provide a mostly unexplored method for deciphering mechanisms of transcription initiation in cells. We propose a simple kinetic model of transcription initiation and elongation from which we calculate nascent RNA copy-number fluctuations. To demonstrate the usefulness of this approach, we test our theory against published nascent RNA data for twelve constitutively expressed yeast genes. Rather than transcription being initiated through a single rate limiting step, as it had been previously proposed, our single-cell analysis reveals the presence of at least two rate limiting steps. Surprisingly, half of the genes analyzed have nearly identical rates of transcription initiation, suggesting a common mechanism. Our analytical framework can be used to extract quantitative information about dynamics of transcription from single-cell sequencing data, as well as from single-molecule imaging and electron micrographs of fixed cells, and provides the mathematical means to exploit the quantitative power of these technologies. PMID:26544860

  10. Spatially coordinated dynamic gene transcription in living pituitary tissue

    PubMed Central

    Featherstone, Karen; Hey, Kirsty; Momiji, Hiroshi; McNamara, Anne V; Patist, Amanda L; Woodburn, Joanna; Spiller, David G; Christian, Helen C; McNeilly, Alan S; Mullins, John J; Finkenstädt, Bärbel F; Rand, David A; White, Michael RH; Davis, Julian RE

    2016-01-01

    Transcription at individual genes in single cells is often pulsatile and stochastic. A key question emerges regarding how this behaviour contributes to tissue phenotype, but it has been a challenge to quantitatively analyse this in living cells over time, as opposed to studying snap-shots of gene expression state. We have used imaging of reporter gene expression to track transcription in living pituitary tissue. We integrated live-cell imaging data with statistical modelling for quantitative real-time estimation of the timing of switching between transcriptional states across a whole tissue. Multiple levels of transcription rate were identified, indicating that gene expression is not a simple binary ‘on-off’ process. Immature tissue displayed shorter durations of high-expressing states than the adult. In adult pituitary tissue, direct cell contacts involving gap junctions allowed local spatial coordination of prolactin gene expression. Our findings identify how heterogeneous transcriptional dynamics of single cells may contribute to overall tissue behaviour. DOI: http://dx.doi.org/10.7554/eLife.08494.001 PMID:26828110

  11. Towards a Quantitative Understanding of Single-Gene Transcription

    NASA Astrophysics Data System (ADS)

    O'Maoiléidigh, Dáibhid

    2008-03-01

    The transcription of the genetic information in DNA into RNA is the first step in protein synthesis. This process is highly regulated and is carried out by RNA polymerase (RNAP), a complex molecular motor. Here we discuss some of the consequences of a Brownian ratchet model of transcription, which incorporates internal structural degrees of freedom of RNAP and kinetic barriers to backtracking of RNAP resulting from steric clashes with co-transcriptionally folded RNA. This approach was previously used (a) to successfully predict sequence dependent positions of pauses during the elongation process [1,2]; (b) to study the behavior of a number of mutants of RNAP, with different elongation behaviors, believed to involve different internal motions of the enzyme [3]; and (c) to gain insight into the interpretation of single-molecule transcription elongation experiments [2]. The same model can be used to characterize the stability of the elongation complex at specific termination sequences, places along DNA where, with high probability, RNAP releases the RNA transcript and disengages from the template. Recent experimental results on termination reinforce a picture of the elongation complex as a flexible structure, not a rigid body [4]. In more general terms, some of the modeling to be presented raises fundamental issues related to ``model comparison'' and ``model selection,'' the problem of identifying and characterizing quantitative models on the basis of limited sets of experimental data [5]. [1] Tadigotla V. R., 'O Maoil'eidigh D., Sengupta A. M., Epshtein V., Ebright R. H., Nudler E., Ruckenstein A. E., Thermodynamic and Kinetic Modeling of Transcriptional Pausing. Proc Natl Acad Sci U S A,03:4439-4444 (2006). [2] D. 'O Maoil'eidigh, Ph.D. Thesis, Rutgers University, 2006 [3] Bar-Nahum, G., Epshtein, V., Ruckenstein, A. E., Rafikov, R., Mustaev, A. and Nudler E., A Ratchet Mechanism of Transcription Elongation and its Control. Cell, 120:183-193 (2005). [4] Epshtein, V

  12. Quantification of Yeast and Bacterial Gene Transcripts in Retail Cheeses by Reverse Transcription-Quantitative PCR

    PubMed Central

    Straub, Cécile; Castellote, Jessie; Onesime, Djamila; Bonnarme, Pascal; Irlinger, Françoise

    2013-01-01

    The cheese microbiota contributes to a large extent to the development of the typical color, flavor, and texture of the final product. Its composition is not well defined in most cases and varies from one cheese to another. The aim of the present study was to establish procedures for gene transcript quantification in cheeses by reverse transcription-quantitative PCR. Total RNA was extracted from five smear-ripened cheeses purchased on the retail market, using a method that does not involve prior separation of microbial cells. 16S rRNA and malate:quinone oxidoreductase gene transcripts of Corynebacterium casei, Brevibacterium aurantiacum, and Arthrobacter arilaitensis and 26S rRNA and beta tubulin gene transcripts of Geotrichum candidum and Debaryomyces hansenii could be detected and quantified in most of the samples. Three types of normalization were applied: against total RNA, against the amount of cheese, and against a reference gene. For the first two types of normalization, differences of reverse transcription efficiencies from one sample to another were taken into account by analysis of exogenous control mRNA. No good correlation was found between the abundances of target mRNA or rRNA transcripts and the viable cell concentration of the corresponding species. However, in most cases, no mRNA transcripts were detected for species that did not belong to the dominant species. The applications of gene expression measurement in cheeses containing an undefined microbiota, as well as issues concerning the strategy of normalization and the assessment of amplification specificity, are discussed. PMID:23124230

  13. Crowding, dynamics and transcription (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Szleifer, Igal

    2016-03-01

    Biophotonic studies based on partial wave spectroscopy have shown that early carcinogenesis is characterized by a change in the nanoscale molecular organization of the cell nuclii. These finding suggest that cancer is associated with change in macromolecular crowding. In this presentation we will discuss a recent approach that we have developed to incorporate molecular scale information into a systems based approach to study the role of macromolecular crowding on different phenomena ranging from protein diffusion to gene transcription. Macromolecular crowding affects both dynamics and equilibrium properties. We will show that transcription is a non-monotonic function of crowders concentration in the cell nuclei. Furthermore, we will show how changes in macromolecular crowding in the nuclei and in the cytoplasm lead to different changes in the oscillatory behavior on NF-κB upon stimuli. Our results show the important regulatory role that non-specific interactions play in biological systems.

  14. Transcriptional rewiring over evolutionary timescales changes quantitative and qualitative properties of gene expression

    PubMed Central

    Dalal, Chiraj K; Zuleta, Ignacio A; Mitchell, Kaitlin F; Andes, David R; El-Samad, Hana; Johnson, Alexander D

    2016-01-01

    Evolutionary changes in transcription networks are an important source of diversity across species, yet the quantitative consequences of network evolution have rarely been studied. Here we consider the transcriptional ‘rewiring’ of the three GAL genes that encode the enzymes needed for cells to convert galactose to glucose. In Saccharomyces cerevisiae, the transcriptional regulator Gal4 binds and activates these genes. In the human pathogen Candida albicans (which last shared a common ancestor with S. cerevisiae some 300 million years ago), we show that different regulators, Rtg1 and Rtg3, activate the three GAL genes. Using single-cell dynamics and RNA-sequencing, we demonstrate that although the overall logic of regulation is the same in both species—the GAL genes are induced by galactose—there are major differences in both the quantitative response of these genes to galactose and in the position of these genes in the overall transcription network structure of the two species. DOI: http://dx.doi.org/10.7554/eLife.18981.001 PMID:27614020

  15. Dynamic Encounters of Genes and Transcripts with the Nuclear Pore.

    PubMed

    Ben-Yishay, Rakefet; Ashkenazy, Asaf J; Shav-Tal, Yaron

    2016-07-01

    Transcribed mRNA molecules must reach the cytoplasm to undergo translation. Technological developments in imaging have placed mRNAs under the spotlight, allowing the quantitative study of the spatial and temporal dynamics of the nucleocytoplasmic mRNA export process. Here, we discuss studies that have used such experimental approaches to demonstrate that gene tethering at the nuclear pore complex (NPC) regulates mRNA expression, and to characterize mRNA dynamics during transport in real time. The paths taken by mRNAs as they move from their sites of transcription and travel through the nucleoplasm, in between chromatin domains, and finally through the NPC, can now be observed in detail. PMID:27185238

  16. Cardiovascular and pulmonary dynamics by quantitative imaging

    NASA Technical Reports Server (NTRS)

    Wood, E. H.

    1976-01-01

    The accuracy and range of studies on cardiovascular and pulmonary functions can be greatly facilitated if the motions of the underlying organ systems throughout individual cycles can be directly visualized and readily measured with minimum or preferably no effect on these motions. Achievement of this objective requires development of techniques for quantitative noninvasive or minimally invasive dynamic and stop-action imaging of the organ systems. A review of advances in dynamic quantitative imaging of moving organs reveals that the revolutionary value of cross-sectional and three-dimensional images produced by various types of radiant energy such as X-rays and gamma rays, positrons, electrons, protons, light, and ultrasound for clinical diagnostic and biomedical research applications is just beginning to be realized. The fabrication of a clinically useful cross-section reconstruction device with sensing capabilities for both anatomical structural composition and chemical composition may be possible and awaits future development.

  17. Single Cell Analysis of Transcriptional Activation Dynamics

    PubMed Central

    Rafalska-Metcalf, Ilona U.; Powers, Sara Lawrence; Joo, Lucy M.; LeRoy, Gary; Janicki, Susan M.

    2010-01-01

    Background Gene activation is thought to occur through a series of temporally defined regulatory steps. However, this process has not been completely evaluated in single living mammalian cells. Methodology/Principal Findings To investigate the timing and coordination of gene activation events, we tracked the recruitment of GCN5 (histone acetyltransferase), RNA polymerase II, Brd2 and Brd4 (acetyl-lysine binding proteins), in relation to a VP16-transcriptional activator, to a transcription site that can be visualized in single living cells. All accumulated rapidly with the VP16 activator as did the transcribed RNA. RNA was also detected at significantly more transcription sites in cells expressing the VP16-activator compared to a p53-activator. After α-amanitin pre-treatment, the VP16-activator, GCN5, and Brd2 are still recruited to the transcription site but the chromatin does not decondense. Conclusions/Significance This study demonstrates that a strong activator can rapidly overcome the condensed chromatin structure of an inactive transcription site and supercede the expected requirement for regulatory events to proceed in a temporally defined order. Additionally, activator strength determines the number of cells in which transcription is induced as well as the extent of chromatin decondensation. As chromatin decondensation is significantly reduced after α-amanitin pre-treatment, despite the recruitment of transcriptional activation factors, this provides further evidence that transcription drives large-scale chromatin decondensation. PMID:20422051

  18. Quantification of transcript levels with quantitative RT-PCR.

    PubMed

    Carleton, Karen L

    2011-01-01

    Differential gene expression is a key factor driving phenotypic divergence. Determining when and where gene expression has diverged between organisms requires a quantitative method. While large-scale approaches such as microarrays or high-throughput mRNA sequencing can identify candidates, quantitative RT-PCR is the definitive method for confirming gene expression differences. Here, we describe the steps for performing qRT-PCR including extracting total RNA, reverse-transcribing it to make a pool of cDNA, and then quantifying relative expression of a few candidate genes using real-time or quantitative PCR.

  19. Dynamics of transcription-translation networks

    NASA Astrophysics Data System (ADS)

    Hudson, D.; Edwards, R.

    2016-09-01

    A theory for qualitative models of gene regulatory networks has been developed over several decades, generally considering transcription factors to regulate directly the expression of other transcription factors, without any intermediate variables. Here we explore a class of models that explicitly includes both transcription and translation, keeping track of both mRNA and protein concentrations. We mainly deal with transcription regulation functions that are steep sigmoids or step functions, as is often done in protein-only models, though translation is governed by a linear term. We extend many aspects of the protein-only theory to this new context, including properties of fixed points, description of trajectories by mappings between switching points, qualitative analysis via a state-transition diagram, and a result on periodic orbits for negative feedback loops. We find that while singular behaviour in switching domains is largely avoided, non-uniqueness of solutions can still occur in the step-function limit.

  20. Stochastic expression dynamics of a transcription factor revealed by single-molecule noise analysis.

    PubMed

    Hensel, Zach; Feng, Haidong; Han, Bo; Hatem, Christine; Wang, Jin; Xiao, Jie

    2012-08-01

    Gene expression is inherently stochastic; precise gene regulation by transcription factors is important for cell-fate determination. Many transcription factors regulate their own expression, suggesting that autoregulation counters intrinsic stochasticity in gene expression. Using a new strategy, cotranslational activation by cleavage (CoTrAC), we probed the stochastic expression dynamics of cI, which encodes the bacteriophage λ repressor CI, a fate-determining transcription factor. CI concentration fluctuations influence both lysogenic stability and induction of bacteriophage λ. We found that the intrinsic stochasticity in cI expression was largely determined by CI expression level irrespective of autoregulation. Furthermore, extrinsic, cell-to-cell variation was primarily responsible for CI concentration fluctuations, and negative autoregulation minimized CI concentration heterogeneity by counteracting extrinsic noise and introducing memory. This quantitative study of transcription factor expression dynamics sheds light on the mechanisms cells use to control noise in gene regulatory networks. PMID:22751020

  1. DNA dynamics play a role as a basal transcription factor in the positioning and regulation of gene transcription initiation

    PubMed Central

    Alexandrov, Boian S.; Gelev, Vladimir; Yoo, Sang Wook; Alexandrov, Ludmil B.; Fukuyo, Yayoi; Bishop, Alan R.; Rasmussen, Kim Ø.; Usheva, Anny

    2010-01-01

    We assess the role of DNA breathing dynamics as a determinant of promoter strength and transcription start site (TSS) location. We compare DNA Langevin dynamic profiles of representative gene promoters, calculated with the extended non-linear PBD model of DNA with experimental data on transcription factor binding and transcriptional activity. Our results demonstrate that DNA dynamic activity at the TSS can be suppressed by mutations that do not affect basal transcription factor binding–DNA contacts. We use this effect to establish the separate contributions of transcription factor binding and DNA dynamics to transcriptional activity. Our results argue against a purely ‘transcription factor-centric’ view of transcription initiation, suggesting that both DNA dynamics and transcription factor binding are necessary conditions for transcription initiation. PMID:20019064

  2. Dynamics of Transcription Factor Binding Site Evolution

    PubMed Central

    Tuğrul, Murat; Paixão, Tiago; Barton, Nicholas H.; Tkačik, Gašper

    2015-01-01

    Evolution of gene regulation is crucial for our understanding of the phenotypic differences between species, populations and individuals. Sequence-specific binding of transcription factors to the regulatory regions on the DNA is a key regulatory mechanism that determines gene expression and hence heritable phenotypic variation. We use a biophysical model for directional selection on gene expression to estimate the rates of gain and loss of transcription factor binding sites (TFBS) in finite populations under both point and insertion/deletion mutations. Our results show that these rates are typically slow for a single TFBS in an isolated DNA region, unless the selection is extremely strong. These rates decrease drastically with increasing TFBS length or increasingly specific protein-DNA interactions, making the evolution of sites longer than ∼ 10 bp unlikely on typical eukaryotic speciation timescales. Similarly, evolution converges to the stationary distribution of binding sequences very slowly, making the equilibrium assumption questionable. The availability of longer regulatory sequences in which multiple binding sites can evolve simultaneously, the presence of “pre-sites” or partially decayed old sites in the initial sequence, and biophysical cooperativity between transcription factors, can all facilitate gain of TFBS and reconcile theoretical calculations with timescales inferred from comparative genomics. PMID:26545200

  3. Transcription dynamics of inducible genes modulated by negative regulations.

    PubMed

    Li, Yanyan; Tang, Moxun; Yu, Jianshe

    2015-06-01

    Gene transcription is a stochastic process in single cells, in which genes transit randomly between active and inactive states. Transcription of many inducible genes is also tightly regulated: It is often stimulated by extracellular signals, activated through signal transduction pathways and later repressed by negative regulations. In this work, we study the nonlinear dynamics of the mean transcription level of inducible genes modulated by the interplay of the intrinsic transcriptional randomness and the repression by negative regulations. In our model, we integrate negative regulations into gene activation process, and make the conventional assumption on the production and degradation of transcripts. We show that, whether or not the basal transcription is temporarily terminated when cells are stimulated, the mean transcription level grows in the typical up and down pattern commonly observed in immune response genes. With the help of numerical simulations, we clarify the delicate impact of the system parameters on the transcription dynamics, and demonstrate how our model generates the distinct temporal gene-induction patterns in mouse fibroblasts discerned in recent experiments.

  4. Effect of soil clay content on RNA isolation and on detection and quantification of bacterial gene transcripts in soil by quantitative reverse transcription-PCR.

    PubMed

    Novinscak, A; Filion, M

    2011-09-01

    In this study, we evaluated the effect of soil clay content on RNA isolation and on quantitative reverse transcription-PCR (qRT-PCR) quantification of microbial gene transcripts. The amount of clay significantly altered RNA isolation yields and qRT-PCR analyses. Recommendations are made for quantifying microbial gene transcripts in soil samples varying in clay content.

  5. Aligning transcript of historical documents using dynamic programming

    NASA Astrophysics Data System (ADS)

    Rabaev, Irina; Cohen, Rafi; El-Sana, Jihad; Kedem, Klara

    2015-01-01

    We present a simple and accurate approach for aligning historical documents with their corresponding transcription. First, a representative of each letter in the historical document is cropped. Then, the transcription is transformed to synthetic word images by representing the letters in the transcription by the cropped letters. These synthetic word images are aligned to groups of connected components in the original text, along each line, using dynamic programming. For measuring image similarities we experimented with a variety of feature extraction and matching methods. The presented alignment algorithm was tested on two historical datasets and provided excellent results.

  6. An Essential Viral Transcription Activator Modulates Chromatin Dynamics

    PubMed Central

    Gibeault, Rebecca L.; Bildersheim, Michael D.

    2016-01-01

    Although ICP4 is the only essential transcription activator of herpes simplex virus 1 (HSV-1), its mechanisms of action are still only partially understood. We and others propose a model in which HSV-1 genomes are chromatinized as a cellular defense to inhibit HSV-1 transcription. To counteract silencing, HSV-1 would have evolved proteins that prevent or destabilize chromatinization to activate transcription. These proteins should act as HSV-1 transcription activators. We have shown that HSV-1 genomes are organized in highly dynamic nucleosomes and that histone dynamics increase in cells infected with wild type HSV-1. We now show that whereas HSV-1 mutants encoding no functional ICP0 or VP16 partially enhanced histone dynamics, mutants encoding no functional ICP4 did so only minimally. Transient expression of ICP4 was sufficient to enhance histone dynamics in the absence of other HSV-1 proteins or HSV-1 DNA. The dynamics of H3.1 were increased in cells expressing ICP4 to a greater extent than those of H3.3. The dynamics of H2B were increased in cells expressing ICP4, whereas those of canonical H2A were not. ICP4 preferentially targets silencing H3.1 and may also target the silencing H2A variants. In infected cells, histone dynamics were increased in the viral replication compartments, where ICP4 localizes. These results suggest a mechanism whereby ICP4 activates transcription by disrupting, or preventing the formation of, stable silencing nucleosomes on HSV-1 genomes. PMID:27575707

  7. Cdk1 activity acts as a quantitative platform for coordinating cell cycle progression with periodic transcription

    PubMed Central

    Banyai, Gabor; Baïdi, Feriel; Coudreuse, Damien; Szilagyi, Zsolt

    2016-01-01

    Cell proliferation is regulated by cyclin-dependent kinases (Cdks) and requires the periodic expression of particular gene clusters in different cell cycle phases. However, the interplay between the networks that generate these transcriptional oscillations and the core cell cycle machinery remains largely unexplored. In this work, we use a synthetic regulable Cdk1 module to demonstrate that periodic expression is governed by quantitative changes in Cdk1 activity, with different clusters directly responding to specific activity levels. We further establish that cell cycle events neither participate in nor interfere with the Cdk1-driven transcriptional program, provided that cells are exposed to the appropriate Cdk1 activities. These findings contrast with current models that propose self-sustained and Cdk1-independent transcriptional oscillations. Our work therefore supports a model in which Cdk1 activity serves as a quantitative platform for coordinating cell cycle transitions with the expression of critical genes to bring about proper cell cycle progression. PMID:27045731

  8. Theory on the dynamic memory in the transcription-factor-mediated transcription activation

    NASA Astrophysics Data System (ADS)

    Murugan, R.

    2011-04-01

    We develop a theory to explain the origin of the static and dynamical memory effects in transcription-factor-mediated transcription activation. Our results suggest that the following inequality conditions should be satisfied to observe such memory effects: (a) τL≫max(τR,τE), (b) τLT≫τT, and (c) τI⩾(τEL+τTR) where τL is the average time required for the looping-mediated spatial interactions of enhancer—transcription-factor complex with the corresponding promoter—RNA-polymerase or eukaryotic RNA polymerase type II (PolII in eukaryotes) complex that is located L base pairs away from the cis-acting element, (τR,τE) are respectively the search times required for the site-specific binding of the RNA polymerase and the transcription factor with the respective promoter and the cis-regulatory module, τLT is the time associated with the relaxation of the looped-out segment of DNA that connects the cis-acting site and promoter, τT is the time required to generate a complete transcript, τI is the transcription initiation time, τEL is the elongation time, and τTR is the termination time. We have theoretically derived the expressions for the various searching, looping, and loop-relaxation time components. Using the experimentally determined values of various time components we further show that the dynamical memory effects cannot be experimentally observed whenever the segment of DNA that connects the cis-regulatory element with the promoter is not loaded with bulky histone bodies. Our analysis suggests that the presence of histone-mediated compaction of the connecting segment of DNA can result in higher values of looping and loop-relaxation times, which is the origin of the static memory in the transcription activation that is mediated by the memory gene loops in eukaryotes.

  9. Circuitry and dynamics of human transcription factor regulatory networks

    PubMed Central

    Neph, Shane; Stergachis, Andrew B.; Reynolds, Alex; Sandstrom, Richard; Borenstein, Elhanan; Stamatoyannopoulos, John A.

    2012-01-01

    SUMMARY The combinatorial cross-regulation of hundreds of sequence-specific transcription factors defines a regulatory network that underlies cellular identity and function. Here we use genome-wide maps of in vivo DNaseI footprints to assemble an extensive core human regulatory network comprising connections among 475 sequence-specific transcription factors, and to analyze the dynamics of these connections across 41 diverse cell and tissue types. We find that human transcription factor networks are highly cell-selective and are driven by cohorts of factors that include regulators with previously unrecognized roles in control of cellular identity. Moreover, we identify many widely expressed factors that impact transcriptional regulatory networks in a cell-selective manner. Strikingly, in spite of their inherent diversity, all cell type regulatory networks independently converge on a common architecture that closely resembles the topology of living neuronal networks. Together, our results provide the first description of the circuitry, dynamics, and organizing principles of the human transcription factor regulatory network. PMID:22959076

  10. Quantitative Real-Time PCR Analysis of Gene Transcripts of Mosquito Follicles.

    PubMed

    Telang, Aparna

    2016-01-01

    Real-time (quantitative) PCR, or QPCR, has become an indispensible tool for characterizing gene expression. Depending on the experimental design, researchers can use either the relative or absolute (standard curve) method to quantify transcript abundance. Characterizing the expression of genes in mosquito ovaries will require use of the standard curve method of quantification. Here, I describe reagents and equipment necessary to run standard curve QPCR. I also provide details on the construction of the standard linear curve and calculations required to determine transcript abundance. PMID:27557577

  11. Analysis of liver connexin expression using reverse transcription quantitative real-time polymerase chain reaction

    PubMed Central

    Maes, Michaël; Willebrords, Joost; Crespo Yanguas, Sara; Cogliati, Bruno; Vinken, Mathieu

    2016-01-01

    Summary Although connexin production is mainly regulated at the protein level, altered connexin gene expression has been identified as the underlying mechanism of several pathologies. When studying the latter, appropriate methods to quantify connexin mRNA levels are required. The present chapter describes a well-established reverse transcription quantitative real-time polymerase chain reaction procedure optimized for analysis of hepatic connexins. The method includes RNA extraction and subsequent quantification, generation of complementary DNA, quantitative real-time polymerase chain reaction and data analysis. PMID:27207283

  12. Analysis of Liver Connexin Expression Using Reverse Transcription Quantitative Real-Time Polymerase Chain Reaction.

    PubMed

    Maes, Michaël; Willebrords, Joost; Crespo Yanguas, Sara; Cogliati, Bruno; Vinken, Mathieu

    2016-01-01

    Although connexin production is mainly regulated at the protein level, altered connexin gene expression has been identified as the underlying mechanism of several pathologies. When studying the latter, appropriate methods to quantify connexin RNA levels are required. The present chapter describes a well-established reverse transcription quantitative real-time polymerase chain reaction procedure optimized for analysis of hepatic connexins. The method includes RNA extraction and subsequent quantification, generation of complementary DNA, quantitative real-time polymerase chain reaction, and data analysis. PMID:27207283

  13. Workshop on quantitative dynamic stratigraphy. Final conference report

    SciTech Connect

    Cross, T.A.

    1988-04-01

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

  14. Validation of Reference Genes for Transcriptional Analyses in Pleurotus ostreatus by Using Reverse Transcription-Quantitative PCR.

    PubMed

    Castanera, Raúl; López-Varas, Leticia; Pisabarro, Antonio G; Ramírez, Lucía

    2015-06-15

    Recently, the lignin-degrading basidiomycete Pleurotus ostreatus has become a widely used model organism for fungal genomic and transcriptomic analyses. The increasing interest in this species has led to an increasing number of studies analyzing the transcriptional regulation of multigene families that encode extracellular enzymes. Reverse transcription (RT) followed by real-time PCR is the most suitable technique for analyzing the expression of gene sets under multiple culture conditions. In this work, we tested the suitability of 13 candidate genes for their use as reference genes in P. ostreatus time course cultures for enzyme production. We applied three different statistical algorithms and obtained a combination of stable reference genes for optimal normalization of RT-quantitative PCR assays. This reference index can be used for future transcriptomic analyses and validation of transcriptome sequencing or microarray data. Moreover, we analyzed the expression patterns of a laccase and a manganese peroxidase (lacc10 and mnp3, respectively) in lignocellulose and glucose-based media using submerged, semisolid, and solid-state fermentation. By testing different normalization strategies, we demonstrate that the use of nonvalidated reference genes as internal controls leads to biased results and misinterpretations of the biological responses underlying expression changes. PMID:25862220

  15. Dynamic, Quantitative Assays of Phagosomal Function

    PubMed Central

    Podinovskaia, Maria; VanderVen, Brian C.; Yates, Robin M.; Glennie, Sarah; Fullerton, Duncan; Mwandumba, Henry C.; Russell, David G.

    2013-01-01

    Much of the activity of the macrophage as an effector cell is performed within its phagocytic compartment. This ranges from the degradation of tissue debris as part of its homeostatic function, to the generation of the superoxide burst as part of its microbicidal response to infection. We have developed a range of real-time readouts of phagosomal function that enables these activities to be rigorously quantified. This chapter contains the description of several of these assays assessed by different methods of quantitation; including a Fluorescence Resonance Emission Transfer (FRET) assay for phagosome/lysosome fusion measured by spectrofluorometer, a fluorogenic assay for the superoxide burst measured by flow cytometry, and a fluorogenic assay for bulk proteolysis measure by confocal microscope. These assays illustrate both the range parameters that can be quantified as well as the flexibility of instrumentation that can be exploited for their quantitation. PMID:24510516

  16. An experimental approach to identify dynamical models of transcriptional regulation in living cells

    NASA Astrophysics Data System (ADS)

    Fiore, G.; Menolascina, F.; di Bernardo, M.; di Bernardo, D.

    2013-06-01

    We describe an innovative experimental approach, and a proof of principle investigation, for the application of System Identification techniques to derive quantitative dynamical models of transcriptional regulation in living cells. Specifically, we constructed an experimental platform for System Identification based on a microfluidic device, a time-lapse microscope, and a set of automated syringes all controlled by a computer. The platform allows delivering a time-varying concentration of any molecule of interest to the cells trapped in the microfluidics device (input) and real-time monitoring of a fluorescent reporter protein (output) at a high sampling rate. We tested this platform on the GAL1 promoter in the yeast Saccharomyces cerevisiae driving expression of a green fluorescent protein (Gfp) fused to the GAL1 gene. We demonstrated that the System Identification platform enables accurate measurements of the input (sugars concentrations in the medium) and output (Gfp fluorescence intensity) signals, thus making it possible to apply System Identification techniques to obtain a quantitative dynamical model of the promoter. We explored and compared linear and nonlinear model structures in order to select the most appropriate to derive a quantitative model of the promoter dynamics. Our platform can be used to quickly obtain quantitative models of eukaryotic promoters, currently a complex and time-consuming process.

  17. Establishment of real-time quantitative reverse transcription polymerase chain reaction assay for transcriptional analysis of duck enteritis virus UL55 gene

    PubMed Central

    2011-01-01

    Background Real-time quantitative reverse transcription polymerase chain reaction assay (qRT-PCR) has become the benchmark for detection and quantification of target gene expression level and been utilized increasingly in detection of viral load and therapy monitoring. The dynamic transcription variation of duck enteritis virus UL55 gene during the life cycle of duck enteritis virus in infected cells has not been reported yet. Results The newly identified duck enteritis virus UL55 gene was amplified and cloned into pMD18-T vector after digestion to generate a recombinant plasmid pMD18-T/UL55 for the establishment of qRT-PCR as standard DNA. The results of agarose gel electrophoresis and melting curve analysis demonstrated the primers we designed for qRT-PCR were specific and available. We used β-actin as a reference gene for normalization and established two standard curves based on pMD18-T/UL55 and pMD18-T/β-actin successfully. Based on that, the transcriptional analysis of DEV UL55 gene was performed, and the result suggested the expression of UL55 mRNA was at a low level from 0 to 8 h post-infection(p.i.), then accumulated quickly since 12 h p.i. and peaked at 36 h p.i., it can be detected till 60 h p.i.. Nucleic acid inhibition test was carried out for analyzing a temporal regulation condition of DEV UL55 gene, result revealed that it was sensitive to ganciclovir. Synthesis procedures of DEV UL55 gene can be inhibited by ganciclovir. Conclusions The method we established in this paper can provide quantitative values reflecting the amounts of measured mRNA in samples. It's available for detection and quantification, also can be used in DEV diagnosis. The DEV UL55 gene was produced most abundantly during the late phase of replication in DEV-infected cells and the transcription of it depended on the synthesized DNA. DEV UL55 gene is a γ2 gene which occurs last and have a strict requirement for viral DNA synthesis. PMID:21631934

  18. How Many Microorganisms Are Present? Quantitative Reverse Transcription PCR (qRT-PCR)

    NASA Astrophysics Data System (ADS)

    Price, Andy; Álvarez, Laura Acuña; Whitby, Corinne; Larsen, Jan

    Quantitative reverse transcription PCR (qRT-PCR) is a variation of conventional quantitative or real-time PCR, whereby mRNA is first converted into the complementary DNA (cDNA) by reverse transcription, the cDNA is then subsequently quantified by qPCR. The use of mRNA as the initial template allows the quantification of gene transcripts, rather than gene copy numbers. mRNA is only produced by actively metabolising cells and is produced by its corresponding gene to provide a 'blueprint' in order for a cell to manufacture a specific protein. Conventional qPCR detects not only DNA present in actively metabolising cells but also inactive and dead cells. qRT-PCR has the advantage that only actively metabolising cells are detected, hence provides a more reliable measure of microbial activity in oilfield samples. When qRT-PCR is combined with primers and probes for specific genes, the activity of microbial processes important in the oilfield, such as sulphate reduction, methanogenesis and nitrate reduction can be monitored.

  19. Quantitative characterization of the interactions among c-myc transcriptional regulators FUSE, FBP, and FIR.

    PubMed

    Hsiao, Hsin-Hao; Nath, Abhinav; Lin, Chi-Yen; Folta-Stogniew, Ewa J; Rhoades, Elizabeth; Braddock, Demetrios T

    2010-06-01

    Human c-myc is critical for cell homeostasis and growth but is a potent oncogenic factor if improperly regulated. The c-myc far-upstream element (FUSE) melts into single-stranded DNA upon active transcription, and the noncoding strand FUSE recruits an activator [the FUSE-binding protein (FBP)] and a repressor [the FBP-interacting repressor (FIR)] to fine-tune c-myc transcription in a real-time manner. Despite detailed biological experiments describing this unique mode of transcriptional regulation, quantitative measurements of the physical constants regulating the protein-DNA interactions remain lacking. Here, we first demonstrate that the two FUSE strands adopt different conformations upon melting, with the noncoding strand DNA in an extended, linear form. FBP binds to the linear noncoding FUSE with a dissociation constant in the nanomolar range. FIR binds to FUSE more weakly, having its modest dissociation constants in the low micromolar range. FIR is monomeric under near-physiological conditions but upon binding of FUSE dimerizes into a 2:1 FIR(2)-FUSE complex mediated by the RRMs. In the tripartite interaction, our analysis suggests a stepwise addition of FIR onto an activating FBP-FUSE complex to form a quaternary FIR(2)-FBP-FUSE inhibitory complex. Our quantitative characterization enhances understanding of DNA strand preference and the mechanism of the stepwise complex formation in the FUSE-FBP-FIR regulatory system.

  20. Dynamic Transcriptional and Epigenetic Regulation of Human Epidermal Keratinocyte Differentiation

    PubMed Central

    Cavazza, Alessia; Miccio, Annarita; Romano, Oriana; Petiti, Luca; Malagoli Tagliazucchi, Guidantonio; Peano, Clelia; Severgnini, Marco; Rizzi, Ermanno; De Bellis, Gianluca; Bicciato, Silvio; Mavilio, Fulvio

    2016-01-01

    Summary Human skin is maintained by the differentiation and maturation of interfollicular stem and progenitors cells. We used DeepCAGE, genome-wide profiling of histone modifications and retroviral integration analysis, to map transcripts, promoters, enhancers, and super-enhancers (SEs) in prospectively isolated keratinocytes and transit-amplifying progenitors, and retrospectively defined keratinocyte stem cells. We show that >95% of the active promoters are in common and differentially regulated in progenitors and differentiated keratinocytes, while approximately half of the enhancers and SEs are stage specific and account for most of the epigenetic changes occurring during differentiation. Transcription factor (TF) motif identification and correlation with TF binding site maps allowed the identification of TF circuitries acting on enhancers and SEs during differentiation. Overall, our study provides a broad, genome-wide description of chromatin dynamics and differential enhancer and promoter usage during epithelial differentiation, and describes a novel approach to identify active regulatory elements in rare stem cell populations. PMID:27050947

  1. Quantitative and temporal definition of the Mla transcriptional regulon during barley-powdery mildew interactions.

    PubMed

    Moscou, Matthew J; Lauter, Nick; Caldo, Rico A; Nettleton, Dan; Wise, Roger P

    2011-06-01

    Barley Mildew resistance locus a (Mla) is a major determinant of immunity to the powdery mildew pathogen, Blumeria graminis f. sp. hordei. Alleles of Mla encode cytoplasmic- and membrane-localized coiled-coil, nucleotide binding site, leucine-rich repeat proteins that mediate resistance when complementary avirulence effectors (AVR(a)) are present in the pathogen. Presence of an appropriate AVR(a) protein triggers nuclear relocalization of MLA, in which MLA binds repressing host transcription factors. Timecourse expression profiles of plants harboring Mla1, Mla6, and Mla12 wild-type alleles versus paired loss-of-function mutants were compared to discover conserved transcriptional targets of MLA and downstream signaling cascades. Pathogen-dependent gene expression was equivalent or stronger in susceptible plants at 20 h after inoculation (HAI) and was attenuated at later timepoints, whereas resistant plants exhibited a time-dependent strengthening of the transcriptional response, increasing in both fold change and the number of genes differentially expressed. Deregulation at 20 HAI implicated 16 HAI as a crucial point in determining the future trajectory of this interaction and was interrogated by quantitative analysis. In total, 28 potential transcriptional targets of the MLA regulon were identified. These candidate targets possess a diverse set of predicted functions, suggesting that multiple pathways are required to mediate the hypersensitive reaction.

  2. Alphaherpesvirus Latency: A Dynamic State of Transcription and Reactivation.

    PubMed

    Bloom, David C

    2016-01-01

    Alphaherpesviruses infect a variety of species from sea turtles to man and can cause significant disease in mammals including humans and livestock. These viruses are characterized by a lytic and latent state in nerve ganglia, with the ability to establish a lifelong latent infection that is interrupted by periodic reactivation. Previously, it was accepted that latency was a dominant state and that only during relatively infrequent reactivation episodes did latent genomes within ganglia become transcriptionally active. Here, we review recent data, focusing mainly on Herpes Simplex Virus type 1 which indicate that the latent state is more dynamic than recently appreciated. PMID:26997590

  3. Complementary Quantitative Proteomics Reveals that Transcription Factor AP-4 Mediates E-box-dependent Complex Formation for Transcriptional Repression of HDM2*

    PubMed Central

    Ku, Wei-Chi; Chiu, Sung-Kay; Chen, Yi-Ju; Huang, Hsin-Hung; Wu, Wen-Guey; Chen, Yu-Ju

    2009-01-01

    Transcription factor activating enhancer-binding protein 4 (AP-4) is a basic helix-loop-helix protein that binds to E-box elements. AP-4 has received increasing attention for its regulatory role in cell growth and development, including transcriptional repression of the human homolog of murine double minute 2 (HDM2), an important oncoprotein controlling cell growth and survival, by an unknown mechanism. Here we demonstrate that AP-4 binds to an E-box located in the HDM2-P2 promoter and represses HDM2 transcription in a p53-independent manner. Incremental truncations of AP-4 revealed that the C-terminal Gln/Pro-rich domain was essential for transcriptional repression of HDM2. To further delineate the molecular mechanism(s) of AP-4 transcriptional control and its potential implications, we used DNA-affinity purification followed by complementary quantitative proteomics, cICAT and iTRAQ labeling methods, to identify a previously unknown E-box-bound AP-4 protein complex containing 75 putative components. The two labeling methods complementarily quantified differentially AP-4-enriched proteins, including the most significant recruitment of DNA damage response proteins, followed by transcription factors, transcriptional repressors/corepressors, and histone-modifying proteins. Specific interaction of AP-4 with CCCTC binding factor, stimulatory protein 1, and histone deacetylase 1 (an AP-4 corepressor) was validated using AP-4 truncation mutants. Importantly, inclusion of trichostatin A did not alleviate AP-4-mediated repression of HDM2 transcription, suggesting a previously unidentified histone deacetylase-independent repression mechanism. In contrast, the complementary quantitative proteomics study suggested that transcription repression occurs via coordination of AP-4 with other transcription factors, histone methyltransferases, and/or a nucleosome remodeling SWI·SNF complex. In addition to previously known functions of AP-4, our data suggest that AP-4 participates in

  4. Cellular dynamics of the negative transcription elongation factor NELF

    SciTech Connect

    Yung, Tetsu M.C.; Narita, Takashi; Komori, Toshiharu; Yamaguchi, Yuki; Handa, Hiroshi

    2009-06-10

    Negative Elongation Factor (NELF) is a transcription factor discovered based on its biochemical activity to suppress transcription elongation, and has since been implicated in various diseases ranging from neurological disorders to cancer. Besides its role in promoter-proximal pausing of RNA polymerase II during early stages of transcription, recently we found that it also plays important roles in the 3'-end processing of histone mRNA. Furthermore, NELF has been found to form a distinct subnuclear structure, which we named NELF bodies. These recent developments point to a wide range of potential functions for NELF, and, as most studies on NELF thus far had been carried out in vitro, here, we prepared a complete set of fusion protein constructs of NELF subunits and carried out a general cell biological study of the intracellular dynamics of NELF. Our data show that NELF subunits exhibit highly specific subcellular localizations, such as in NELF bodies or in midbodies, and some shuttle actively between the nucleus and cytoplasm. We further show that loss of NELF from cells can lead to enlarged and/or multiple nuclei. This work serves as a foundation and starting point for further cell biological investigations of NELF in the future.

  5. A comprehensive collection of experimentally validated primers for Polymerase Chain Reaction quantitation of murine transcript abundance

    PubMed Central

    Spandidos, Athanasia; Wang, Xiaowei; Wang, Huajun; Dragnev, Stefan; Thurber, Tara; Seed, Brian

    2008-01-01

    Background Quantitative polymerase chain reaction (QPCR) is a widely applied analytical method for the accurate determination of transcript abundance. Primers for QPCR have been designed on a genomic scale but non-specific amplification of non-target genes has frequently been a problem. Although several online databases have been created for the storage and retrieval of experimentally validated primers, only a few thousand primer pairs are currently present in existing databases and the primers are not designed for use under a common PCR thermal profile. Results We previously reported the implementation of an algorithm to predict PCR primers for most known human and mouse genes. We now report the use of that resource to identify 17483 pairs of primers that have been experimentally verified to amplify unique sequences corresponding to distinct murine transcripts. The primer pairs have been validated by gel electrophoresis, DNA sequence analysis and thermal denaturation profile. In addition to the validation studies, we have determined the uniformity of amplification using the primers and the technical reproducibility of the QPCR reaction using the popular and inexpensive SYBR Green I detection method. Conclusion We have identified an experimentally validated collection of murine primer pairs for PCR and QPCR which can be used under a common PCR thermal profile, allowing the evaluation of transcript abundance of a large number of genes in parallel. This feature is increasingly attractive for confirming and/or making more precise data trends observed from experiments performed with DNA microarrays. PMID:19108745

  6. Quantitative confocal fluorescence microscopy of dynamic processes by multifocal fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Krmpot, Aleksandar J.; Nikolić, Stanko N.; Vitali, Marco; Papadopoulos, Dimitrios K.; Oasa, Sho; Thyberg, Per; Tisa, Simone; Kinjo, Masataka; Nilsson, Lennart; Gehring, Walter J.; Terenius, Lars; Rigler, Rudolf; Vukojevic, Vladana

    2015-07-01

    Quantitative confocal fluorescence microscopy imaging without scanning is developed for the study of fast dynamical processes. The method relies on the use of massively parallel Fluorescence Correlation Spectroscopy (mpFCS). Simultaneous excitation of fluorescent molecules across the specimen is achieved by passing a single laser beam through a Diffractive Optical Element (DOE) to generate a quadratic illumination matrix of 32×32 light sources. Fluorescence from 1024 illuminated spots is detected in a confocal arrangement by a matching matrix detector consisting of the same number of single-photon avalanche photodiodes (SPADs). Software was developed for data acquisition and fast autoand cross-correlation analysis by parallel signal processing using a Graphic Processing Unit (GPU). Instrumental performance was assessed using a conventional single-beam FCS instrument as a reference. Versatility of the approach for application in biomedical research was evaluated using ex vivo salivary glands from Drosophila third instar larvae expressing a fluorescently-tagged transcription factor Sex Combs Reduced (Scr) and live PC12 cells stably expressing the fluorescently tagged mu-opioid receptor (MOPeGFP). We show that quantitative mapping of local concentration and mobility of transcription factor molecules across the specimen can be achieved using this approach, which paves the way for future quantitative characterization of dynamical reaction-diffusion landscapes across live cells/tissue with a submillisecond temporal resolution (presently 21 μs/frame) and single-molecule sensitivity.

  7. Dynamic multidrug recognition by multidrug transcriptional repressor LmrR.

    PubMed

    Takeuchi, Koh; Tokunaga, Yuji; Imai, Misaki; Takahashi, Hideo; Shimada, Ichio

    2014-11-18

    LmrR is a multidrug transcriptional repressor that controls the expression of a major multidrug transporter, LmrCD, in Lactococcus lactis. However, the molecular mechanism by which LmrR binds to structurally unrelated compounds and is released from the promoter region remains largely unknown. Here, we structurally and dynamically characterized LmrR in the apo, compound-bound and promoter-bound states. The compound-binding site of LmrR exhibits ps-μs dynamics in the apo state, and compound ligation shifts the preexisting conformational equilibrium to varying extents to achieve multidrug recognition. Meanwhile, the compound binding induces redistribution of ps-ns dynamics to the allosteric sites, which entropically favors the high-affinity recognition. Furthermore, the reciprocal compound/promoter binding by LmrR is achieved by the incompatible conformational ensembles between the compound- and promoter-bound states. Collectively, the data show how LmrR can dynamically exert its functions through promiscuous multi-target interactions, in a manner that cannot be understood by a static structural view.

  8. Qualitative and Quantitative Change in the Dynamics of Motor Learning

    ERIC Educational Resources Information Center

    Liu, Yeou-Teh; Mayer-Kress, Gottfried; Newell, Karl M.

    2006-01-01

    The experiments examined qualitative and quantitative changes in the dynamics of learning a novel motor skill (roller ball task) as a function of the manipulation of a control parameter (initial ball speed). The focus was on the relation between the rates of change in performance over practice time and the changing time scales of the evolving…

  9. Quantitative Phase Microscopy of Live Biological Cell Dynamics

    NASA Astrophysics Data System (ADS)

    Shaked, Natan T.; Wax, Adam

    2010-04-01

    Interferometric phase microscopy of biological cell dynamics has the potential to provide a label-free quantitative tool for cell biology, as well as for medical diagnosis and monitoring. The current state of the art of this field, the open questions, and specific solutions developed in our laboratory will be presented.

  10. Quantitative characterisation of audio data by ordinal symbolic dynamics

    NASA Astrophysics Data System (ADS)

    Aschenbrenner, T.; Monetti, R.; Amigó, J. M.; Bunk, W.

    2013-06-01

    Ordinal symbolic dynamics has developed into a valuable method to describe complex systems. Recently, using the concept of transcripts, the coupling behaviour of systems was assessed, combining the properties of the symmetric group with information theoretic ideas. In this contribution, methods from the field of ordinal symbolic dynamics are applied to the characterisation of audio data. Coupling complexity between frequency bands of solo violin music, as a fingerprint of the instrument, is used for classification purposes within a support vector machine scheme. Our results suggest that coupling complexity is able to capture essential characteristics, sufficient to distinguish among different violins.

  11. Ultrasensitive in-house reverse transcription-competitive PCR for quantitation of HIV-1 RNA in plasma.

    PubMed

    Venturi, G; Ferruzzi, R; Romano, L; Catucci, M; Valensin, P E; Zazzi, M

    2000-06-01

    An ultrasensitive version of an 'in-house' reverse transcription-competitive polymerase chain reaction assay described previously for quantitation of human immunodeficiency virus type 1 (HIV-1) RNA in plasma was developed. The increase in sensitivity from 400 to 50 HIV-1 RNA copies/ml was achieved by pelleting virus particles from 1.8 ml plasma by centrifugation prior to RNA extraction, modifying competitor DNA structure and amounts, and redesigning primers. Quantitation of HIV-1 RNA in 130 samples tested previously by the standard assay showed that the two procedures yield comparable results (mean absolute difference, 0.26+/-0.20 log) and that the ultrasensitive version detects HIV-1 RNA below the threshold of sensitivity of the standard method. The ultrasensitive 'in-house assay' and the reference QUANTIPLEX HIV-1 RNA 3.0 had the same sensitivity and gave equivalent results (mean absolute difference, 0.19+/-0.11 log), as shown by parallel blinded testing of 47 plasma samples. Titration experiments with reconstructed plasma samples allowed the determination of a dynamic range of 50-500000 HIV-1 RNA copies/ml for the 'in-house' system. The interassay coefficient of variation for samples nominally containing 200, 4000 and 80000 HIV-1 RNA copies/ml were 33.4, 22.9 and 38.2%, respectively. The performance, turnaround time, and cost-effectiveness of this system make it suitable for medium-scale clinical application.

  12. Strand-Specific Quantitative Reverse Transcription-Polymerase Chain Reaction Assay for Measurement of Arenavirus Genomic and Antigenomic RNAs

    PubMed Central

    Haist, Kelsey; Ziegler, Christopher; Botten, Jason

    2015-01-01

    Arenaviruses are bi-segmented, single-stranded RNA viruses that cause significant human disease. The manner in which they regulate the replication of their genome is not well-understood. This is partly due to the absence of a highly sensitive assay to measure individual species of arenavirus replicative RNAs. To overcome this obstacle, we designed a quantitative reverse transcription (RT)-PCR assay for selective quantitation of each of the lymphocytic choriomeningitis virus (LCMV) genomic or antigenomic RNAs. During the course of assay design, we identified a nonspecific priming phenomenon whereby, in the absence of an RT primer, cDNAs complementary to each of the LCMV replicative RNA species are generated during RT. We successfully circumvented this nonspecific priming event through the use of biotinylated primers in the RT reaction, which permitted affinity purification of primer-specific cDNAs using streptavidin-coated magnetic beads. As proof of principle, we used the assay to map the dynamics of LCMV replication at acute and persistent time points and to determine the quantities of genomic and antigenomic RNAs that are incorporated into LCMV particles. This assay can be adapted to measure total S or L segment-derived viral RNAs and therefore represents a highly sensitive diagnostic platform to screen for LCMV infection in rodent and human tissue samples and can also be used to quantify virus-cell attachment. PMID:25978311

  13. Dynamic regulation of transcription factors by nucleosome remodeling.

    PubMed

    Li, Ming; Hada, Arjan; Sen, Payel; Olufemi, Lola; Hall, Michael A; Smith, Benjamin Y; Forth, Scott; McKnight, Jeffrey N; Patel, Ashok; Bowman, Gregory D; Bartholomew, Blaine; Wang, Michelle D

    2015-06-05

    The chromatin landscape and promoter architecture are dominated by the interplay of nucleosome and transcription factor (TF) binding to crucial DNA sequence elements. However, it remains unclear whether nucleosomes mobilized by chromatin remodelers can influence TFs that are already present on the DNA template. In this study, we investigated the interplay between nucleosome remodeling, by either yeast ISW1a or SWI/SNF, and a bound TF. We found that a TF serves as a major barrier to ISW1a remodeling, and acts as a boundary for nucleosome repositioning. In contrast, SWI/SNF was able to slide a nucleosome past a TF, with concurrent eviction of the TF from the DNA, and the TF did not significantly impact the nucleosome positioning. Our results provide direct evidence for a novel mechanism for both nucleosome positioning regulation by bound TFs and TF regulation via dynamic repositioning of nucleosomes.

  14. Deep proteomics of mouse skeletal muscle enables quantitation of protein isoforms, metabolic pathways, and transcription factors.

    PubMed

    Deshmukh, Atul S; Murgia, Marta; Nagaraj, Nagarjuna; Treebak, Jonas T; Cox, Jürgen; Mann, Matthias

    2015-04-01

    Skeletal muscle constitutes 40% of individual body mass and plays vital roles in locomotion and whole-body metabolism. Proteomics of skeletal muscle is challenging because of highly abundant contractile proteins that interfere with detection of regulatory proteins. Using a state-of-the art MS workflow and a strategy to map identifications from the C2C12 cell line model to tissues, we identified a total of 10,218 proteins, including skeletal muscle specific transcription factors like myod1 and myogenin and circadian clock proteins. We obtain absolute abundances for proteins expressed in a muscle cell line and skeletal muscle, which should serve as a valuable resource. Quantitation of protein isoforms of glucose uptake signaling pathways and in glucose and lipid metabolic pathways provides a detailed metabolic map of the cell line compared with tissue. This revealed unexpectedly complex regulation of AMP-activated protein kinase and insulin signaling in muscle tissue at the level of enzyme isoforms.

  15. Deep Proteomics of Mouse Skeletal Muscle Enables Quantitation of Protein Isoforms, Metabolic Pathways, and Transcription Factors*

    PubMed Central

    Deshmukh, Atul S.; Murgia, Marta; Nagaraj, Nagarjuna; Treebak, Jonas T.; Cox, Jürgen; Mann, Matthias

    2015-01-01

    Skeletal muscle constitutes 40% of individual body mass and plays vital roles in locomotion and whole-body metabolism. Proteomics of skeletal muscle is challenging because of highly abundant contractile proteins that interfere with detection of regulatory proteins. Using a state-of-the art MS workflow and a strategy to map identifications from the C2C12 cell line model to tissues, we identified a total of 10,218 proteins, including skeletal muscle specific transcription factors like myod1 and myogenin and circadian clock proteins. We obtain absolute abundances for proteins expressed in a muscle cell line and skeletal muscle, which should serve as a valuable resource. Quantitation of protein isoforms of glucose uptake signaling pathways and in glucose and lipid metabolic pathways provides a detailed metabolic map of the cell line compared with tissue. This revealed unexpectedly complex regulation of AMP-activated protein kinase and insulin signaling in muscle tissue at the level of enzyme isoforms. PMID:25616865

  16. Molecular Dynamics of "Fuzzy" Transcriptional Activator-Coactivator Interactions

    PubMed Central

    Scholes, Natalie S.; Weinzierl, Robert O. J.

    2016-01-01

    Transcriptional activation domains (ADs) are generally thought to be intrinsically unstructured, but capable of adopting limited secondary structure upon interaction with a coactivator surface. The indeterminate nature of this interface made it hitherto difficult to study structure/function relationships of such contacts. Here we used atomistic accelerated molecular dynamics (aMD) simulations to study the conformational changes of the GCN4 AD and variants thereof, either free in solution, or bound to the GAL11 coactivator surface. We show that the AD-coactivator interactions are highly dynamic while obeying distinct rules. The data provide insights into the constant and variable aspects of orientation of ADs relative to the coactivator, changes in secondary structure and energetic contributions stabilizing the various conformers at different time points. We also demonstrate that a prediction of α-helical propensity correlates directly with the experimentally measured transactivation potential of a large set of mutagenized ADs. The link between α-helical propensity and the stimulatory activity of ADs has fundamental practical and theoretical implications concerning the recruitment of ADs to coactivators. PMID:27175900

  17. Quantitative spatial analysis of transcripts in multinucleate cells using single-molecule FISH.

    PubMed

    Lee, ChangHwan; Roberts, Samantha E; Gladfelter, Amy S

    2016-04-01

    mRNA positioning in the cell is important for diverse cellular functions and proper development of multicellular organisms. Single-molecule RNA FISH (smFISH) enables quantitative investigation of mRNA localization and abundance at the level of individual molecules in the context of cellular features. Details about spatial mRNA patterning at various times, in different genetic backgrounds, at different developmental stages, and under varied environmental conditions provide invaluable insights into the mechanisms and functions of spatial regulation. Here, we describe detailed methods for performing smFISH along with immunofluorescence for two large, multinucleate cell types: the fungus Ashbya gossypii and cultured mouse myotubes. We also put forward a semi-automated image processing tool that systematically detects mRNAs from smFISH data and statistically analyzes the spatial pattern of mRNAs using a customized MATLAB code. These protocols and image analysis tools can be adapted to a wide variety of transcripts and cell types for systematically and quantitatively analyzing mRNA distribution in three-dimensional space. PMID:26690072

  18. Normalization of Reverse Transcription Quantitative PCR Data During Ageing in Distinct Cerebral Structures.

    PubMed

    Bruckert, G; Vivien, D; Docagne, F; Roussel, B D

    2016-04-01

    Reverse transcription quantitative-polymerase chain reaction (RT-qPCR) has become a routine method in many laboratories. Normalization of data from experimental conditions is critical for data processing and is usually achieved by the use of a single reference gene. Nevertheless, as pointed by the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, several reference genes should be used for reliable normalization. Ageing is a physiological process that results in a decline of many expressed genes. Reliable normalization of RT-qPCR data becomes crucial when studying ageing. Here, we propose a RT-qPCR study from four mouse brain regions (cortex, hippocampus, striatum and cerebellum) at different ages (from 8 weeks to 22 months) in which we studied the expression of nine commonly used reference genes. With the use of two different algorithms, we found that all brain structures need at least two genes for a good normalization step. We propose specific pairs of gene for efficient data normalization in the four brain regions studied. These results underline the importance of reliable reference genes for specific brain regions in ageing.

  19. Dynamic regulation of eve stripe 2 expression reveals transcriptional bursts in living Drosophila embryos.

    PubMed

    Bothma, Jacques P; Garcia, Hernan G; Esposito, Emilia; Schlissel, Gavin; Gregor, Thomas; Levine, Michael

    2014-07-22

    We present the use of recently developed live imaging methods to examine the dynamic regulation of even-skipped (eve) stripe 2 expression in the precellular Drosophila embryo. Nascent transcripts were visualized via MS2 RNA stem loops. The eve stripe 2 transgene exhibits a highly dynamic pattern of de novo transcription, beginning with a broad domain of expression during nuclear cycle 12 (nc12), and progressive refinement during nc13 and nc14. The mature stripe 2 pattern is surprisingly transient, constituting just ∼15 min of the ∼90-min period of expression. Nonetheless, this dynamic transcription profile faithfully predicts the limits of the mature stripe visualized by conventional in situ detection methods. Analysis of individual transcription foci reveals intermittent bursts of de novo transcription, with duration cycles of 4-10 min. We discuss a multistate model of transcription regulation and speculate on its role in the dynamic repression of the eve stripe 2 expression pattern during development.

  20. Post-translational Control of the Temporal Dynamics of Transcription Factor Activity Regulates Neurogenesis.

    PubMed

    Quan, Xiao-Jiang; Yuan, Liqun; Tiberi, Luca; Claeys, Annelies; De Geest, Natalie; Yan, Jiekun; van der Kant, Rob; Xie, Wei R; Klisch, Tiemo J; Shymkowitz, Joost; Rousseau, Frederic; Bollen, Mathieu; Beullens, Monique; Zoghbi, Huda Y; Vanderhaeghen, Pierre; Hassan, Bassem A

    2016-01-28

    Neurogenesis is initiated by the transient expression of the highly conserved proneural proteins, bHLH transcriptional regulators. Here, we discover a conserved post-translational switch governing the duration of proneural protein activity that is required for proper neuronal development. Phosphorylation of a single Serine at the same position in Scute and Atonal proneural proteins governs the transition from active to inactive forms by regulating DNA binding. The equivalent Neurogenin2 Threonine also regulates DNA binding and proneural activity in the developing mammalian neocortex. Using genome editing in Drosophila, we show that Atonal outlives its mRNA but is inactivated by phosphorylation. Inhibiting the phosphorylation of the conserved proneural Serine causes quantitative changes in expression dynamics and target gene expression resulting in neuronal number and fate defects. Strikingly, even a subtle change from Serine to Threonine appears to shift the duration of Atonal activity in vivo, resulting in neuronal fate defects. PMID:26824657

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

    PubMed Central

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

    2016-01-01

    Drying and aging paint dispersions display a wealth of complex phenomena that make their study fascinating yet challenging. To meet the growing demand for sustainable, high-quality paints, it is essential to unravel the microscopic mechanisms underlying these phenomena. Visualising the governing dynamics is, however, intrinsically difficult because the dynamics are typically heterogeneous and span a wide range of time scales. Moreover, the high turbidity of paints precludes conventional imaging techniques from reaching deep inside the paint. To address these challenges, we apply a scattering technique, Laser Speckle Imaging, as a versatile and quantitative tool to elucidate the internal dynamics, with microscopic resolution and spanning seven decades of time. We present a toolbox of data analysis and image processing methods that allows a tailored investigation of virtually any turbid dispersion, regardless of the geometry and substrate. Using these tools we watch a variety of paints dry and age with unprecedented detail. PMID:27682840

  2. RNA sequencing analysis identifies novel spliced transcripts but does not indicate quantitative or qualitative changes of viral transcripts during progression of cottontail rabbit papillomavirus-induced tumours.

    PubMed

    Probst-Hunczek, Sonja; Jäger, Günter; Schneider, Markus; Notz, Ekaterina; Stubenrauch, Frank; Iftner, Thomas

    2015-10-01

    Persistent infections with high-risk human papillomaviruses (HPVs) can result in the development of cancer of the cervix uteri and other malignancies. The underlying molecular mechanisms leading to the progression of HPV-induced lesions are, however, not well understood. Cottontail rabbit papillomavirus (CRPV) induces papillomas in domestic rabbits which progress at a very high rate to cancer. Using this model, we compared the transcriptional patterns of CRPV in papillomas and carcinomas by RNA sequencing (RNA-seq). The most abundant transcripts can encode E7, short E6 and E1^E4, followed by full-length E6, E2, E1 and E9^E2C. In addition, we identified two rare, novel splice junctions 7810/3714 and 1751/3065 in both papillomas and carcinomas which have been described for other papillomaviruses. Neither RNA-seq nor quantitative real-time PCR-based assays identified qualitative or quantitative changes of viral transcription between papillomas and carcinomas. In summary, our analyses confirmed that papillomaviruses have highly similar transcriptional patterns, but they do not suggest that changes in these patterns contribute to the progression of CRPV-induced tumours. PMID:26297146

  3. Evaluation of quantitative polymerase chain reaction-based approaches for determining gene copy and gene transcript numbers in environmental samples.

    PubMed

    Smith, Cindy J; Nedwell, David B; Dong, Liang F; Osborn, A Mark

    2006-05-01

    Quantitative polymerase chain reaction (Q-PCR) amplification is widely applied for determining gene and transcript numbers within environmental samples. This research evaluated Q-PCR reproducibility via TaqMan assays quantifying 16S rRNA gene and transcript numbers in sediments, within and between replicate Q-PCR assays. Intra-assay variation in 16S rRNA gene numbers in replicate DNA samples was low (coefficients of variation; CV from 3.2 to 5.2%). However, variability increased using replicated standard curves within separate Q-PCR assays (CV from 11.2% to 26%), indicating absolute comparison of gene numbers between Q-PCR assays was less reliable. 16S rRNA transcript quantification was evaluated using standard curves of diluted RNA or cDNA (before, or following, reverse transcription). These standard curves were statistically different with cDNA-derived curves giving higher r(2) values and Q-PCR efficiencies. Template concentrations used in Q-PCR also affected 16S rRNA gene and transcript numbers. For DNA, 10(-3) dilutions yielded higher gene numbers than 10(-1) and 10(-2) dilutions. Conversely, RNA template dilution reduced numbers of transcripts detected. Finally, different nucleic acid isolation methods also resulted in gene and transcript number variability. This research demonstrates Q-PCR determination of absolute numbers of genes and transcripts using environmental nucleic acids should be treated cautiously.

  4. Dynamic mitochondrial localization of nuclear transcription factor HMGA1

    SciTech Connect

    Dement, Gregory A.; Treff, Nathan R.; Magnuson, Nancy S.; Franceschi, Vincent; Reeves, Raymond . E-mail: reevesr@mail.wsu.edu

    2005-07-15

    It has been well established that high mobility group A1 (HMGA1) proteins act within the nucleus of mammalian cells as architectural transcription factors that regulate the expression of numerous genes. Here, however, we report on the unexpected cytoplasmic/mitochondrial localization of the HMGA1 proteins within multiple cell types. Indirect immunofluorescence, electron microscopic immunolocalization, and Western blot studies revealed that, in addition to the nucleus, HMGA1 proteins could also be found in both the cytoplasm and mitochondria of randomly dividing populations of wild-type murine NIH3T3 cells and transgenic human MCF-7 breast cancer epithelial cells expressing a hemagglutinin tagged-HMGA1a fusion protein. While the molecular mechanisms underlying these novel subcellular localization patterns have not yet been determined, initial synchronization studies revealed a dynamic, cell cycle-dependent translocation of HMGA1 proteins from the nucleus into the cytoplasm and mitochondria of NIH3T3 cells. Furthermore, preliminary functionality studies utilizing a modified 'chromatin' immunoprecipitation protocol revealed that HMGA1 retains its DNA binding capabilities within the mitochondria and associates with the regulatory D-loop region in vivo. We discuss potential new biological roles for the classically nuclear HMGA1 proteins with regard to the observed nucleocytoplasmic translocation, mitochondrial internalization, and regulatory D-loop DNA binding.

  5. A novel approach to quantitating leukemia fusion transcripts by qRT-PCR without the need for standard curves.

    PubMed

    Schumacher, Jonathan A; Scott Reading, N; Szankasi, Philippe; Matynia, Anna P; Kelley, Todd W

    2015-08-01

    Acute myeloid leukemia patients with recurrent cytogenetic abnormalities including inv(16);CBFB-MYH11 and t(15;17);PML-RARA may be assessed by monitoring the levels of the corresponding abnormal fusion transcripts by quantitative reverse transcription-PCR (qRT-PCR). Such testing is important for evaluating the response to therapy and for the detection of early relapse. Existing qRT-PCR methods are well established and in widespread use in clinical laboratories but they are laborious and require the generation of standard curves. Here, we describe a new method to quantitate fusion transcripts in acute myeloid leukemia by qRT-PCR without the need for standard curves. Our approach uses a plasmid calibrator containing both a fusion transcript sequence and a reference gene sequence, representing a perfect normalized copy number (fusion transcript copy number/reference gene transcript copy number; NCN) of 1.0. The NCN of patient specimens can be calculated relative to that of the single plasmid calibrator using experimentally derived PCR efficiency values. We compared the data obtained using the plasmid calibrator method to commercially available assays using standard curves and found that the results obtained by both methods are comparable over a broad range of values with similar sensitivities. Our method has the advantage of simplicity and is therefore lower in cost and may be less subject to errors that may be introduced during the generation of standard curves.

  6. A general quantitative theory of forest structure and dynamics.

    PubMed

    West, Geoffrey B; Enquist, Brian J; Brown, James H

    2009-04-28

    We present the first part of a quantitative theory for the structure and dynamics of forests at demographic and resource steady state. The theory uses allometric scaling relations, based on metabolism and biomechanics, to quantify how trees use resources, fill space, and grow. These individual-level traits and properties scale up to predict emergent properties of forest stands, including size-frequency distributions, spacing relations, resource flux rates, and canopy configurations. Two insights emerge from this analysis: (i) The size structure and spatial arrangement of trees in the entire forest are emergent manifestations of the way that functionally invariant xylem elements are bundled together to conduct water and nutrients up from the trunks, through the branches, to the leaves of individual trees. (ii) Geometric and dynamic properties of trees in a forest and branches in trees scale identically, so that the entire forest can be described mathematically and behaves structurally and functionally like a scaled version of the branching networks in the largest tree. This quantitative framework uses a small number of parameters to predict numerous structural and dynamical properties of idealized forests.

  7. Quantitative fluorescent speckle microscopy (QFSM) to measure actin dynamics.

    PubMed

    Mendoza, Michelle C; Besson, Sebastien; Danuser, Gaudenz

    2012-10-01

    Quantitative fluorescent speckle microscopy (QFSM) is a live-cell imaging method to analyze the dynamics of macromolecular assemblies with high spatial and temporal resolution. Its greatest successes were in the analysis of actin filament and adhesion dynamics in the context of cell migration and microtubule dynamics in interphase and the meiotic/mitotic spindle. Here, focus is on the former application to illustrate the procedures of FSM imaging and the computational image processing that extracts quantitative information from these experiments. QFSM is advantageous over other methods because it measures the movement and turnover kinetics of the actin filament (F-actin) network in living cells across the entire field of view. Experiments begin with the microinjection of fluorophore-labeled actin into cells, which generate a low ratio of fluorescently labeled to endogenously unlabeled actin monomers. Spinning disk confocal or wide-field imaging then visualizes fluorophore clusters (two to eight actin monomers) within the assembled F-actin network as speckles. QFSM software identifies and computationally tracks and utilizes the location, appearance, and disappearance of speckles to derive network flows and maps of the rate of filament assembly and disassembly. PMID:23042526

  8. Global, quantitative and dynamic mapping of protein subcellular localization

    PubMed Central

    Itzhak, Daniel N; Tyanova, Stefka; Cox, Jürgen; Borner, Georg HH

    2016-01-01

    Subcellular localization critically influences protein function, and cells control protein localization to regulate biological processes. We have developed and applied Dynamic Organellar Maps, a proteomic method that allows global mapping of protein translocation events. We initially used maps statically to generate a database with localization and absolute copy number information for over 8700 proteins from HeLa cells, approaching comprehensive coverage. All major organelles were resolved, with exceptional prediction accuracy (estimated at >92%). Combining spatial and abundance information yielded an unprecedented quantitative view of HeLa cell anatomy and organellar composition, at the protein level. We subsequently demonstrated the dynamic capabilities of the approach by capturing translocation events following EGF stimulation, which we integrated into a quantitative model. Dynamic Organellar Maps enable the proteome-wide analysis of physiological protein movements, without requiring any reagents specific to the investigated process, and will thus be widely applicable in cell biology. DOI: http://dx.doi.org/10.7554/eLife.16950.001 PMID:27278775

  9. Lessons Learned from Quantitative Dynamical Modeling in Systems Biology

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2013-01-01

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

  11. A simple, inexpensive method for preparing cell lysates suitable for downstream reverse transcription quantitative PCR

    PubMed Central

    Shatzkes, Kenneth; Teferedegne, Belete; Murata, Haruhiko

    2014-01-01

    Sample nucleic acid purification can often be rate-limiting for conventional quantitative PCR (qPCR) workflows. We recently developed high-throughput virus microneutralization assays using an endpoint assessment approach based on reverse transcription qPCR (RT-qPCR). The need for cumbersome RNA purification is circumvented in our assays by making use of a commercial reagent that can easily generate crude cell lysates amenable to direct analysis by one-step RT-qPCR. In the present study, we demonstrate that a simple buffer containing a non-ionic detergent can serve as an inexpensive alternative to commercially available reagents for the purpose of generating RT-qPCR-ready cell lysates from MDCK cells infected with influenza virus. We have found that addition of exogenous RNase inhibitor as a buffer component is not essential in order to maintain RNA integrity, even following stress at 37°C incubation for 1–2 hours, in cell-lysate samples either freshly prepared or previously stored frozen at −80°C. PMID:24722424

  12. Dynamic Mechanism for the Transcription Apparatus Orchestrating Reliable Responses to Activators

    NASA Astrophysics Data System (ADS)

    Wang, Yaolai; Liu, Feng; Wang, Wei

    2012-05-01

    The transcription apparatus (TA) is a huge molecular machine. It detects the time-varying concentrations of transcriptional activators and initiates mRNA transcripts at appropriate rates. Based on the general structural organizations of the TA, we propose how the TA dynamically orchestrates transcriptional responses. The activators rapidly cycle in and out of a clamp-like space temporarily formed between the enhancer and the Mediator, with the concentration of activators encoded as their temporal occupancy rate (RTOR) within the space. The entry of activators into this space induces allostery in the Mediator, resulting in a facilitated circumstance for transcriptional reinitiation. The reinitiation rate is much larger than the cycling rate of activators, thereby RTOR guiding the amount of transcripts. Based on this mechanism, stochastic simulations can qualitatively reproduce and interpret multiple features of gene expression, e.g., transcriptional bursting is not mere noise as traditionally believed, but rather the basis of reliable transcriptional responses.

  13. VITELLOGENIN GENE TRANSCRIPTION: A RELATIVE QUANTITATIVE EXPOSURE INDICATOR OF ENVIRONMENTAL ESTROGENS

    EPA Science Inventory

    We report the development of a quantifiable exposure indicator for measuring the presence of environmental estrogens in aquatic systems. Synthetic oligonucleotides, designed specifically for the vitellogenin gene (Vg) transcription product, were used in a Reverse Transcription Po...

  14. Dynamic Zebrafish Interactome Reveals Transcriptional Mechanisms of Dioxin Toxicity

    PubMed Central

    Alexeyenko, Andrey; Wassenberg, Deena M.; Lobenhofer, Edward K.; Yen, Jerry; Linney, Elwood; Sonnhammer, Erik L. L.; Meyer, Joel N.

    2010-01-01

    Background In order to generate hypotheses regarding the mechanisms by which 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) causes toxicity, we analyzed global gene expression changes in developing zebrafish embryos exposed to this potent toxicant in the context of a dynamic gene network. For this purpose, we also computationally inferred a zebrafish (Danio rerio) interactome based on orthologs and interaction data from other eukaryotes. Methodology/Principal Findings Using novel computational tools to analyze this interactome, we distinguished between dioxin-dependent and dioxin-independent interactions between proteins, and tracked the temporal propagation of dioxin-dependent transcriptional changes from a few genes that were altered initially, to large groups of biologically coherent genes at later times. The most notable processes altered at later developmental stages were calcium and iron metabolism, embryonic morphogenesis including neuronal and retinal development, a variety of mitochondria-related functions, and generalized stress response (not including induction of antioxidant genes). Within the interactome, many of these responses were connected to cytochrome P4501A (cyp1a) as well as other genes that were dioxin-regulated one day after exposure. This suggests that cyp1a may play a key role initiating the toxic dysregulation of those processes, rather than serving simply as a passive marker of dioxin exposure, as suggested by earlier research. Conclusions/Significance Thus, a powerful microarray experiment coupled with a flexible interactome and multi-pronged interactome tools (which are now made publicly available for microarray analysis and related work) suggest the hypothesis that dioxin, best known in fish as a potent cardioteratogen, has many other targets. Many of these types of toxicity have been observed in mammalian species and are potentially caused by alterations to cyp1a. PMID:20463971

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

    PubMed

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

    2014-02-28

    Two methods for quantitation of triacetone triperoxide (TATP) vapor using a programmable temperature vaporization (PTV) inlet coupled to a gas chromatography/mass spectrometer (GC/MS) have been demonstrated. The dynamic headspace of bulk TATP was mixed with clean humid air to produce a TATP vapor stream. Sampling via a heated transfer line to a PTV inlet with a Tenax-TA™ filled liner allowed for direct injection of the vapor stream to a GC/MS for vapor quantitation. TATP was extracted from the vapor stream and subsequently desorbed from the PTV liner for splitless injection on the GC column. Calibration curves were prepared using solution standards with a standard split/splitless GC inlet for quantitation of the TATP vapor. Alternatively, vapor was sampled onto a Tenax-TA™ sample tube and placed into a thermal desorption system. In this instance, vapor was desorbed from the tube and subsequently trapped on a liquid nitrogen cooled PTV inlet. Calibration curves for this method were prepared from direct liquid injection of standards onto samples tube with the caveat that a vacuum is applied to the tube during deposition to ensure that the volatile TATP penetrates into the tube. Vapor concentration measurements, as determined by either GC/MS analysis or mass gravimetry of the bulk TATP, were statistically indistinguishable. Different approaches to broaden the TATP vapor dynamic range, including diluent air flow, sample chamber temperature, sample vial orifice size, and sample size are discussed. Vapor concentrations between 50 and 5400ngL(-1) are reported, with stable vapor generation observed for as long as 60 consecutive hours. PMID:24508355

  16. Xist and Tsix Transcription Dynamics Is Regulated by the X-to-Autosome Ratio and Semistable Transcriptional States

    PubMed Central

    Loos, Friedemann; Maduro, Cheryl; Loda, Agnese; Lehmann, Johannes; Kremers, Gert-Jan; ten Berge, Derk; Grootegoed, J. Anton

    2016-01-01

    In female mammals, X chromosome inactivation (XCI) is a key process in the control of gene dosage compensation between X-linked genes and autosomes. Xist and Tsix, two overlapping antisense-transcribed noncoding genes, are central elements of the X inactivation center (Xic) regulating XCI. Xist upregulation results in the coating of the entire X chromosome by Xist RNA in cis, whereas Tsix transcription acts as a negative regulator of Xist. Here, we generated Xist and Tsix reporter mouse embryonic stem (ES) cell lines to study the genetic and dynamic regulation of these genes upon differentiation. Our results revealed mutually antagonistic roles for Tsix on Xist and vice versa and indicate the presence of semistable transcriptional states of the Xic locus predicting the outcome of XCI. These transcriptional states are instructed by the X-to-autosome ratio, directed by regulators of XCI, and can be modulated by tissue culture conditions. PMID:27528619

  17. [Selective detection of viable pathogenic bacteria in water using reverse transcription quantitative PCR].

    PubMed

    Lin, Yi-Wen; Li, Dan; Wu, Shu-Xu; He, Miao; Yang, Tian

    2012-11-01

    A reverse transcription q quantitative PCR (RT-qPCR) assay method was established, which can quantify the copy numbers of RNA in pathogenic bacteria of E. coli and Enterococcus faecium. The results showed that cDNA was generated with the RT-PCR reagents, target gene was quantified with the qPCR, the copy numbers of RNA were stable at about 1 copies x CFU(-1) for E. coli and 7.98 x 10(2) copies x CFU(-1) for Enterococcus faecium respectively during the stationary grow phase for the both indicator bacteria [E. coli (6-18 h) and Enterococcus faecium (10-38 h)]. The established RT-qPCR method can quantify the numbers of viable bacteria through detecting bacterial RNA targets. Through detecting the heat-treated E. coli and Enterococcus faecium by three methods (culture method, qPCR, RT-qPCR), we found that the qPCR and RT-qPCR can distinguish 1.43 lg copy non-viable E. coli and 2.5 lg copy non-viable Enterococcus faecium. These results indicated that the established methods could effectively distinguish viable bacteria from non-viable bacteria. Finally we used this method to evaluate the real effluents of the secondary sedimentation of wastewater treatment plant (WWTP), the results showed that the correlation coefficients (R2) between RT-qPCR and culture method were 0.930 (E. coli) and 0.948 (Enterococcus faecium), and this established RT-PCR method can rapidly detect viable pathogenic bacteria in genuine waters.

  18. Sensitive quantitative detection of commensal bacteria by rRNA-targeted reverse transcription-PCR.

    PubMed

    Matsuda, Kazunori; Tsuji, Hirokazu; Asahara, Takashi; Kado, Yukiko; Nomoto, Koji

    2007-01-01

    A sensitive rRNA-targeted reverse transcription-quantitative PCR (RT-qPCR) method was developed for exact and sensitive enumeration of subdominant bacterial populations. Using group- or species-specific primers for 16S or 23S rRNA, analytical curves were constructed for Escherichia coli, Enterococcus faecalis, Staphylococcus aureus, Clostridium perfringens, and Pseudomonas aeruginosa, and the threshold cycle value was found to be linear up to an RNA amount of 10(-3) cell per RT-PCR. The number of bacteria in culture was determined by RT-qPCR, and the results correlated well with the CFU count over the range from 10(0) to 10(5) CFU. The bacterial counts obtained by RT-qPCR were the same as the CFU counts irrespective of the growth phase in vitro, except for C. perfringens during starvation periods; the viable cell counts obtained by using a combination of 4',6-diamidino-2-phenylindole (DAPI) staining and SYTO9-propidium iodide double staining were in good agreement with the RT-qPCR counts rather than with the CFU counts. The RT-qPCR method could detect endogenous Enterobacteriaceae and P. aeruginosa in feces of hospitalized patients (n = 38) at a level of 10(3) cells per g of feces, and for enumeration of S. aureus or P. aeruginosa spiked into human peripheral blood, the lower detection limit for RT-qPCR quantification of the bacteria was 2 cells per ml of blood, suggesting that this method was equivalent to the conventional culture method. As only 5 h was needed for RT-qPCR quantification, we suggest that rRNA-targeted RT-qPCR assays provide a sensitive and convenient system for quantification of commensal bacteria and for examining their possible invasion of a host.

  19. Fractional dynamics of globally slow transcription and its impact on deterministic genetic oscillation.

    PubMed

    Wei, Kun; Gao, Shilong; Zhong, Suchuan; Ma, Hong

    2012-01-01

    In dynamical systems theory, a system which can be described by differential equations is called a continuous dynamical system. In studies on genetic oscillation, most deterministic models at early stage are usually built on ordinary differential equations (ODE). Therefore, gene transcription which is a vital part in genetic oscillation is presupposed to be a continuous dynamical system by default. However, recent studies argued that discontinuous transcription might be more common than continuous transcription. In this paper, by appending the inserted silent interval lying between two neighboring transcriptional events to the end of the preceding event, we established that the running time for an intact transcriptional event increases and gene transcription thus shows slow dynamics. By globally replacing the original time increment for each state increment by a larger one, we introduced fractional differential equations (FDE) to describe such globally slow transcription. The impact of fractionization on genetic oscillation was then studied in two early stage models--the Goodwin oscillator and the Rössler oscillator. By constructing a "dual memory" oscillator--the fractional delay Goodwin oscillator, we suggested that four general requirements for generating genetic oscillation should be revised to be negative feedback, sufficient nonlinearity, sufficient memory and proper balancing of timescale. The numerical study of the fractional Rössler oscillator implied that the globally slow transcription tends to lower the chance of a coupled or more complex nonlinear genetic oscillatory system behaving chaotically.

  20. Preparation of cell lines for single-cell analysis of transcriptional activation dynamics.

    PubMed

    Rafalska-Metcalf, Ilona U; Janicki, Susan M

    2013-01-01

    Imaging molecularly defined regions of chromatin in single living cells during transcriptional activation has the potential to provide new insight into gene regulatory mechanisms. Here, we describe a method for isolating cell lines with multi-copy arrays of reporter transgenes, which can be used for real-time high-resolution imaging of transcriptional activation dynamics in single cells.

  1. Development of quantitative RT-PCR assays for detection of three classes of HHV-6B gene transcripts.

    PubMed

    Ihira, Masaru; Enomoto, Yoshihiko; Kawamura, Yoshiki; Nakai, Hidetaka; Sugata, Ken; Asano, Yoshizo; Tsuzuki, Motohiro; Emi, Nobuhiko; Goto, Tatsunori; Miyamura, Koichi; Matsumoto, Kimikazu; Kato, Koji; Takahashi, Yoshiyuki; Kojima, Seiji; Yoshikawa, Tetsushi

    2012-09-01

    The monitoring of active human herpesvirus 6 (HHV-6) B infection is important for distinguishing between the reactivation and latent state of the virus. The aim of this present study is to develop a quantitative reverse transcription polymerase chain reaction (RT-PCR) assay for diagnosis of active viral infection. Primers and probes for in house quantitative RT-PCR methods were designed to detect the three kinetic classes of HHV-6B mRNAs (U90, U12, U100). Stored PBMCs samples collected from 10 patients with exanthem subitum (primary HHV-6B infection) and 15 hematopoietic stem cell transplant recipients with HHV-6B reactivation were used to evaluate reliability for testing clinical samples. Excellent linearity was obtained with high correlation efficiency between the diluted RNA (1-100 ng/reaction) and C(t) value of each gene transcript. The U90 and U12 gene transcripts were detected in all of the peripheral blood mononuclear cells (PBMCs) samples collected in acute period of primary HHV-6B infection. Only one convalescent PBMCs sample was positive for the U90 gene transcript. Additionally, the reliability of HHV-6B quantitative RT-PCRs for diagnosis of viral reactivation in hematopoietic transplant recipients was evaluated. Relative to virus culture, U90 quantitative RT-PCR demonstrated the highest assay sensitivity, specificity, positive predictive value, and negative predictive value. Thus, this method could be a rapid and lower cost alternative to virus culture, which is difficult to perform generally, for identifying active HHV-6B infection. PMID:22825817

  2. Transcription dynamically patterns the meiotic chromosome-axis interface

    PubMed Central

    Sun, Xiaoji; Huang, Lingzhi; Markowitz, Tovah E; Blitzblau, Hannah G; Chen, Doris; Klein, Franz; Hochwagen, Andreas

    2015-01-01

    Meiotic chromosomes are highly compacted yet remain transcriptionally active. To understand how chromosome folding accommodates transcription, we investigated the assembly of the axial element, the proteinaceous structure that compacts meiotic chromosomes and promotes recombination and fertility. We found that the axial element proteins of budding yeast are flexibly anchored to chromatin by the ring-like cohesin complex. The ubiquitous presence of cohesin at sites of convergent transcription provides well-dispersed points for axis attachment and thus chromosome compaction. Axis protein enrichment at these sites directly correlates with the propensity for recombination initiation nearby. A separate modulating mechanism that requires the conserved axial-element component Hop1 biases axis protein binding towards small chromosomes. Importantly, axis anchoring by cohesin is adjustable and readily displaced in the direction of transcription by the transcriptional machinery. We propose that such robust but flexible tethering allows the axial element to promote recombination while easily adapting to changes in chromosome activity. DOI: http://dx.doi.org/10.7554/eLife.07424.001 PMID:26258962

  3. Quantitative Vibrational Dynamics of Iron in Carbonyl Porphyrins

    PubMed Central

    Leu, Bogdan M.; Silvernail, Nathan J.; Zgierski, Marek Z.; Wyllie, Graeme R. A.; Ellison, Mary K.; Scheidt, W. Robert; Zhao, Jiyong; Sturhahn, Wolfgang; Alp, E. Ercan; Sage, J. Timothy

    2007-01-01

    We use nuclear resonance vibrational spectroscopy and computational predictions based on density functional theory (DFT) to explore the vibrational dynamics of 57Fe in porphyrins that mimic the active sites of histidine-ligated heme proteins complexed with carbon monoxide. Nuclear resonance vibrational spectroscopy yields the complete vibrational spectrum of a Mössbauer isotope, and provides a valuable probe that is not only selective for protein active sites but quantifies the mean-squared amplitude and direction of the motion of the probe nucleus, in addition to vibrational frequencies. Quantitative comparison of the experimental results with DFT calculations provides a detailed, rigorous test of the vibrational predictions, which in turn provide a reliable description of the observed vibrational features. In addition to the well-studied stretching vibration of the Fe-CO bond, vibrations involving the Fe-imidazole bond, and the Fe-Npyr bonds to the pyrrole nitrogens of the porphyrin contribute prominently to the observed experimental signal. All of these frequencies show structural sensitivity to the corresponding bond lengths, but previous studies have failed to identify the latter vibrations, presumably because the coupling to the electronic excitation is too small in resonance Raman measurements. We also observe the FeCO bending vibrations, which are not Raman active for these unhindered model compounds. The observed Fe amplitude is strongly inconsistent with three-body oscillator descriptions of the FeCO fragment, but agrees quantitatively with DFT predictions. Over the past decade, quantum chemical calculations have suggested revised estimates of the importance of steric distortion of the bound CO in preventing poisoning of heme proteins by carbon monoxide. Quantitative agreement with the predicted frequency, amplitude, and direction of Fe motion for the FeCO bending vibrations provides direct experimental support for the quantum chemical description of the

  4. Quantitative characterization and analysis of the dynamic NF-κB response in microglia

    PubMed Central

    2011-01-01

    Background Activation of the NF-κB transcription factor and its associated gene expression in microglia is a key component in the response to brain injury. Its activation is dynamic and is part of a network of biochemical species with multiple feedback regulatory mechanisms. Mathematical modeling, which has been instrumental for understanding the NF-κB response in other cell types, offers a valuable tool to investigate the regulation of NF-κB activation in microglia at a systems level. Results We quantify the dynamic response of NF-κB activation and activation of the upstream kinase IKK using ELISA measurements of a microglial cell line following treatment with the pro-inflammatory cytokine TNFα. A new mathematical model is developed based on these data sets using a modular procedure that exploits the feedback structure of the network. We show that the new model requires previously unmodeled dynamics involved in the stimulus-induced degradation of the inhibitor IκBα in order to properly describe microglial NF-κB activation in a statistically consistent manner. This suggests a more prominent role for the ubiquitin-proteasome system in regulating the activation of NF-κB to inflammatory stimuli. We also find that the introduction of nonlinearities in the kinetics of IKK activation and inactivation is essential for proper characterization of transient IKK activity and corresponds to known biological mechanisms. Numerical analyses of the model highlight key regulators of the microglial NF-κB response, as well as those governing IKK activation. Results illustrate the dynamic regulatory mechanisms and the robust yet fragile nature of the negative feedback regulated network. Conclusions We have developed a new mathematical model that incorporates previously unmodeled dynamics to characterize the dynamic response of the NF-κB signaling network in microglia. This model is the first of its kind for microglia and provides a tool for the quantitative, systems level

  5. Printing 2-dimentional droplet array for single-cell reverse transcription quantitative PCR assay with a microfluidic robot.

    PubMed

    Zhu, Ying; Zhang, Yun-Xia; Liu, Wen-Wen; Ma, Yan; Fang, Qun; Yao, Bo

    2015-01-01

    This paper describes a nanoliter droplet array-based single-cell reverse transcription quantitative PCR (RT-qPCR) assay method for quantifying gene expression in individual cells. By sequentially printing nanoliter-scale droplets on microchip using a microfluidic robot, all liquid-handling operations including cell encapsulation, lysis, reverse transcription, and quantitative PCR with real-time fluorescence detection, can be automatically achieved. The inhibition effect of cell suspension buffer on RT-PCR assay was comprehensively studied to achieve high-sensitivity gene quantification. The present system was applied in the quantitative measurement of expression level of mir-122 in single Huh-7 cells. A wide distribution of mir-122 expression in single cells from 3061 copies/cell to 79998 copies/cell was observed, showing a high level of cell heterogeneity. With the advantages of full-automation in liquid-handling, simple system structure, and flexibility in achieving multi-step operations, the present method provides a novel liquid-handling mode for single cell gene expression analysis, and has significant potentials in transcriptional identification and rare cell analysis. PMID:25828383

  6. Quantitative Reactivity Scales for Dynamic Covalent and Systems Chemistry.

    PubMed

    Zhou, Yuntao; Li, Lijie; Ye, Hebo; Zhang, Ling; You, Lei

    2016-01-13

    Dynamic covalent chemistry (DCC) has become a powerful tool for the creation of molecular assemblies and complex systems in chemistry and materials science. Herein we developed for the first time quantitative reactivity scales capable of correlation and prediction of the equilibrium of dynamic covalent reactions (DCRs). The reference reactions are based upon universal DCRs between imines, one of the most utilized structural motifs in DCC, and a series of O-, N-, and S- mononucleophiles. Aromatic imines derived from pyridine-2-carboxyaldehyde exhibit capability for controlling the equilibrium through distinct substituent effects. Electron-donating groups (EDGs) stabilize the imine through quinoidal resonance, while electron-withdrawing groups (EWGs) stabilize the adduct by enhancing intramolecular hydrogen bonding, resulting in curvature in Hammett analysis. Notably, unique nonlinearity induced by both EDGs and EWGs emerged in Hammett plot when cyclic secondary amines were used. This is the first time such a behavior is observed in a thermodynamically controlled system, to the best of our knowledge. Unified quantitative reactivity scales were proposed for DCC and defined by the correlation log K = S(N) (R(N) + R(E)). Nucleophilicity parameters (R(N) and S(N)) and electrophilicity parameters (R(E)) were then developed from DCRs discovered. Furthermore, the predictive power of those parameters was verified by successful correlation of other DCRs, validating our reactivity scales as a general and useful tool for the evaluation and modeling of DCRs. The reactivity parameters proposed here should be complementary to well-established kinetics based parameters and find applications in many aspects, such as DCR discovery, bioconjugation, and catalysis. PMID:26652793

  7. Noninvasive, quantitative respirator fit testing through dynamic pressure measurement.

    PubMed

    Carpenter, D R; Willeke, K

    1988-10-01

    A new method has been invented for the noninvasive and quantitative determination of fit for a respirator. The test takes a few seconds and requires less expensive instrumentation than presently used for invasive testing. In this test, the breath is held at a negative pressure for a few seconds, and the leak-induced pressure decay inside the respirator cavity is monitored. A dynamic pressure sensor is attached to a modified cartridge of an air-purifying respirator or built into the respirator body or into the air supply line of an air-supplied respirator. The method is noninvasive in that the modified cartridge can be mounted onto any air-purifying respirator. The pressure decay during testing quantifies the airflow entered through the leak site. An equation has been determined which gives the air leakage as a function of pressure decay slope, respirator volume and the pressure differential during actual wear--all of which are determined by the dynamic pressure sensor. Thus, the ratio of air inhaled through the filters or via the air supply line to the leak rate is a measure of respirator fit, independent of aerosol deposition in the lung and aerosol distribution in the respirator cavity as found for quantitative fit testing with aerosols. The new method is shown to be independent of leak and sensor locations. The concentration and distribution of aerosols entered through the leak site is dependent only on the physical dimensions of the leak site and the air velocity in it, which can be determined independently.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3189157

  8. Quantitative vibrational dynamics of iron in nitrosyl porphyrins.

    PubMed

    Leu, Bogdan M; Zgierski, Marek Z; Wyllie, Graeme R A; Scheidt, W Robert; Sturhahn, Wolfgang; Alp, E Ercan; Durbin, Stephen M; Sage, J Timothy

    2004-04-01

    We use quantitative experimental and theoretical approaches to characterize the vibrational dynamics of the Fe atom in porphyrins designed to model heme protein active sites. Nuclear resonance vibrational spectroscopy (NRVS) yields frequencies, amplitudes, and directions for 57Fe vibrations in a series of ferrous nitrosyl porphyrins, which provide a benchmark for evaluation of quantum chemical vibrational calculations. Detailed normal mode predictions result from DFT calculations on ferrous nitrosyl tetraphenylporphyrin Fe(TPP)(NO), its cation [Fe(TPP)(NO)]+, and ferrous nitrosyl porphine Fe(P)(NO). Differing functionals lead to significant variability in the predicted Fe-NO bond length and frequency for Fe(TPP)(NO). Otherwise, quantitative comparison of calculated and measured Fe dynamics on an absolute scale reveals good overall agreement, suggesting that DFT calculations provide a reliable guide to the character of observed Fe vibrational modes. These include a series of modes involving Fe motion in the plane of the porphyrin, which are rarely identified using infrared and Raman spectroscopies. The NO binding geometry breaks the four-fold symmetry of the Fe environment, and the resulting frequency splittings of the in-plane modes predicted for Fe(TPP)(NO) agree with observations. In contrast to expectations of a simple three-body model, mode energy remains localized on the FeNO fragment for only two modes, an N-O stretch and a mode with mixed Fe-NO stretch and FeNO bend character. Bending of the FeNO unit also contributes to several of the in-plane modes, but no primary FeNO bending mode is identified for Fe(TPP)(NO). Vibrations associated with hindered rotation of the NO and heme doming are predicted at low frequencies, where Fe motion perpendicular to the heme is identified experimentally at 73 and 128 cm-1. Identification of the latter two modes is a crucial first step toward quantifying the reactive energetics of Fe porphyrins and heme proteins.

  9. The Dynamics of DNA Damage Repair and Transcription

    PubMed Central

    Shanbhag, Niraj M.; Greenberg, Roger A.

    2013-01-01

    Recent advances have led to several systems to study transcription from defined loci in living cells. It has now become possible to address long-standing questions regarding the interplay between the processes of DNA damage repair and transcription—two disparate processes that can occur on the same stretch of chromatin and which both lead to extensive chromatin change. Here we describe the development of a system to create enzymatically induced DNA double-strand breaks (DSBs) at a site of inducible transcription and methods to study the interplay between these processes. PMID:23980011

  10. A Nonlinear Discrete Dynamical Model for Transcriptional Regulation: Construction and Properties

    PubMed Central

    Goutsias, John; Kim, Seungchan

    2004-01-01

    Transcriptional regulation is a fundamental mechanism of living cells, which allows them to determine their actions and properties, by selectively choosing which proteins to express and by dynamically controlling the amounts of those proteins. In this article, we revisit the problem of mathematically modeling transcriptional regulation. First, we adopt a biologically motivated continuous model for gene transcription and mRNA translation, based on first-order rate equations, coupled with a set of nonlinear equations that model cis-regulation. Then, we view the processes of transcription and translation as being discrete, which, together with the need to use computational techniques for large-scale analysis and simulation, motivates us to model transcriptional regulation by means of a nonlinear discrete dynamical system. Classical arguments from chemical kinetics allow us to specify the nonlinearities underlying cis-regulation and to include both activators and repressors as well as the notion of regulatory modules in our formulation. We show that the steady-state behavior of the proposed discrete dynamical system is identical to that of the continuous model. We discuss several aspects of our model, related to homeostatic and epigenetic regulation as well as to Boolean networks, and elaborate on their significance. Simulations of transcriptional regulation of a hypothetical metabolic pathway illustrate several properties of our model, and demonstrate that a nonlinear discrete dynamical system may be effectively used to model transcriptional regulation in a biologically relevant way. PMID:15041638

  11. Transcriptional Dynamics of the Embryonic Stem Cell Switch

    PubMed Central

    Chickarmane, Vijay; Troein, Carl; Nuber, Ulrike A; Sauro, Herbert M; Peterson, Carsten

    2006-01-01

    Recent ChIP experiments of human and mouse embryonic stem cells have elucidated the architecture of the transcriptional regulatory circuitry responsible for cell determination, which involves the transcription factors OCT4, SOX2, and NANOG. In addition to regulating each other through feedback loops, these genes also regulate downstream target genes involved in the maintenance and differentiation of embryonic stem cells. A search for the OCT4–SOX2–NANOG network motif in other species reveals that it is unique to mammals. With a kinetic modeling approach, we ascribe function to the observed OCT4–SOX2–NANOG network by making plausible assumptions about the interactions between the transcription factors at the gene promoter binding sites and RNA polymerase (RNAP), at each of the three genes as well as at the target genes. We identify a bistable switch in the network, which arises due to several positive feedback loops, and is switched on/off by input environmental signals. The switch stabilizes the expression levels of the three genes, and through their regulatory roles on the downstream target genes, leads to a binary decision: when OCT4, SOX2, and NANOG are expressed and the switch is on, the self-renewal genes are on and the differentiation genes are off. The opposite holds when the switch is off. The model is extremely robust to parameter changes. In addition to providing a self-consistent picture of the transcriptional circuit, the model generates several predictions. Increasing the binding strength of NANOG to OCT4 and SOX2, or increasing its basal transcriptional rate, leads to an irreversible bistable switch: the switch remains on even when the activating signal is removed. Hence, the stem cell can be manipulated to be self-renewing without the requirement of input signals. We also suggest tests that could discriminate between a variety of feedforward regulation architectures of the target genes by OCT4, SOX2, and NANOG. PMID:16978048

  12. Encoding four gene expression programs in the activation dynamics of a single transcription factor.

    PubMed

    Hansen, Anders S; O'Shea, Erin K

    2016-04-01

    Cellular signaling response pathways often exhibit a bow-tie topology [1,2]: multiple upstream stress signals converge on a single shared transcription factor, which is thought to induce different downstream gene expression programs (Figure 1A). However, if several different signals activate the same transcription factor, can each signal then induce a specific gene expression response? A growing body of literature supports a temporal coding theory where information about environmental signals can be encoded, at least partially, in the temporal dynamics of the shared transcription factor [1,2]. For example, in the case of the budding yeast transcription factor Msn2, different stresses induce distinct Msn2 activation dynamics: Msn2 shows pulsatile nuclear activation with dose-dependent frequency under glucose limitation, but sustained nuclear activation with dose-dependent amplitude under oxidative stress [3]. These dynamic patterns can then lead to differential gene expression responses [3-5], but it is not known how much specificity can be obtained. Thus, a major question of this temporal coding theory is how many gene response programs or cellular functions can be robustly encoded by dynamic control of a single transcription factor. Here we provide the first direct evidence that, simply by regulating the activation dynamics of a single transcription factor, it is possible to preferentially induce four distinct gene expression programs. PMID:27046808

  13. Characterization and Improvement of RNA-Seq Precision in Quantitative Transcript Expression Profiling

    SciTech Connect

    Labaj, Pawel P.; Leparc, German G.; Linggi, Bryan E.; Markillie, Lye Meng; Wiley, H. S.; Kreil, David P.

    2011-07-01

    Measurement precision determines the power of any analysis to reliably identify significant signals, such as in screens for differential expression, independent of whether the experimental design incorporates replicates or not. With the compilation of large scale RNA-Seq data sets with technical replicate samples, however, we can now, for the first time, perform a systematic analysis of the precision of expression level estimates from massively parallel sequencing technology. This then allows considerations for its improvement by computational or experimental means. Results: We report on a comprehensive study of target coverage and measurement precision, including their dependence on transcript expression levels, read depth and other parameters. In particular, an impressive target coverage of 84% of the estimated true transcript population could be achieved with 331 million 50 bp reads, with diminishing returns from longer read lengths and even less gains from increased sequencing depths. Most of the measurement power (75%) is spent on only 7% of the known transcriptome, however, making less strongly expressed transcripts harder to measure. Consequently, less than 30% of all transcripts could be quantified reliably with a relative error < 20%. Based on established tools, we then introduce a new approach for mapping and analyzing sequencing reads that yields substantially improved performance in gene expression profiling, increasing the number of transcripts that can reliably be quantified to over 40%. Extrapolations to higher sequencing depths highlight the need for efficient complementary steps. In discussion we outline possible experimental and computational strategies for further improvements in quantification precision.

  14. In Vivo Transcription Dynamics of the Galactose Operon: A Study on the Promoter Transition from P1 to P2 at Onset of Stationary Phase

    PubMed Central

    Ji, Sang Chun; Wang, Xun; Yun, Sang Hoon; Jeon, Heung Jin; Lee, Hee Jung; Kim, Hackjin; Lim, Heon M.

    2011-01-01

    Quantitative analyses of the 5′ end of gal transcripts indicate that transcription from the galactose operon P1 promoter is higher during cell division. When cells are no longer dividing, however, transcription is initiated more often from the P2 promoter. Escherichia coli cells divide six times before the onset of the stationary phase when grown in LB containing 0.5% galactose at 37°C. Transcription from the two promoters increases, although at different rates, during early exponential phase (until the third cell division, OD600 0.4), and then reaches a plateau. The steady-state transcription from P1 continues in late exponential phase (the next three cell divisions, OD600 3.0), after which transcription from this promoter decreases. However, steady-state transcription from P2 continues 1 h longer into the stationary phase, before decreasing. This longer steady-state P2 transcription constitutes the promoter transition from P1 to P2 at the onset of the stationary phase. The intracellular cAMP concentration dictates P1 transcription dynamics; therefore, promoter transition may result from a lack of cAMP-CRP complex binding to the gal operon. The decay rate of gal-specific transcripts is constant through the six consecutive cell divisions that comprise the exponential growth phase, increases at the onset of the stationary phase, and is too low to be measured during the stationary phase. These data suggest that a regulatory mechanism coordinates the synthesis and decay of gal mRNAs to maintain the observed gal transcription. Our analysis indicates that the increase in P1 transcription is the result of cAMP-CRP binding to increasing numbers of galactose operons in the cell population. PMID:21445255

  15. Quantitative high dynamic range beam profiling for fluorescence microscopy

    SciTech Connect

    Mitchell, T. J. Saunter, C. D.; O’Nions, W.; Girkin, J. M.; Love, G. D.

    2014-10-15

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

  16. Dynamic Regulation of AP-1 Transcriptional Complexes Directs Trophoblast Differentiation

    PubMed Central

    Kent, Lindsey N.; Rumi, M. A. Karim; Roby, Katherine F.

    2015-01-01

    Placentation is a process that establishes the maternal-fetal interface and is required for successful pregnancy. The epithelial component of the placenta consists of trophoblast cells, which possess the capacity for multilineage differentiation and are responsible for placenta-specific functions. FOS-like antigen 1 (FOSL1), a component of AP-1 transcription factor complexes, contributes to the regulation of placental development. FOSL1 expression is restricted to trophoblast giant cells and invasive trophoblast cells. In the present study, we characterized the FOSL1 regulatory pathway in rat trophoblast cells. Transcriptome profiling in control and FOSL1 knockdown cells identified FOSL1-dependent gene sets linked to endocrine and invasive functions. FOSL1 was shown to occupy AP-1 binding sites within these gene loci, as determined by chromatin immunoprecipitation (ChIP). Complementary in vivo experiments using trophoblast-specific lentiviral delivery of FOSL1 short hairpin RNAs (shRNAs) provided in vivo validation of FOSL1 targets. FOSL1 actions require a dimerization partner. Coimmunoprecipitation, coimmunolocalization, and ChIP analyses showed that FOSL1 interacts with JUNB and, to a lesser extent, JUN in differentiating trophoblast cells. Knockdown of FOSL1 and JUNB expression inhibited both endocrine and invasive properties of trophoblast cells. In summary, FOSL1 recruits JUNB to form AP-1 transcriptional complexes that specifically regulate the endocrine and invasive trophoblast phenotypes. PMID:26149388

  17. Dynamic phosphorylation of RelA on Ser42 and Ser45 in response to TNFα stimulation regulates DNA binding and transcription

    PubMed Central

    Lanucara, Francesco; Lam, Connie; Mann, Jelena; Monie, Tom P.; Colombo, Stefano A. P.; Holman, Stephen W.; Boyd, James; Dange, Manohar C.; Mann, Derek A.; White, Michael R. H.

    2016-01-01

    The NF-κB signalling module controls transcription through a network of protein kinases such as the IKKs, as well as inhibitory proteins (IκBs) and transcription factors including RelA/p65. Phosphorylation of the NF-κB subunits is critical for dictating system dynamics. Using both non-targeted discovery and quantitative selected reaction monitoring-targeted proteomics, we show that the cytokine TNFα induces dynamic multisite phosphorylation of RelA at a number of previously unidentified residues. Putative roles for many of these phosphorylation sites on RelA were predicted by modelling of various crystal structures. Stoichiometry of phosphorylation determination of Ser45 and Ser42 revealed preferential early phosphorylation of Ser45 in response to TNFα. Quantitative analyses subsequently confirmed differential roles for pSer42 and pSer45 in promoter-specific DNA binding and a role for both of these phosphosites in regulating transcription from the IL-6 promoter. These temporal dynamics suggest that RelA-mediated transcription is likely to be controlled by functionally distinct NF-κB proteoforms carrying different combinations of modifications, rather than a simple ‘one modification, one effect’ system. PMID:27466442

  18. A dynamic CTCF chromatin binding landscape promotes DNA hydroxymethylation and transcriptional induction of adipocyte differentiation

    PubMed Central

    Dubois-Chevalier, Julie; Oger, Frédérik; Dehondt, Hélène; Firmin, François F.; Gheeraert, Céline; Staels, Bart; Lefebvre, Philippe; Eeckhoute, Jérôme

    2014-01-01

    CCCTC-binding factor (CTCF) is a ubiquitously expressed multifunctional transcription factor characterized by chromatin binding patterns often described as largely invariant. In this context, how CTCF chromatin recruitment and functionalities are used to promote cell type-specific gene expression remains poorly defined. Here, we show that, in addition to constitutively bound CTCF binding sites (CTS), the CTCF cistrome comprises a large proportion of sites showing highly dynamic binding patterns during the course of adipogenesis. Interestingly, dynamic CTCF chromatin binding is positively linked with changes in expression of genes involved in biological functions defining the different stages of adipogenesis. Importantly, a subset of these dynamic CTS are gained at cell type-specific regulatory regions, in line with a requirement for CTCF in transcriptional induction of adipocyte differentiation. This relates to, at least in part, CTCF requirement for transcriptional activation of both the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARG) and its target genes. Functionally, we show that CTCF interacts with TET methylcytosine dioxygenase (TET) enzymes and promotes adipogenic transcriptional enhancer DNA hydroxymethylation. Our study reveals a dynamic CTCF chromatin binding landscape required for epigenomic remodeling of enhancers and transcriptional activation driving cell differentiation. PMID:25183525

  19. Establishment and Validation of a Non-Radioactive Method for In Vitro Transcription Assay Using Primer Extension and Quantitative Real Time PCR.

    PubMed

    Wang, Juan; Zhao, Shasha; Zhou, Ying; Wei, Yun; Deng, Wensheng

    2015-01-01

    Primer extension-dependent in vitro transcription assay is one of the most important approaches in the research field of gene transcription. However, conventional in vitro transcription assays incorporates radioactive isotopes that cause environmental and health concerns and restricts its scope of application. Here we report a novel non-radioactive method for in vitro transcription analysis by combining primer extension with quantitative real time PCR (qPCR). We show that the DNA template within the transcription system can be effectively eliminated to a very low level by our specially designed approach, and that the primers uniquely designed for primer extension and qPCR can specifically recognize the RNA transcripts. Quantitative PCR data demonstrate that the novel method has successfully been applied to in vitro transcription analyses using the adenovirus E4 and major late promoters. Furthermore, we show that the TFIIB recognition element inhibits transcription of TATA-less promoters using both conventional and nonradioactive in vitro transcription assays. Our method will benefit the laboratories that need to perform in vitro transcription but either lack of or choose to avoid radioactive facilities.

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

    PubMed Central

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

    2015-01-01

    Motivation: Recent progress in live-cell imaging and modeling techniques has resulted in generation of a large amount of quantitative data (from experimental measurements and computer simulations) on spatiotemporal dynamics of biological objects such as molecules, cells and organisms. Although many research groups have independently dedicated their efforts to developing software tools for visualizing and analyzing these data, these tools are often not compatible with each other because of different data formats. Results: We developed an open unified format, Biological Dynamics Markup Language (BDML; current version: 0.2), which provides a basic framework for representing quantitative biological dynamics data for objects ranging from molecules to cells to organisms. BDML is based on Extensible Markup Language (XML). Its advantages are machine and human readability and extensibility. BDML will improve the efficiency of development and evaluation of software tools for data visualization and analysis. Availability and implementation: A specification and a schema file for BDML are freely available online at http://ssbd.qbic.riken.jp/bdml/. Contact: sonami@riken.jp Supplementary Information: Supplementary data are available at Bioinformatics online. PMID:25414366

  1. Identification of quantitative trait transcripts for growth traits in the large scales of liver and muscle samples.

    PubMed

    Xiong, Xinwei; Yang, Hui; Yang, Bin; Chen, Congying; Huang, Lusheng

    2015-07-01

    Growth-related traits are economically important traits to the pig industry. Identification of causative gene and mutation responsible for growth-related QTL will facilitate the improvement of pig growth through marker-assisted selection. In this study, we applied whole genome gene expression and quantitative trait transcript (QTT) analyses in 497 liver and 586 longissimus dorsi muscle samples to identify candidate genes and dissect the genetic basis of pig growth in a white Duroc × Erhualian F2 resource population. A total of 20,108 transcripts in liver and 23,728 transcripts in muscle with expression values were used for association analysis between gene expression level and phenotypic value. At the significance threshold of P < 0.0005, we identified a total of 169 and 168 QTTs for nine growth-related traits in liver and muscle, respectively. We also found that some QTTs were correlated to more than one trait. The QTTs identified here showed high tissue specificity. We did not identify any QTTs that were associated with one trait in both liver and muscle. Through an integrative genomic approach, we identified SDR16C5 as the important candidate gene in pig growth trait. These findings contribute to further identification of the causative genes for porcine growth traits and facilitate improvement of pig breeding.

  2. Cloning, characterisation, and comparative quantitative expression analyses of receptor for advanced glycation end products (RAGE) transcript forms.

    PubMed

    Sterenczak, Katharina A; Willenbrock, Saskia; Barann, Matthias; Klemke, Markus; Soller, Jan T; Eberle, Nina; Nolte, Ingo; Bullerdiek, Jörn; Murua Escobar, Hugo

    2009-04-01

    RAGE is a member of the immunoglobulin superfamily of cell surface molecules playing key roles in pathophysiological processes, e.g. immune/inflammatory disorders, Alzheimer's disease, diabetic arteriosclerosis and tumourigenesis. In humans 19 naturally occurring RAGE splicing variants resulting in either N-terminally or C-terminally truncated proteins were identified and are lately discussed as mechanisms for receptor regulation. Accordingly, deregulation of sRAGE levels has been associated with several diseases e.g. Alzheimer's disease, Type 1 diabetes, and rheumatoid arthritis. Administration of recombinant sRAGE to animal models of cancer blocked tumour growth successfully. In spite of its obvious relationship to cancer and metastasis data focusing sRAGE deregulation and tumours is rare. In this study we screened a set of tumours, healthy tissues and various cancer cell lines for RAGE splicing variants and analysed their structure. Additionally, we analysed the ratio of the mainly found transcript variants using quantitative Real-Time PCR. In total we characterised 24 previously not described canine and 4 human RAGE splicing variants, analysed their structure, classified their characteristics, and derived their respective protein forms. Interestingly, the healthy and the neoplastic tissue samples showed in majority RAGE transcripts coding for the complete receptor and transcripts showing insertions of intron 1. PMID:19061941

  3. Rapid Transcriptional Pulsing Dynamics of High Expressing Retroviral Transgenes in Embryonic Stem Cells

    PubMed Central

    Lo, Mandy Y. M.; Rival-Gervier, Sylvie; Pasceri, Peter; Ellis, James

    2012-01-01

    Single cell imaging studies suggest that transcription is not continuous and occurs as discrete pulses of gene activity. To study mechanisms by which retroviral transgenes can transcribe to high levels, we used the MS2 system to visualize transcriptional dynamics of high expressing proviral integration sites in embryonic stem (ES) cells. We established two ES cell lines each bearing a single copy, self-inactivating retroviral vector with a strong ubiquitous human EF1α gene promoter directing expression of mRFP fused to an MS2-stem-loop array. Transfection of MS2-EGFP generated EGFP focal dots bound to the mRFP-MS2 stem loop mRNA. These transcription foci colocalized with the transgene integration site detected by immunoFISH. Live tracking of single cells for 20 minutes detected EGFP focal dots that displayed frequent and rapid fluctuations in transcription over periods as short as 25 seconds. Similarly rapid fluctuations were detected from focal doublet signals that colocalized with replicated proviral integration sites by immunoFISH, consistent with transcriptional pulses from sister chromatids. We concluded that retroviral transgenes experience rapid transcriptional pulses in clonal ES cell lines that exhibit high level expression. These events are directed by a constitutive housekeeping gene promoter and may provide precedence for rapid transcriptional pulsing at endogenous genes in mammalian stem cells. PMID:22606340

  4. Quantitative Models of the Mechanisms That Control Genome-Wide Patterns of Transcription Factor Binding during Early Drosophila Development

    PubMed Central

    Kaplan, Tommy; Li, Xiao-Yong; Sabo, Peter J.; Thomas, Sean; Stamatoyannopoulos, John A.; Biggin, Mark D.; Eisen, Michael B.

    2011-01-01

    Transcription factors that drive complex patterns of gene expression during animal development bind to thousands of genomic regions, with quantitative differences in binding across bound regions mediating their activity. While we now have tools to characterize the DNA affinities of these proteins and to precisely measure their genome-wide distribution in vivo, our understanding of the forces that determine where, when, and to what extent they bind remains primitive. Here we use a thermodynamic model of transcription factor binding to evaluate the contribution of different biophysical forces to the binding of five regulators of early embryonic anterior-posterior patterning in Drosophila melanogaster. Predictions based on DNA sequence and in vitro protein-DNA affinities alone achieve a correlation of ∼0.4 with experimental measurements of in vivo binding. Incorporating cooperativity and competition among the five factors, and accounting for spatial patterning by modeling binding in every nucleus independently, had little effect on prediction accuracy. A major source of error was the prediction of binding events that do not occur in vivo, which we hypothesized reflected reduced accessibility of chromatin. To test this, we incorporated experimental measurements of genome-wide DNA accessibility into our model, effectively restricting predicted binding to regions of open chromatin. This dramatically improved our predictions to a correlation of 0.6–0.9 for various factors across known target genes. Finally, we used our model to quantify the roles of DNA sequence, accessibility, and binding competition and cooperativity. Our results show that, in regions of open chromatin, binding can be predicted almost exclusively by the sequence specificity of individual factors, with a minimal role for protein interactions. We suggest that a combination of experimentally determined chromatin accessibility data and simple computational models of transcription factor binding may be

  5. A Regulatory Hierarchy Controls the Dynamic Transcriptional Response to Extreme Oxidative Stress in Archaea

    PubMed Central

    Gulli, Jordan G.; Sharma, Kriti; Schmid, Amy K.

    2015-01-01

    Networks of interacting transcription factors are central to the regulation of cellular responses to abiotic stress. Although the architecture of many such networks has been mapped, their dynamic function remains unclear. Here we address this challenge in archaea, microorganisms possessing transcription factors that resemble those of both eukaryotes and bacteria. Using genome-wide DNA binding location analysis integrated with gene expression and cell physiological data, we demonstrate that a bacterial-type transcription factor (TF), called RosR, and five TFIIB proteins, homologs of eukaryotic TFs, combinatorially regulate over 100 target genes important for the response to extremely high levels of peroxide. These genes include 20 other transcription factors and oxidative damage repair genes. RosR promoter occupancy is surprisingly dynamic, with the pattern of target gene expression during the transition from rapid growth to stress correlating strongly with the pattern of dynamic binding. We conclude that a hierarchical regulatory network orchestrated by TFs of hybrid lineage enables dynamic response and survival under extreme stress in archaea. This raises questions regarding the evolutionary trajectory of gene networks in response to stress. PMID:25569531

  6. Increased transcripts for B-type natriuretic peptide in spontaneously hypertensive rats. Quantitative polymerase chain reaction for atrial and brain natriuretic peptide transcripts.

    PubMed

    Dagnino, L; Lavigne, J P; Nemer, M

    1992-11-01

    The cardiac natriuretic peptide family includes atrial natriuretic factor and brain or B-type natriuretic peptide, also known as iso-atrial natriuretic factor (isoANF). Although these peptides contribute to cardiovascular homeostasis, their respective roles remain unclear. To study regulation of atrial natriuretic factor and isoANF gene expression during progression of hypertension, we developed a quantitative polymerase chain reaction protocol to measure their transcript level in spontaneously hypertensive rat (SHR) hearts. At the onset of hypertension, atrial natriuretic factor transcripts in 5-week-old SHR were 50% of those of age-matched Wistar-Kyoto (WKY) rats, whereas the level of isoANF transcripts was similar in atria and twofold higher in ventricles. Because atria are the major sites of atrial natriuretic factor gene expression and ventricles contribute predominantly to cardiac isoANF synthesis, total atrial natriuretic factor messenger RNA (mRNA) in the hearts of 5-week-old SHR was about 50% of that in WKY rats, and total isoANF mRNA content was already higher than in control rats. In left ventricles and ventricular septa, progression of hypertension led to a maximal increase of twofold and fourfold in atrial natriuretic factor and isoANF mRNA levels, respectively, with no detectable change in right ventricles. In the atria of older SHR, atrial natriuretic factor and isoANF mRNA levels were comparable to those of age-matched controls. These data indicate that, although increased blood pressure stimulates both atrial natriuretic factor and isoANF gene expression, regulation of the two natriuretic peptide genes is not temporally coordinated in all cardiac compartments. Furthermore, isoANF mRNA is already induced in the ventricles at the onset of the hypertensive stage, and in older SHR, the isoANF gene is hyperresponsive to progression of hypertension compared with atrial natriuretic factor. Thus, isoANF might represent a very sensitive marker of cardiac

  7. Dynamics and rRNA transcriptional activity of lactococci and lactobacilli during Cheddar cheese ripening.

    PubMed

    Desfossés-Foucault, Émilie; LaPointe, Gisèle; Roy, Denis

    2013-08-16

    Cheddar cheese is a complex ecosystem where both the bacterial population and the cheese making process contribute to flavor and texture development. The aim of this study was to use molecular methods to evaluate the impact of milk heat treatment and ripening temperature on starter lactococci and non-starter lactic acid bacteria (NSLAB) throughout ripening of Cheddar cheese. Eight Cheddar cheese batches were manufactured (four with thermized and four with pasteurized milk) and ripened at 4, 7 and 12°C to analyze the bacterial composition and rRNA transcriptional activity reflecting the ability of lactococci and lactobacilli to synthesize proteins. Abundance and rRNA transcription of lactococci and lactobacilli were quantified after DNA and RNA extraction by using quantitative PCR (qPCR) and reverse transcription-quantitative PCR (RT-qPCR) targeting the 16S rRNA gene, respectively. Results showed that lactococci remained dominant throughout ripening, although 16S rRNA genome and cDNA copies/g of cheese decreased by four and two log copy numbers, respectively. Abundance and rRNA transcription of Lactobacillus paracasei, Lactobacillus buchneri/parabuchneri, Lactobacillus rhamnosus, Lactobacillus brevis, and Lactobacillus coryniformis as well as total lactobacilli were also estimated using specific 16S rRNA primers. L. paracasei and L. buchneri/parabuchneri concomitantly grew in cheese made from thermized milk at 7 and 12°C, although L. paracasei displayed the most rRNA transcription among Lactobacillus species. This work showed that rRNA transcriptional activity of lactococci decreased throughout ripening and supports the usefulness of RNA analysis to assess which bacterial species have the ability to synthesize proteins during ripening, and could thereby contribute to cheese quality. PMID:23850855

  8. Dynamic Transcriptional Response of Escherichia coli to Inclusion Body Formation

    PubMed Central

    Baig, Faraz; Fernando, Lawrence P.; Salazar, Mary Alice; Powell, Rhonda R.; Bruce, Terri F.; Harcum, Sarah W.

    2014-01-01

    Escherichia coli is used intensively for recombinant protein production, but one key challenge with recombinant E. coli is the tendency of recombinant proteins to misfold and aggregate into insoluble inclusion bodies (IBs). IBs contain high concentrations of inactive recombinant protein that require recovery steps to salvage a functional recombinant protein. Currently, no universally effective method exists to prevent IB formation in recombinant E. coli. In this study, DNA microarrays were used to compare the E. coli gene expression response dynamics to soluble and insoluble recombinant protein production. As expected and previously reported, the classical heat-shock genes had increased expression due to IB formation, including protein folding chaperones and proteases. Gene expression levels for protein synthesis-related and energy-synthesis pathways were also increased. Many transmembrane transporter and corresponding catabolic pathways genes had decreased expression for substrates not present in the culture medium. Additionally, putative genes represented over one-third of the genes identified to have significant expression changes due to IB formation, indicating many important cellular responses to IB formation still need to be characterized. Interestingly, cells grown in 3% ethanol had significantly reduced gene expression responses due to IB formation. Taken together, these results indicate that IB formation is complex, stimulates the heat-shock response, increases protein and energy synthesis needs, and streamlines transport and catabolic processes, while ethanol diminished all of these responses. PMID:24338599

  9. Integrated microfluidic approach for quantitative high-throughput measurements of transcription factor binding affinities

    PubMed Central

    Glick, Yair; Orenstein, Yaron; Chen, Dana; Avrahami, Dorit; Zor, Tsaffrir; Shamir, Ron; Gerber, Doron

    2016-01-01

    Protein binding to DNA is a fundamental process in gene regulation. Methodologies such as ChIP-Seq and mapping of DNase I hypersensitive sites provide global information on this regulation in vivo. In vitro methodologies provide valuable complementary information on protein–DNA specificities. However, current methods still do not measure absolute binding affinities. There is a real need for large-scale quantitative protein–DNA affinity measurements. We developed QPID, a microfluidic application for measuring protein–DNA affinities. A single run is equivalent to 4096 gel-shift experiments. Using QPID, we characterized the different affinities of ATF1, c-Jun, c-Fos and AP-1 to the CRE consensus motif and CRE half-site in two different genomic sequences on a single device. We discovered that binding of ATF1, but not of AP-1, to the CRE half-site is highly affected by its genomic context. This effect was highly correlated with ATF1 ChIP-seq and PBM experiments. Next, we characterized the affinities of ATF1 and ATF3 to 128 genomic CRE and CRE half-site sequences. Our affinity measurements explained that in vivo binding differences between ATF1 and ATF3 to CRE and CRE half-sites are partially mediated by differences in the minor groove width. We believe that QPID would become a central tool for quantitative characterization of biophysical aspects affecting protein–DNA binding. PMID:26635393

  10. Integrated microfluidic approach for quantitative high-throughput measurements of transcription factor binding affinities.

    PubMed

    Glick, Yair; Orenstein, Yaron; Chen, Dana; Avrahami, Dorit; Zor, Tsaffrir; Shamir, Ron; Gerber, Doron

    2016-04-01

    Protein binding to DNA is a fundamental process in gene regulation. Methodologies such as ChIP-Seq and mapping of DNase I hypersensitive sites provide global information on this regulation in vivo In vitro methodologies provide valuable complementary information on protein-DNA specificities. However, current methods still do not measure absolute binding affinities. There is a real need for large-scale quantitative protein-DNA affinity measurements. We developed QPID, a microfluidic application for measuring protein-DNA affinities. A single run is equivalent to 4096 gel-shift experiments. Using QPID, we characterized the different affinities of ATF1, c-Jun, c-Fos and AP-1 to the CRE consensus motif and CRE half-site in two different genomic sequences on a single device. We discovered that binding of ATF1, but not of AP-1, to the CRE half-site is highly affected by its genomic context. This effect was highly correlated with ATF1 ChIP-seq and PBM experiments. Next, we characterized the affinities of ATF1 and ATF3 to 128 genomic CRE and CRE half-site sequences. Our affinity measurements explained that in vivo binding differences between ATF1 and ATF3 to CRE and CRE half-sites are partially mediated by differences in the minor groove width. We believe that QPID would become a central tool for quantitative characterization of biophysical aspects affecting protein-DNA binding.

  11. Uncovering a Macrophage Transcriptional Program by Integrating Evidence from Motif Scanning and Expression Dynamics

    PubMed Central

    Ramsey, Stephen A.; Klemm, Sandy L.; Zak, Daniel E.; Kennedy, Kathleen A.; Thorsson, Vesteinn; Li, Bin; Gilchrist, Mark; Gold, Elizabeth S.; Johnson, Carrie D.; Litvak, Vladimir; Navarro, Garnet; Roach, Jared C.; Rosenberger, Carrie M.; Rust, Alistair G.; Yudkovsky, Natalya; Aderem, Alan; Shmulevich, Ilya

    2008-01-01

    Macrophages are versatile immune cells that can detect a variety of pathogen-associated molecular patterns through their Toll-like receptors (TLRs). In response to microbial challenge, the TLR-stimulated macrophage undergoes an activation program controlled by a dynamically inducible transcriptional regulatory network. Mapping a complex mammalian transcriptional network poses significant challenges and requires the integration of multiple experimental data types. In this work, we inferred a transcriptional network underlying TLR-stimulated murine macrophage activation. Microarray-based expression profiling and transcription factor binding site motif scanning were used to infer a network of associations between transcription factor genes and clusters of co-expressed target genes. The time-lagged correlation was used to analyze temporal expression data in order to identify potential causal influences in the network. A novel statistical test was developed to assess the significance of the time-lagged correlation. Several associations in the resulting inferred network were validated using targeted ChIP-on-chip experiments. The network incorporates known regulators and gives insight into the transcriptional control of macrophage activation. Our analysis identified a novel regulator (TGIF1) that may have a role in macrophage activation. PMID:18369420

  12. A dynamic model of proteome changes reveals new roles for transcript alteration in yeast

    PubMed Central

    Lee, M Violet; Topper, Scott E; Hubler, Shane L; Hose, James; Wenger, Craig D; Coon, Joshua J; Gasch, Audrey P

    2011-01-01

    The transcriptome and proteome change dynamically as cells respond to environmental stress; however, prior proteomic studies reported poor correlation between mRNA and protein, rendering their relationships unclear. To address this, we combined high mass accuracy mass spectrometry with isobaric tagging to quantify dynamic changes in ∼2500 Saccharomyces cerevisiae proteins, in biological triplicate and with paired mRNA samples, as cells acclimated to high osmolarity. Surprisingly, while transcript induction correlated extremely well with protein increase, transcript reduction produced little to no change in the corresponding proteins. We constructed a mathematical model of dynamic protein changes and propose that the lack of protein reduction is explained by cell-division arrest, while transcript reduction supports redistribution of translational machinery. Furthermore, the transient ‘burst' of mRNA induction after stress serves to accelerate change in the corresponding protein levels. We identified several classes of post-transcriptional regulation, but show that most of the variance in protein changes is explained by mRNA. Our results present a picture of the coordinated physiological responses at the levels of mRNA, protein, protein-synthetic capacity, and cellular growth. PMID:21772262

  13. Sequential use of transcriptional profiling, expression quantitative trait mapping, and gene association implicates MMP20 in human kidney aging.

    PubMed

    Wheeler, Heather E; Metter, E Jeffrey; Tanaka, Toshiko; Absher, Devin; Higgins, John; Zahn, Jacob M; Wilhelmy, Julie; Davis, Ronald W; Singleton, Andrew; Myers, Richard M; Ferrucci, Luigi; Kim, Stuart K

    2009-10-01

    Kidneys age at different rates, such that some people show little or no effects of aging whereas others show rapid functional decline. We sequentially used transcriptional profiling and expression quantitative trait loci (eQTL) mapping to narrow down which genes to test for association with kidney aging. We first performed whole-genome transcriptional profiling to find 630 genes that change expression with age in the kidney. Using two methods to detect eQTLs, we found 101 of these age-regulated genes contain expression-associated SNPs. We tested the eQTLs for association with kidney aging, measured by glomerular filtration rate (GFR) using combined data from the Baltimore Longitudinal Study of Aging (BLSA) and the InCHIANTI study. We found a SNP association (rs1711437 in MMP20) with kidney aging (uncorrected p = 3.6 x 10(-5), empirical p = 0.01) that explains 1%-2% of the variance in GFR among individuals. The results of this sequential analysis may provide the first evidence for a gene association with kidney aging in humans.

  14. Sequential Use of Transcriptional Profiling, Expression Quantitative Trait Mapping, and Gene Association Implicates MMP20 in Human Kidney Aging

    PubMed Central

    Wheeler, Heather E.; Metter, E. Jeffrey; Tanaka, Toshiko; Absher, Devin; Higgins, John; Zahn, Jacob M.; Wilhelmy, Julie; Davis, Ronald W.; Singleton, Andrew; Myers, Richard M.; Ferrucci, Luigi; Kim, Stuart K.

    2009-01-01

    Kidneys age at different rates, such that some people show little or no effects of aging whereas others show rapid functional decline. We sequentially used transcriptional profiling and expression quantitative trait loci (eQTL) mapping to narrow down which genes to test for association with kidney aging. We first performed whole-genome transcriptional profiling to find 630 genes that change expression with age in the kidney. Using two methods to detect eQTLs, we found 101 of these age-regulated genes contain expression-associated SNPs. We tested the eQTLs for association with kidney aging, measured by glomerular filtration rate (GFR) using combined data from the Baltimore Longitudinal Study of Aging (BLSA) and the InCHIANTI study. We found a SNP association (rs1711437 in MMP20) with kidney aging (uncorrected p = 3.6×10−5, empirical p = 0.01) that explains 1%–2% of the variance in GFR among individuals. The results of this sequential analysis may provide the first evidence for a gene association with kidney aging in humans. PMID:19834535

  15. Selection of reference genes for reverse transcription quantitative real-time PCR normalization in black rockfish (Sebastes schlegeli).

    PubMed

    Liman, Ma; Wenji, Wang; Conghui, Liu; Haiyang, Yu; Zhigang, Wang; Xubo, Wang; Jie, Qi; Quanqi, Zhang

    2013-09-01

    Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a technique widely used for quantification of mRNA transcription. Data normalization is an indispensable process for RT-qPCR and reference genes are most commonly used to normalize RT-qPCR and to reduce possible errors generated in the quantification of genes among several proposed methods. To date, RT-qPCR has been used in terms of gene expression studies in black rockfish (Sebastes schlegeli) but the majority of published RT-qPCR studies still lack proper validation of the reference genes. In the present study, mRNA transcription profiles of eight putative reference genes (18S rRNA, ACTB, GAPDH, TUBA, RPL17, EF1A, HPRT, and B2M) were examined using RT-qPCR in different tissues and larvae developmental stages of black rockfish. Three common statistical algorithms (geNorm, NormFinder, and BestKeeper) were used to assess expression stability and select the most stable genes for gene normalization. Two reference genes, RPL17 and EF1A showed high stability in black rockfish tissue analysis, while GAPDH was the least stable gene. During larvae developmental stages, EF1A, RPL17 and ACTB were identified as the optimal reference genes for data normalization, whereas B2M appeared unsuitable as the reference gene. In summary, our results could provide a useful guideline for reference gene selection and enable more accurate normalization of gene expression data in gene expression studies of black rockfish.

  16. RNA Enrichment Method for Quantitative Transcriptional Analysis of Pathogens In Vivo Applied to the Fungus Candida albicans

    PubMed Central

    Amorim-Vaz, Sara; Tran, Van Du T.; Pradervand, Sylvain; Pagni, Marco; Coste, Alix T.

    2015-01-01

    ABSTRACT In vivo transcriptional analyses of microbial pathogens are often hampered by low proportions of pathogen biomass in host organs, hindering the coverage of full pathogen transcriptome. We aimed to address the transcriptome profiles of Candida albicans, the most prevalent fungal pathogen in systemically infected immunocompromised patients, during systemic infection in different hosts. We developed a strategy for high-resolution quantitative analysis of the C. albicans transcriptome directly from early and late stages of systemic infection in two different host models, mouse and the insect Galleria mellonella. Our results show that transcriptome sequencing (RNA-seq) libraries were enriched for fungal transcripts up to 1,600-fold using biotinylated bait probes to capture C. albicans sequences. This enrichment biased the read counts of only ~3% of the genes, which can be identified and removed based on a priori criteria. This allowed an unprecedented resolution of C. albicans transcriptome in vivo, with detection of over 86% of its genes. The transcriptional response of the fungus was surprisingly similar during infection of the two hosts and at the two time points, although some host- and time point-specific genes could be identified. Genes that were highly induced during infection were involved, for instance, in stress response, adhesion, iron acquisition, and biofilm formation. Of the in vivo-regulated genes, 10% are still of unknown function, and their future study will be of great interest. The fungal RNA enrichment procedure used here will help a better characterization of the C. albicans response in infected hosts and may be applied to other microbial pathogens. PMID:26396240

  17. Selection of Valid Reference Genes for Reverse Transcription Quantitative PCR Analysis in Heliconius numata (Lepidoptera: Nymphalidae)

    PubMed Central

    Chouteau, Mathieu; Whibley, Annabel; Joron, Mathieu; Llaurens, Violaine

    2016-01-01

    Identifying the genetic basis of adaptive variation is challenging in non-model organisms and quantitative real time PCR. is a useful tool for validating predictions regarding the expression of candidate genes. However, comparing expression levels in different conditions requires rigorous experimental design and statistical analyses. Here, we focused on the neotropical passion-vine butterflies Heliconius, non-model species studied in evolutionary biology for their adaptive variation in wing color patterns involved in mimicry and in the signaling of their toxicity to predators. We aimed at selecting stable reference genes to be used for normalization of gene expression data in RT-qPCR analyses from developing wing discs according to the minimal guidelines described in Minimum Information for publication of Quantitative Real-Time PCR Experiments (MIQE). To design internal RT-qPCR controls, we studied the stability of expression of nine candidate reference genes (actin, annexin, eF1α, FK506BP, PolyABP, PolyUBQ, RpL3, RPS3A, and tubulin) at two developmental stages (prepupal and pupal) using three widely used programs (GeNorm, NormFinder and BestKeeper). Results showed that, despite differences in statistical methods, genes RpL3, eF1α, polyABP, and annexin were stably expressed in wing discs in late larval and pupal stages of Heliconius numata. This combination of genes may be used as a reference for a reliable study of differential expression in wings for instance for genes involved in important phenotypic variation, such as wing color pattern variation. Through this example, we provide general useful technical recommendations as well as relevant statistical strategies for evolutionary biologists aiming to identify candidate-genes involved adaptive variation in non-model organisms. PMID:27271971

  18. Selection of Valid Reference Genes for Reverse Transcription Quantitative PCR Analysis in Heliconius numata (Lepidoptera: Nymphalidae).

    PubMed

    Piron Prunier, Florence; Chouteau, Mathieu; Whibley, Annabel; Joron, Mathieu; Llaurens, Violaine

    2016-01-01

    Identifying the genetic basis of adaptive variation is challenging in non-model organisms and quantitative real time PCR. is a useful tool for validating predictions regarding the expression of candidate genes. However, comparing expression levels in different conditions requires rigorous experimental design and statistical analyses. Here, we focused on the neotropical passion-vine butterflies Heliconius, non-model species studied in evolutionary biology for their adaptive variation in wing color patterns involved in mimicry and in the signaling of their toxicity to predators. We aimed at selecting stable reference genes to be used for normalization of gene expression data in RT-qPCR analyses from developing wing discs according to the minimal guidelines described in Minimum Information for publication of Quantitative Real-Time PCR Experiments (MIQE). To design internal RT-qPCR controls, we studied the stability of expression of nine candidate reference genes (actin, annexin, eF1α, FK506BP, PolyABP, PolyUBQ, RpL3, RPS3A, and tubulin) at two developmental stages (prepupal and pupal) using three widely used programs (GeNorm, NormFinder and BestKeeper). Results showed that, despite differences in statistical methods, genes RpL3, eF1α, polyABP, and annexin were stably expressed in wing discs in late larval and pupal stages of Heliconius numata This combination of genes may be used as a reference for a reliable study of differential expression in wings for instance for genes involved in important phenotypic variation, such as wing color pattern variation. Through this example, we provide general useful technical recommendations as well as relevant statistical strategies for evolutionary biologists aiming to identify candidate-genes involved adaptive variation in non-model organisms.

  19. Selection of Valid Reference Genes for Reverse Transcription Quantitative PCR Analysis in Heliconius numata (Lepidoptera: Nymphalidae).

    PubMed

    Piron Prunier, Florence; Chouteau, Mathieu; Whibley, Annabel; Joron, Mathieu; Llaurens, Violaine

    2016-01-01

    Identifying the genetic basis of adaptive variation is challenging in non-model organisms and quantitative real time PCR. is a useful tool for validating predictions regarding the expression of candidate genes. However, comparing expression levels in different conditions requires rigorous experimental design and statistical analyses. Here, we focused on the neotropical passion-vine butterflies Heliconius, non-model species studied in evolutionary biology for their adaptive variation in wing color patterns involved in mimicry and in the signaling of their toxicity to predators. We aimed at selecting stable reference genes to be used for normalization of gene expression data in RT-qPCR analyses from developing wing discs according to the minimal guidelines described in Minimum Information for publication of Quantitative Real-Time PCR Experiments (MIQE). To design internal RT-qPCR controls, we studied the stability of expression of nine candidate reference genes (actin, annexin, eF1α, FK506BP, PolyABP, PolyUBQ, RpL3, RPS3A, and tubulin) at two developmental stages (prepupal and pupal) using three widely used programs (GeNorm, NormFinder and BestKeeper). Results showed that, despite differences in statistical methods, genes RpL3, eF1α, polyABP, and annexin were stably expressed in wing discs in late larval and pupal stages of Heliconius numata This combination of genes may be used as a reference for a reliable study of differential expression in wings for instance for genes involved in important phenotypic variation, such as wing color pattern variation. Through this example, we provide general useful technical recommendations as well as relevant statistical strategies for evolutionary biologists aiming to identify candidate-genes involved adaptive variation in non-model organisms. PMID:27271971

  20. Dynamic transcriptional events in embryonic stem cells mediated by the super elongation complex (SEC)

    PubMed Central

    Lin, Chengqi; Garrett, Alexander S.; De Kumar, Bony; Smith, Edwin R.; Gogol, Madelaine; Seidel, Christopher; Krumlauf, Robb; Shilatifard, Ali

    2011-01-01

    Transcriptional regulation of developmentally controlled genes is at the heart of differentiation and organogenesis. In this study, we performed global genomic analyses in murine embryonic stem (ES) cells and in human cells in response to activation signals. We identified an essential role for the ELL (eleven–nineteen lysine-rich leukemia gene)/P-TEFb (positive transcription elongation factor)-containing super elongation complex (SEC) in the regulation of gene expression, including several genes bearing paused RNA polymerase II (Pol II). Paused Pol II has been proposed to be associated with loci that respond rapidly to environmental stimuli. However, our studies in ES cells also identified a requirement for SEC at genes without paused Pol II, which also respond dynamically to differentiation signals. Our findings suggest that SEC is a major class of active P-TEFb-containing complexes required for transcriptional activation in response to environmental cues such as differentiation signals. PMID:21764852

  1. Digital holographic microscopy for quantitative cell dynamic evaluation during laser microsurgery

    PubMed Central

    Yu, Lingfeng; Mohanty, Samarendra; Zhang, Jun; Genc, Suzanne; Kim, Myung K.; Berns, Michael W.; Chen, Zhongping

    2010-01-01

    Digital holographic microscopy allows determination of dynamic changes in the optical thickness profile of a transparent object with subwavelength accuracy. Here, we report a quantitative phase laser microsurgery system for evaluation of cellular/ sub-cellular dynamic changes during laser micro-dissection. The proposed method takes advantage of the precise optical manipulation by the laser microbeam and quantitative phase imaging by digital holographic microscopy with high spatial and temporal resolution. This system will permit quantitative evaluation of the damage and/or the repair of the cell or cell organelles in real time. PMID:19582118

  2. Transfer RNA Post-Transcriptional Processing, Turnover, and Subcellular Dynamics in the Yeast Saccharomyces cerevisiae

    PubMed Central

    Hopper, Anita K.

    2013-01-01

    Transfer RNAs (tRNAs) are essential for protein synthesis. In eukaryotes, tRNA biosynthesis employs a specialized RNA polymerase that generates initial transcripts that must be subsequently altered via a multitude of post-transcriptional steps before the tRNAs beome mature molecules that function in protein synthesis. Genetic, genomic, biochemical, and cell biological approaches possible in the powerful Saccharomyces cerevisiae system have led to exciting advances in our understandings of tRNA post-transcriptional processing as well as to novel insights into tRNA turnover and tRNA subcellular dynamics. tRNA processing steps include removal of transcribed leader and trailer sequences, addition of CCA to the 3′ mature sequence and, for tRNAHis, addition of a 5′ G. About 20% of yeast tRNAs are encoded by intron-containing genes. The three-step splicing process to remove the introns surprisingly occurs in the cytoplasm in yeast and each of the splicing enzymes appears to moonlight in functions in addition to tRNA splicing. There are 25 different nucleoside modifications that are added post-transcriptionally, creating tRNAs in which ∼15% of the residues are nucleosides other than A, G, U, or C. These modified nucleosides serve numerous important functions including tRNA discrimination, translation fidelity, and tRNA quality control. Mature tRNAs are very stable, but nevertheless yeast cells possess multiple pathways to degrade inappropriately processed or folded tRNAs. Mature tRNAs are also dynamic in cells, moving from the cytoplasm to the nucleus and back again to the cytoplasm; the mechanism and function of this retrograde process is poorly understood. Here, the state of knowledge for tRNA post-transcriptional processing, turnover, and subcellular dynamics is addressed, highlighting the questions that remain. PMID:23633143

  3. Development of one-step quantitative reverse transcription PCR for the rapid detection of flaviviruses

    PubMed Central

    2013-01-01

    Background The genus Flavivirus includes several pathogenic agents that cause severe illness in humans. Re-emergence of West Nile virus in Europe and continuous spread of certain flaviviruses such as dengue, yellow fever and Japanese encephalitis viruses represent a global danger to public health. Therefore, a rapid and accurate molecular method is required for diagnostics and epidemiological surveillance of flaviviruses. Methods A Pan-Flavi quantitative RT-PCR assay using a Locked-Nucleic Acid probe targeting the flavivirus NS5 gene was developed and optimized to detect a wide range of flaviviruses simultaneously. The specificity and sensitivity of the Pan-Flavi assay were tested using RNA of different flaviviruses and non-flaviviruses. Furthermore, the assay was compared directly to flavivirus species-specific assays for the ability to detect flaviviruses sensitively. Results Two degenerate primers and one Locked-Nucleic Acids probe were designed to amplify most of the flaviviruses. To increase the specificity and fluorescence signal of the Pan-Flavi assay for detection of yellow fever virus and dengue virus 4, additional primers and probes were included. Viral RNA of thirty different flaviviruses was detected, verifying the broad range specificity. The testing of this assay was successful, using standard plasmid and RNA dilutions of yellow fever virus vaccine strain, dengue virus 1 and tick-borne encephalitis virus, with a sensitivity limit of 10–100 genome copies/reaction. Also comparatively good results were achieved for detecting different flaviviruses by the Pan-Flavi assay when compared to the flavivirus species-specific assays. Conclusion The assay is rapid, broad-range flavivirus-specific and highly sensitive making it a valuable tool for rapid detection of flaviviruses in livestock samples, epidemiological studies or as useful complement to single flavivirus-specific assays for clinical diagnosis. PMID:23410000

  4. Characterization of the gut microbiota of Papua New Guineans using reverse transcription quantitative PCR.

    PubMed

    Greenhill, Andrew R; Tsuji, Hirokazu; Ogata, Kiyohito; Natsuhara, Kazumi; Morita, Ayako; Soli, Kevin; Larkins, Jo-Ann; Tadokoro, Kiyoshi; Odani, Shingo; Baba, Jun; Naito, Yuichi; Tomitsuka, Eriko; Nomoto, Koji; Siba, Peter M; Horwood, Paul F; Umezaki, Masahiro

    2015-01-01

    There has been considerable interest in composition of gut microbiota in recent years, leading to a better understanding of the role the gut microbiota plays in health and disease. Most studies have been limited in their geographical and socioeconomic diversity to high-income settings, and have been conducted using small sample sizes. To date, few analyses have been conducted in low-income settings, where a better understanding of the gut microbiome could lead to the greatest return in terms of health benefits. Here, we have used quantitative real-time polymerase chain reaction targeting dominant and sub-dominant groups of microorganisms associated with human gut microbiome in 115 people living a subsistence lifestyle in rural areas of Papua New Guinea. Quantification of Clostridium coccoides group, C. leptum subgroup, C. perfringens, Bacteroides fragilis group, Bifidobacterium, Atopobium cluster, Prevotella, Enterobacteriaceae, Enterococcus, Staphylococcus, and Lactobacillus spp. was conducted. Principle coordinates analysis (PCoA) revealed two dimensions with Prevotella, clostridia, Atopobium, Enterobacteriaceae, Enterococcus and Staphylococcus grouping in one dimension, while B. fragilis, Bifidobacterium and Lactobacillus grouping in the second dimension. Highland people had higher numbers of most groups of bacteria detected, and this is likely a key factor for the differences revealed by PCoA between highland and lowland study participants. Age and sex were not major determinants in microbial population composition. The study demonstrates a gut microbial composition with some similarities to those observed in other low-income settings where traditional diets are consumed, which have previously been suggested to favor energy extraction from a carbohydrate rich diet.

  5. Detection and quantitation of the CBFbeta/MYH11 transcripts associated with the inv(16) in presentation and follow-up samples from patients with AML.

    PubMed

    Evans, P A; Short, M A; Jack, A S; Norfolk, D R; Child, J A; Shiach, C R; Davies, F; Tobal, K; Liu Yin, J A; Morgan, G J

    1997-03-01

    We have developed a competitor-based RT-PCR technique which will detect and quantitate the CBFbeta/MYH11 transcripts associated with inv(16)(q22;p13) and have used it to study presentation and follow-up samples of acute myeloid leukaemia (AML). The levels of the leukaemia-specific transcripts are expressed as a ratio to a ubiquitously expressed mRNA species (Abl) which controls for RNA degradation. This technique has been applied to 75 consecutive patients presenting with either de novo AML or tMDS; 6/75 patients analysed were positive for the inv(16), all were confirmed by conventional cytogenetics. The inv(16) has a strong association with M4Eo, but we found only 2/6-positive patients to have this diagnosis (two patients with M2, one patient M1 and one patient had MDS). At presentation the levels of CBFbeta/MYH11 transcripts were 0.1-10/Abl transcript (mean 3.3/Abl transcript). Seventeen follow-up samples were available on 5/6 of these patients, and on two further patients in whom stored material was available. Following the first cycle of chemotherapy the level of transcripts was at least 10(-2) lower (0.1-10 x 10(-2)/abl transcript) than their presentation sample. Subsequent samples on these patients when in remission gave transcript levels in the range (1.0 x 10(-4) - 2 x 10(-3)/abl transcript), and three long-term follow-up samples were negative. We have developed a quantitative test which opens the possibility of predicting relapse by detecting changes in the numbers of leukaemia-specific transcripts. PMID:9067575

  6. Dynamic expression of transcription factor Brn3b during mouse cranial nerve development.

    PubMed

    Sajgo, Szilard; Ali, Seid; Popescu, Octavian; Badea, Tudor Constantin

    2016-04-01

    During development, transcription factor combinatorial codes define a large variety of morphologically and physiologically distinct neurons. Such a combinatorial code has been proposed for the differentiation of projection neurons of the somatic and visceral components of cranial nerves. It is possible that individual neuronal cell types are not specified by unique transcription factors but rather emerge through the intersection of their expression domains. Brn3a, Brn3b, and Brn3c, in combination with each other and/or transcription factors of other families, can define subgroups of retinal ganglion cells (RGC), spiral and vestibular ganglia, inner ear and vestibular hair cell neurons in the vestibuloacoustic system, and groups of somatosensory neurons in the dorsal root ganglia. The present study investigates the expression and potential role of the Brn3b transcription factor in cranial nerves and associated nuclei of the brainstem. We report the dynamic expression of Brn3b in the somatosensory component of cranial nerves II, V, VII, and VIII and visceromotor nuclei of nerves VII, IX, and X as well as other brainstem nuclei during different stages of development into adult stage. We find that genetically identified Brn3b(KO) RGC axons show correct but delayed pathfinding during the early stages of embryonic development. However, loss of Brn3b does not affect the anatomy of the other cranial nerves normally expressing this transcription factor.

  7. Spatio-temporal dynamics of replication and transcription sites in the mammalian cell nucleus.

    PubMed

    Malyavantham, Kishore S; Bhattacharya, Sambit; Alonso, William D; Acharya, Raj; Berezney, Ronald

    2008-12-01

    To study when and where active genes replicated in early S phase are transcribed, a series of pulse-chase experiments are performed to label replicating chromatin domains (RS) in early S phase and subsequently transcription sites (TS) after chase periods of 0 to 24 h. Surprisingly, transcription activity throughout these chase periods did not show significant colocalization with early RS chromatin domains. Application of novel image segmentation and proximity algorithms, however, revealed close proximity of TS with the labeled chromatin domains independent of chase time. In addition, RNA polymerase II was highly proximal and showed significant colocalization with both TS and the chromatin domains. Based on these findings, we propose that chromatin activated for transcription dynamically unfolds or "loops out" of early RS chromatin domains where it can interact with RNA polymerase II and other components of the transcriptional machinery. Our results further suggest that the early RS chromatin domains are transcribing genes throughout the cell cycle and that multiple chromatin domains are organized around the same transcription factory.

  8. Dynamic Metabolite Profiling in an Archaeon Connects Transcriptional Regulation to Metabolic Consequences

    PubMed Central

    Todor, Horia; Gooding, Jessica; Ilkayeva, Olga R.; Schmid, Amy K.

    2015-01-01

    Previous work demonstrated that the TrmB transcription factor is responsible for regulating the expression of many enzyme-coding genes in the hypersaline-adapted archaeon Halobacterium salinarum via a direct interaction with a cis-regulatory sequence in their promoters. This interaction is abolished in the presence of glucose. Although much is known about the effects of TrmB at the transcriptional level, it remains unclear whether and to what extent changes in mRNA levels directly affect metabolite levels. In order to address this question, here we performed a high-resolution metabolite profiling time course during a change in nutrients using a combination of targeted and untargeted methods in wild-type and ΔtrmB strain backgrounds. We found that TrmB-mediated transcriptional changes resulted in widespread and significant changes to metabolite levels across the metabolic network. Additionally, the pattern of growth complementation using various purines suggests that the mis-regulation of gluconeogenesis in the ΔtrmB mutant strain in the absence of glucose results in low phosphoribosylpyrophosphate (PRPP) levels. We confirmed these low PRPP levels using a quantitative mass spectrometric technique and found that they are associated with a metabolic block in de novo purine synthesis, which is partially responsible for the growth defect of the ΔtrmB mutant strain in the absence of glucose. In conclusion, we show how transcriptional regulation of metabolism affects metabolite levels and ultimately, phenotypes. PMID:26284786

  9. Dynamic Metabolite Profiling in an Archaeon Connects Transcriptional Regulation to Metabolic Consequences.

    PubMed

    Todor, Horia; Gooding, Jessica; Ilkayeva, Olga R; Schmid, Amy K

    2015-01-01

    Previous work demonstrated that the TrmB transcription factor is responsible for regulating the expression of many enzyme-coding genes in the hypersaline-adapted archaeon Halobacterium salinarum via a direct interaction with a cis-regulatory sequence in their promoters. This interaction is abolished in the presence of glucose. Although much is known about the effects of TrmB at the transcriptional level, it remains unclear whether and to what extent changes in mRNA levels directly affect metabolite levels. In order to address this question, here we performed a high-resolution metabolite profiling time course during a change in nutrients using a combination of targeted and untargeted methods in wild-type and ΔtrmB strain backgrounds. We found that TrmB-mediated transcriptional changes resulted in widespread and significant changes to metabolite levels across the metabolic network. Additionally, the pattern of growth complementation using various purines suggests that the mis-regulation of gluconeogenesis in the ΔtrmB mutant strain in the absence of glucose results in low phosphoribosylpyrophosphate (PRPP) levels. We confirmed these low PRPP levels using a quantitative mass spectrometric technique and found that they are associated with a metabolic block in de novo purine synthesis, which is partially responsible for the growth defect of the ΔtrmB mutant strain in the absence of glucose. In conclusion, we show how transcriptional regulation of metabolism affects metabolite levels and ultimately, phenotypes.

  10. Dynamic long-range chromatin interactions control Myb proto-oncogene transcription during erythroid development

    PubMed Central

    Stadhouders, Ralph; Thongjuea, Supat; Andrieu-Soler, Charlotte; Palstra, Robert-Jan; Bryne, Jan Christian; van den Heuvel, Anita; Stevens, Mary; de Boer, Ernie; Kockx, Christel; van der Sloot, Antoine; van den Hout, Mirjam; van IJcken, Wilfred; Eick, Dirk; Lenhard, Boris; Grosveld, Frank; Soler, Eric

    2012-01-01

    The key haematopoietic regulator Myb is essential for coordinating proliferation and differentiation. ChIP-Sequencing and Chromosome Conformation Capture (3C)-Sequencing were used to characterize the structural and protein-binding dynamics of the Myb locus during erythroid differentiation. In proliferating cells expressing Myb, enhancers within the Myb-Hbs1l intergenic region were shown to form an active chromatin hub (ACH) containing the Myb promoter and first intron. This first intron was found to harbour the transition site from transcription initiation to elongation, which takes place around a conserved CTCF site. Upon erythroid differentiation, Myb expression is downregulated and the ACH destabilized. We propose a model for Myb activation by distal enhancers dynamically bound by KLF1 and the GATA1/TAL1/LDB1 complex, which primarily function as a transcription elongation element through chromatin looping. PMID:22157820

  11. Dynamic long-range chromatin interactions control Myb proto-oncogene transcription during erythroid development.

    PubMed

    Stadhouders, Ralph; Thongjuea, Supat; Andrieu-Soler, Charlotte; Palstra, Robert-Jan; Bryne, Jan Christian; van den Heuvel, Anita; Stevens, Mary; de Boer, Ernie; Kockx, Christel; van der Sloot, Antoine; van den Hout, Mirjam; van Ijcken, Wilfred; Eick, Dirk; Lenhard, Boris; Grosveld, Frank; Soler, Eric

    2012-02-15

    The key haematopoietic regulator Myb is essential for coordinating proliferation and differentiation. ChIP-Sequencing and Chromosome Conformation Capture (3C)-Sequencing were used to characterize the structural and protein-binding dynamics of the Myb locus during erythroid differentiation. In proliferating cells expressing Myb, enhancers within the Myb-Hbs1l intergenic region were shown to form an active chromatin hub (ACH) containing the Myb promoter and first intron. This first intron was found to harbour the transition site from transcription initiation to elongation, which takes place around a conserved CTCF site. Upon erythroid differentiation, Myb expression is downregulated and the ACH destabilized. We propose a model for Myb activation by distal enhancers dynamically bound by KLF1 and the GATA1/TAL1/LDB1 complex, which primarily function as a transcription elongation element through chromatin looping. PMID:22157820

  12. Dynamics of chromatin accessibility and gene regulation by MADS-domain transcription factors in flower development

    PubMed Central

    2014-01-01

    Background Development of eukaryotic organisms is controlled by transcription factors that trigger specific and global changes in gene expression programs. In plants, MADS-domain transcription factors act as master regulators of developmental switches and organ specification. However, the mechanisms by which these factors dynamically regulate the expression of their target genes at different developmental stages are still poorly understood. Results We characterized the relationship of chromatin accessibility, gene expression, and DNA binding of two MADS-domain proteins at different stages of Arabidopsis flower development. Dynamic changes in APETALA1 and SEPALLATA3 DNA binding correlated with changes in gene expression, and many of the target genes could be associated with the developmental stage in which they are transcriptionally controlled. We also observe dynamic changes in chromatin accessibility during flower development. Remarkably, DNA binding of APETALA1 and SEPALLATA3 is largely independent of the accessibility status of their binding regions and it can precede increases in DNA accessibility. These results suggest that APETALA1 and SEPALLATA3 may modulate chromatin accessibility, thereby facilitating access of other transcriptional regulators to their target genes. Conclusions Our findings indicate that different homeotic factors regulate partly overlapping, yet also distinctive sets of target genes in a partly stage-specific fashion. By combining the information from DNA-binding and gene expression data, we are able to propose models of stage-specific regulatory interactions, thereby addressing dynamics of regulatory networks throughout flower development. Furthermore, MADS-domain TFs may regulate gene expression by alternative strategies, one of which is modulation of chromatin accessibility. PMID:24581456

  13. Non-linear longitudinal compression effect on dynamics of the transcription bubble in DNA.

    PubMed

    Shikhovtseva, E S; Nazarov, V N

    2016-01-01

    The dependence of the dynamics of transcription bubble on the parameters of non-linear longitudinal compression is presented on the base of simple model of soliton-like conformational switchings in two-component bistable polymer molecules with energetically non-equivalent stable states. It has been shown that under certain conditions the longitudinal compression may be a trap for a conformational switching. PMID:27232455

  14. Dynamic regulation of the transcription initiation landscape at single nucleotide resolution during vertebrate embryogenesis

    PubMed Central

    Nepal, Chirag; Hadzhiev, Yavor; Previti, Christopher; Haberle, Vanja; Li, Nan; Takahashi, Hazuki; Suzuki, Ana Maria M.; Sheng, Ying; Abdelhamid, Rehab F.; Anand, Santosh; Gehrig, Jochen; Akalin, Altuna; Kockx, Christel E.M.; van der Sloot, Antoine A.J.; van IJcken, Wilfred F.J.; Armant, Olivier; Rastegar, Sepand; Watson, Craig; Strähle, Uwe; Stupka, Elia; Carninci, Piero; Lenhard, Boris; Müller, Ferenc

    2013-01-01

    Spatiotemporal control of gene expression is central to animal development. Core promoters represent a previously unanticipated regulatory level by interacting with cis-regulatory elements and transcription initiation in different physiological and developmental contexts. Here, we provide a first and comprehensive description of the core promoter repertoire and its dynamic use during the development of a vertebrate embryo. By using cap analysis of gene expression (CAGE), we mapped transcription initiation events at single nucleotide resolution across 12 stages of zebrafish development. These CAGE-based transcriptome maps reveal genome-wide rules of core promoter usage, structure, and dynamics, key to understanding the control of gene regulation during vertebrate ontogeny. They revealed the existence of multiple classes of pervasive intra- and intergenic post-transcriptionally processed RNA products and their developmental dynamics. Among these RNAs, we report splice donor site-associated intronic RNA (sRNA) to be specific to genes of the splicing machinery. For the identification of conserved features, we compared the zebrafish data sets to the first CAGE promoter map of Tetraodon and the existing human CAGE data. We show that a number of features, such as promoter type, newly discovered promoter properties such as a specialized purine-rich initiator motif, as well as sRNAs and the genes in which they are detected, are conserved in mammalian and Tetraodon CAGE-defined promoter maps. The zebrafish developmental promoterome represents a powerful resource for studying developmental gene regulation and revealing promoter features shared across vertebrates. PMID:24002785

  15. Personal genomes, quantitative dynamic omics and personalized medicine

    PubMed Central

    Mias, George I.; Snyder, Michael

    2015-01-01

    The rapid technological developments following the Human Genome Project have made possible the availability of personalized genomes. As the focus now shifts from characterizing genomes to making personalized disease associations, in combination with the availability of other omics technologies, the next big push will be not only to obtain a personalized genome, but to quantitatively follow other omics. This will include transcriptomes, proteomes, metabolomes, antibodyomes, and new emerging technologies, enabling the profiling of thousands of molecular components in individuals. Furthermore, omics profiling performed longitudinally can probe the temporal patterns associated with both molecular changes and associated physiological health and disease states. Such data necessitates the development of computational methodology to not only handle and descriptively assess such data, but also construct quantitative biological models. Here we describe the availability of personal genomes and developing omics technologies that can be brought together for personalized implementations and how these novel integrated approaches may effectively provide a precise personalized medicine that focuses on not only characterization and treatment but ultimately the prevention of disease. PMID:25798291

  16. Protein–DNA binding dynamics predict transcriptional response to nutrients in archaea

    PubMed Central

    Todor, Horia; Sharma, Kriti; Pittman, Adrianne M. C.; Schmid, Amy K.

    2013-01-01

    Organisms across all three domains of life use gene regulatory networks (GRNs) to integrate varied stimuli into coherent transcriptional responses to environmental pressures. However, inferring GRN topology and regulatory causality remains a central challenge in systems biology. Previous work characterized TrmB as a global metabolic transcription factor in archaeal extremophiles. However, it remains unclear how TrmB dynamically regulates its ∼100 metabolic enzyme-coding gene targets. Using a dynamic perturbation approach, we elucidate the topology of the TrmB metabolic GRN in the model archaeon Halobacterium salinarum. Clustering of dynamic gene expression patterns reveals that TrmB functions alone to regulate central metabolic enzyme-coding genes but cooperates with various regulators to control peripheral metabolic pathways. Using a dynamical model, we predict gene expression patterns for some TrmB-dependent promoters and infer secondary regulators for others. Our data suggest feed-forward gene regulatory topology for cobalamin biosynthesis. In contrast, purine biosynthesis appears to require TrmB-independent regulators. We conclude that TrmB is an important component for mediating metabolic modularity, integrating nutrient status and regulating gene expression dynamics alone and in concert with secondary regulators. PMID:23892291

  17. Protein-DNA binding dynamics predict transcriptional response to nutrients in archaea.

    PubMed

    Todor, Horia; Sharma, Kriti; Pittman, Adrianne M C; Schmid, Amy K

    2013-10-01

    Organisms across all three domains of life use gene regulatory networks (GRNs) to integrate varied stimuli into coherent transcriptional responses to environmental pressures. However, inferring GRN topology and regulatory causality remains a central challenge in systems biology. Previous work characterized TrmB as a global metabolic transcription factor in archaeal extremophiles. However, it remains unclear how TrmB dynamically regulates its ∼100 metabolic enzyme-coding gene targets. Using a dynamic perturbation approach, we elucidate the topology of the TrmB metabolic GRN in the model archaeon Halobacterium salinarum. Clustering of dynamic gene expression patterns reveals that TrmB functions alone to regulate central metabolic enzyme-coding genes but cooperates with various regulators to control peripheral metabolic pathways. Using a dynamical model, we predict gene expression patterns for some TrmB-dependent promoters and infer secondary regulators for others. Our data suggest feed-forward gene regulatory topology for cobalamin biosynthesis. In contrast, purine biosynthesis appears to require TrmB-independent regulators. We conclude that TrmB is an important component for mediating metabolic modularity, integrating nutrient status and regulating gene expression dynamics alone and in concert with secondary regulators.

  18. Use of the growing environment as a source of variation to identify the quantitative trait transcripts and modules of co-expressed genes that determine chlorogenic acid accumulation

    PubMed Central

    JOËT, THIERRY; SALMONA, JORDI; LAFFARGUE, ANDRÉINA; DESCROIX, FRÉDÉRIC; DUSSERT, STÉPHANE

    2010-01-01

    Developing Coffea arabica seeds accumulate large amounts of chlorogenic acids (CGAs) as a storage form of phenylpropanoid derivatives, making coffee a valuable model to investigate the metabolism of these widespread plant phenolics. However, developmental and environmental regulations of CGA metabolism are poorly understood. In the present work, the expression of selected phenylpropanoid genes, together with CGA isomer profiles, was monitored throughout seed development across a wide set of contrasted natural environments. Although CGA metabolism was controlled by major developmental factors, the mean temperature during seed development had a direct impact on the time-window of CGA biosynthesis, as well as on final CGA isomer composition through subtle transcriptional regulations. We provide evidence that the variability induced by the environment is a useful tool to test whether CGA accumulation is quantitatively modulated at the transcriptional level, hence enabling detection of rate-limiting transcriptional steps [quantitative trait transcripts (QTTs)] for CGA biosynthesis. Variations induced by the environment also enabled a better description of the phenylpropanoid gene transcriptional network throughout seed development, as well as the detection of three temporally distinct modules of quantitatively co-expressed genes. Finally, analysis of metabolite-to-metabolite relationships revealed new biochemical characteristics of the isomerization steps that remain uncharacterized at the gene level. PMID:20199615

  19. A quantitative model of honey bee colony population dynamics.

    PubMed

    Khoury, David S; Myerscough, Mary R; Barron, Andrew B

    2011-01-01

    Since 2006 the rate of honey bee colony failure has increased significantly. As an aid to testing hypotheses for the causes of colony failure we have developed a compartment model of honey bee colony population dynamics to explore the impact of different death rates of forager bees on colony growth and development. The model predicts a critical threshold forager death rate beneath which colonies regulate a stable population size. If death rates are sustained higher than this threshold rapid population decline is predicted and colony failure is inevitable. The model also predicts that high forager death rates draw hive bees into the foraging population at much younger ages than normal, which acts to accelerate colony failure. The model suggests that colony failure can be understood in terms of observed principles of honey bee population dynamics, and provides a theoretical framework for experimental investigation of the problem. PMID:21533156

  20. A Quantitative Model of Honey Bee Colony Population Dynamics

    PubMed Central

    Khoury, David S.; Myerscough, Mary R.; Barron, Andrew B.

    2011-01-01

    Since 2006 the rate of honey bee colony failure has increased significantly. As an aid to testing hypotheses for the causes of colony failure we have developed a compartment model of honey bee colony population dynamics to explore the impact of different death rates of forager bees on colony growth and development. The model predicts a critical threshold forager death rate beneath which colonies regulate a stable population size. If death rates are sustained higher than this threshold rapid population decline is predicted and colony failure is inevitable. The model also predicts that high forager death rates draw hive bees into the foraging population at much younger ages than normal, which acts to accelerate colony failure. The model suggests that colony failure can be understood in terms of observed principles of honey bee population dynamics, and provides a theoretical framework for experimental investigation of the problem. PMID:21533156

  1. Development of duplex SYBR Green I-based real-time quantitative reverse-transcription PCR for detection and discrimination of grapevine viruses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A SYBR® Green-based real-time quantitative reverse transcription PCR (qRT-PCR) assay in combination with melt curve analysis (MCA) was developed for the detection of nine grapevine viruses. The detection limits for singleplex qRT-PCR for all nine grapevine viruses were determined to be in the range ...

  2. Comparison of propidium monoazide-quantitative PCR and reverse transcription quantitative PCR for viability detection of fresh Cryptosporidium oocysts following disinfection and after long-term storage in water samples

    EPA Science Inventory

    Purified oocysts of Cryptosporidium parvum were used to evaluate applicability of two quantitative PCR (qPCR) viability detection methods in raw surface water and disinfection treated water. Propidium monoazide-qPCR targeting hsp70 gene was compared to reverse transcription (RT)-...

  3. A quantitative evolutionary theory of adaptive behavior dynamics.

    PubMed

    McDowell, J J

    2013-10-01

    The idea that behavior is selected by its consequences in a process analogous to organic evolution has been discussed for over 100 years. A recently proposed theory instantiates this idea by means of a genetic algorithm that operates on a population of potential behaviors. Behaviors in the population are represented by numbers in decimal integer (phenotypic) and binary bit string (genotypic) forms. One behavior from the population is emitted at random each time tick, after which a new population of potential behaviors is constructed by recombining parent behavior bit strings. If the emitted behavior produced a benefit to the organism, then parents are chosen on the basis of their phenotypic similarity to the emitted behavior; otherwise, they are chosen at random. After parent behavior recombination, the population is subjected to a small amount of mutation by flipping random bits in the population's bit strings. The behavior generated by this process of selection, reproduction, and mutation reaches equilibrium states that conform to every empirically valid equation of matching theory, exactly and without systematic error. These equations are known to describe the behavior of many vertebrate species, including humans, in a variety of experimental, naturalistic, natural, and social environments. The evolutionary theory also generates instantaneous dynamics and patterns of preference change in constantly changing environments that are consistent with the dynamics of live-organism behavior. These findings support the assertion that the world of behavior we observe and measure is generated by evolutionary dynamics. PMID:24219847

  4. A quantitative evolutionary theory of adaptive behavior dynamics.

    PubMed

    McDowell, J J

    2013-10-01

    The idea that behavior is selected by its consequences in a process analogous to organic evolution has been discussed for over 100 years. A recently proposed theory instantiates this idea by means of a genetic algorithm that operates on a population of potential behaviors. Behaviors in the population are represented by numbers in decimal integer (phenotypic) and binary bit string (genotypic) forms. One behavior from the population is emitted at random each time tick, after which a new population of potential behaviors is constructed by recombining parent behavior bit strings. If the emitted behavior produced a benefit to the organism, then parents are chosen on the basis of their phenotypic similarity to the emitted behavior; otherwise, they are chosen at random. After parent behavior recombination, the population is subjected to a small amount of mutation by flipping random bits in the population's bit strings. The behavior generated by this process of selection, reproduction, and mutation reaches equilibrium states that conform to every empirically valid equation of matching theory, exactly and without systematic error. These equations are known to describe the behavior of many vertebrate species, including humans, in a variety of experimental, naturalistic, natural, and social environments. The evolutionary theory also generates instantaneous dynamics and patterns of preference change in constantly changing environments that are consistent with the dynamics of live-organism behavior. These findings support the assertion that the world of behavior we observe and measure is generated by evolutionary dynamics.

  5. Quantitative evolutionary dynamics using high-resolution lineage tracking.

    PubMed

    Levy, Sasha F; Blundell, Jamie R; Venkataram, Sandeep; Petrov, Dmitri A; Fisher, Daniel S; Sherlock, Gavin

    2015-03-12

    Evolution of large asexual cell populations underlies ∼30% of deaths worldwide, including those caused by bacteria, fungi, parasites, and cancer. However, the dynamics underlying these evolutionary processes remain poorly understood because they involve many competing beneficial lineages, most of which never rise above extremely low frequencies in the population. To observe these normally hidden evolutionary dynamics, we constructed a sequencing-based ultra high-resolution lineage tracking system in Saccharomyces cerevisiae that allowed us to monitor the relative frequencies of ∼500,000 lineages simultaneously. In contrast to some expectations, we found that the spectrum of fitness effects of beneficial mutations is neither exponential nor monotonic. Early adaptation is a predictable consequence of this spectrum and is strikingly reproducible, but the initial small-effect mutations are soon outcompeted by rarer large-effect mutations that result in variability between replicates. These results suggest that early evolutionary dynamics may be deterministic for a period of time before stochastic effects become important.

  6. The RosR transcription factor is required for gene expression dynamics in response to extreme oxidative stress in a hypersaline-adapted archaeon

    PubMed Central

    2012-01-01

    Background Previous work has shown that the hypersaline-adapted archaeon, Halobacterium salinarum NRC-1, is highly resistant to oxidative stress caused by exposure to hydrogen peroxide, UV, and gamma radiation. Dynamic alteration of the gene regulatory network (GRN) has been implicated in such resistance. However, the molecular functions of transcription regulatory proteins involved in this response remain unknown. Results Here we have reanalyzed several existing GRN and systems biology datasets for H. salinarum to identify and characterize a novel winged helix-turn-helix transcription factor, VNG0258H, as a regulator required for reactive oxygen species resistance in this organism. This protein appears to be unique to the haloarchaea at the primary sequence level. High throughput quantitative growth assays in a deletion mutant strain implicate VNG0258H in extreme oxidative stress resistance. According to time course gene expression analyses, this transcription factor is required for the appropriate dynamic response of nearly 300 genes to reactive oxygen species damage from paraquat and hydrogen peroxide. These genes are predicted to function in repair of oxidative damage to proteins and DNA. In vivo DNA binding assays demonstrate that VNG0258H binds DNA to mediate gene regulation. Conclusions Together these results suggest that VNG0258H is a novel archaeal transcription factor that regulates gene expression to enable adaptation to the extremely oxidative, hypersaline niche of H. salinarum. We have therefore renamed VNG0258H as RosR, for reactive oxygen species regulator. PMID:22846541

  7. Transcription factors dynamically control the spatial organization of the yeast genome

    PubMed Central

    Randise-Hinchliff, Carlo; Brickner, Jason H.

    2016-01-01

    ABSTRACT In yeast, inducible genes such as INO1, PRM1 and HIS4 reposition from the nucleoplasm to nuclear periphery upon activation. This leads to a physical interaction with nuclear pore complex (NPC), interchromosomal clustering, and stronger transcription. Repositioning to the nuclear periphery is controlled by cis-acting transcription factor (TF) binding sites located within the promoters of these genes and the TFs that bind to them. Such elements are both necessary and sufficient to control positioning of genes to the nuclear periphery. We have identified 4 TFs capable of controlling the regulated positioning of genes to the nuclear periphery in budding yeast under different conditions: Put3, Cbf1, Gcn4 and Ste12. In each case, we have defined the molecular basis of regulated relocalization to the nuclear periphery. Put3- and Cbf1-mediated targeting to nuclear periphery is regulated through local recruitment of Rpd3(L) histone deacetylase complex by transcriptional repressors. Rpd3(L), through its histone deacetylase activity, prevents TF-mediated gene positioning by blocking TF binding. Many yeast transcriptional repressors were capable of blocking Put3-mediated recruitment; 11 of these required Rpd3. Thus, it is a general function of transcription repressors to regulate TF-mediated recruitment. However, Ste12 and Gcn4-mediated recruitment is regulated independently of Rpd3(L) and transcriptional repressors. Ste12-mediated recruitment is regulated by phosphorylation of an inhibitor called Dig2, and Gcn4-mediated gene targeting is up-regulated by increasing Gcn4 protein levels. The ability to control spatial position of genes in yeast represents a novel function for TFs and different regulatory strategies provide dynamic control of the yeast genome through different time scales. PMID:27442220

  8. In vivo quantification of formulated and chemically modified small interfering RNA by heating-in-Triton quantitative reverse transcription polymerase chain reaction (HIT qRT-PCR)

    PubMed Central

    2010-01-01

    Background While increasing numbers of small interfering RNA (siRNA) therapeutics enter into clinical trials, the quantification of siRNA from clinical samples for pharmacokinetic studies remains a challenge. This challenge is even more acute for the quantification of chemically modified and formulated siRNAs such as those typically required for systemic delivery. Results Here, we describe a novel method, heating-in-Triton quantitative reverse transcription PCR (HIT qRT-PCR) that improves upon the stem-loop RT-PCR technique for the detection of formulated and chemically modified siRNAs from plasma and tissue. The broad dynamic range of this assay spans five orders of magnitude and can detect as little as 70 pg duplex in 1 g of liver or in 1 ml of plasma. We have used this assay to quantify intravenously administrated siRNA in rodents and have reliably correlated target reduction with tissue drug concentrations. We were able to detect siRNA in rat liver for at least 10 days post injection and determined that for a modified factor VII (FVII) siRNA, on average, approximately 500 siRNA molecules per cell are required to achieve a 50% target reduction. Conclusions HIT qRT-PCR is a novel approach that simplifies the in vivo quantification of siRNA and provides a highly sensitive and reproducible tool to measure the silencing efficiency of chemically modified and formulated siRNAs. PMID:20731861

  9. The RootScope: a simple high-throughput screening system for quantitating gene expression dynamics in plant roots

    PubMed Central

    2013-01-01

    Background High temperature stress responses are vital for plant survival. The mechanisms that plants use to sense high temperatures are only partially understood and involve multiple sensing and signaling pathways. Here we describe the development of the RootScope, an automated microscopy system for quantitating heat shock responses in plant roots. Results The promoter of Hsp17.6 was used to build a Hsp17.6p:GFP transcriptional reporter that is induced by heat shock in Arabidopsis. An automated fluorescence microscopy system which enables multiple roots to be imaged in rapid succession was used to quantitate Hsp17.6p:GFP response dynamics. Hsp17.6p:GFP signal increased with temperature increases from 28°C to 37°C. At 40°C the kinetics and localization of the response are markedly different from those at 37°C. This suggests that different mechanisms mediate heat shock responses above and below 37°C. Finally, we demonstrate that Hsp17.6p:GFP expression exhibits wave like dynamics in growing roots. Conclusions The RootScope system is a simple and powerful platform for investigating the heat shock response in plants. PMID:24119322

  10. Dynamic transcriptome analysis reveals AP2/ERF transcription factors responsible for cold stress in rapeseed (Brassica napus L.).

    PubMed

    Du, Chunfang; Hu, Kaining; Xian, Shuanshi; Liu, Chunqing; Fan, Jianchun; Tu, Jinxing; Fu, Tingdong

    2016-06-01

    The APETALA2/ethylene response factor (AP2/ERF) transcription factor (TF) superfamily plays an important regulatory role in signal transduction of the plant responses to various stresses including low temperature. Significant progress has been made in understanding the mechanism of cold resistance in Brassica napus, an important oilseed crop. However, comprehensive studies on the induction and activity of these TFs under low temperature have been lacking. In this study, 132 AP2/ERF genes were identified by transcriptome sequencing of rapeseed leaves exposed to 0, 2, 6, 12, and 24 h of low (4 °C) temperature stress. The genes were classified into 4 subfamilies (AP2, DREB, ERF, and RAV) and 13 subgroups, among which the DREB subfamily and ERF subfamily contained 114 genes, no genes were assigned to soloist or DREB A3 subgroups. One hundred and eighteen genes were located on chromosomes A1 to C9. GO functional analysis and promoter sequence analysis revealed that these genes are involved in many molecular pathways that may enhance cold resistance in plants, such as the low-temperature responsiveness, methyl jasmonate, abscisic acid, and ethylene-responsiveness pathways. Their expression patterns revealed dynamic control at different times following initiation of cold stress; the RAV and DREB subfamilies were expressed at the early stage of cold stress, whereas the AP2 subfamily was expressed later. Quantitative PCR analyses of 13 cold-induced AP2/ERF TFs confirmed the accuracy of above results. This study is the first dynamic analysis of the AP2/ERF TFs responsible for cold stress in rapeseed. These findings will serve as a reference for future functional research on transcription in rapeseed. PMID:26728151

  11. Extending the dynamic range of transcription factor action by translational regulation

    NASA Astrophysics Data System (ADS)

    Sokolowski, Thomas R.; Walczak, Aleksandra M.; Bialek, William; Tkačik, Gašper

    2016-02-01

    A crucial step in the regulation of gene expression is binding of transcription factor (TF) proteins to regulatory sites along the DNA. But transcription factors act at nanomolar concentrations, and noise due to random arrival of these molecules at their binding sites can severely limit the precision of regulation. Recent work on the optimization of information flow through regulatory networks indicates that the lower end of the dynamic range of concentrations is simply inaccessible, overwhelmed by the impact of this noise. Motivated by the behavior of homeodomain proteins, such as the maternal morphogen Bicoid in the fruit fly embryo, we suggest a scheme in which transcription factors also act as indirect translational regulators, binding to the mRNA of other regulatory proteins. Intuitively, each mRNA molecule acts as an independent sensor of the input concentration, and averaging over these multiple sensors reduces the noise. We analyze information flow through this scheme and identify conditions under which it outperforms direct transcriptional regulation. Our results suggest that the dual role of homeodomain proteins is not just a historical accident, but a solution to a crucial physics problem in the regulation of gene expression.

  12. Top-level dynamics and the regulated gene response of feed-forward loop transcriptional motifs

    NASA Astrophysics Data System (ADS)

    Mayo, Michael; Abdelzaher, Ahmed; Perkins, Edward J.; Ghosh, Preetam

    2014-09-01

    Feed-forward loops are hierarchical three-node transcriptional subnetworks, wherein a top-level protein regulates the activity of a target gene via two paths: a direct-regulatory path, and an indirect route, whereby the top-level proteins act implicitly through an intermediate transcription factor. Using a transcriptional network of the model bacterium Escherichia coli, we confirmed that nearly all types of feed-forward loop were significantly overrepresented in the bacterial network. We then used mathematical modeling to study their dynamics by manipulating the rise times of the top-level protein concentration, termed the induction time, through alteration of the protein destruction rates. Rise times of the regulated proteins exhibited two qualitatively different regimes, depending on whether top-level inductions were "fast" or "slow." In the fast regime, rise times were nearly independent of rapid top-level inductions, indicative of biological robustness, and occurred when RNA production rate-limits the protein yield. Alternatively, the protein rise times were dependent upon slower top-level inductions, greater than approximately one bacterial cell cycle. An equation is given for this crossover, which depends upon three parameters of the direct-regulatory path: transcriptional cooperation at the DNA-binding site, a protein-DNA dissociation constant, and the relative magnitude of the top-level protien concentration.

  13. Top-level dynamics and the regulated gene response of feed-forward loop transcriptional motifs.

    PubMed

    Mayo, Michael; Abdelzaher, Ahmed; Perkins, Edward J; Ghosh, Preetam

    2014-09-01

    Feed-forward loops are hierarchical three-node transcriptional subnetworks, wherein a top-level protein regulates the activity of a target gene via two paths: a direct-regulatory path, and an indirect route, whereby the top-level proteins act implicitly through an intermediate transcription factor. Using a transcriptional network of the model bacterium Escherichia coli, we confirmed that nearly all types of feed-forward loop were significantly overrepresented in the bacterial network. We then used mathematical modeling to study their dynamics by manipulating the rise times of the top-level protein concentration, termed the induction time, through alteration of the protein destruction rates. Rise times of the regulated proteins exhibited two qualitatively different regimes, depending on whether top-level inductions were "fast" or "slow." In the fast regime, rise times were nearly independent of rapid top-level inductions, indicative of biological robustness, and occurred when RNA production rate-limits the protein yield. Alternatively, the protein rise times were dependent upon slower top-level inductions, greater than approximately one bacterial cell cycle. An equation is given for this crossover, which depends upon three parameters of the direct-regulatory path: transcriptional cooperation at the DNA-binding site, a protein-DNA dissociation constant, and the relative magnitude of the top-level protien concentration.

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

    PubMed

    Cordier, Florence; Chaffotte, Alain; Wolff, Nicolas

    2015-05-01

    The dual lipid and protein phosphatase PTEN is a tumor suppressor controlling key biological processes, such as cell growth, proliferation and neuro-survival. Its activity and intracellular trafficking is finely regulated notably by multi-site phosphorylation of its C-terminal tail. The reversible and highly dynamic character of these regulatory events confers a temporal dimension to the cell for triggering crucial decisions. In this review, we describe how a recently developed time-resolved NMR spectroscopy approach unveils the dynamic establishment of the phosphorylation events of PTEN C-terminal tail controlled by CK2 and GSK3β kinases. Two cascades of reactions have been identified, in vitro and in extracts of human neuroblastoma cells. They are triggered independently on two nearby clusters of sites (S380-S385 and S361-S370) and occur on different timescales. In each cascade, the reactions follow an ordered model with a distributive kinetic mechanism. The vision of these cascades as two delay timers activating distinct or time-delayed regulatory responses gives a temporal dimension on PTEN regulation and is discussed in relation to the known functional roles of each cluster. PMID:25449899

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

    PubMed

    Lyons, S Kathleen

    2005-06-01

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

  16. The GATA transcription factor GtaC regulates early developmental gene expression dynamics in Dictyostelium

    PubMed Central

    Santhanam, Balaji; Cai, Huaqing; Devreotes, Peter N.; Shaulsky, Gad; Katoh-Kurasawa, Mariko

    2015-01-01

    In many systems, including the social amoeba Dictyostelium discoideum, development is often marked by dynamic morphological and transcriptional changes orchestrated by key transcription factors. However, efforts to examine sequential genome-wide changes of gene regulation in developmental processes have been fairly limited. Here we report the developmental regulatory dynamics of GtaC, a GATA-type zinc-finger transcription factor, through the analyses of serial ChIP- and RNA-sequencing data. GtaC is essential for developmental progression, decoding extracellular cAMP pulses during early development and may play a role in mediating cell-type differentiation at later stages. We find that GtaC exhibits temporally distinctive DNA-binding patterns concordant with each developmental stage. We identify direct GtaC targets and observe cotemporaneous GtaC-binding and developmental expression regulation. Our results suggest that GtaC regulates multiple physiological processes as Dictyostelium transitions from a group of unicellular amoebae to an integrated multicellular organism. PMID:26144553

  17. Quantifying the dynamics of the oligomeric transcription factor STAT3 by pair correlation of molecular brightness

    PubMed Central

    Hinde, Elizabeth; Pandžić, Elvis; Yang, Zhengmin; Ng, Ivan H. W.; Jans, David A.; Bogoyevitch, Marie A.; Gratton, Enrico; Gaus, Katharina

    2016-01-01

    Oligomerization of transcription factors controls their translocation into the nucleus and DNA-binding activity. Here we present a fluorescence microscopy analysis termed pCOMB (pair correlation of molecular brightness) that tracks the mobility of different oligomeric species within live cell nuclear architecture. pCOMB amplifies the signal from the brightest species present and filters the dynamics of the extracted oligomeric population based on arrival time between two locations. We use this method to demonstrate a dependence of signal transducer and activator of transcription 3 (STAT3) mobility on oligomeric state. We find that on entering the nucleus STAT3 dimers must first bind DNA to form STAT3 tetramers, which are also DNA-bound but exhibit a different mobility signature. Examining the dimer-to-tetramer transition by a cross-pair correlation analysis (cpCOMB) reveals that chromatin accessibility modulates STAT3 tetramer formation. Thus, the pCOMB approach is suitable for mapping the impact oligomerization on transcription factor dynamics. PMID:27009358

  18. The dynamic nature and territory of transcriptional machinery in the bacterial chromosome

    PubMed Central

    Jin, Ding J.; Cagliero, Cedric; Martin, Carmen M.; Izard, Jerome; Zhou, Yan N.

    2015-01-01

    Our knowledge of the regulation of genes involved in bacterial growth and stress responses is extensive; however, we have only recently begun to understand how environmental cues influence the dynamic, three-dimensional distribution of RNA polymerase (RNAP) in Escherichia coli on the level of single cell, using wide-field fluorescence microscopy and state-of-the-art imaging techniques. Live-cell imaging using either an agarose-embedding procedure or a microfluidic system further underscores the dynamic nature of the distribution of RNAP in response to changes in the environment and highlights the challenges in the study. A general agreement between live-cell and fixed-cell images has validated the formaldehyde-fixing procedure, which is a technical breakthrough in the study of the cell biology of RNAP. In this review we use a systems biology perspective to summarize the advances in the cell biology of RNAP in E. coli, including the discoveries of the bacterial nucleolus, the spatial compartmentalization of the transcription machinery at the periphery of the nucleoid, and the segregation of the chromosome territories for the two major cellular functions of transcription and replication in fast-growing cells. Our understanding of the coupling of transcription and bacterial chromosome (or nucleoid) structure is also summarized. Using E. coli as a simple model system, co-imaging of RNAP with DNA and other factors during growth and stress responses will continue to be a useful tool for studying bacterial growth and adaptation in changing environment. PMID:26052320

  19. Transcription-Driven Twin Supercoiling of a DNA Loop: A Brownian Dynamics Study

    SciTech Connect

    Mielke, S P; Fink, W H; Krishnan, K; Gronbech-Jensen, N; Benham, C J

    2004-06-30

    The torque generated by RNA polymerase as it tracks along double-stranded DNA can potentially induce long-range structural deformations integral to mechanisms of biological significance in both prokaryotes and eukaryotes. In this report, we introduce a dynamic computer model for investigating this phenomenon. Duplex DNA is represented as a chain of hydrodynamic bends interacting through elastic potentials. The chain, linear when relaxed, is looped to form two open but topologically constrained subdomains. This permits the dynamic introduction of torsional stress via a centrally applied torque. We simulate by Brownian dynamics the 100 {micro}s response of a 477-basepair B-DNA template to the localized torque generated by the prokaryotic transcription ensemble. Following a sharp rise at early times, the distributed twist assumes a nearly constant value in both subdomains, and a succession of supercoiling deformations occurs as superhelical stress is increasingly partitioned to writhe. The magnitude of writhe surpasses that of twist before also leveling off when the structure reaches mechanical equilibrium with the torsional load. Superhelicity is simultaneously right-handed in one subdomain and left-handed in the other. The properties of the chain at the onset of writhing agree well with predictions from theory, and the generated stress is ample for driving secondary structural transitions in physiological DNA. These results suggest that the torsional stress generated by transcription can significantly deform the DNA template over short times. This highlights the potential of transcription and other tracking processes to play a central role in gene regulation, and prompts further investigation of dynamically-generated supercoiling.

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

    NASA Astrophysics Data System (ADS)

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

    2015-11-01

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

  1. Structural and dynamic studies of the transcription factor ERG reveal DNA binding is allosterically autoinhibited

    PubMed Central

    Regan, Michael C.; Horanyi, Peter S.; Pryor, Edward E.; Sarver, Jessica L.; Cafiso, David S.; Bushweller, John H.

    2013-01-01

    The Ets-Related Gene (ERG) belongs to the Ets family of transcription factors and is critically important for maintenance of the hematopoietic stem cell population. A chromosomal translocation observed in the majority of human prostate cancers leads to the aberrant overexpression of ERG. We have identified regions flanking the ERG Ets domain responsible for autoinhibition of DNA binding and solved crystal structures of uninhibited, autoinhibited, and DNA-bound ERG. NMR-based measurements of backbone dynamics show that uninhibited ERG undergoes substantial dynamics on the millisecond-to-microsecond timescale but autoinhibited and DNA-bound ERG do not. We propose a mechanism whereby the allosteric basis of ERG autoinhibition is mediated predominantly by the regulation of Ets-domain dynamics with only modest structural changes. PMID:23898196

  2. Multiple visual quantitative cues enhance discrimination of dynamic stimuli during infancy.

    PubMed

    Baker, Joseph M; Mahamane, Salif P; Jordan, Kerry E

    2014-06-01

    Infants possess basic capabilities to assess various quantitative properties such as number, size, and time. Preverbal discriminations are approximate, however, and are similarly limited across these dimensions. Here, we present the first evidence that multiple sources of quantitative unisensory information about dynamic stimuli-namely, simultaneous visual cues to changes in both number and surface area-may accelerate 6-month-olds' quantitative competence. Using a habituation-dishabituation paradigm, results from Experiment 1 demonstrate that, when provided with such visual cues to multiple quantitative properties that occur in the same direction, infants make more precise discriminations than has been shown when they receive information about either cue alone. Moreover, Experiment 2 demonstrates that infants' discrimination also benefits from simultaneous visual cues to quantitative changes that occur in opposite directions. Finally, Experiment 3 demonstrates that these findings are not driven by infants' ability to discriminate a 2:3 ratio change in surface area of a dynamic stimulus alone. Thus, we hypothesize that enhanced quantitative discrimination occurs because simultaneous visual quantitative changes may be more salient than single-source information, which could better recruit attention and result in more precise learning and remembering.

  3. Dynamic transcription factor activity and networks during ErbB2 breast oncogenesis and targeted therapy.

    PubMed

    Weiss, M S; Peñalver Bernabé, B; Shin, S; Asztalos, S; Dubbury, S J; Mui, M D; Bellis, A D; Bluver, D; Tonetti, D A; Saez-Rodriguez, J; Broadbelt, L J; Jeruss, J S; Shea, L D

    2014-12-01

    Tissue development and disease progression are multi-stage processes controlled by an evolving set of key regulatory factors, and identifying these factors necessitates a dynamic analysis spanning relevant time scales. Current omics approaches depend on incomplete biological databases to identify critical cellular processes. Herein, we present TRACER (TRanscriptional Activity CEll aRrays), which was employed to quantify the dynamic activity of numerous transcription factor (TFs) simultaneously in 3D and networks for TRACER (NTRACER), a computational algorithm that allows for cellular rewiring to establish dynamic regulatory networks based on activity of TF reporter constructs. We identified major hubs at various stages of culture associated with normal and abnormal tissue growth (i.e., ELK-1 and E2F1, respectively) and the mechanism of action for a targeted therapeutic, lapatinib, through GATA-1, which were confirmed in human ErbB2 positive breast cancer patients and human ErbB2 positive breast cancer cell lines that were either sensitive or resistant to lapatinib.

  4. Contributions of transcription and mRNA decay to gene expression dynamics of fission yeast in response to oxidative stress

    PubMed Central

    Marguerat, Samuel; Lawler, Katherine; Brazma, Alvis; Bähler, Jürg

    2014-01-01

    The cooperation of transcriptional and post-transcriptional levels of control to shape gene regulation is only partially understood. Here we show that a combination of two simple and non-invasive genomic techniques, coupled with kinetic mathematical modeling, affords insight into the intricate dynamics of RNA regulation in response to oxidative stress in the fission yeast Schizosaccharomyces pombe. This study reveals a dominant role of transcriptional regulation in response to stress, but also points to the first minutes after stress induction as a critical time when the coordinated control of mRNA turnover can support the control of transcription for rapid gene regulation. In addition, we uncover specialized gene expression strategies associated with distinct functional gene groups, such as simultaneous transcriptional repression and mRNA destabilization for genes encoding ribosomal proteins, delayed mRNA destabilization with varying contribution of transcription for ribosome biogenesis genes, dominant roles of mRNA stabilization for genes functioning in protein degradation, and adjustment of both transcription and mRNA turnover during the adaptation to stress. We also show that genes regulated independently of the bZIP transcription factor Atf1p are predominantly controlled by mRNA turnover, and identify putative cis-regulatory sequences that are associated with different gene expression strategies during the stress response. This study highlights the intricate and multi-faceted interplay between transcription and RNA turnover during the dynamic regulatory response to stress. PMID:25007214

  5. Quantitative comparisons of analogue models of brittle wedge dynamics

    NASA Astrophysics Data System (ADS)

    Schreurs, Guido

    2010-05-01

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

  6. Quantitative analysis of rib movement based on dynamic chest bone images: preliminary results

    NASA Astrophysics Data System (ADS)

    Tanaka, R.; Sanada, S.; Oda, M.; Mitsutaka, M.; Suzuki, K.; Sakuta, K.; Kawashima, H.

    2014-03-01

    Rib movement during respiration is one of the diagnostic criteria in pulmonary impairments. In general, the rib movement is assessed in fluoroscopy. However, the shadows of lung vessels and bronchi overlapping ribs prevent accurate quantitative analysis of rib movement. Recently, an image-processing technique for separating bones from soft tissue in static chest radiographs, called "bone suppression technique", has been developed. Our purpose in this study was to evaluate the usefulness of dynamic bone images created by the bone suppression technique in quantitative analysis of rib movement. Dynamic chest radiographs of 10 patients were obtained using a dynamic flat-panel detector (FPD). Bone suppression technique based on a massive-training artificial neural network (MTANN) was applied to the dynamic chest images to create bone images. Velocity vectors were measured in local areas on the dynamic bone images, which formed a map. The velocity maps obtained with bone and original images for scoliosis and normal cases were compared to assess the advantages of bone images. With dynamic bone images, we were able to quantify and distinguish movements of ribs from those of other lung structures accurately. Limited rib movements of scoliosis patients appeared as reduced rib velocity vectors. Vector maps in all normal cases exhibited left-right symmetric distributions, whereas those in abnormal cases showed nonuniform distributions. In conclusion, dynamic bone images were useful for accurate quantitative analysis of rib movements: Limited rib movements were indicated as a reduction of rib movement and left-right asymmetric distribution on vector maps. Thus, dynamic bone images can be a new diagnostic tool for quantitative analysis of rib movements without additional radiation dose.

  7. SND1 transcription factor-directed quantitative functional hierarchical genetic regulatory network in wood formation in Populus trichocarpa.

    PubMed

    Lin, Ying-Chung; Li, Wei; Sun, Ying-Hsuan; Kumari, Sapna; Wei, Hairong; Li, Quanzi; Tunlaya-Anukit, Sermsawat; Sederoff, Ronald R; Chiang, Vincent L

    2013-11-01

    Wood is an essential renewable raw material for industrial products and energy. However, knowledge of the genetic regulation of wood formation is limited. We developed a genome-wide high-throughput system for the discovery and validation of specific transcription factor (TF)-directed hierarchical gene regulatory networks (hGRNs) in wood formation. This system depends on a new robust procedure for isolation and transfection of Populus trichocarpa stem differentiating xylem protoplasts. We overexpressed Secondary Wall-Associated NAC Domain 1s (Ptr-SND1-B1), a TF gene affecting wood formation, in these protoplasts and identified differentially expressed genes by RNA sequencing. Direct Ptr-SND1-B1-DNA interactions were then inferred by integration of time-course RNA sequencing data and top-down Graphical Gaussian Modeling-based algorithms. These Ptr-SND1-B1-DNA interactions were verified to function in differentiating xylem by anti-PtrSND1-B1 antibody-based chromatin immunoprecipitation (97% accuracy) and in stable transgenic P. trichocarpa (90% accuracy). In this way, we established a Ptr-SND1-B1-directed quantitative hGRN involving 76 direct targets, including eight TF and 61 enzyme-coding genes previously unidentified as targets. The network can be extended to the third layer from the second-layer TFs by computation or by overexpression of a second-layer TF to identify a new group of direct targets (third layer). This approach would allow the sequential establishment, one two-layered hGRN at a time, of all layers involved in a more comprehensive hGRN. Our approach may be particularly useful to study hGRNs in complex processes in plant species resistant to stable genetic transformation and where mutants are unavailable.

  8. Selection of reference genes for quantitative reverse-transcription polymerase chain reaction normalization in Brassica napus under various stress conditions.

    PubMed

    Wang, Zheng; Chen, Yu; Fang, Hedi; Shi, Haifeng; Chen, Keping; Zhang, Zhiyan; Tan, Xiaoli

    2014-10-01

    Data normalization is essential for reliable output of quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) assays, as the unsuitable choice of reference gene(s), whose expression might be influenced by exogenous treatments in plant tissues, could cause misinterpretation of results. To date, no systematic studies on reference genes have been performed in stressed Brassica napus. In this study, we investigated the expression variations of nine candidate reference genes in 40 samples of B. napus leaves subjected to various exogenous treatments. Parallel analyses by geNorm and NormFinder revealed that optimal reference genes differed across the different sets of samples. The best-ranked reference genes were PP2A and TIP41 for salt stress, TIP41 and ACT7 for heavy metal (Cr(6+)) stress, PP2A and UBC21 for drought stress, F-box and SAND for cold stress, F-box and ZNF for salicylic acid stress, TIP41, ACT7, and PP2A for methyl jasmonate stress, TIP41 and ACT7 for abscisic acid stress, and TIP41, UBC21, and PP2A for Sclerotinia sclerotiorum stress. Two newly employed reference genes, TIP41 and PP2A, showed better performances, suggesting their suitability in multiple conditions. To further validate the suitability of the reference genes, the expression patterns of BnWRKY40 and BnMKS1 were studied in parallel. This study is the first systematic analysis of reference gene selection for qRT-PCR normalization in B. napus, an agriculturally important crop, under different stress conditions. The results will contribute toward more accurate and widespread use of qRT-PCR in gene analysis of the genus Brassica. PMID:24770781

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

    PubMed

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

    2015-05-19

    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.

  10. ETS family transcriptional regulators drive chromatin dynamics and malignancy in squamous cell carcinomas

    PubMed Central

    Adam, Rene C; Keyes, Brice E; Wang, Ping; Zheng, Deyou; Fuchs, Elaine

    2015-01-01

    Tumor-initiating stem cells (SCs) exhibit distinct patterns of transcription factors and gene expression compared to healthy counterparts. Here, we show that dramatic shifts in large open-chromatin domain (super-enhancer) landscapes underlie these differences and reflect tumor microenvironment. By in vivo super-enhancer and transcriptional profiling, we uncover a dynamic cancer-specific epigenetic network selectively enriched for binding motifs of a transcription factor cohort expressed in squamous cell carcinoma SCs (SCC-SCs). Many of their genes, including Ets2 and Elk3, are themselves regulated by SCC-SC super-enhancers suggesting a cooperative feed-forward loop. Malignant progression requires these genes, whose knockdown severely impairs tumor growth and prohibits progression from benign papillomas to SCCs. ETS2-deficiency disrupts the SCC-SC super-enhancer landscape and downstream cancer genes while ETS2-overactivation in epidermal-SCs induces hyperproliferation and SCC super-enhancer-associated genes Fos, Junb and Klf5. Together, our findings unearth an essential regulatory network required for the SCC-SC chromatin landscape and unveil its importance in malignant progression. DOI: http://dx.doi.org/10.7554/eLife.10870.001 PMID:26590320

  11. Dynamic control of gene regulatory logic by seemingly redundant transcription factors

    PubMed Central

    AkhavanAghdam, Zohreh; Sinha, Joydeb; Tabbaa, Omar P; Hao, Nan

    2016-01-01

    Many transcription factors co-express with their homologs to regulate identical target genes, however the advantages of such redundancies remain elusive. Using single-cell imaging and microfluidics, we study the yeast general stress response transcription factor Msn2 and its seemingly redundant homolog Msn4. We find that gene regulation by these two factors is analogous to logic gate systems. Target genes with fast activation kinetics can be fully induced by either factor, behaving as an 'OR' gate. In contrast, target genes with slow activation kinetics behave as an 'AND' gate, requiring distinct contributions from both factors, upon transient stimulation. Furthermore, such genes become an 'OR' gate when the input duration is prolonged, suggesting that the logic gate scheme is not static but rather dependent on the input dynamics. Therefore, Msn2 and Msn4 enable a time-based mode of combinatorial gene regulation that might be applicable to homologous transcription factors in other organisms. DOI: http://dx.doi.org/10.7554/eLife.18458.001 PMID:27690227

  12. 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.

  13. Development of a quantitative real-time RT-PCR for kinetic analysis of immediate-early transcripts of rat cytomegalovirus.

    PubMed

    Loh, H S; Mohd-Azmi, M L

    2009-01-01

    One-step real-time RT-PCR assay was developed for quantification of the immediate-early (IE), namely IE1 and IE2 transcripts of Rat cytomegalovirus (RCMV), strain ALL-03 in rat embryonic fibroblast cells (REF). This in-house SYBR Green I based RT-PCR was shown to have higher amplification efficiency and detection limit as compared to a commercially available real-time RT-PCR kit in quantifying these two transcripts. The quantification histogram revealed the divergence of transcription activities of the two IE genes. The IE1 transcript had a concentration peak at 7 hrs post infection (p.i.), whereas IE2 transcript at 20 hrs p.i. Regulation of IE expression is critical for determination, whether the infection is going to be abortive, lytic or latent. Therefore, this in-house developed quantitative RT-PCR assay offers an alternative for diagnosis and monitoring of the acute cytomegalovirus (CMV) infection directed at IE transcript detection.

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

    NASA Astrophysics Data System (ADS)

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

    2015-03-01

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

  15. Quantitative Proteomics Reveals Factors Regulating RNA Biology as Dynamic Targets of Stress-induced SUMOylation in Arabidopsis *

    PubMed Central

    Miller, Marcus J.; Scalf, Mark; Rytz, Thérèse C.; Hubler, Shane L.; Smith, Lloyd M.; Vierstra, Richard D.

    2013-01-01

    The stress-induced attachment of small ubiquitin-like modifier (SUMO) to a diverse collection of nuclear proteins regulating chromatin architecture, transcription, and RNA biology has been implicated in protecting plants and animals against numerous environmental challenges. In order to better understand stress-induced SUMOylation, we combined stringent purification of SUMO conjugates with isobaric tag for relative and absolute quantification mass spectrometry and an advanced method to adjust for sample-to-sample variation so as to study quantitatively the SUMOylation dynamics of intact Arabidopsis seedlings subjected to stress. Inspection of 172 SUMO substrates during and after heat shock (37 °C) revealed that stress mostly increases the abundance of existing conjugates, as opposed to modifying new targets. Some of the most robustly up-regulated targets participate in RNA processing and turnover and RNA-directed DNA modification, thus implicating SUMO as a regulator of the transcriptome during stress. Many of these targets were also strongly SUMOylated during ethanol and oxidative stress, suggesting that their modification is crucial for general stress tolerance. Collectively, our quantitative data emphasize the importance of SUMO to RNA-related processes protecting plants from adverse environments. PMID:23197790

  16. Transcriptional dynamics reveal critical roles for non-coding RNAs in the immediate-early response.

    PubMed

    Aitken, Stuart; Magi, Shigeyuki; Alhendi, Ahmad M N; Itoh, Masayoshi; Kawaji, Hideya; Lassmann, Timo; Daub, Carsten O; Arner, Erik; Carninci, Piero; Forrest, Alistair R R; Hayashizaki, Yoshihide; Khachigian, Levon M; Okada-Hatakeyama, Mariko; Semple, Colin A

    2015-04-01

    The immediate-early response mediates cell fate in response to a variety of extracellular stimuli and is dysregulated in many cancers. However, the specificity of the response across stimuli and cell types, and the roles of non-coding RNAs are not well understood. Using a large collection of densely-sampled time series expression data we have examined the induction of the immediate-early response in unparalleled detail, across cell types and stimuli. We exploit cap analysis of gene expression (CAGE) time series datasets to directly measure promoter activities over time. Using a novel analysis method for time series data we identify transcripts with expression patterns that closely resemble the dynamics of known immediate-early genes (IEGs) and this enables a comprehensive comparative study of these genes and their chromatin state. Surprisingly, these data suggest that the earliest transcriptional responses often involve promoters generating non-coding RNAs, many of which are produced in advance of canonical protein-coding IEGs. IEGs are known to be capable of induction without de novo protein synthesis. Consistent with this, we find that the response of both protein-coding and non-coding RNA IEGs can be explained by their transcriptionally poised, permissive chromatin state prior to stimulation. We also explore the function of non-coding RNAs in the attenuation of the immediate early response in a small RNA sequencing dataset matched to the CAGE data: We identify a novel set of microRNAs responsible for the attenuation of the IEG response in an estrogen receptor positive cancer cell line. Our computational statistical method is well suited to meta-analyses as there is no requirement for transcripts to pass thresholds for significant differential expression between time points, and it is agnostic to the number of time points per dataset. PMID:25885578

  17. Transcriptional dynamics reveal critical roles for non-coding RNAs in the immediate-early response.

    PubMed

    Aitken, Stuart; Magi, Shigeyuki; Alhendi, Ahmad M N; Itoh, Masayoshi; Kawaji, Hideya; Lassmann, Timo; Daub, Carsten O; Arner, Erik; Carninci, Piero; Forrest, Alistair R R; Hayashizaki, Yoshihide; Khachigian, Levon M; Okada-Hatakeyama, Mariko; Semple, Colin A

    2015-04-01

    The immediate-early response mediates cell fate in response to a variety of extracellular stimuli and is dysregulated in many cancers. However, the specificity of the response across stimuli and cell types, and the roles of non-coding RNAs are not well understood. Using a large collection of densely-sampled time series expression data we have examined the induction of the immediate-early response in unparalleled detail, across cell types and stimuli. We exploit cap analysis of gene expression (CAGE) time series datasets to directly measure promoter activities over time. Using a novel analysis method for time series data we identify transcripts with expression patterns that closely resemble the dynamics of known immediate-early genes (IEGs) and this enables a comprehensive comparative study of these genes and their chromatin state. Surprisingly, these data suggest that the earliest transcriptional responses often involve promoters generating non-coding RNAs, many of which are produced in advance of canonical protein-coding IEGs. IEGs are known to be capable of induction without de novo protein synthesis. Consistent with this, we find that the response of both protein-coding and non-coding RNA IEGs can be explained by their transcriptionally poised, permissive chromatin state prior to stimulation. We also explore the function of non-coding RNAs in the attenuation of the immediate early response in a small RNA sequencing dataset matched to the CAGE data: We identify a novel set of microRNAs responsible for the attenuation of the IEG response in an estrogen receptor positive cancer cell line. Our computational statistical method is well suited to meta-analyses as there is no requirement for transcripts to pass thresholds for significant differential expression between time points, and it is agnostic to the number of time points per dataset.

  18. Transcriptional Dynamics Reveal Critical Roles for Non-coding RNAs in the Immediate-Early Response

    PubMed Central

    Aitken, Stuart; Magi, Shigeyuki; Alhendi, Ahmad M. N.; Itoh, Masayoshi; Kawaji, Hideya; Lassmann, Timo; Daub, Carsten O.; Arner, Erik; Carninci, Piero; Forrest, Alistair R. R.; Hayashizaki, Yoshihide; Khachigian, Levon M.; Okada-Hatakeyama, Mariko; Semple, Colin A.

    2015-01-01

    The immediate-early response mediates cell fate in response to a variety of extracellular stimuli and is dysregulated in many cancers. However, the specificity of the response across stimuli and cell types, and the roles of non-coding RNAs are not well understood. Using a large collection of densely-sampled time series expression data we have examined the induction of the immediate-early response in unparalleled detail, across cell types and stimuli. We exploit cap analysis of gene expression (CAGE) time series datasets to directly measure promoter activities over time. Using a novel analysis method for time series data we identify transcripts with expression patterns that closely resemble the dynamics of known immediate-early genes (IEGs) and this enables a comprehensive comparative study of these genes and their chromatin state. Surprisingly, these data suggest that the earliest transcriptional responses often involve promoters generating non-coding RNAs, many of which are produced in advance of canonical protein-coding IEGs. IEGs are known to be capable of induction without de novo protein synthesis. Consistent with this, we find that the response of both protein-coding and non-coding RNA IEGs can be explained by their transcriptionally poised, permissive chromatin state prior to stimulation. We also explore the function of non-coding RNAs in the attenuation of the immediate early response in a small RNA sequencing dataset matched to the CAGE data: We identify a novel set of microRNAs responsible for the attenuation of the IEG response in an estrogen receptor positive cancer cell line. Our computational statistical method is well suited to meta-analyses as there is no requirement for transcripts to pass thresholds for significant differential expression between time points, and it is agnostic to the number of time points per dataset. PMID:25885578

  19. Transcription factor p63 bookmarks and regulates dynamic enhancers during epidermal differentiation

    PubMed Central

    Kouwenhoven, Evelyn N; Oti, Martin; Niehues, Hanna; van Heeringen, Simon J; Schalkwijk, Joost; Stunnenberg, Hendrik G; van Bokhoven, Hans; Zhou, Huiqing

    2015-01-01

    The transcription factor p63 plays a pivotal role in keratinocyte proliferation and differentiation in the epidermis. However, how p63 regulates epidermal genes during differentiation is not yet clear. Using epigenome profiling of differentiating human primary epidermal keratinocytes, we characterized a catalog of dynamically regulated genes and p63-bound regulatory elements that are relevant for epithelial development and related diseases. p63-bound regulatory elements occur as single or clustered enhancers, and remarkably, only a subset is active as defined by the co-presence of the active enhancer mark histone modification H3K27ac in epidermal keratinocytes. We show that the dynamics of gene expression correlates with the activity of p63-bound enhancers rather than with p63 binding itself. The activity of p63-bound enhancers is likely determined by other transcription factors that cooperate with p63. Our data show that inactive p63-bound enhancers in epidermal keratinocytes may be active during the development of other epithelial-related structures such as limbs and suggest that p63 bookmarks genomic loci during the commitment of the epithelial lineage and regulates genes through temporal- and spatial-specific active enhancers. PMID:26034101

  20. Tracing the molecular basis of transcriptional dynamics in noisy data by using an experiment-based mathematical model.

    PubMed

    Rybakova, Katja N; Tomaszewska, Aleksandra; van Mourik, Simon; Blom, Joke; Westerhoff, Hans V; Carlberg, Carsten; Bruggeman, Frank J

    2015-01-01

    Changes in transcription factor levels, epigenetic status, splicing kinetics and mRNA degradation can each contribute to changes in the mRNA dynamics of a gene. We present a novel method to identify which of these processes is changed in cells in response to external signals or as a result of a diseased state. The method employs a mathematical model, for which the kinetics of gene regulation, splicing, elongation and mRNA degradation were estimated from experimental data of transcriptional dynamics. The time-dependent dynamics of several species of adipose differentiation-related protein (ADRP) mRNA were measured in response to ligand activation of the transcription factor peroxisome proliferator-activated receptor δ (PPARδ). We validated the method by monitoring the mRNA dynamics upon gene activation in the presence of a splicing inhibitor. Our mathematical model correctly identifies splicing as the inhibitor target, despite the noise in the data.

  1. Expression dynamics of the pea rbcS multigene family and organ distribution of the transcripts

    PubMed Central

    Fluhr, Robert; Moses, Phyllis; Morelli, Giorgio; Coruzzi, Gloria; Chua, Nam-Hai

    1986-01-01

    We have determined the nucleotide sequence of two members (rbcS-3A and -3C) of the pea nuclear gene family encoding the small subunit (rbcS) of ribulose-1,5-bisphosphate carboxylase. Both rbcS-3A and -3C are interrupted by two introns located at the same positions as those of the other three pea rbcS genes. Compared with the other pea rbcS genes the rbcS-3C gene has the most divergent 5'- and 3'-flanking sequences while the rbcS-3A gene has a larger and highly divergent intron 1. All five pea rbcS genes are conserved in their coding regions but show considerable sequence differences in their 3'-untranslated portion. The 3' sequence divergence of the rbcS genes has allowed us to use S1 nuclease mapping procedures to compare their expression levels in different organs and during light induction. All the rbcS genes are differentially expressed in various organs of the pea plants; moreover, specific rbcS transcripts are under-represented in seeds and petals. In leaves there is a 10-fold difference between the highest and lowest specific rbcS transcript levels. By quantitating the distribution of rbcS transcripts during light, phytochrome and blue light induction of immature (etiolated), and mature (green), pea leaves, we show that the genes are differentially activated during leaf development. ImagesFig. 3.Fig. 4.Fig. 5.Fig. 6.Fig. 7.Fig. 8. PMID:16453702

  2. Performance of the COBAS AMPLICOR HCV MONITOR Test, Version 2.0, an Automated Reverse Transcription-PCR Quantitative System for Hepatitis C Virus Load Determination

    PubMed Central

    Gerken, G.; Rothaar, T.; Rumi, M. G.; Soffredini, R.; Trippler, M.; Blunk, M. J.; Butcher, A.; Soviero, S.; Colucci, G.

    2000-01-01

    A clinical evaluation of an automated quantitative PCR assay, the COBAS AMPLICOR HCV MONITOR test, version 2.0 (v2.0), was carried out to assess the performance of this test in comparison with that of the previous, manual version, the AMPLICOR HCV MONITOR test, and with that of nested PCR. Serial dilutions of serum samples infected with genotype 1b, 2a, or 3, as well as synthetic RNA transcripts and serum samples derived from 87 patients with chronic hepatitis C and infected with genotype 1a, 1b, 2a, 2b, 3a, 3b, 4, or 5, were analyzed to determine the ability of the system to efficiently quantify various hepatitis C virus (HCV) genotypes. These experiments showed that the COBAS AMPLICOR HCV MONITOR test, v2.0, has mean intra-assay, interassay, and interoperator coefficients of variation that range from 22 to 34.5% and a 3-logarithm dynamic range, which spans from 103 to 106 copies/ml. Compared to the previous, manual version of the test, the COBAS AMPLICOR HCV MONITOR test, v2.0, showed an improved efficacy for all genotypes, especially genotypes 2, 3, and 4, whose estimated concentrations were on average 1 logarithm higher. When used to monitor patients under treatment, however, both versions showed the same patterns of viremia, indicating that the COBAS AMPLICOR HCV MONITOR test, v2.0, and the AMPLICOR HCV MONITOR test were equally effective at detecting relative viremia changes in serial samples. As expected, the automated test was less sensitive than nested PCR; among specimens from a cohort of patients treated with interferon, nested PCR identified three more viremic specimens, which probably contained very low concentrations of HCV RNA. PMID:10834978

  3. Generation of HIV-1 and Internal Control Transcripts as Standards for an In-House Quantitative Competitive RT-PCR Assay to Determine HIV-1 Viral Load

    PubMed Central

    Armas Cayarga, Anny; Perea Hernández, Yenitse; González González, Yaimé J.; Dueñas Carrera, Santiago; González Pérez, Idania; Robaina Álvarez, René

    2011-01-01

    Human immunodeficiency virus type-1 (HIV-1) viral load is useful for monitoring disease progression in HIV-infected individuals. We generated RNA standards of HIV-1 and internal control (IC) by in vitro transcription and evaluated its performance in a quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay. HIV-1 and IC standards were obtained at high RNA concentrations, without DNA contamination. When these transcripts were included as standards in a qRT-PCR assay, it was obtained a good accuracy (±0.5 log10 unit of the expected results) in the quantification of the HIV-1 RNA international standard and controls. The lower limit detection achieved using these standards was 511.0 IU/mL. A high correlation (r = 0.925) was obtained between the in-house qRT-PCR assay and the NucliSens easyQ HIV-1 test (bioMerieux) for HIV-1 RNA quantitation with clinical samples (N = 14). HIV-1 and IC RNA transcripts, generated in this study, proved to be useful as standards in an in-house qRT-PCR assay for determination of HIV-1 viral load. PMID:21766036

  4. Haplotype and quantitative transcript analyses of Portuguese breast/ovarian cancer families with the BRCA1 R71G founder mutation of Galician origin.

    PubMed

    Santos, Catarina; Peixoto, Ana; Rocha, Patrícia; Vega, Ana; Soares, Maria José; Cerveira, Nuno; Bizarro, Susana; Pinheiro, Manuela; Pereira, Deolinda; Rodrigues, Helena; Castro, Fernando; Henrique, Rui; Teixeira, Manuel R

    2009-01-01

    We investigated the functional effect of the missense variant c.211A>G (R71G) localized at position -2 of exon 5 donor splice site in the BRCA1 gene and evaluated whether Portuguese and Galician families with this mutation share a common ancestry. Three unrelated Portuguese breast/ovarian cancer families carrying this variant were studied through qualitative and quantitative transcript analyses. We also evaluated the presence of loss of heterozigosity and the histopathologic characteristics of the carcinomas in those families. Informative families (two from Portugal and one from Galicia) were genotyped for polymorphic microsatellite markers flanking BRCA1 to reconstruct haplotypes. Qualitative RNA analysis revealed the presence of two alternative transcripts both in carriers of the BRCA1 R71G variant and in controls. Semi-quantitative fragment analysis and real-time RT-PCR showed a significant increase of the transcript with an out of frame deletion of the last 22nt of exon 5 (BRCA1-Delta22ntex5) and a decrease of the full-length transcript (BRCA1-ex5FL) in patients carrying the R71G mutation as compared to controls, whereas no significant differences were found for the transcript with in frame skipping of exon 5 (BRCA1-Deltaex5). One haplotype was found to segregate in the two informative Portuguese families and in the Galician family. We demonstrate that disruption of alternative transcript ratios is the mechanism causing hereditary breast/ovarian cancer associated with the BRCA1 R71G mutation. Furthermore, our findings indicate a common ancestry of the Portuguese and Galician families sharing this mutation. PMID:19123044

  5. Construction and analysis of dynamic transcription factor regulatory networks in the progression of glioma.

    PubMed

    Li, Yongsheng; Shao, Tingting; Jiang, Chunjie; Bai, Jing; Wang, Zishan; Zhang, Jinwen; Zhang, Lili; Zhao, Zheng; Xu, Juan; Li, Xia

    2015-11-03

    The combinatorial cross-regulation of transcription factors (TFs) plays an important role in cellular identity and function; however, the dynamic regulation of TFs during glioma progression remains largely unknown. Here, we used the genome-wide expression of TFs to construct an extensive human TF network comprising interactions among 513 TFs and to analyse the dynamics of the TF-TF network during glioma progression. We found that the TF regulatory networks share a common architecture and that the topological structures are conserved. Strikingly, despite the conservation of the network architecture, TF regulatory networks are highly grade specific, and TF circuitry motifs are dynamically rewired during glioma progression. In addition, the most frequently observed structure in the grade-specific TF networks was the feedforward loop (FFL). We described applications that show how investigating the behaviour of FFLs in glioblastoma can reveal FFLs (such as RARG-NR1I2-CDX2) that are associated with prognosis. We constructed comprehensive TF-TF networks and systematically analysed the circuitry, dynamics, and topological principles of the networks during glioma progression, which will further enhance our understanding of the functions of TFs in glioma.

  6. Rapid dynamics of general transcription factor TFIIB binding during preinitiation complex assembly revealed by single-molecule analysis

    PubMed Central

    Zhang, Zhengjian; English, Brian P.; Grimm, Jonathan B.; Kazane, Stephanie A.; Hu, Wenxin; Tsai, Albert; Inouye, Carla; You, Changjiang; Piehler, Jacob; Schultz, Peter G.; Lavis, Luke D.; Revyakin, Andrey; Tjian, Robert

    2016-01-01

    Transcription of protein-encoding genes in eukaryotic cells requires the coordinated action of multiple general transcription factors (GTFs) and RNA polymerase II (Pol II). A “step-wise” preinitiation complex (PIC) assembly model has been suggested based on conventional ensemble biochemical measurements, in which protein factors bind stably to the promoter DNA sequentially to build a functional PIC. However, recent dynamic measurements in live cells suggest that transcription factors mostly interact with chromatin DNA rather transiently. To gain a clearer dynamic picture of PIC assembly, we established an integrated in vitro single-molecule transcription platform reconstituted from highly purified human transcription factors and complemented it by live-cell imaging. Here we performed real-time measurements of the hierarchal promoter-specific binding of TFIID, TFIIA, and TFIIB. Surprisingly, we found that while promoter binding of TFIID and TFIIA is stable, promoter binding by TFIIB is highly transient and dynamic (with an average residence time of 1.5 sec). Stable TFIIB–promoter association and progression beyond this apparent PIC assembly checkpoint control occurs only in the presence of Pol II–TFIIF. This transient-to-stable transition of TFIIB-binding dynamics has gone undetected previously and underscores the advantages of single-molecule assays for revealing the dynamic nature of complex biological reactions. PMID:27798851

  7. Dynamic Quantitative T1 Mapping in Orthotopic Brain Tumor Xenografts1

    PubMed Central

    Herrmann, Kelsey; Erokwu, Bernadette O.; Johansen, Mette L.; Basilion, James P.; Gulani, Vikas; Griswold, Mark A.; Flask, Chris A.; Brady-Kalnay, Susann M.

    2016-01-01

    Human brain tumors such as glioblastomas are typically detected using conventional, nonquantitative magnetic resonance imaging (MRI) techniques, such as T2-weighted and contrast enhanced T1-weighted MRI. In this manuscript, we tested whether dynamic quantitative T1 mapping by MRI can localize orthotopic glioma tumors in an objective manner. Quantitative T1 mapping was performed by MRI over multiple time points using the conventional contrast agent Optimark. We compared signal differences to determine the gadolinium concentration in tissues over time. The T1 parametric maps made it easy to identify the regions of contrast enhancement and thus tumor location. Doubling the typical human dose of contrast agent resulted in a clearer demarcation of these tumors. Therefore, T1 mapping of brain tumors is gadolinium dose dependent and improves detection of tumors by MRI. The use of T1 maps provides a quantitative means to evaluate tumor detection by gadolinium-based contrast agents over time. This dynamic quantitative T1 mapping technique will also enable future quantitative evaluation of various targeted MRI contrast agents. PMID:27084431

  8. Dynamic phase imaging of host cells attacked by Vibrio vulnificus using quantitative phase microscopy

    NASA Astrophysics Data System (ADS)

    Lee, Seungrag; Yang, Wenzhong; Lee, Ji Yong; Cha, Mi Hye; Kim, Young Ran; Kim, Dug Young

    2010-02-01

    We present the real time quantitative analysis of Vibrio vulnificus-infected host cells using high stability quantitative phase microscopy (HSQPM). It provides the ability to retrieve the phase or optical path length distribution over the cell from a single interferogram image, which has been measured with nanometer path length sensitivity for long periods of time. We have applied HSQPM to study dynamic cell morphologic changes and to quantify noninvasively cell volumes of rat basophilic leukemia RBL-2H3 cells infected with pathogenic bacteria V. vulnificus strains, wild type (MO6-24/O) and RTX toxin mutant (CMM770). During the process of V. vulnificus wild type infection to RBL-2H3 cells, the dynamic changes of quantitative phase images, cell volumes and areas were observed in real time using HSQPM. In contrast, the dramatic changes were not detected in RBL-2H3 cells infected with RTX toxin mutant. The results showed the good correlation between HSQPM analysis and biochemical assays such as lactate dehydrogenase (LDH) assay and β-hexosaminidase release assay. We suggest that HSQPM is useful real time quantitative method to study the dynamic process of host cells infected with pathogen in a noninvasive manner.

  9. Application of Long-Range and Binding Reverse Transcription-Quantitative PCR To Indicate the Viral Integrities of Noroviruses

    PubMed Central

    De Keuckelaere, Ann; Uyttendaele, Mieke

    2014-01-01

    This study intends to establish and apply methods evaluating both viral capsid and genome integrities of human noroviruses (NoVs), which thus far remain nonculturable. Murine norovirus 1 (MNV-1) and human NoV GII.4 in phosphate-buffered saline suspensions were treated with heat, UV light, or ethanol and detected by reverse transcription-quantitative PCR (RT-qPCR), long-range RT-qPCR, binding RT-qPCR, and binding long-range RT-qPCR. For MNV-1 heated at 60°C for 2 and 30 min, limited reductions of genomic copies (<0.3-log) were obtained by RT-qPCR and long-range RT-qPCR, while the cell-binding pretreatments obtained higher reductions (>1.89-log reduction after 60°C for 30 min by binding long-range RT-qPCR). The human NoV GII.4 was found to be more heat resistant than MNV-1. For both MNV-1 and human NoV GII.4 after UV treatments of 20 and 200 mJ/cm2, no significant difference (P > 0.05) was observed between the dose-dependent reductions obtained by the four detection methodologies. Treatment of 70% ethanol for 1 min was shown to be more effective for inactivation of both MNV-1 and human NoV GII.4 than the heat and UV treatments used in this study. Subsequently, eight raspberry and four shellfish samples previously shown to be naturally contaminated with human NoVs by RT-qPCR (GI and GII; thus, 24 RT-qPCR signals) were subjected to comparison by this method. RT-qPCR, long-range RT-qPCR, binding RT-qPCR, and binding long-range RT-qPCR detected 20/24, 14/24, 24/24, and 23/24 positive signals, respectively, indicating the abundant presence of intact NoV particles. PMID:25107982

  10. A simple analytical and experimental procedure for selection of reference genes for reverse-transcription quantitative PCR normalization data.

    PubMed

    Manjarin, R; Trottier, N L; Weber, P S; Liesman, J S; Taylor, N P; Steibel, J P

    2011-10-01

    Variation in cellular activity in a tissue induces changes in RNA concentration, which affects the validity of gene mRNA abundance analyzed by reverse transcription quantitative PCR (RT-qPCR). A common way of accounting for such variation consists of the use of reference genes for normalization. Programs such as geNorm may be used to select suitable reference genes, although a large set of genes that are not co-regulated must be analyzed to obtain accurate results. The objective of this study was to propose an alternative experimental and analytical protocol to assess the invariance of reference genes in porcine mammary tissue using mammary RNA and DNA concentrations as correction factors. Mammary glands were biopsied from 4 sows on d 110 of gestation (prepartum), on d 5 (early) and 17 (peak) of lactation, and on d 5 after weaning (postweaning). Relative expression of 7 potential reference genes, API5, MRPL39, VAPB, ACTB, GAPDH, RPS23, and MTG1, and one candidate gene, SLC7A1, was quantified by RT-qPCR using a relative standard curve approach. Variation in gene expression levels, measured as cycles to threshold at each stage of mammary physiological activity, was tested using a linear mixed model fitting RNA and DNA concentrations as covariates. Results were compared with those obtained with geNorm analysis, and genes selected by each method were used to normalize SLC7A1. Quantified relative mRNA abundance of GAPDH and MRPL39 remained unchanged across stages of mammary physiological activity after accounting for changes in tissue RNA and DNA concentration. In contrast, geNorm analysis selected MTG1, MRPL39, and VAPB as the best reference genes. However, when target gene SLC7A1 was normalized with genes selected either based on our proposed protocol or by geNorm, fold changes in mRNA abundance did not differ. In conclusion, the proposed analytical protocol assesses expression invariance of potential reference genes by accounting for variation in tissue RNA and DNA

  11. Ammonia quantitative analysis model based on miniaturized Al ionization gas sensor and non-linear bistable dynamic model

    PubMed Central

    Ma, Rongfei

    2015-01-01

    In this paper, ammonia quantitative analysis based on miniaturized Al ionization gas sensor and non-linear bistable dynamic model was proposed. Al plate anodic gas-ionization sensor was used to obtain the current-voltage (I-V) data. Measurement data was processed by non-linear bistable dynamics model. Results showed that the proposed method quantitatively determined ammonia concentrations. PMID:25975362

  12. Transcript and metabolite analysis in Trincadeira cultivar reveals novel information regarding the dynamics of grape ripening

    PubMed Central

    2011-01-01

    Background Grapes (Vitis vinifera L.) are economically the most important fruit crop worldwide. However, the complexity of molecular and biochemical events that lead to the onset of ripening of nonclimacteric fruits is not fully understood which is further complicated in grapes due to seasonal and cultivar specific variation. The Portuguese wine variety Trincadeira gives rise to high quality wines but presents extremely irregular berry ripening among seasons probably due to high susceptibility to abiotic and biotic stresses. Results Ripening of Trincadeira grapes was studied taking into account the transcriptional and metabolic profilings complemented with biochemical data. The mRNA expression profiles of four time points spanning developmental stages from pea size green berries, through véraison and mature berries (EL 32, EL 34, EL 35 and EL 36) and in two seasons (2007 and 2008) were compared using the Affymetrix GrapeGen® genome array containing 23096 probesets corresponding to 18726 unique sequences. Over 50% of these probesets were significantly differentially expressed (1.5 fold) between at least two developmental stages. A common set of modulated transcripts corresponding to 5877 unigenes indicates the activation of common pathways between years despite the irregular development of Trincadeira grapes. These unigenes were assigned to the functional categories of "metabolism", "development", "cellular process", "diverse/miscellanenous functions", "regulation overview", "response to stimulus, stress", "signaling", "transport overview", "xenoprotein, transposable element" and "unknown". Quantitative RT-PCR validated microarrays results being carried out for eight selected genes and five developmental stages (EL 32, EL 34, EL 35, EL 36 and EL 38). Metabolic profiling using 1H NMR spectroscopy associated to two-dimensional techniques showed the importance of metabolites related to oxidative stress response, amino acid and sugar metabolism as well as secondary

  13. Identification and Evaluation of Suitable Reference Genes for Gene Expression Studies in the Whitefly Bemisia tabaci (Asia I) by Reverse Transcription Quantitative Real-Time PCR

    PubMed Central

    Collins, Carl; Patel, Mitulkumar V.; Colvin, John; Bailey, David; Seal, Susan

    2014-01-01

    This study presents a reliable method for performing reverse transcription quantitative real-time PCR (RT-qPCR) to measure gene expression in the whitefly Bemisia tabaci (Asia I) (Gennadius) (Hemiptera: Aleyrodidae), utilising suitable reference genes for data normalisation. We identified orthologs of commonly used reference genes (actin (ACT), cyclophilin 1 (CYP1), elongation factor 1α (EF1A), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), ribosomal protein L13a (RPL13A), and α-tubulin (TUB1A)), measured the levels of their transcripts by RT-qPCR during development and in response to thermal stress, and evaluated their suitability as endogenous controls using geNorm, BestKeeper, and NormFinder programs. Overall, TUB1A, RPL13A, and CYP1 were the most stable reference genes during B. tabaci development, and TUB1A, GAPDH, and RPL13A were the most stable reference genes in the context of thermal stress. An analysis of the effects of reference gene choice on the transcript profile of a developmentally-regulated gene encoding vitellogenin demonstrated the importance of selecting the correct endogenous controls for RT-qPCR studies. We propose the use of TUB1A, RPL13A, and CYP1 as endogenous controls for transcript profiling studies of B. tabaci development, whereas the combination of TUB1A, GAPDH, and RPL13A should be employed for studies into thermal stress. The data presented here will assist future transcript profiling studies in whiteflies. PMID:25373210

  14. Identification and evaluation of suitable reference genes for gene expression studies in the whitefly Bemisia tabaci (Asia I) by reverse transcription quantitative realtime PCR.

    PubMed

    Collins, Carl; Patel, Mitulkumar V; Colvin, John; Bailey, David; Seal, Susan

    2014-05-02

    This study presents a reliable method for performing reverse transcription quantitative realtime PCR (RT-qPCR) to measure gene expression in the whitefly Bemisia tabaci (Asia I) (Gennadius) (Hemiptera: Aleyrodidae), utilising suitable reference genes for data normalisation. We identified orthologs of commonly used reference genes (actin (ACT), cyclophilin 1 (CYP1), elongation factor 1α (EF1A), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), ribosomal protein L13a (RPL13A), and α-tubulin (TUB1A)), measured the levels of their transcripts by RT-qPCR during development and in response to thermal stress, and evaluated their suitability as endogenous controls using geNorm, BestKeeper, and NormFinder programs. Overall, TUB1A, RPL13A, and CYP1 were the most stable reference genes during B. tabaci development, and TUB1A, GAPDH, and RPL13A were the most stable reference genes in the context of thermal stress. An analysis of the effects of reference gene choice on the transcript profile of a developmentally-regulated gene encoding vitellogenin demonstrated the importance of selecting the correct endogenous controls for RT-qPCR studies. We propose the use of TUB1A, RPL13A, and CYP1 as endogenous controls for transcript profiling studies of B. tabaci development, whereas the combination of TUB1A, GAPDH, and RPL13A should be employed for studies into thermal stress. The data pre- sented here will assist future transcript profiling studies in whiteflies.

  15. Quantitative Decomposition of Dynamics of Mathematical Cell Models: Method and Application to Ventricular Myocyte Models.

    PubMed

    Shimayoshi, Takao; Cha, Chae Young; Amano, Akira

    2015-01-01

    Mathematical cell models are effective tools to understand cellular physiological functions precisely. For detailed analysis of model dynamics in order to investigate how much each component affects cellular behaviour, mathematical approaches are essential. This article presents a numerical analysis technique, which is applicable to any complicated cell model formulated as a system of ordinary differential equations, to quantitatively evaluate contributions of respective model components to the model dynamics in the intact situation. The present technique employs a novel mathematical index for decomposed dynamics with respect to each differential variable, along with a concept named instantaneous equilibrium point, which represents the trend of a model variable at some instant. This article also illustrates applications of the method to comprehensive myocardial cell models for analysing insights into the mechanisms of action potential generation and calcium transient. The analysis results exhibit quantitative contributions of individual channel gating mechanisms and ion exchanger activities to membrane repolarization and of calcium fluxes and buffers to raising and descending of the cytosolic calcium level. These analyses quantitatively explicate principle of the model, which leads to a better understanding of cellular dynamics. PMID:26091413

  16. Quantitative Decomposition of Dynamics of Mathematical Cell Models: Method and Application to Ventricular Myocyte Models

    PubMed Central

    Shimayoshi, Takao; Cha, Chae Young; Amano, Akira

    2015-01-01

    Mathematical cell models are effective tools to understand cellular physiological functions precisely. For detailed analysis of model dynamics in order to investigate how much each component affects cellular behaviour, mathematical approaches are essential. This article presents a numerical analysis technique, which is applicable to any complicated cell model formulated as a system of ordinary differential equations, to quantitatively evaluate contributions of respective model components to the model dynamics in the intact situation. The present technique employs a novel mathematical index for decomposed dynamics with respect to each differential variable, along with a concept named instantaneous equilibrium point, which represents the trend of a model variable at some instant. This article also illustrates applications of the method to comprehensive myocardial cell models for analysing insights into the mechanisms of action potential generation and calcium transient. The analysis results exhibit quantitative contributions of individual channel gating mechanisms and ion exchanger activities to membrane repolarization and of calcium fluxes and buffers to raising and descending of the cytosolic calcium level. These analyses quantitatively explicate principle of the model, which leads to a better understanding of cellular dynamics. PMID:26091413

  17. Counteracting H3K4 methylation modulators Set1 and Jhd2 co-regulate chromatin dynamics and gene transcription

    PubMed Central

    Ramakrishnan, Saravanan; Pokhrel, Srijana; Palani, Sowmiya; Pflueger, Christian; Parnell, Timothy J.; Cairns, Bradley R.; Bhaskara, Srividya; Chandrasekharan, Mahesh B.

    2016-01-01

    Histone H3K4 methylation is connected to gene transcription from yeast to humans, but its mechanistic roles in transcription and chromatin dynamics remain poorly understood. We investigated the functions for Set1 and Jhd2, the sole H3K4 methyltransferase and H3K4 demethylase, respectively, in S. cerevisiae. Here, we show that Set1 and Jhd2 predominantly co-regulate genome-wide transcription. We find combined activities of Set1 and Jhd2 via H3K4 methylation contribute to positive or negative transcriptional regulation. Providing mechanistic insights, our data reveal that Set1 and Jhd2 together control nucleosomal turnover and occupancy during transcriptional co-regulation. Moreover, we find a genome-wide co-regulation of chromatin structure by Set1 and Jhd2 at different groups of transcriptionally active or inactive genes and at different regions within yeast genes. Overall, our study puts forth a model wherein combined actions of Set1 and Jhd2 via modulating H3K4 methylation−demethylation together control chromatin dynamics during various facets of transcriptional regulation. PMID:27325136

  18. Analysis of dynamic changes in retinoid-induced transcription and epigenetic profiles of murine Hox clusters in ES cells

    PubMed Central

    De Kumar, Bony; Parrish, Mark E.; Slaughter, Brian D.; Unruh, Jay R.; Gogol, Madelaine; Seidel, Christopher; Paulson, Ariel; Li, Hua; Gaudenz, Karin; Peak, Allison; McDowell, William; Fleharty, Brian; Ahn, Youngwook; Lin, Chengqi; Smith, Edwin; Shilatifard, Ali; Krumlauf, Robb

    2015-01-01

    The clustered Hox genes, which are highly conserved across metazoans, encode homeodomain-containing transcription factors that provide a blueprint for segmental identity along the body axis. Recent studies have underscored that in addition to encoding Hox genes, the homeotic clusters contain key noncoding RNA genes that play a central role in development. In this study, we have taken advantage of genome-wide approaches to provide a detailed analysis of retinoic acid (RA)-induced transcriptional and epigenetic changes within the homeotic clusters of mouse embryonic stem cells. Although there is a general colinear response, our analyses suggest a lack of strict colinearity for several genes in the HoxA and HoxB clusters. We have identified transcribed novel noncoding RNAs (ncRNAs) and their cis-regulatory elements that function in response to RA and demonstrated that the expression of these ncRNAs from both strands represent some of the most rapidly induced transcripts in ES cells. Finally, we have provided dynamic analyses of chromatin modifications for the coding and noncoding genes expressed upon activation and suggest that active transcription can occur in the presence of chromatin modifications and machineries associated with repressed transcription state over the clusters. Overall, our data provide a resource for a better understanding of the dynamic nature of the coding and noncoding transcripts and their associated chromatin marks in the regulation of homeotic gene transcription during development. PMID:26025802

  19. Analysis of specific RNA in cultured cells through quantitative integration of q-PCR and N-SIM single cell FISH images: Application to hormonal stimulation of StAR transcription.

    PubMed

    Lee, Jinwoo; Foong, Yee Hoon; Musaitif, Ibrahim; Tong, Tiegang; Jefcoate, Colin

    2016-07-01

    The steroidogenic acute regulatory protein (StAR) has been proposed to serve as the switch that can turn on/off steroidogenesis. We investigated the events that facilitate dynamic StAR transcription in response to cAMP stimulation in MA-10 Leydig cells, focusing on splicing anomalies at StAR gene loci. We used 3' reverse primers in a single reaction to respectively quantify StAR primary (p-RNA), spliced (sp-RNA/mRNA), and extended 3' untranslated region (UTR) transcripts, which were quantitatively imaged by high-resolution fluorescence in situ hybridization (FISH). This approach delivers spatio-temporal resolution of initiation and splicing at single StAR loci, and transfers individual mRNA molecules to cytoplasmic sites. Gene expression was biphasic, initially showing slow splicing, transitioning to concerted splicing. The alternative 3.5-kb mRNAs were distinguished through the use of extended 3'UTR probes, which exhibited distinctive mitochondrial distribution. Combining quantitative PCR and FISH enables imaging of localization of RNA expression and analysis of RNA processing rates.

  20. Developmental roles of 21 Drosophila transcription factors are determined by quantitative differences in binding to an overlapping set of thousands of genomic regions

    SciTech Connect

    MacArthur, Stewart; Li, Xiao-Yong; Li, Jingyi; Brown, James B.; Chu, Hou Cheng; Zeng, Lucy; Grondona, Brandi P.; Hechmer, Aaron; Simirenko, Lisa; Keranen, Soile V.E.; Knowles, David W.; Stapleton, Mark; Bickel, Peter; Biggin, Mark D.; Eisen, Michael B.

    2009-05-15

    BACKGROUND: We previously established that six sequence-specific transcription factors that initiate anterior/posterior patterning in Drosophila bind to overlapping sets of thousands of genomic regions in blastoderm embryos. While regions bound at high levels include known and probable functional targets, more poorly bound regions are preferentially associated with housekeeping genes and/or genes not transcribed in the blastoderm, and are frequently found in protein coding sequences or in less conserved non-coding DNA, suggesting that many are likely non-functional. RESULTS: Here we show that an additional 15 transcription factors that regulate other aspects of embryo patterning show a similar quantitative continuum of function and binding to thousands of genomic regions in vivo. Collectively, the 21 regulators show a surprisingly high overlap in the regions they bind given that they belong to 11 DNA binding domain families, specify distinct developmental fates, and can act via different cis-regulatory modules. We demonstrate, however, that quantitative differences in relative levels of binding to shared targets correlate with the known biological and transcriptional regulatory specificities of these factors. CONCLUSIONS: It is likely that the overlap in binding of biochemically and functionally unrelated transcription factors arises from the high concentrations of these proteins in nuclei, which, coupled with their broad DNA binding specificities, directs them to regions of open chromatin. We suggest that most animal transcription factors will be found to show a similar broad overlapping pattern of binding in vivo, with specificity achieved by modulating the amount, rather than the identity, of bound factor.

  1. Integrating Single-Molecule Experiments and Discrete Stochastic Models to Understand Heterogeneous Gene Transcription Dynamics

    PubMed Central

    Munsky, Brian; Fox, Zachary; Neuert, Gregor

    2015-01-01

    The production and degradation of RNA transcripts is inherently subject to biological noise that arises from small gene copy numbers in individual cells. As a result, cellular RNA levels can exhibit large fluctuations over time and from one cell to the next. This article presents a range of precise single-molecule experimental techniques, based upon RNA fluorescence in situ hybridization, which can be used to measure the fluctuations of RNA at the single-cell level. A class of models for gene activation and deactivation is postulated in order to capture complex stochastic effects of chromatin modifications or transcription factor interactions. A computational tool, known the Finite State Projection approach, is introduced to accurately and efficiently analyze these models in order to predict how probability distributions of RNA change over time in response to changing environmental conditions. These single-molecule experiments, discrete stochastic models, and computational analyses are systematically integrated to identify models of gene regulation dynamics. To illustrate the power and generality of our integrated experimental and computational approach, we explore cases that include different models for three different RNA types (sRNA, mRNA and nascent RNA), three different experimental techniques and three different biological species (bacteria, yeast and human cells). PMID:26079925

  2. Subunit dynamics and nucleotide-dependent asymmetry of an AAA(+) transcription complex.

    PubMed

    Zhang, Nan; Gordiyenko, Yuliya; Joly, Nicolas; Lawton, Edward; Robinson, Carol V; Buck, Martin

    2014-01-01

    Bacterial enhancer binding proteins (bEBPs) are transcription activators that belong to the AAA(+) protein family. They form higher-order self-assemblies to regulate transcription initiation at stress response and pathogenic promoters. The precise mechanism by which these ATPases utilize ATP binding and hydrolysis energy to remodel their substrates remains unclear. Here we employed mass spectrometry of intact complexes to investigate subunit dynamics and nucleotide occupancy of the AAA(+) domain of one well-studied bEBP in complex with its substrate, the σ(54) subunit of RNA polymerase. Our results demonstrate that the free AAA(+) domain undergoes significant changes in oligomeric states and nucleotide occupancy upon σ(54) binding. Such changes likely correlate with one transition state of ATP and are associated with an open spiral ring formation that is vital for asymmetric subunit function and interface communication. We confirmed that the asymmetric subunit functionality persists for open promoter complex formation using single-chain forms of bEBP lacking the full complement of intact ATP hydrolysis sites. Outcomes reconcile low- and high-resolution structures and yield a partial sequential ATP hydrolysis model for bEBPs. PMID:24055699

  3. Comparative transcript profiling by SuperSAGE identifies novel candidate genes for controlling potato quantitative resistance to late blight not compromised by late maturity

    PubMed Central

    Draffehn, Astrid M.; Li, Li; Krezdorn, Nicolas; Ding, Jia; Lübeck, Jens; Strahwald, Josef; Muktar, Meki S.; Walkemeier, Birgit; Rotter, Björn; Gebhardt, Christiane

    2013-01-01

    Resistance to pathogens is essential for survival of wild and cultivated plants. Pathogen susceptibility causes major losses of crop yield and quality. Durable field resistance combined with high yield and other superior agronomic characters are therefore, important objectives in every crop breeding program. Precision and efficacy of resistance breeding can be enhanced by molecular diagnostic tools, which result from knowledge of the molecular basis of resistance and susceptibility. Breeding uses resistance conferred by single R genes and polygenic quantitative resistance. The latter is partial but considered more durable. Molecular mechanisms of plant pathogen interactions are elucidated mainly in experimental systems involving single R genes, whereas most genes important for quantitative resistance in crops like potato are unknown. Quantitative resistance of potato to Phytophthora infestans causing late blight is often compromised by late plant maturity, a negative agronomic character. Our objective was to identify candidate genes for quantitative resistance to late blight not compromised by late plant maturity. We used diagnostic DNA-markers to select plants with different field levels of maturity corrected resistance (MCR) to late blight and compared their leaf transcriptomes before and after infection with P. infestans using SuperSAGE (serial analysis of gene expression) technology and next generation sequencing. We identified 2034 transcripts up or down regulated upon infection, including a homolog of the kiwi fruit allergen kiwellin. 806 transcripts showed differential expression between groups of genotypes with contrasting MCR levels. The observed expression patterns suggest that MCR is in part controlled by differential transcript levels in uninfected plants. Functional annotation suggests that, besides biotic and abiotic stress responses, general cellular processes such as photosynthesis, protein biosynthesis, and degradation play a role in MCR. PMID

  4. How motif environment influences transcription factor search dynamics: Finding a needle in a haystack.

    PubMed

    Dror, Iris; Rohs, Remo; Mandel-Gutfreund, Yael

    2016-07-01

    Transcription factors (TFs) have to find their binding sites, which are distributed throughout the genome. Facilitated diffusion is currently the most widely accepted model for this search process. Based on this model the TF alternates between one-dimensional sliding along the DNA, and three-dimensional bulk diffusion. In this view, the non-specific associations between the proteins and the DNA play a major role in the search dynamics. However, little is known about how the DNA properties around the motif contribute to the search. Accumulating evidence showing that TF binding sites are embedded within a unique environment, specific to each TF, leads to the hypothesis that the search process is facilitated by favorable DNA features that help to improve the search efficiency. Here, we review the field and present the hypothesis that TF-DNA recognition is dictated not only by the motif, but is also influenced by the environment in which the motif resides. PMID:27192961

  5. How motif environment influences transcription factor search dynamics: Finding a needle in a haystack.

    PubMed

    Dror, Iris; Rohs, Remo; Mandel-Gutfreund, Yael

    2016-07-01

    Transcription factors (TFs) have to find their binding sites, which are distributed throughout the genome. Facilitated diffusion is currently the most widely accepted model for this search process. Based on this model the TF alternates between one-dimensional sliding along the DNA, and three-dimensional bulk diffusion. In this view, the non-specific associations between the proteins and the DNA play a major role in the search dynamics. However, little is known about how the DNA properties around the motif contribute to the search. Accumulating evidence showing that TF binding sites are embedded within a unique environment, specific to each TF, leads to the hypothesis that the search process is facilitated by favorable DNA features that help to improve the search efficiency. Here, we review the field and present the hypothesis that TF-DNA recognition is dictated not only by the motif, but is also influenced by the environment in which the motif resides.

  6. A quantitative dynamic systems model of health-related quality of life among older adults

    PubMed Central

    Roppolo, Mattia; Kunnen, E Saskia; van Geert, Paul L; Mulasso, Anna; Rabaglietti, Emanuela

    2015-01-01

    Health-related quality of life (HRQOL) is a person-centered concept. The analysis of HRQOL is highly relevant in the aged population, which is generally suffering from health decline. Starting from a conceptual dynamic systems model that describes the development of HRQOL in individuals over time, this study aims to develop and test a quantitative dynamic systems model, in order to reveal the possible dynamic trends of HRQOL among older adults. The model is tested in different ways: first, with a calibration procedure to test whether the model produces theoretically plausible results, and second, with a preliminary validation procedure using empirical data of 194 older adults. This first validation tested the prediction that given a particular starting point (first empirical data point), the model will generate dynamic trajectories that lead to the observed endpoint (second empirical data point). The analyses reveal that the quantitative model produces theoretically plausible trajectories, thus providing support for the calibration procedure. Furthermore, the analyses of validation show a good fit between empirical and simulated data. In fact, no differences were found in the comparison between empirical and simulated final data for the same subgroup of participants, whereas the comparison between different subgroups of people resulted in significant differences. These data provide an initial basis of evidence for the dynamic nature of HRQOL during the aging process. Therefore, these data may give new theoretical and applied insights into the study of HRQOL and its development with time in the aging population. PMID:26604722

  7. A quantitative dynamic systems model of health-related quality of life among older adults.

    PubMed

    Roppolo, Mattia; Kunnen, E Saskia; van Geert, Paul L; Mulasso, Anna; Rabaglietti, Emanuela

    2015-01-01

    Health-related quality of life (HRQOL) is a person-centered concept. The analysis of HRQOL is highly relevant in the aged population, which is generally suffering from health decline. Starting from a conceptual dynamic systems model that describes the development of HRQOL in individuals over time, this study aims to develop and test a quantitative dynamic systems model, in order to reveal the possible dynamic trends of HRQOL among older adults. The model is tested in different ways: first, with a calibration procedure to test whether the model produces theoretically plausible results, and second, with a preliminary validation procedure using empirical data of 194 older adults. This first validation tested the prediction that given a particular starting point (first empirical data point), the model will generate dynamic trajectories that lead to the observed endpoint (second empirical data point). The analyses reveal that the quantitative model produces theoretically plausible trajectories, thus providing support for the calibration procedure. Furthermore, the analyses of validation show a good fit between empirical and simulated data. In fact, no differences were found in the comparison between empirical and simulated final data for the same subgroup of participants, whereas the comparison between different subgroups of people resulted in significant differences. These data provide an initial basis of evidence for the dynamic nature of HRQOL during the aging process. Therefore, these data may give new theoretical and applied insights into the study of HRQOL and its development with time in the aging population. PMID:26604722

  8. Dynamic transcription factor networks in epithelial-mesenchymal transition in breast cancer models.

    PubMed

    Siletz, Anaar; Schnabel, Michael; Kniazeva, Ekaterina; Schumacher, Andrew J; Shin, Seungjin; Jeruss, Jacqueline S; Shea, Lonnie D

    2013-01-01

    The epithelial-mesenchymal transition (EMT) is a complex change in cell differentiation that allows breast carcinoma cells to acquire invasive properties. EMT involves a cascade of regulatory changes that destabilize the epithelial phenotype and allow mesenchymal features to manifest. As transcription factors (TFs) are upstream effectors of the genome-wide expression changes that result in phenotypic change, understanding the sequential changes in TF activity during EMT provides rich information on the mechanism of this process. Because molecular interactions will vary as cells progress from an epithelial to a mesenchymal differentiation program, dynamic networks are needed to capture the changing context of molecular processes. In this study we applied an emerging high-throughput, dynamic TF activity array to define TF activity network changes in three cell-based models of EMT in breast cancer based on HMLE Twist ER and MCF-7 mammary epithelial cells. The TF array distinguished conserved from model-specific TF activity changes in the three models. Time-dependent data was used to identify pairs of TF activities with significant positive or negative correlation, indicative of interdependent TF activity throughout the six-day study period. Dynamic TF activity patterns were clustered into groups of TFs that change along a time course of gene expression changes and acquisition of invasive capacity. Time-dependent TF activity data was combined with prior knowledge of TF interactions to construct dynamic models of TF activity networks as epithelial cells acquire invasive characteristics. These analyses show EMT from a unique and targetable vantage and may ultimately contribute to diagnosis and therapy.

  9. A mass spectrometry-based method for comprehensive quantitative determination of post-transcriptional RNA modifications: the complete chemical structure of Schizosaccharomyces pombe ribosomal RNAs

    PubMed Central

    Taoka, Masato; Nobe, Yuko; Hori, Masayuki; Takeuchi, Aiko; Masaki, Shunpei; Yamauchi, Yoshio; Nakayama, Hiroshi; Takahashi, Nobuhiro; Isobe, Toshiaki

    2015-01-01

    We present a liquid chromatography–mass spectrometry (LC-MS)-based method for comprehensive quantitative identification of post-transcriptional modifications (PTMs) of RNA. We incorporated an in vitro-transcribed, heavy isotope-labeled reference RNA into a sample RNA solution, digested the mixture with a number of RNases and detected the post-transcriptionally modified oligonucleotides quantitatively based on shifts in retention time and the MS signal in subsequent LC-MS. This allowed the determination and quantitation of all PTMs in Schizosaccharomyces pombe ribosomal (r)RNAs and generated the first complete PTM maps of eukaryotic rRNAs at single-nucleotide resolution. There were 122 modified sites, most of which appear to locate at the interface of ribosomal subunits where translation takes place. We also identified PTMs at specific locations in rRNAs that were altered in response to growth conditions of yeast cells, suggesting that the cells coordinately regulate the modification levels of RNA. PMID:26013808

  10. New insights into the Saccharomyces cerevisiae fermentation switch: Dynamic transcriptional response to anaerobicity and glucose-excess

    PubMed Central

    van den Brink, Joost; Daran-Lapujade, Pascale; Pronk, Jack T; de Winde, Johannes H

    2008-01-01

    Background The capacity of respiring cultures of Saccharomyces cerevisiae to immediately switch to fast alcoholic fermentation upon a transfer to anaerobic sugar-excess conditions is a key characteristic of Saccharomyces cerevisiae in many of its industrial applications. This transition was studied by exposing aerobic glucose-limited chemostat cultures grown at a low specific growth rate to two simultaneous perturbations: oxygen depletion and relief of glucose limitation. Results The shift towards fully fermentative conditions caused a massive transcriptional reprogramming, where one third of all genes within the genome were transcribed differentially. The changes in transcript levels were mostly driven by relief from glucose-limitation. After an initial strong response to the addition of glucose, the expression profile of most transcriptionally regulated genes displayed a clear switch at 30 minutes. In this respect, a striking difference was observed between the transcript profiles of genes encoding ribosomal proteins and those encoding ribosomal biogenesis components. Not all regulated genes responded with this binary profile. A group of 87 genes showed a delayed and steady increase in expression that specifically responded to anaerobiosis. Conclusion Our study demonstrated that, despite the complexity of this multiple-input perturbation, the transcriptional responses could be categorized and biologically interpreted. By comparing this study with public datasets representing dynamic and steady conditions, 14 up-regulated and 11 down-regulated genes were determined to be anaerobic specific. Therefore, these can be seen as true "signature" transcripts for anaerobicity under dynamic as well as under steady state conditions. PMID:18304306

  11. Spatial Organization and Dynamics of Transcription Elongation and Pre-mRNA Processing in Live Cells.

    PubMed

    Sánchez-Álvarez, Miguel; Sánchez-Hernández, Noemí; Suñé, Carlos

    2011-01-01

    During the last 30 years, systematic biochemical and functional studies have significantly expanded our knowledge of the transcriptional molecular components and the pre-mRNA processing machinery of the cell. However, our current understanding of how these functions take place spatiotemporally within the highly compartmentalized eukaryotic nucleus remains limited. Moreover, it is increasingly clear that "the whole is more than the sum of its parts" and that an understanding of the dynamic coregulation of genes is essential for fully characterizing complex biological phenomena and underlying diseases. Recent technological advances in light microscopy in addition to novel cell and molecular biology approaches have led to the development of new tools, which are being used to address these questions and may contribute to achieving an integrated and global understanding of how the genome works at a cellular level. Here, we review major hallmarks and novel insights in RNA polymerase II activity and pre-mRNA processing in the context of nuclear organization, as well as new concepts and challenges arising from our ability to gather extensive dynamic information at the single-cell resolution.

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

    PubMed

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

    2016-04-01

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

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

    NASA Technical Reports Server (NTRS)

    Hess, R. A.

    1994-01-01

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

  14. Quantitative sampling of conformational heterogeneity of a DNA hairpin using molecular dynamics simulations and ultrafast fluorescence spectroscopy

    PubMed Central

    Voltz, Karine; Léonard, Jérémie; Touceda, Patricia Tourón; Conyard, Jamie; Chaker, Ziyad; Dejaegere, Annick; Godet, Julien; Mély, Yves; Haacke, Stefan; Stote, Roland H.

    2016-01-01

    Molecular dynamics (MD) simulations and time resolved fluorescence (TRF) spectroscopy were combined to quantitatively describe the conformational landscape of the DNA primary binding sequence (PBS) of the HIV-1 genome, a short hairpin targeted by retroviral nucleocapsid proteins implicated in the viral reverse transcription. Three 2-aminopurine (2AP) labeled PBS constructs were studied. For each variant, the complete distribution of fluorescence lifetimes covering 5 orders of magnitude in timescale was measured and the populations of conformers experimentally observed to undergo static quenching were quantified. A binary quantification permitted the comparison of populations from experimental lifetime amplitudes to populations of aromatically stacked 2AP conformers obtained from simulation. Both populations agreed well, supporting the general assumption that quenching of 2AP fluorescence results from pi-stacking interactions with neighboring nucleobases and demonstrating the success of the proposed methodology for the combined analysis of TRF and MD data. Cluster analysis of the latter further identified predominant conformations that were consistent with the fluorescence decay times and amplitudes, providing a structure-based rationalization for the wide range of fluorescence lifetimes. Finally, the simulations provided evidence of local structural perturbations induced by 2AP. The approach presented is a general tool to investigate fine structural heterogeneity in nucleic acid and nucleoprotein assemblies. PMID:26896800

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

    PubMed

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

    2014-04-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2014-04-01

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

  17. Quantitative Expression Analysis in Brassica napus by Northern Blot Analysis and Reverse Transcription-Quantitative PCR in a Complex Experimental Setting

    PubMed Central

    Rumlow, Annekathrin; Keunen, Els; Klein, Jan; Pallmann, Philip; Riemenschneider, Anja; Cuypers, Ann

    2016-01-01

    Analysis of gene expression is one of the major ways to better understand plant reactions to changes in environmental conditions. The comparison of many different factors influencing plant growth challenges the gene expression analysis for specific gene-targeted experiments, especially with regard to the choice of suitable reference genes. The aim of this study is to compare expression results obtained by Northern blot, semi-quantitative PCR and RT-qPCR, and to identify a reliable set of reference genes for oilseed rape (Brassica napus L.) suitable for comparing gene expression under complex experimental conditions. We investigated the influence of several factors such as sulfur deficiency, different time points during the day, varying light conditions, and their interaction on gene expression in oilseed rape plants. The expression of selected reference genes was indeed influenced under these conditions in different ways. Therefore, a recently developed algorithm, called GrayNorm, was applied to validate a set of reference genes for normalizing results obtained by Northern blot analysis. After careful comparison of the three methods mentioned above, Northern blot analysis seems to be a reliable and cost-effective alternative for gene expression analysis under a complex growth regime. For using this method in a quantitative way a number of references was validated revealing that for our experiment a set of three references provides an appropriate normalization. Semi-quantitative PCR was prone to many handling errors and difficult to control while RT-qPCR was very sensitive to expression fluctuations of the reference genes. PMID:27685087

  18. Molecular Dynamics and Monte Carlo simulations in the microcanonical ensemble: Quantitative comparison and reweighting techniques.

    PubMed

    Schierz, Philipp; Zierenberg, Johannes; Janke, Wolfhard

    2015-10-01

    Molecular Dynamics (MD) and Monte Carlo (MC) simulations are the most popular simulation techniques for many-particle systems. Although they are often applied to similar systems, it is unclear to which extent one has to expect quantitative agreement of the two simulation techniques. In this work, we present a quantitative comparison of MD and MC simulations in the microcanonical ensemble. For three test examples, we study first- and second-order phase transitions with a focus on liquid-gas like transitions. We present MD analysis techniques to compensate for conservation law effects due to linear and angular momentum conservation. Additionally, we apply the weighted histogram analysis method to microcanonical histograms reweighted from MD simulations. By this means, we are able to estimate the density of states from many microcanonical simulations at various total energies. This further allows us to compute estimates of canonical expectation values.

  19. Surfactant Uptake Dynamics in Mammalian Cells Elucidated with Quantitative Coherent Anti-Stokes Raman Scattering Microspectroscopy

    PubMed Central

    Okuno, Masanari; Kano, Hideaki; Fujii, Kenkichi; Bito, Kotatsu; Naito, Satoru; Leproux, Philippe; Couderc, Vincent; Hamaguchi, Hiro-o

    2014-01-01

    The mechanism of surfactant-induced cell lysis has been studied with quantitative coherent anti-Stokes Raman scattering (CARS) microspectroscopy. The dynamics of surfactant molecules as well as intracellular biomolecules in living Chinese Hamster Lung (CHL) cells has been examined for a low surfactant concentration (0.01 w%). By using an isotope labeled surfactant having CD bonds, surfactant uptake dynamics in living cells has been traced in detail. The simultaneous CARS imaging of the cell itself and the internalized surfactant has shown that the surfactant molecules is first accumulated inside a CHL cell followed by a sudden leak of cytosolic components such as proteins to the outside of the cell. This finding indicates that surfactant uptake occurs prior to the cell lysis, contrary to what has been believed: surface adsorption of surfactant molecules has been thought to occur first with subsequent disruption of cell membranes. Quantitative CARS microspectroscopy enables us to determine the molecular concentration of the surfactant molecules accumulated in a cell. We have also investigated the effect of a drug, nocodazole, on the surfactant uptake dynamics. As a result of the inhibition of tubulin polymerization by nocodazole, the surfactant uptake rate is significantly lowered. This fact suggests that intracellular membrane trafficking contributes to the surfactant uptake mechanism. PMID:24710120

  20. Integrating discrete stochastic models and single-cell experiments to infer predictive models of MAPK-induced transcription dynamics

    NASA Astrophysics Data System (ADS)

    Munsky, Brian

    2015-03-01

    MAPK signal-activated transcription plays central roles in myriad biological processes including stress adaptation responses and cell fate decisions. Recent single-cell and single-molecule experiments have advanced our ability to quantify the spatial, temporal, and stochastic fluctuations for such signals and their downstream effects on transcription regulation. This talk explores how integrating such experiments with discrete stochastic computational analyses can yield quantitative and predictive understanding of transcription regulation in both space and time. We use single-molecule mRNA fluorescence in situ hybridization (smFISH) experiments to reveal locations and numbers of multiple endogenous mRNA species in 100,000's of individual cells, at different times and under different genetic and environmental perturbations. We use finite state projection methods to precisely and efficiently compute the full joint probability distributions of these mRNA, which capture measured spatial, temporal and correlative fluctuations. By combining these experimental and computational tools with uncertainty quantification, we systematically compare models of varying complexity and select those which give optimally precise and accurate predictions in new situations. We use these tools to explore two MAPK-activated gene regulation pathways. In yeast adaptation to osmotic shock, we analyze Hog1 kinase activation of transcription for three different genes STL1 (osmotic stress), CTT1 (oxidative stress) and HSP12 (heat shock). In human osteosarcoma cells under serum induction, we analyze ERK activation of c-Fos transcription.

  1. Quantitative analysis of binding of transcription factor complex to biotinylated DNA probe by a streptavidin-agarose pulldown assay.

    PubMed

    Deng, Wu-Guo; Zhu, Ying; Montero, Alberto; Wu, Kenneth K

    2003-12-01

    Gene expression is regulated by a large complex of proteins that bind to the promoter/enhancer region of a gene. We determined whether a streptavidin-bead binding assay might be useful in detecting individual proteins in the complex comprising transactivators, coactivators, mediators, and general transcription factors. We used biotinylated cyclooxygenase-2 promoter probes as a model. Nuclear extracts obtained from human fibroblasts treated with or without an agonist were incubated with a 5(')-biotinylated probe and streptavidin-agarose beads at room temperature for 1h. After centrifugation, the pellet was washed and proteins in the complex were assessed by immunoblots. An array of transcription factors was detectable concurrently in the same batch of pellets at basal state. p300 and its associated factor PCAF levels but not Srb7, Med7, or TFII(B) were increased by phorbol ester or tumor necrosis factor alpha stimulation. Only trace of CREB-binding protein was detected. These results suggest that p300 and PCAF are the predominant coactivators for COX-2 promoter activation. Our findings indicate that the streptavidin-bead pulldown assay is valuable for determining the binding of a large number of transcription factors to promoter/enhancer and evaluating the relationship of protein binding with regulation of gene expression.

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

    PubMed Central

    Tang, Xuxiang; Liu, Fuqi

    2015-01-01

    In this paper, a novel benzene quantitative analysis method utilizing miniaturized metal ionization gas sensor and non-linear bistable dynamic system was investigated. Al plate anodic gas-ionization sensor was installed for electrical current-voltage data measurement. Measurement data was analyzed by non-linear bistable dynamics system. Results demonstrated that this method realized benzene concentration quantitative determination. This method is promising in laboratory safety management in benzene leak detection. PMID:26218927

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

    PubMed

    Tang, Xuxiang; Liu, Fuqi

    2015-01-01

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

  4. 4D PET iterative deconvolution with spatiotemporal regularization for quantitative dynamic PET imaging.

    PubMed

    Reilhac, Anthonin; Charil, Arnaud; Wimberley, Catriona; Angelis, Georgios; Hamze, Hasar; Callaghan, Paul; Garcia, Marie-Paule; Boisson, Frederic; Ryder, Will; Meikle, Steven R; Gregoire, Marie-Claude

    2015-09-01

    Quantitative measurements in dynamic PET imaging are usually limited by the poor counting statistics particularly in short dynamic frames and by the low spatial resolution of the detection system, resulting in partial volume effects (PVEs). In this work, we present a fast and easy to implement method for the restoration of dynamic PET images that have suffered from both PVE and noise degradation. It is based on a weighted least squares iterative deconvolution approach of the dynamic PET image with spatial and temporal regularization. Using simulated dynamic [(11)C] Raclopride PET data with controlled biological variations in the striata between scans, we showed that the restoration method provides images which exhibit less noise and better contrast between emitting structures than the original images. In addition, the method is able to recover the true time activity curve in the striata region with an error below 3% while it was underestimated by more than 20% without correction. As a result, the method improves the accuracy and reduces the variability of the kinetic parameter estimates calculated from the corrected images. More importantly it increases the accuracy (from less than 66% to more than 95%) of measured biological variations as well as their statistical detectivity. PMID:26080302

  5. Genome shuffling of Saccharomyces cerevisiae for enhanced glutathione yield and relative gene expression analysis using fluorescent quantitation reverse transcription polymerase chain reaction.

    PubMed

    Yin, Hua; Ma, Yanlin; Deng, Yang; Xu, Zhenbo; Liu, Junyan; Zhao, Junfeng; Dong, Jianjun; Yu, Junhong; Chang, Zongming

    2016-08-01

    Genome shuffling is an efficient and promising approach for the rapid improvement of microbial phenotypes. In this study, genome shuffling was applied to enhance the yield of glutathione produced by Saccharomyces cerevisiae YS86. Six isolates with subtle improvements in glutathione yield were obtained from populations generated by ultraviolet (UV) irradiation and nitrosoguanidine (NTG) mutagenesis. These yeast strains were then subjected to recursive pool-wise protoplast fusion. A strain library that was likely to yield positive colonies was created by fusing the lethal protoplasts obtained from both UV irradiation and heat treatments. After two rounds of genome shuffling, a high-yield recombinant YSF2-19 strain that exhibited 3.2- and 3.3-fold increases in glutathione production in shake flask and fermenter respectively was obtained. Comparative analysis of synthetase gene expression was conducted between the initial and shuffled strains using FQ (fluorescent quantitation) RT-PCR (reverse transcription polymerase chain reaction). Delta CT (threshold cycle) relative quantitation analysis revealed that glutathione synthetase gene (GSH-I) expression at the transcriptional level in the YSF2-19 strain was 9.9-fold greater than in the initial YS86. The shuffled yeast strain has a potential application in brewing, other food, and pharmaceutical industries. Simultaneously, the analysis of improved phenotypes will provide more valuable data for inverse metabolic engineering. PMID:27302037

  6. Mediator facilitates transcriptional activation and dynamic long-range contacts at the IgH locus during class switch recombination.

    PubMed

    Thomas-Claudepierre, Anne-Sophie; Robert, Isabelle; Rocha, Pedro P; Raviram, Ramya; Schiavo, Ebe; Heyer, Vincent; Bonneau, Richard; Luo, Vincent M; Reddy, Janardan K; Borggrefe, Tilman; Skok, Jane A; Reina-San-Martin, Bernardo

    2016-03-01

    Immunoglobulin (Ig) class switch recombination (CSR) is initiated by the transcription-coupled recruitment of activation-induced cytidine deaminase (AID) to Ig switch regions (S regions). During CSR, the IgH locus undergoes dynamic three-dimensional structural changes in which promoters, enhancers, and S regions are brought to close proximity. Nevertheless, little is known about the underlying mechanisms. In this study, we show that Med1 and Med12, two subunits of the mediator complex implicated in transcription initiation and long-range enhancer/promoter loop formation, are dynamically recruited to the IgH locus enhancers and the acceptor regions during CSR and that their knockdown in CH12 cells results in impaired CSR. Furthermore, we show that conditional inactivation of Med1 in B cells results in defective CSR and reduced acceptor S region transcription. Finally, we show that in B cells undergoing CSR, the dynamic long-range contacts between the IgH enhancers and the acceptor regions correlate with Med1 and Med12 binding and that they happen at a reduced frequency in Med1-deficient B cells. Our results implicate the mediator complex in the mechanism of CSR and are consistent with a model in which mediator facilitates the long-range contacts between S regions and the IgH locus enhancers during CSR and their transcriptional activation.

  7. Mediator facilitates transcriptional activation and dynamic long-range contacts at the IgH locus during class switch recombination

    PubMed Central

    Thomas-Claudepierre, Anne-Sophie; Robert, Isabelle; Rocha, Pedro P.; Raviram, Ramya; Schiavo, Ebe; Heyer, Vincent; Bonneau, Richard; Luo, Vincent M.; Reddy, Janardan K.; Borggrefe, Tilman; Skok, Jane A.

    2016-01-01

    Immunoglobulin (Ig) class switch recombination (CSR) is initiated by the transcription-coupled recruitment of activation-induced cytidine deaminase (AID) to Ig switch regions (S regions). During CSR, the IgH locus undergoes dynamic three-dimensional structural changes in which promoters, enhancers, and S regions are brought to close proximity. Nevertheless, little is known about the underlying mechanisms. In this study, we show that Med1 and Med12, two subunits of the mediator complex implicated in transcription initiation and long-range enhancer/promoter loop formation, are dynamically recruited to the IgH locus enhancers and the acceptor regions during CSR and that their knockdown in CH12 cells results in impaired CSR. Furthermore, we show that conditional inactivation of Med1 in B cells results in defective CSR and reduced acceptor S region transcription. Finally, we show that in B cells undergoing CSR, the dynamic long-range contacts between the IgH enhancers and the acceptor regions correlate with Med1 and Med12 binding and that they happen at a reduced frequency in Med1-deficient B cells. Our results implicate the mediator complex in the mechanism of CSR and are consistent with a model in which mediator facilitates the long-range contacts between S regions and the IgH locus enhancers during CSR and their transcriptional activation. PMID:26903242

  8. Regulation of flavonol content and composition in (Syrah×Pinot Noir) mature grapes: integration of transcriptional profiling and metabolic quantitative trait locus analyses

    PubMed Central

    Malacarne, Giulia; Costantini, Laura; Coller, Emanuela; Battilana, Juri; Velasco, Riccardo; Vrhovsek, Urska; Grando, Maria Stella; Moser, Claudio

    2015-01-01

    Flavonols are a ubiquitous class of flavonoids that accumulate preferentially in flowers and mature berries. Besides their photo-protective function, they play a fundamental role during winemaking, stabilizing the colour by co-pigmentation with anthocyanins and contributing to organoleptic characteristics. Although the general flavonol pathway has been genetically and biochemically elucidated, the genetic control of flavonol content and composition at harvest is still not clear. To this purpose, the grapes of 170 segregating F1 individuals from a ‘Syrah’×’Pinot Noir’ population were evaluated at the mature stage for the content of six flavonol aglycons in four seasons. Metabolic data in combination with genetic data enabled the identification of 16 mQTLs (metabolic quantitative trait loci). For the first time, major genetic control by the linkage group 2 (LG 2)/MYBA region on flavonol variation, in particular of tri-hydroxylated flavonols, is demonstrated. Moreover, seven regions specifically associated with the fine control of flavonol biosynthesis are identified. Gene expression profiling of two groups of individuals significantly divergent for their skin flavonol content identified a large set of differentially modulated transcripts. Among these, the transcripts coding for MYB and bZIP transcription factors, methyltranferases, and glucosyltranferases specific for flavonols, proteins, and factors belonging to the UV-B signalling pathway and co-localizing with the QTL regions are proposed as candidate genes for the fine regulation of flavonol content and composition in mature grapes. PMID:26071529

  9. Quantitative determination of electric field strengths within dynamically operated devices using EBIC analysis in the SEM.

    PubMed

    Pugatschow, Anton; Heiderhoff, Ralf; Balk, Ludwig J

    2008-01-01

    Although electron beam-induced current (EBIC) technique was invented in the seventies, it is still a powerful technique for failure analysis and reliability investigations of modern materials and devices. Time-resolved and stroboscopic microanalyses using sampling Fourier components decomposed by modulated charge carrier excitation are introduced. Quantitative determination of electric field strengths within dynamically operated devices in the scanning electron microscope (SEM) will be demonstrated. This technique allows investigations of diffusion and drift processes and of variations of electric field distributions inside active devices.

  10. A quantitative method based on total relative change for dynamic electrical impedance tomography.

    PubMed

    You, Fusheng; Shi, Xuetao; Dong, Xiuzhen; Fu, Feng; Liu, Ruigang; Shuai, Wanjun; Li, Zheng

    2008-03-01

    We proposed a new method based on total relative change (TRC) from measured boundary voltages to quantify the volume changes of fluid during electrical impedance tomography (EIT) monitoring. The results showed that TRC linearly correlated with the volume of infused saline solution into a phantom, and the slope of TRC changes was approximately linear with the infusion speed. A inserted copper tube at different positions did not affect TRC significantly. The linear relationship between TRC and volume change indicates that TRC could be a good quantitative index for dynamic EIT.

  11. Transcriptional dynamics of Phytophthora infestans during sequential stages of hemibiotrophic infection of tomato.

    PubMed

    Zuluaga, Andrea P; Vega-Arreguín, Julio C; Fei, Zhangjun; Ponnala, Lalit; Lee, Sang Jik; Matas, Antonio J; Patev, Sean; Fry, William E; Rose, Jocelyn K C

    2016-01-01

    Hemibiotrophic plant pathogens, such as the oomycete Phytophthora infestans, employ a biphasic infection strategy, initially behaving as biotrophs, where minimal symptoms are exhibited by the plant, and subsequently as necrotrophs, feeding on dead plant tissue. The regulation of this transition and the breadth of molecular mechanisms that modulate plant defences are not well understood, although effector proteins secreted by the pathogen are thought to play a key role. We examined the transcriptional dynamics of P. infestans in a compatible interaction with its host tomato (Solanum lycopersicum) at three infection stages: biotrophy; the transition from biotrophy to necrotrophy; and necrotrophy. The expression data suggest a tight temporal regulation of many pathways associated with the suppression of plant defence mechanisms and pathogenicity, including the induction of putative cytoplasmic and apoplastic effectors. Twelve of these were experimentally evaluated to determine their ability to suppress necrosis caused by the P. infestans necrosis-inducing protein PiNPP1.1 in Nicotiana benthamiana. Four effectors suppressed necrosis, suggesting that they might prolong the biotrophic phase. This study suggests that a complex regulation of effector expression modulates the outcome of the interaction.

  12. Mapping replication dynamics in Trypanosoma brucei reveals a link with telomere transcription and antigenic variation

    PubMed Central

    Devlin, Rebecca; Marques, Catarina A; Paape, Daniel; Prorocic, Marko; Zurita-Leal, Andrea C; Campbell, Samantha J; Lapsley, Craig; Dickens, Nicholas; McCulloch, Richard

    2016-01-01

    Survival of Trypanosoma brucei depends upon switches in its protective Variant Surface Glycoprotein (VSG) coat by antigenic variation. VSG switching occurs by frequent homologous recombination, which is thought to require locus-specific initiation. Here, we show that a RecQ helicase, RECQ2, acts to repair DNA breaks, including in the telomeric site of VSG expression. Despite this, RECQ2 loss does not impair antigenic variation, but causes increased VSG switching by recombination, arguing against models for VSG switch initiation through direct generation of a DNA double strand break (DSB). Indeed, we show DSBs inefficiently direct recombination in the VSG expression site. By mapping genome replication dynamics, we reveal that the transcribed VSG expression site is the only telomeric site that is early replicating – a differential timing only seen in mammal-infective parasites. Specific association between VSG transcription and replication timing reveals a model for antigenic variation based on replication-derived DNA fragility. DOI: http://dx.doi.org/10.7554/eLife.12765.001 PMID:27228154

  13. Theory on the Dynamics of Oscillatory Loops in the Transcription Factor Networks

    PubMed Central

    Murugan, Rajamanickam

    2014-01-01

    We develop a detailed theoretical framework for various types of transcription factor gene oscillators. We further demonstrate that one can build genetic-oscillators which are tunable and robust against perturbations in the critical control parameters by coupling two or more independent Goodwin-Griffith oscillators through either -OR- or -AND- type logic. Most of the coupled oscillators constructed in the literature so far seem to be of -OR- type. When there are transient perturbations in one of the -OR- type coupled-oscillators, then the overall period of the system remains constant (period-buffering) whereas in case of -AND- type coupling the overall period of the system moves towards the perturbed oscillator. Though there is a period-buffering, the amplitudes of oscillators coupled through -OR- type logic are more sensitive to perturbations in the parameters associated with the promoter state dynamics than -AND- type. Further analysis shows that the period of -AND- type coupled dual-feedback oscillators can be tuned without conceding on the amplitudes. Using these results we derive the basic design principles governing the robust and tunable synthetic gene oscillators without compromising on their amplitudes. PMID:25111803

  14. Direct observation of transcription activator-like effector (TALE) protein dynamics

    NASA Astrophysics Data System (ADS)

    Cuculis, Luke; Abil, Zhanar; Zhao, Huimin; Schroeder, Charles M.

    2014-03-01

    In this work, we describe a single molecule assay to probe the site-search dynamics of transcription activator-like effector (TALE) proteins along DNA. In modern genetics, the ability to selectively edit the human genome is an unprecedented development, driven by recent advances in targeted nuclease proteins. Specific gene editing can be accomplished using TALE proteins, which are programmable DNA-binding proteins that can be fused to a nuclease domain. In this way, TALENs are a leading technology that has shown great success in the genomic editing of pluripotent stem cells. A major hurdle facing clinical implementation, however, is the potential for deleterious off-target binding events. For these reasons, a molecular-level understanding of TALE binding and target sequence search on DNA is essential. To this end, we developed a single-molecule fluorescence imaging assay that provides a first-of-its-kind view of the 1-D diffusion of TALE proteins along stretched DNA. Taken together with co-crystal structures of DNA-bound TALEs, our results suggest a rotationally-coupled, major groove tracking model for diffusion. We further report diffusion constants for TALE proteins as a function of salt concentration, consistent with previously described models of 1-D protein diffusion.

  15. Changes in transcriptional pausing modify the folding dynamics of the pH-responsive RNA element

    PubMed Central

    Nechooshtan, Gal; Elgrably-Weiss, Maya; Altuvia, Shoshy

    2014-01-01

    Previously, we described a novel pH-responsive RNA element in Escherichia coli that resides in the 5′ untranslated region of the alx gene and controls its translation in a pH-dependent manner. Under normal growth conditions, this RNA region forms a translationally inactive structure, but when transcribed under alkaline conditions, it forms an active structure producing the Alx protein. We identified two distinct transcriptional pause sites and proposed that pausing at these sites interfered with the formation of the inactive structure while facilitating folding of the active one. Alkali increases the longevity of pausing at these sites, thereby promoting folding of the translationally active form of alx RNA. We show here that mutations that modify the extent and/or position of pausing, although silent with regard to structure stability per se, greatly influence the dynamics of folding and thereby translation. Our data illustrate the mechanistic design of alx regulation, relying on precise temporal and spatial characteristics. We propose that this unique design provides an opportunity for environmental signals such as pH to introduce structural changes in the RNA and thereby modulate expression. PMID:24078087

  16. Quantitative assessment of molecular dynamics-grown amorphous silicon and germanium films on silicon (111)

    NASA Astrophysics Data System (ADS)

    Käshammer, Peter; Borgardt, Nikolai I.; Seibt, Michael; Sinno, Talid

    2016-09-01

    Molecular dynamics based on the empirical Tersoff potential was used to simulate the deposition of amorphous silicon and germanium on silicon(111) at various deposition rates and temperatures. The resulting films were analyzed quantitatively by comparing one-dimensional atomic density profiles to experimental measurements. It is found that the simulations are able to capture well the structural features of the deposited films, which exhibit a gradual loss of crystalline order over several monolayers. A simple mechanistic model is used to demonstrate that the simulation temperature may be used to effectively accelerate the surface relaxation processes during deposition, leading to films that are consistent with experimental samples grown at deposition rates many orders-of-magnitude slower than possible in a molecular dynamics simulation.

  17. Communication patterns in a psychotherapy following traumatic brain injury: A quantitative case study based on symbolic dynamics

    PubMed Central

    2011-01-01

    Background The role of psychotherapy in the treatment of traumatic brain injury is receiving increased attention. The evaluation of psychotherapy with these patients has been conducted largely in the absence of quantitative data concerning the therapy itself. Quantitative methods for characterizing the sequence-sensitive structure of patient-therapist communication are now being developed with the objective of improving the effectiveness of psychotherapy following traumatic brain injury. Methods The content of three therapy session transcripts (sessions were separated by four months) obtained from a patient with a history of several motor vehicle accidents who was receiving dialectical behavior therapy was scored and analyzed using methods derived from the mathematical theory of symbolic dynamics. Results The analysis of symbol frequencies was largely uninformative. When repeated triples were examined a marked pattern of change in content was observed over the three sessions. The context free grammar complexity and the Lempel-Ziv complexity were calculated for each therapy session. For both measures, the rate of complexity generation, expressed as bits per minute, increased longitudinally during the course of therapy. The between-session increases in complexity generation rates are consistent with calculations of mutual information. Taken together these results indicate that there was a quantifiable increase in the variability of patient-therapist verbal behavior during the course of therapy. Comparison of complexity values against values obtained from equiprobable random surrogates established the presence of a nonrandom structure in patient-therapist dialog (P = .002). Conclusions While recognizing that only limited conclusions can be based on a case history, it can be noted that these quantitative observations are consistent with qualitative clinical observations of increases in the flexibility of discourse during therapy. These procedures can be of particular

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

    PubMed Central

    Bhaduri, Anirban; Ghosh, Dipak

    2016-01-01

    The cardiac dynamics during meditation is explored quantitatively with two chaos-based non-linear techniques viz. multi-fractal detrended fluctuation analysis and visibility network analysis techniques. The data used are the instantaneous heart rate (in beats/minute) of subjects performing Kundalini Yoga and Chi meditation from PhysioNet. The results show consistent differences between the quantitative parameters obtained by both the analysis techniques. This indicates an interesting phenomenon of change in the complexity of the cardiac dynamics during meditation supported with quantitative parameters. The results also produce a preliminary evidence that these techniques can be used as a measure of physiological impact on subjects performing meditation. PMID:26909045

  19. Analysis of expression of chorionic gonadotrophin transcripts in prostate cancer by quantitative Taqman and a modified molecular beacon RT-PCR.

    PubMed

    Span, P N; Thomas, C M G; Heuvel, J J; Bosch, R R; Schalken, J A; vd Locht, L; Mensink, E J B M; Sweep, C G J

    2002-03-01

    Expression of human chorionic gonadotrophin (hCG) is associated with trophoblastic, testicular and other malignancies such as bladder, pancreatic, cervical, breast and prostate cancer. In the prostate, however, hCG expression, associated with neuroendocrine cells, is also found in normal tissue. Of the six highly homologous genes that all encode the beta-subunit of hCG, the beta 7 gene is reportedly the only gene expressed in several non-transformed tissues. The beta 3, 5 and 8 genes would be variably expressed in malignant tissue and placenta, but not in normal tissue. To assess to what extent this expression difference can also be found in the prostate, we compared the levels of the different hCG beta transcripts in concurrent normal and cancerous prostate tissues obtained from 17 patients. To this end, we developed a Taqman real-time fluorescent RT-PCR assay for hCG beta, and a quantitative assay specific for the beta 3, 5 and 8 genes, modified from the molecular beacon principle. This latter assay proved highly specific and capable of reliably distinguishing between these hCG beta transcripts that differ in only one nucleotide. Surprisingly, median expression levels of hCG beta were lower in prostate cancer when compared with normal tissue from the same patient. In contrast, hCG beta 3, 5 and 8 transcripts were found in normal tissue and did not differ in prostate cancer, arguing against a specific role of these transcripts in the development of prostate cancer.

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

    PubMed Central

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

    2012-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2012-03-01

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

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

    PubMed

    Saitou, Takashi; Imamura, Takeshi

    2016-01-01

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

  3. Quantitative Analysis of Axonal Branch Dynamics in the Developing Nervous System

    PubMed Central

    Scott, Ethan K.; Goodhill, Geoffrey J.

    2016-01-01

    Branching is an important mechanism by which axons navigate to their targets during neural development. For instance, in the developing zebrafish retinotectal system, selective branching plays a critical role during both initial pathfinding and subsequent arborisation once the target zone has been reached. Here we show how quantitative methods can help extract new information from time-lapse imaging about the nature of the underlying branch dynamics. First, we introduce Dynamic Time Warping to this domain as a method for automatically matching branches between frames, replacing the effort required for manual matching. Second, we model branch dynamics as a birth-death process, i.e. a special case of a continuous-time Markov process. This reveals that the birth rate for branches from zebrafish retinotectal axons, as they navigate across the tectum, increased over time. We observed no significant change in the death rate for branches over this time period. However, blocking neuronal activity with TTX slightly increased the death rate, without a detectable change in the birth rate. Third, we show how the extraction of these rates allows computational simulations of branch dynamics whose statistics closely match the data. Together these results reveal new aspects of the biology of retinotectal pathfinding, and introduce computational techniques which are applicable to the study of axon branching more generally. PMID:26998842

  4. [A novel quantitative approach to study dynamic anaerobic process at micro scale].

    PubMed

    Zhang, Zhong-Liang; Wu, Jing; Jiang, Jian-Kai; Jiang, Jie; Li, Huai-Zhi

    2012-11-01

    Anaerobic digestion is attracting more and more interests because of its advantages such as low cost and recovery of clean energy etc. In order to overcome the drawbacks of the existed methods to study the dynamic anaerobic process, a novel microscopical quantitative approach at the granule level was developed combining both the microdevice and the quantitative image analysis techniques. This experiment displayed the process and characteristics of the gas production at static state for the first time and the results indicated that the method was of satisfactory repeatability. The gas production process at static state could be divided into three stages including rapid linear increasing stage, decelerated increasing stage and slow linear increasing stage. The rapid linear increasing stage was long and the biogas rate was high under high initial organic loading rate. The results showed that it was feasible to make the anaerobic process to be carried out in the microdevice; furthermore this novel method was reliable and could clearly display the dynamic process of the anaerobic reaction at the micro scale. The results are helpful to understand the anaerobic process.

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

    NASA Astrophysics Data System (ADS)

    Xu, Xiangqun; Zhu, Jiang; Chen, Zhongping

    2016-04-01

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

  6. Portable low-coherence interferometry for quantitatively imaging fast dynamics with extended field of view

    NASA Astrophysics Data System (ADS)

    Shaked, Natan T.; Girshovitz, Pinhas; Frenklach, Irena

    2014-06-01

    We present our recent advances in the development of compact, highly portable and inexpensive wide-field interferometric modules. By a smart design of the interferometric system, including the usage of low-coherence illumination sources and common-path off-axis geometry of the interferometers, spatial and temporal noise levels of the resulting quantitative thickness profile can be sub-nanometric, while processing the phase profile in real time. In addition, due to novel experimentally-implemented multiplexing methods, we can capture low-coherence off-axis interferograms with significantly extended field of view and in faster acquisition rates. Using these techniques, we quantitatively imaged rapid dynamics of live biological cells including sperm cells and unicellular microorganisms. Then, we demonstrated dynamic profiling during lithography processes of microscopic elements, with thicknesses that may vary from several nanometers to hundreds of microns. Finally, we present new algorithms for fast reconstruction (including digital phase unwrapping) of off-axis interferograms, which allow real-time processing in more than video rate on regular single-core computers.

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

    PubMed

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

    2015-10-13

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

  8. Quantitative analysis of dynamic adhesion properties in human hepatocellular carcinoma cells with fullerenol.

    PubMed

    Liu, Yang; Wang, Zuobin; Wang, Xinyue; Huang, Yanhong

    2015-12-01

    In this study, the effect of fullerenol (C60(OH)24) on the cellular dynamic biomechanical behaviors of living human hepatocellular carcinoma (SMCC-7721) cancer cells were investigated by atomic force microscope (AFM) nanoindentation. As an important biomarker of cellular information, the cell adhesion is essential to maintain proper functioning as well as links with the pathogenesis and canceration. Nonetheless, it is challenging to properly evaluate the complex adhesion properties as all the biomechanical parameters interfere with each other. To investigate the dynamic adhesion changes, especially in the case of the fullerenol treatment, the detachment force and work, adhesion events, and membrane tether properties were measured and analyzed systematically with the proposed quantitative method. The statistical analyses suggest that, under the same operating parameters of AFM, the dependence of adhesion energy on the tip-cell contact area is weakened after the fullerenol treatment and the probability of adhesion decreases significantly from 30.6% to 4.2%. In addition, the disruption of the cytoskeleton resulted in a 34% decrease of the average membrane tether force and a 21% increase of the average tether length. Benefiting from the quantitative method, this work contributes to revealing the effects of fullerenol on the cellular biomechanical properties of the living SMCC-7721 cells in a precise and rigorous way and additionally is further instructive to interpret the interaction mechanism of other potential nanomedicines with living cells.

  9. A novel duplex real time quantitative reverse transcription polymerase chain reaction for rubella virus with armored RNA as a noncompetitive internal positive control.

    PubMed

    Zhao, Lihong; Li, Ruiying; Liu, Aihua; Zhao, Shuping

    2015-07-01

    The objective of this study was to build and apply a duplex real time quantitative reverse transcription-polymerase chain reaction (RT-PCR) for rubella virus. Firstly, a 60-bp-long armored RV RNA was constructed in the laboratory. Secondly, a duplex real time RT-PCR assay was established. Thirdly, the 60-bp-long armored RV RNA was used as an internal positive control (IPC) for the duplex real time RT-PCR. And finally the duplex real time RT-PCR assay was applied to detect RV RNA in clinical specimens. The in-house assay has a high amplification efficiency (0.99), a high analytical sensitivity (200 copies/mL), and a good reproducibility. The diagnostic specificity and sensitivity of the in-house assay were both 100%, due to the monitoring of the armored RV RNA IPC. Therefore, the in-house duplex real time quantitative RT-PCR assay is a specific, sensitive, reproducible and accurate assay for quantitation of RV RNA in clinical specimens. And noncompetitive armored RV RNA IPC can monitor RT-PCR inhibition and prevent false-negative and inaccurate results in the real time detection system.

  10. Integrative analysis of time course microarray data and DNA sequence data via log-linear models for identifying dynamic transcriptional regulatory networks.

    PubMed

    Choi, Hyung-Seok; Kim, Youngchul; Cho, Kwang-Hyun; Park, Taesung

    2013-01-01

    Since eukaryotic transcription is regulated by sets of Transcription Factors (TFs) having various transcriptional time delays, identification of temporal combinations of activated TFs is important to reconstruct Transcriptional Regulatory Networks (TRNs). Our methods combine time course microarray data, information on physical binding between the TFs and their targets and the regulatory sequences of genes using a log-linear model to reconstruct dynamic functional TRNs of the yeast cell cycle and human apoptosis. In conclusion, our results suggest that the proposed dynamic motif search method is more effective in reconstructing TRNs than the static motif search method.

  11. Characteristics of quantitative perfusion parameters on dynamic contrast-enhanced MRI in mammographically occult breast cancer.

    PubMed

    Ryu, Jung Kyu; Rhee, Sun Jung; Song, Jeong Yoon; Cho, Soo Hyun; Jahng, Geon-Ho

    2016-01-01

    The purpose of this study was to compare the characteristics of quantitative per-fusion parameters obtained from dynamic contrast-enhanced (DCE) magnetic resonance imaging (MRI) in patients with mammographically occult (MO) breast cancers and those with mammographically visible (MV) breast cancers. Quantitative parameters (AUC, Ktrans, kep, ve, vp, and wi) from 13 MO breast cancers and 16 MV breast cancers were mapped after the DCE-MRI data were acquired. Various prog-nostic factors, including axillary nodal status, estrogen receptor (ER), progesterone receptor (PR), Ki-67, p53, E-cadherin, and human epidermal growth factor receptor 2 (HER2) were obtained in each group. Fisher's exact test was used to compare any differences of the various prognostic factors between the two groups. The Mann- Whitney U test was applied to compare the quantitative parameters between these two groups. Finally, Spearman's correlation was used to investigate the relation-ships between perfusion indices and four factors - age, tumor size, Ki-67, and p53 - for each group. Although age, tumor size, and the prognostic factors were not statistically different between the two groups, the mean values of the quantitative parameters, except wi in the MV group, were higher than those in the MO group without statistical significance (p = 0.219). The kep value was significantly differ-ent between the two groups (p = 0.048), but the other parameters were not. In the MO group, vp with size, ve with p53, and Ktrans and vp with Ki-67 had significant correlations (p < 0.05). However, in the MV group, only kep showed significant correlation with age. The kep value was only the perfusion parameter of statistical significance between MO and MV breast cancers. PMID:27685105

  12. Quantitative RT-PCR comparison of the urea and nitric oxide cycle gene transcripts in adult human tissues.

    PubMed

    Neill, Meaghan Anne; Aschner, Judy; Barr, Frederick; Summar, Marshall L

    2009-06-01

    The urea cycle and nitric oxide cycle play significant roles in complex biochemical and physiologic reactions. These cycles have distinct biochemical goals including the clearance of waste nitrogen; the production of the intermediates ornithine, citrulline, and arginine for the urea cycle; and the production of nitric oxide for the nitric oxide pathway. Despite their disparate functions, the two pathways share two enzymes, argininosuccinic acid synthase and argininosuccinic acid lyase, and a transporter, citrin. Studying the gene expression of these enzymes is paramount in understanding these complex biochemical pathways. Here, we examine the expression of genes involved in the urea cycle and the nitric oxide cycle in a panel of eleven different tissue samples obtained from individual adults without known inborn errors of metabolism. In this study, the pattern of co-expressed enzymes provides a global view of the metabolic activity of the urea and nitric oxide cycles in human tissues. Our results show that these transcripts are differentially expressed in different tissues. Using the co-expression profiles, we discovered that the combination of expression of enzyme transcripts as detected in our study, might serve to fulfill specific physiologic function(s) including urea production/nitrogen removal, arginine/citrulline production, nitric oxide production, and ornithine production. Our study reveals the importance of studying not only the expression profile of an enzyme of interest, but also studying the expression profiles of the other enzymes involved in a particular pathway so as to better understand the context of expression. The tissue patterns we observed highlight the variety of important functions of these enzymes and provides insight into the many clinical observations that result from their disruption. These results have implications for the management of urea cycle patients and raise considerations for the care of those patients receiving liver

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

    NASA Technical Reports Server (NTRS)

    Cheng, Rendy P.

    1995-01-01

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

  14. Comparative quantitative analysis of BCR-ABL transcripts with the T315I mutant clone by polymerase chain reaction (PCR)-Invader method.

    PubMed

    Tadokoro, Kenichi; Ishikawa, Maho; Suzuki, Makoto; Saito, Tomoyoshi; Suzuki, Yoshie; Yamaguchi, Toshikazu; Yagasaki, Fumiharu

    2011-09-01

    Drug resistance is a serious complication in the treatment of chronic myeloid leukemia (CML). The most common and best-characterized mechanism of secondary imatinib resistance in CML is the development of kinase domain mutations in the BCR-ABL gene. Second-generation tyrosine kinase inhibitors, such as dasatinib or nilotinib, overcome most of these mutations, but they are not effective against the T315I mutant. To determine whether these mutations contribute to clinical resistance, it is necessary to monitor the ratio of the mutant and wild-type forms. Here, we developed a polymerase chain reaction (PCR)-Invader assay for comparative quantitative analysis (qPI assay) of BCR-ABL transcripts with the T315I mutant clone. T315I ratios were calculated for the wild-type and mutant fold-over-zero (FOZ) values. In examination with 2 kinds of plasmids containing wild-type or T315I mutant PCR amplicons, mutant FOZ values were detected down to 1% of the total. The results of 12 serial samples from 2 patients (case A: Philadelphia-positive acute lymphoblastic leukemia and case B: CML) with the T315I mutant clone were compared with those of direct sequencing or 2 kinds of allele-specific oligonucleotide (ASO)-PCR. All samples showed the T315I mutation by qPI assay and ASO-PCR, and 10 samples showed it by direct sequencing. Significant correlation (correlation coefficient; r2 = 0.951) was noted between the qPI assay and quantitative ASO-PCR to analyze T315I mutant ratios. Thus, the qPI assay is a useful method for evaluating the T315I mutant clone in BCR-ABL transcripts.

  15. Comparative quantitative analysis of BCR-ABL transcripts with the T315I mutant clone by polymerase chain reaction (PCR)-Invader method.

    PubMed

    Tadokoro, Kenichi; Ishikawa, Maho; Suzuki, Makoto; Saito, Tomoyoshi; Suzuki, Yoshie; Yamaguchi, Toshikazu; Yagasaki, Fumiharu

    2011-09-01

    Drug resistance is a serious complication in the treatment of chronic myeloid leukemia (CML). The most common and best-characterized mechanism of secondary imatinib resistance in CML is the development of kinase domain mutations in the BCR-ABL gene. Second-generation tyrosine kinase inhibitors, such as dasatinib or nilotinib, overcome most of these mutations, but they are not effective against the T315I mutant. To determine whether these mutations contribute to clinical resistance, it is necessary to monitor the ratio of the mutant and wild-type forms. Here, we developed a polymerase chain reaction (PCR)-Invader assay for comparative quantitative analysis (qPI assay) of BCR-ABL transcripts with the T315I mutant clone. T315I ratios were calculated for the wild-type and mutant fold-over-zero (FOZ) values. In examination with 2 kinds of plasmids containing wild-type or T315I mutant PCR amplicons, mutant FOZ values were detected down to 1% of the total. The results of 12 serial samples from 2 patients (case A: Philadelphia-positive acute lymphoblastic leukemia and case B: CML) with the T315I mutant clone were compared with those of direct sequencing or 2 kinds of allele-specific oligonucleotide (ASO)-PCR. All samples showed the T315I mutation by qPI assay and ASO-PCR, and 10 samples showed it by direct sequencing. Significant correlation (correlation coefficient; r2 = 0.951) was noted between the qPI assay and quantitative ASO-PCR to analyze T315I mutant ratios. Thus, the qPI assay is a useful method for evaluating the T315I mutant clone in BCR-ABL transcripts. PMID:21867983

  16. Comparative analysis of quantitative reverse transcription real-time PCR and commercial enzyme imunoassays for detection of enterotoxigenic Bacillus thuringiensis isolates.

    PubMed

    Kaminska, Paulina S; Yernazarova, Aliya; Murawska, Emilia; Swiecicki, Jakub; Fiedoruk, Krzysztof; Bideshi, Dennis K; Swiecicka, Izabela

    2014-08-01

    Entomopathogenic Bacillus thuringiensis is closely related to Bacillus cereus, a human pathogen known to cause emesis and diarrhea. Standard detection methods do not distinguish these bacilli. Hemolysin BL (hbl) and non-hemolytic enterotoxin (nhe) genes that encode, respectively, HBL and NHE enterotoxins, are known to be harbored in both bacterial species, suggesting that differentiation of these bacilli is clinically and epidemiologically relevant. In this study the reliability of quantitative reverse transcription real-time PCR (qRT-PCR) and enzyme immunoassays (EIAs) in detecting hbl and nhe transcripts and corresponding toxins in environmental B. thuringiensis isolates was assessed. At least one enterotoxin gene was present in each isolate, and nhe or hbl genes were found in 85% and 55% of the strains, respectively. Based on statistical analyses, both BCET-RPLA and Duopath detected HBL at similar levels, and TECRA and Duopath can be used interchangeably for the detection of NHE, although TECRA has significantly lower sensitivity than Duopath. Thus, as potential enterotoxic B. thuringiensis strains occur in the natural environment, and EIA results may not correspond with the presence of enterotoxin genes and their expression, we suggest that reliable interpretation will be significantly enhanced by including qRT-PCR to support inferences based on EIAs.

  17. A novel quantitative reverse-transcription PCR (qRT-PCR) for the enumeration of total bacteria, using meat micro-flora as a model.

    PubMed

    Dolan, Anthony; Burgess, Catherine M; Barry, Thomas B; Fanning, Seamus; Duffy, Geraldine

    2009-04-01

    A sensitive quantitative reverse-transcription PCR (qRT-PCR) method was developed for enumeration of total bacteria. Using two sets of primers separately to target the ribonuclease-P (RNase P) RNA transcripts of gram positive and gram negative bacteria. Standard curves were generated using SYBR Green I kits for the LightCycler 2.0 instrument (Roche Diagnostics) to allow quantification of mixed microflora in liquid media. RNA standards were used and extracted from known cell equivalents and subsequently converted to cDNA for the construction of standard curves. The number of mixed bacteria in culture was determined by qRT-PCR, and the results correlated (r(2)=0.88, rsd=0.466) with the total viable count over the range from approx. Log(10) 3 to approx. Log(10) 7 CFU ml(-1). The rapid nature of this assay (8 h) and its potential as an alternative method to the standard plate count method to predict total viable counts and shelf life are discussed.

  18. In-vivo quantitative proteomics reveals a key contribution of post-transcriptional mechanisms to the circadian regulation of liver metabolism.

    PubMed

    Robles, Maria S; Cox, Jürgen; Mann, Matthias

    2014-01-01

    Circadian clocks are endogenous oscillators that drive the rhythmic expression of a broad array of genes, orchestrating metabolism and physiology. Recent evidence indicates that post-transcriptional and post-translational mechanisms play essential roles in modulating temporal gene expression for proper circadian function, particularly for the molecular mechanism of the clock. Due to technical limitations in large-scale, quantitative protein measurements, it remains unresolved to what extent the circadian clock regulates metabolism by driving rhythms of protein abundance. Therefore, we aimed to identify global circadian oscillations of the proteome in the mouse liver by applying in vivo SILAC mouse technology in combination with state of the art mass spectrometry. Among the 3000 proteins accurately quantified across two consecutive cycles, 6% showed circadian oscillations with a defined phase of expression. Interestingly, daily rhythms of one fifth of the liver proteins were not accompanied by changes at the transcript level. The oscillations of almost half of the cycling proteome were delayed by more than six hours with respect to the corresponding, rhythmic mRNA. Strikingly we observed that the length of the time lag between mRNA and protein cycles varies across the day. Our analysis revealed a high temporal coordination in the abundance of proteins involved in the same metabolic process, such as xenobiotic detoxification. Apart from liver specific metabolic pathways, we identified many other essential cellular processes in which protein levels are under circadian control, for instance vesicle trafficking and protein folding. Our large-scale proteomic analysis reveals thus that circadian post-transcriptional and post-translational mechanisms play a key role in the temporal orchestration of liver metabolism and physiology.

  19. Strand-specific community RNA-seq reveals prevalent and dynamic antisense transcription in human gut microbiota

    PubMed Central

    Bao, Guanhui; Wang, Mingjie; Doak, Thomas G.; Ye, Yuzhen

    2015-01-01

    Metagenomics and other meta-omics approaches (including metatranscriptomics) provide insights into the composition and function of microbial communities living in different environments or animal hosts. Metatranscriptomics research provides an unprecedented opportunity to examine gene regulation for many microbial species simultaneously, and more importantly, for the majority that are unculturable microbial species, in their natural environments (or hosts). Current analyses of metatranscriptomic datasets focus on the detection of gene expression levels and the study of the relationship between changes of gene expression and changes of environment. As a demonstration of utilizing metatranscriptomics beyond these common analyses, we developed a computational and statistical procedure to analyze the antisense transcripts in strand-specific metatranscriptomic datasets. Antisense RNAs encoded on the DNA strand opposite a gene’s CDS have the potential to form extensive base-pairing interactions with the corresponding sense RNA, and can have important regulatory functions. Most studies of antisense RNAs in bacteria are rather recent, are mostly based on transcriptome analysis, and have been applied mainly to single bacterial species. Application of our approaches to human gut-associated metatranscriptomic datasets allowed us to survey antisense transcription for a large number of bacterial species associated with human beings. The ratio of protein coding genes with antisense transcription ranges from 0 to 35.8% (median = 10.0%) among 47 species. Our results show that antisense transcription is dynamic, varying between human individuals. Functional enrichment analysis revealed a preference of certain gene functions for antisense transcription, and transposase genes are among the most prominent ones (but we also observed antisense transcription in bacterial house-keeping genes). PMID:26388849

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

    NASA Astrophysics Data System (ADS)

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

    2015-10-01

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

  1. Estimation methods for monthly humidity from dynamical downscaling data for quantitative assessments of climate change impacts

    NASA Astrophysics Data System (ADS)

    Ueyama, Hideki

    2012-07-01

    Methods are proposed to estimate the monthly relative humidity and wet bulb temperature based on observations from a dynamical downscaling coupled general circulation model with a regional climate model (RCM) for a quantitative assessment of climate change impacts. The water vapor pressure estimation model developed was a regression model with a monthly saturated water vapor pressure that used minimum air temperature as a variable. The monthly minimum air temperature correction model for RCM bias was developed by stepwise multiple regression analysis using the difference in monthly minimum air temperatures between observations and RCM output as a dependent variable and geographic factors as independent variables. The wet bulb temperature was estimated using the estimated water vapor pressure, air temperature, and atmospheric pressure at ground level both corrected for RCM bias. Root mean square errors of the data decreased considerably in August.

  2. Mesoscopic dynamics of fermionic cold atoms - Quantitative analysis of transport coefficients and relaxation times

    NASA Astrophysics Data System (ADS)

    Kikuchi, Yuta; Tsumura, Kyosuke; Kunihiro, Teiji

    2016-05-01

    We give a quantitative analysis of the dynamical properties of fermionic cold atomic gases in normal phase, such as the shear viscosity, heat conductivity, and viscous relaxation times, using the novel microscopic expressions derived by the renormalization group (RG) method, where the Boltzmann equation is faithfully solved to extract the hydrodynamics without recourse to any ansatz. In particular, we examine the quantum statistical effects, temperature dependence, and scattering-length dependence of the transport coefficients and the viscous relaxation times. The numerical calculation shows that the relation τπ = η / P, which is derived in the relaxation-time approximation (RTA) and is used in most of the literature, turns out to be satisfied quite well, while the similar relation for the viscous relaxation time τJ of the heat conductivity is satisfied only approximately with a considerable error.

  3. Quantitative agent-based firm dynamics simulation with parameters estimated by financial and transaction data analysis

    NASA Astrophysics Data System (ADS)

    Ikeda, Yuichi; Souma, Wataru; Aoyama, Hideaki; Iyetomi, Hiroshi; Fujiwara, Yoshi; Kaizoji, Taisei

    2007-03-01

    Firm dynamics on a transaction network is considered from the standpoint of econophysics, agent-based simulations, and game theory. In this model, interacting firms rationally invest in a production facility to maximize net present value. We estimate parameters used in the model through empirical analysis of financial and transaction data. We propose two different methods ( analytical method and regression method) to obtain an interaction matrix of firms. On a subset of a real transaction network, we simulate firm's revenue, cost, and fixed asset, which is the accumulated investment for the production facility. The simulation reproduces the quantitative behavior of past revenues and costs within a standard error when we use the interaction matrix estimated by the regression method, in which only transaction pairs are taken into account. Furthermore, the simulation qualitatively reproduces past data of fixed assets.

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

    SciTech Connect

    Lettieri, S.; Capello, V.; Santamaria, L.; Maddalena, P.

    2013-12-09

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

  5. Methods for quantitative evaluation of dynamics of repair proteins within irradiated cells

    NASA Astrophysics Data System (ADS)

    Hable, V.; Dollinger, G.; Greubel, C.; Hauptner, A.; Krücken, R.; Dietzel, S.; Cremer, T.; Drexler, G. A.; Friedl, A. A.; Löwe, R.

    2006-04-01

    Living HeLa cells are irradiated well directed with single 100 MeV oxygen ions by the superconducting ion microprobe SNAKE, the Superconducting Nanoscope for Applied Nuclear (=Kern-) Physics Experiments, at the Munich 14 MV tandem accelerator. Various proteins, which are involved directly or indirectly in repair processes, accumulate as clusters (so called foci) at DNA-double strand breaks (DSBs) induced by the ions. The spatiotemporal dynamics of these foci built by the phosphorylated histone γ-H2AX are studied. For this purpose cells are irradiated in line patterns. The γ-H2AX is made visible under the fluorescence microscope using immunofluorescence techniques. Quantitative analysis methods are developed to evaluate the data of the microscopic images in order to analyze movement of the foci and their changing size.

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

    NASA Astrophysics Data System (ADS)

    Plougonven, Erwan; Bernard, Dominique; Viot, Philippe

    2006-08-01

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

  7. ReAsH as a Quantitative Probe of In-Cell Protein Dynamics.

    PubMed

    Gelman, Hannah; Wirth, Anna Jean; Gruebele, Martin

    2016-04-01

    The tetracysteine (tc) tag/biarsenical dye system (FlAsH or ReAsH) promises to combine the flexibility of fluorescent protein tags with the small size of dye labels, allowing in-cell study of target proteins that are perturbed by large protein tags. Quantitative thermodynamic and kinetic studies in-cell using FlAsH and ReAsH have been hampered by methodological complexities presented by the fluorescence properties of the tag-dye complex probed by either Förster resonance energy transfer (FRET) or direct excitation. We label the model protein phosphoglycerate kinase (PGK) with AcGFP1 and ReAsH for direct comparison with AcGFP1/mCherry-labeled PGK. We find that fast relaxation imaging (FReI), combining millisecond temperature jump kinetics with fluorescence microscopy detection, circumvents many of the difficulties encountered working with the ReAsH system, allowing us to obtain quantitative FRET measurements of protein stability and kinetics both in vitro and in cells. We also demonstrate the to us surprising result that fluorescence from directly excited, unburied ReAsH at the C-terminus of the model protein also reports on folding in vitro and in cells. Comparing the ReAsH-labeled protein to a construct labeled with two fluorescent protein tags allows us to evaluate how a bulkier protein tag affects protein dynamics in cells and in vitro. We find that the average folding rate in the cell is closer to the in vitro rate with the smaller tag, highlighting the effect of tags on quantitative in-cell measurements. PMID:26959408

  8. Quantitative molecular characterization of bovine vitreous and lens with non-invasive dynamic light scattering

    NASA Technical Reports Server (NTRS)

    Ansari, R. R.; Suh, K. I.; Dunker, S.; Kitaya, N.; Sebag, J.

    2001-01-01

    The non-invasive technique of dynamic light scattering (DLS) was used to quantitatively characterize vitreous and lens structure on a molecular level by measuring the sizes of the predominant particles and mapping the three-dimensional topographic distribution of these structural macromolecules in three spatial dimensions. The results of DLS measurements in five fresh adult bovine eyes were compared to DLS measurements in model solutions of hyaluronan (HA) and collagen (Coll). In the bovine eyes DLS measurements were obtained from excised samples of gel and liquid vitreous and compared to the model solutions. Measurements in whole vitreous were obtained at multiple points posterior to the lens to generate a three-dimensional 'map' of molecular structure. The macromolecule distribution in bovine lens was similarly characterized.In each bovine vitreous (Bo Vit) specimen, DLS predominantly detected two distinct particles, which differed in diffusion properties and hence size. Comparisons with model vitreous solutions demonstrated that these most likely corresponded to the Coll and HA components of vitreous. Three-dimensional mapping of Bo Vit found heterogeneity throughout the vitreous body, with different particle size distributions for Coll and HA at different loci. In contrast, the three-dimensional distribution of lens macromolecules was more homogeneous. Thus, the non-invasive DLS technique can quantitate the average sizes of vitreous and lens macromolecules and map their three-dimensional distribution. This method to assess quantitatively the macromolecular structure of vitreous and lens should be useful for clinical as well as experimental applications in health and disease. Copyright 2001 Academic Press.

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

    SciTech Connect

    YANG, CHIN-RANG

    2013-12-11

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

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

    NASA Astrophysics Data System (ADS)

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

    2013-03-01

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

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

    NASA Astrophysics Data System (ADS)

    Suminar, Wulan; Saepuloh, Asep; Meilano, Irwan

    2016-05-01

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

  12. Single-molecule RNA observation in vivo reveals dynamics of co-transcriptional splicing

    NASA Astrophysics Data System (ADS)

    Ferguson, M. L.; Coulon, A.; de Turris, V.; Palangat, M.; Chow, C. C.; Singer, R. H.; Larson, D. R.

    2013-03-01

    The synthesis of pre-mRNA and the splicing of that pre-mRNA to form completed transcripts requires coordination between two large multi-subunit complexes (the transcription elongation complex and the spliceosome). How this coordination occurs in vivo is unknown. Here we report the first experimental observation of transcription and splicing occurring at the same gene in living cells. By utilizing the PP7/MS2 fluorescent RNA reporter system, we can directly observe two distinct regions of the nascent RNA, allowing us to measure the rise and fall time of the intron and exon of a reporter gene stably integrated into a human cell line. The reporter gene consists of a beta globin gene where we have inserted a 24 RNA hairpin cassette into the intron/exon. Upon synthesis, the RNA hairpins are tightly bound by fluorescently-labeled PP7/MS2 bacteriophage coat proteins. After gene induction, a single locus of active transcription in the nucleus shows fluorescence intensity changes characteristic of the synthesis and excision of the intron/exon. Using fluctuation analysis, we determine the elongation rate to be 1.5 kb/min. From the temporal cross correlation function, we determine that splicing of this gene must be co-transcriptional with a splicing time of ~100 seconds before termination and a ~200 second pause at termination. We propose that dual-color RNA imaging may be extended to investigate other mechanisms of transcription, gene regulation, and RNA processing.

  13. The in vivo dynamics of TCERG1, a factor that couples transcriptional elongation with splicing.

    PubMed

    Sánchez-Hernández, Noemí; Boireau, Stéphanie; Schmidt, Ute; Muñoz-Cobo, Juan Pablo; Hernández-Munain, Cristina; Bertrand, Edouard; Suñé, Carlos

    2016-04-01

    Coupling between transcription and RNA processing is key for gene regulation. Using live-cell photobleaching techniques, we investigated the factor TCERG1, which coordinates transcriptional elongation with splicing. We demonstrate that TCERG1 is highly mobile in the nucleoplasm and that this mobility is slightly decreased when it is associated with speckles. Dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) but not α-amanitin treatment reduced the mobility of TCERG1, which suggests interaction with paused transcription elongation complexes. We found that TCERG1 mobility is rapid at the transcription site (TS) of a reporter that splices post-transcriptionally and that TCERG1 is recruited to the active TS independent of the CTD of RNAPII, thus excluding phosphorylated CTD as a requirement for recruiting this factor to the TS. Importantly, the mobility of TCERG1 is reduced when the reporter splices cotranscriptionally, which suggests that TCERG1 forms new macromolecular complexes when splicing occurs cotranscriptionally. In this condition, spliceostatin A has no effect, indicating that TCERG1 rapidly binds and dissociates from stalled spliceosomal complexes and that the mobility properties of TCERG1 do not depend on events occurring after the initial spliceosome formation. Taken together, these data suggest that TCERG1 binds independently to elongation and splicing complexes, thus performing their coupling by transient interactions rather than by stable association with one or the other complexes. This finding has conceptual implications for understanding the coupling between transcription and RNA processing. PMID:26873599

  14. Development of TaqMan real-time reverse transcription-polymerase chain reaction for the detection and quantitation of porcine kobuvirus.

    PubMed

    Zhu, Xiangdong; Wang, Yufei; Chen, Jianfei; Zhang, Xin; Shi, Hongyan; Shi, Da; Gao, Jing; Feng, Li

    2016-08-01

    Porcine kobuvirus (PKV) is a newly emerging virus that has been detected in diarrheic pigs. Presently, reverse transcription-polymerase chain reaction (RT-PCR) and RT-loop-mediated amplification are the only methods that can be used to detect PKV. To develop a TaqMan real-time RT-PCR for the rapid detection and quantitation of PKV nucleic acid in fecal samples, a pair of primers and a probe were designed to amplify the conserved 3D region of the PKV genome. After optimization, the TaqMan real-time RT-PCR was highly specific and ∼1000 times more sensitive than conventional RT-PCR, and the detection limit was as low as 30 DNA copies. Among the 148 intestinal samples from piglets with diarrhea, 136 and 118 were positive based on the TaqMan and conventional RT-PCR methods, respectively, indicating that the TaqMan RT-PCR was more sensitive than conventional RT-PCR, and the total concordance of the two methods was approximately 87.84%. Thus, the TaqMan real-time RT-PCR should be a useful tool for the early detection and quantitation of PKV. PMID:26912233

  15. Quantitative analysis of beta-actin, beta-2-microglobulin and porphobilinogen deaminase mRNA and their comparison as control transcripts for RT-PCR.

    PubMed

    Lupberger, J; Kreuzer, K A; Baskaynak, G; Peters, U R; le Coutre, P; Schmidt, C A

    2002-02-01

    Quantitation of target mRNAs using the reverse-transcription polymerase chain reaction found a widespread field of application in diverse biomedical diagnostic assays. However, the problem of varying sample quality has to be solved by correcting target molecule amounts through detection of an endogenous control template. The choice of an appropriate reference gene is still object of debate as pseudogene co-amplification and expression level variations may limit the usefulness of some currently used reference reactions. We compared quantitative expression levels of the commonly used endogenous reference genes beta-actin (beta-actin), beta-2-microglobulin (beta2-MG) and porphobilinogen deaminase (PBDG) using the TaqMan chemistry. With these assays we investigated the respective expression patterns in K562 cells and leucocytes of normal individuals as well as of malignoma patients. In K562 cells 1544+246 beta-actin, 65+30 beta2-MG and 22+/-8 PBDG copies/cell were detected. In normal leucocytes 491+/-97 beta-actin, 40+/-17 beta2-MG and <1 PBDG copies/cell were quantified. Leucocytes of various malignancies exhibited 84+/-51 beta-actin, 106+/-8 beta2-MG and <1 PBDG copies/cell. We conclude that beta2-MG is the most suitable reference gene tested as its variation between different sample origins and within distinct cell types was acceptable low.

  16. Heterogeneous dynamics in DNA site discrimination by the structurally homologous DNA-binding domains of ETS-family transcription factors.

    PubMed

    He, Gaofei; Tolic, Ana; Bashkin, James K; Poon, Gregory M K

    2015-04-30

    The ETS family of transcription factors exemplifies current uncertainty in how eukaryotic genetic regulators with overlapping DNA sequence preferences achieve target site specificity. PU.1 and Ets-1 represent archetypes for studying site discrimination by ETS proteins because their DNA-binding domains are the most divergent in sequence, yet they share remarkably superimposable DNA-bound structures. To gain insight into the contrasting thermodynamics and kinetics of DNA recognition by these two proteins, we investigated the structure and dynamics of site discrimination by their DNA-binding domains. Electrophoretic mobilities of complexes formed by the two homologs with circularly permuted binding sites showed significant dynamic differences only for DNA complexes of PU.1. Free solution measurements by dynamic light scattering showed PU.1 to be more dynamic than Ets-1; moreover, dynamic changes are strongly coupled to site discrimination by PU.1, but not Ets-1. Interrogation of the protein/DNA interface by DNA footprinting showed similar accessibility to dimethyl sulfate for PU.1/DNA and Ets-1/DNA complexes, indicating that the dynamics of PU.1/DNA complexes reside primarily outside that interface. An information-based analysis of the two homologs' binding motifs suggests a role for dynamic coupling in PU.1's ability to enforce a more stringent sequence preference than Ets-1 and its proximal sequence homologs.

  17. Heterogeneous dynamics in DNA site discrimination by the structurally homologous DNA-binding domains of ETS-family transcription factors.

    PubMed

    He, Gaofei; Tolic, Ana; Bashkin, James K; Poon, Gregory M K

    2015-04-30

    The ETS family of transcription factors exemplifies current uncertainty in how eukaryotic genetic regulators with overlapping DNA sequence preferences achieve target site specificity. PU.1 and Ets-1 represent archetypes for studying site discrimination by ETS proteins because their DNA-binding domains are the most divergent in sequence, yet they share remarkably superimposable DNA-bound structures. To gain insight into the contrasting thermodynamics and kinetics of DNA recognition by these two proteins, we investigated the structure and dynamics of site discrimination by their DNA-binding domains. Electrophoretic mobilities of complexes formed by the two homologs with circularly permuted binding sites showed significant dynamic differences only for DNA complexes of PU.1. Free solution measurements by dynamic light scattering showed PU.1 to be more dynamic than Ets-1; moreover, dynamic changes are strongly coupled to site discrimination by PU.1, but not Ets-1. Interrogation of the protein/DNA interface by DNA footprinting showed similar accessibility to dimethyl sulfate for PU.1/DNA and Ets-1/DNA complexes, indicating that the dynamics of PU.1/DNA complexes reside primarily outside that interface. An information-based analysis of the two homologs' binding motifs suggests a role for dynamic coupling in PU.1's ability to enforce a more stringent sequence preference than Ets-1 and its proximal sequence homologs. PMID:25824951

  18. The transcriptional co-activator LEDGF/p75 displays a dynamic scan-and-lock mechanism for chromatin tethering

    PubMed Central

    Hendrix, Jelle; Gijsbers, Rik; De Rijck, Jan; Voet, Arnout; Hotta, Jun-ichi; McNeely, Melissa; Hofkens, Johan; Debyser, Zeger; Engelborghs, Yves

    2011-01-01

    Nearly all cellular and disease related functions of the transcriptional co-activator lens epithelium-derived growth factor (LEDGF/p75) involve tethering of interaction partners to chromatin via its conserved integrase binding domain (IBD), but little is known about the mechanism of in vivo chromatin binding and tethering. In this work we studied LEDGF/p75 in real-time in living HeLa cells combining different quantitative fluorescence techniques: spot fluorescence recovery after photobleaching (sFRAP) and half-nucleus fluorescence recovery after photobleaching (hnFRAP), continuous photobleaching, fluorescence correlation spectroscopy (FCS) and an improved FCS method to study diffusion dependence of chromatin binding, tunable focus FCS. LEDGF/p75 moves about in nuclei of living cells in a chromatin hopping/scanning mode typical for transcription factors. The PWWP domain of LEDGF/p75 is necessary, but not sufficient for in vivo chromatin binding. After interaction with HIV-1 integrase via its IBD, a general protein–protein interaction motif, kinetics of LEDGF/p75 shift to 75-fold larger affinity for chromatin. The PWWP is crucial for locking the complex on chromatin. We propose a scan-and-lock model for LEDGF/p75, unifying paradoxical notions of transcriptional co-activation and lentiviral integration targeting. PMID:20974633

  19. Quantitative image analysis to characterize the dynamics of Listeria monocytogenes biofilms.

    PubMed

    Mosquera-Fernández, M; Sanchez-Vizuete, P; Briandet, R; Cabo, M L; Balsa-Canto, E

    2016-11-01

    This work shows that the combination of two-dimensional (2D) and three-dimensional (3D) analyses of images acquired by confocal laser scanning microscopy facilitates the quantitative spatiotemporal characterization of architectures formed by Listeria monocytogenes biofilms. In particular, the analysis of structural features such as maximum thickness, biovolume, areal porosity and maximum diffusion distance allowed elucidating differences in biofilm formation of three L. monocytogenes strains (L1A1, CECT5873 and CECT4032). The analysis showed a common sequence for all strains. In the first phase, independent clusters evolve to interconnected clusters and honeycomb-like structures. Flat biofilms characterized the second phase. The structures disappear in the third phase. Nevertheless, the duration of the phases differed from strain to strain. L1A1 strain exhibited the slowest dynamics and the thickest biofilms while the strain CECT4032 presented the faster dynamics and the thinnest biofilms. Also, the number of dead cells varies significantly from strain to strain. From the results of the analysis, it can be concluded that 2D parameters are critical to differentiating morphological features while 3D parameters ease the interpretation and comparative study of the different phases during the life cycle of biofilms.

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

    NASA Astrophysics Data System (ADS)

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

    2015-03-01

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

  1. Dynamic subnuclear relocalization of WRKY40, a potential new mechanism of ABA-dependent transcription factor regulation.

    PubMed

    Geilen, Katja; Böhmer, Maik

    2015-01-01

    The phytohormone ABA plays a major role during plant development, e.g. seed maturation and seed germination, and during adaptation to abiotic stresses like stomatal aperture regulation. The three closely related WRKY transcription factors WRKY18, WRKY40 and WRKY60 function in ABA signal transduction. We recently demonstrated that WRKY18 and WRKY40 but not WRKY60 localize to nuclear bodies in A. thaliana mesophyll protoplasts. WRKY40, a negative regulator of ABA-dependent inhibition of seed germination, relocalizes from PNBs to the nucleoplasm in the presence of ABA in a dynamic and phosphorylation-dependent manner. We propose that subnuclear relocalization of WRKY40 might constitute a new regulatory mechanism of ABA-dependent modulation of transcription factor activity. PMID:26479147

  2. Quantitative modeling of virus evolutionary dynamics and adaptation in serial passages using empirically inferred fitness landscapes.

    PubMed

    Woo, Hyung Jun; Reifman, Jaques

    2014-01-01

    We describe a stochastic virus evolution model representing genomic diversification and within-host selection during experimental serial passages under cell culture or live-host conditions. The model incorporates realistic descriptions of the virus genotypes in nucleotide and amino acid sequence spaces, as well as their diversification from error-prone replications. It quantitatively considers factors such as target cell number, bottleneck size, passage period, infection and cell death rates, and the replication rate of different genotypes, allowing for systematic examinations of how their changes affect the evolutionary dynamics of viruses during passages. The relative probability for a viral population to achieve adaptation under a new host environment, quantified by the rate with which a target sequence frequency rises above 50%, was found to be most sensitive to factors related to sequence structure (distance from the wild type to the target) and selection strength (host cell number and bottleneck size). For parameter values representative of RNA viruses, the likelihood of observing adaptations during passages became negligible as the required number of mutations rose above two amino acid sites. We modeled the specific adaptation process of influenza A H5N1 viruses in mammalian hosts by simulating the evolutionary dynamics of H5 strains under the fitness landscape inferred from multiple sequence alignments of H3 proteins. In light of comparisons with experimental findings, we observed that the evolutionary dynamics of adaptation is strongly affected not only by the tendency toward increasing fitness values but also by the accessibility of pathways between genotypes constrained by the genetic code.

  3. A dynamic model for PC4 coactivator function in RNA polymerase II transcription

    PubMed Central

    Malik, Sohail; Guermah, Mohamed; Roeder, Robert G.

    1998-01-01

    Human positive cofactor (PC4) acts as a general coactivator for activator-dependent transcription by RNA polymerase II. Here we show that PC4 coactivator function, in contrast to basal (activator-independent) transcription, is dependent both on TATA binding protein (TBP)-associated factors (TAFs) in TFIID and on TFIIH. Surprisingly, PC4 strongly represses transcription initiation by minimal preinitiation complexes in the absence of TAFs and TFIIH, while simultaneously promoting the formation of these complexes. Furthermore, TFIIH and TAFII250, the largest subunit of TFIID, can both phosphorylate PC4. These results provide evidence for an inactive, PC4-induced intermediate in preinitiation complex assembly and point to TFIIH and TAF requirements for its progression into a functional preinitiation complex. Thus PC4 coactivator activity is realized in a stepwise series of events reminiscent of prokaryotic activation pathways involving conversion of inactive RNA polymerase-promoter complexes to an initiation-competent state. PMID:9482861

  4. The Transcriptional Response in Human Umbilical Vein Endothelial Cells Exposed to Insulin: A Dynamic Gene Expression Approach

    PubMed Central

    Di Camillo, Barbara; Sanavia, Tiziana; Iori, Elisabetta; Bronte, Vincenzo; Roncaglia, Enrica; Maran, Alberto; Avogaro, Angelo; Toffolo, Gianna; Cobelli, Claudio

    2010-01-01

    Background In diabetes chronic hyperinsulinemia contributes to the instability of the atherosclerotic plaque and stimulates cellular proliferation through the activation of the MAP kinases, which in turn regulate cellular proliferation. However, it is not known whether insulin itself could increase the transcription of specific genes for cellular proliferation in the endothelium. Hence, the characterization of transcriptional modifications in endothelium is an important step for a better understanding of the mechanism of insulin action and the relationship between endothelial cell dysfunction and insulin resistance. Methodology and principal findings The transcriptional response of endothelial cells in the 440 minutes following insulin stimulation was monitored using microarrays and compared to a control condition. About 1700 genes were selected as differentially expressed based on their treated minus control profile, thus allowing the detection of even small but systematic changes in gene expression. Genes were clustered in 7 groups according to their time expression profile and classified into 15 functional categories that can support the biological effects of insulin, based on Gene Ontology enrichment analysis. In terms of endothelial function, the most prominent processes affected were NADH dehydrogenase activity, N-terminal myristoylation domain binding, nitric-oxide synthase regulator activity and growth factor binding. Pathway-based enrichment analysis revealed “Electron Transport Chain” significantly enriched. Results were validated on genes belonging to “Electron Transport Chain” pathway, using quantitative RT-PCR. Conclusions As far as we know, this is the first systematic study in the literature monitoring transcriptional response to insulin in endothelial cells, in a time series microarray experiment. Since chronic hyperinsulinemia contributes to the instability of the atherosclerotic plaque and stimulates cellular proliferation, some of the

  5. Modeling, molecular dynamics, and docking assessment of transcription factor rho: a potential drug target in Brucella melitensis 16M

    PubMed Central

    Pradeepkiran, Jangampalli Adi; Kumar, Konidala Kranthi; Kumar, Yellapu Nanda; Bhaskar, Matcha

    2015-01-01

    The zoonotic disease brucellosis, a chronic condition in humans affecting renal and cardiac systems and causing osteoarthritis, is caused by Brucella, a genus of Gram-negative, facultative, intracellular pathogens. The mode of transmission and the virulence of the pathogens are still enigmatic. Transcription regulatory elements, such as rho proteins, play an important role in the termination of transcription and/or the selection of genes in Brucella. Adverse effects of the transcription inhibitors play a key role in the non-successive transcription challenges faced by the pathogens. In the investigation presented here, we computationally predicted the transcription termination factor rho (TtFRho) inhibitors against Brucella melitensis 16M via a structure-based method. In view the unknown nature of its crystal structure, we constructed a robust three-dimensional homology model of TtFRho’s structure by comparative modeling with the crystal structure of the Escherichia coli TtFRho (Protein Data Bank ID: 1PVO) as a template in MODELLER (v 9.10). The modeled structure was optimized by applying a molecular dynamics simulation for 2 ns with the CHARMM (Chemistry at HARvard Macromolecular Mechanics) 27 force field in NAMD (NAnoscale Molecular Dynamics program; v 2.9) and then evaluated by calculating the stereochemical quality of the protein. The flexible docking for the interaction phenomenon of the template consists of ligand-related inhibitor molecules from the ZINC (ZINC Is Not Commercial) database using a structure-based virtual screening strategy against minimized TtFRho. Docking simulations revealed two inhibitors compounds – ZINC24934545 and ZINC72319544 – that showed high binding affinity among 2,829 drug analogs that bind with key active-site residues; these residues are considered for protein-ligand binding and unbinding pathways via steered molecular dynamics simulations. Arg215 in the model plays an important role in the stability of the protein

  6. Selection of reliable reference genes for expression studies by reverse transcription quantitative real-time PCR in litchi under different experimental conditions.

    PubMed

    Zhong, Hai-Ying; Chen, Jian-Wen; Li, Cai-Qin; Chen, Lei; Wu, Jian-Yang; Chen, Jian-Ye; Lu, Wang-Jin; Li, Jian-Guo

    2011-04-01

    Reverse transcription quantitative real-time PCR (RT-qPCR), a sensitive technique for quantifying gene expression, depends on the stability of the reference gene(s) used for data normalization. Only a few studies on reference genes have been done in fruit trees and none in litchi. In the present study, seven frequently used candidate reference genes, including actin (ACTIN), glyceraldehyde-3-phosphate-dehydrogenase (GADPH), elongation factor 1-alpha (EF-1α), poly ubiquitin enzyme (UBQ), α-tubulin (TUA), β-tubulin (TUB) and RNA polymerase-II transcription factor (RPII), were evaluated for their expression stability in litchi. A total of 78 samples, including different varieties, tissues, organs, developmental stages and treatments, such as NAA, shading and girdling plus defoliation, were addressed in this analysis. Our results showed that GAPDH was the most suitable reference gene among all the tested samples, different organs and NAA treatment. ACTIN was stably expressed in varieties and fruit developmental stages. RPII and UBQ exhibited better expression stability in tissues. EF-1α was the most stable gene in shading and girdling plus defoliation treatments. Moreover, using combination of two genes as reference genes might improve the reliability of gene expression by RT-qPCR in litchi. A better combination was GAPDH + EF-1α or GAPDH + ACTIN for all the examined samples. In addition, the validated reference genes were further relied on to quantify the expression of an interested gene, LcARF13 under different experimental conditions. These results first provide guidelines for reference genes selection under different experimental conditions and also a foundation for more accurate and widespread use of RT-qPCR in litchi. PMID:21301853

  7. Gene expression analysis in biomarker research and early drug development using function tested reverse transcription quantitative real-time PCR assays.

    PubMed

    Lohmann, Sabine; Herold, Andrea; Bergauer, Tobias; Belousov, Anton; Betzl, Gisela; Demario, Mark; Dietrich, Manuel; Luistro, Leopoldo; Poignée-Heger, Manuela; Schostack, Kathy; Simcox, Mary; Walch, Heiko; Yin, Xuefeng; Zhong, Hua; Weisser, Martin

    2013-01-01

    The identification of new biomarkers is essential in the implementation of personalized health care strategies that offer new therapeutic approaches with optimized and individualized treatment. In support of hypothesis generation and testing in the course of our biomarker research an online portal and respective function-tested reverse transcription quantitative real-time PCR assays (RT-qPCR) facilitated the selection of relevant biomarker genes. We have established workflows applicable for convenient high throughput gene expression analysis in biomarker research with cell lines (in vitro studies) and xenograft mouse models (in vivo studies) as well as formalin-fixed paraffin-embedded tissue (FFPET) sections from various human research and clinical tumor samples. Out of 92 putative biomarker candidate genes selected in silico, 35 were shown to exhibit differential expression in various tumor cell lines. These were further analysed by in vivo xenograft mouse models, which identified 13 candidate genes including potential response prediction biomarkers and a potential pharmacodynamic biomarker. Six of these candidate genes were selected for further evaluation in FFPET samples, where optimized RNA isolation, reverse transcription and qPCR assays provided reliable determination of relative expression levels as precondition for differential gene expression analysis of FFPET samples derived from projected clinical studies. Thus, we successfully applied function tested RT-qPCR assays in our biomarker research for hypothesis generation with in vitro and in vivo models as well as for hypothesis testing with human FFPET samples. Hence, appropriate function-tested RT-qPCR assays are available in biomarker research accompanying the different stages of drug development, starting from target identification up to early clinical development. The workflow presented here supports the identification and validation of new biomarkers and may lead to advances in efforts to achieve the

  8. Quantitative steps in the evolution of metabolic organisation as specified by the Dynamic Energy Budget theory.

    PubMed

    Kooijman, S A L M; Troost, T A

    2007-02-01

    The Dynamic Energy Budget (DEB) theory quantifies the metabolic organisation of organisms on the basis of mechanistically inspired assumptions. We here sketch a scenario for how its various modules, such as maintenance, storage dynamics, development, differentiation and life stages could have evolved since the beginning of life. We argue that the combination of homeostasis and maintenance induced the development of reserves and that subsequent increases in the maintenance costs came with increases of the reserve capacity. Life evolved from a multiple reserves - single structure system (prokaryotes, many protoctists) to systems with multiple reserves and two structures (plants) or single reserve and single structure (animals). This had profound consequences for the possible effects of temperature on rates. We present an alternative explanation for what became known as the down-regulation of maintenance at high growth rates in microorganisms; the density of the limiting reserve increases with the growth rate, and reserves do not require maintenance while structure-specific maintenance costs are independent of the growth rate. This is also the mechanism behind the variation of the respiration rate with body size among species. The DEB theory specifies reserve dynamics on the basis of the requirements of weak homeostasis and partitionability. We here present a new and simple mechanism for this dynamics which accounts for the rejection of mobilised reserve by busy maintenance/growth machinery. This module, like quite a few other modules of DEB theory, uses the theory of Synthesising Units; we review recent progress in this field. The plasticity of membranes that evolved in early eukaryotes is a major step forward in metabolic evolution; we discuss quantitative aspects of the efficiency of phagocytosis relative to the excretion of digestive enzymes to illustrate its importance. Some processes of adaptation and gene expression can be understood in terms of allocation

  9. [Quantitative estimation of the dynamics of adventive flora (by the example of the Tula region)].

    PubMed

    Khorun, L V; Zakharov, V G; Sokolov, D D

    2006-01-01

    The rate of enrichment of the Tula region flora with adventive species was quantitatively estimated taking into account the changes of their degree of naturalization during the last 200 years. Numerical score of degree of the naturalization for each species was used to compile the initial database: "0", species absent from the territory; "1", ephemerophyte; "2", colonophyte; "3", epecophyte; "4", argiophyte; "?", lack of data. Non-interpolated integral index of the dynamics of adventive flora NI(t) was calculated from this database. This index displays the sum of the degrees of naturalization of all the adventive species in the flora in some particular year. The interpolation of the initial database, aimed at minimizing the influence of random factors (e.g., gaps in observations or different activity of the researchers in different years), was performed by substituting the "?" symbol by a series of intermediate values based on studies of the data for adjacent territories. Interpolated integral indices I(t) were calculated from the interpolated database. These indices were then leveled out with Morlet wavelets, in order to distinguish random spikes (lasting less than 50 years) from the analyzed signal, and thus approximate the index dynamics to the objective trend that represents the dynamics of the flora and not the rate of activity of the researchers. The dynamics of the adventive flora of the Tula region revealed with this method shows the following facts: 1) average rate of the enrichment of the adventive flora with strange species has been constant for these 200 years and amounted to 15 species per decade; 2) average rate of naturalization was relatively low and constant, amounting to 5 species per decade; 3) fluctuations of the composition and naturalization degree of the Tula region adventive flora species were not shown to be dependant directly on the changes in the territory's economic development during the last two centuries; 4) no periodicity was

  10. T cell activation regulates CD6 alternative splicing by transcription dynamics and SRSF1.

    PubMed

    da Glória, Vânia G; Martins de Araújo, Mafalda; Mafalda Santos, Ana; Leal, Rafaela; de Almeida, Sérgio F; Carmo, Alexandre M; Moreira, Alexandra

    2014-07-01

    The T cell-surface glycoprotein CD6 is a modulator of cellular responses and has been implicated in several autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, and psoriasis. During Ag presentation, CD6 is targeted to the immunological synapse in a ligand binding-dependent manner, in which CD6 domain 3 directly contacts CD166, expressed on the APC. T cell activation results in the induction of CD6Δd3, an alternatively spliced isoform that lacks the ligand-binding domain and thus no longer localizes at the immunological synapse. In this study, we investigated the molecular mechanisms regulating the expression of CD6Δd3 upon human primary T cell activation. Using chromatin immunoprecipitation, we observed an increase in RNA polymerase II occupancy along the CD6 gene and augmented CD6 transcription. We showed that activation leads to transcription-related chromatin modifications, revealed by higher CD6 acetylation levels. Modulation of chromatin conformation using a histone deacetylase inhibitor that increases transcription rate causes an increase of exon 5 skipping. We further showed that the splicing factor SRSF1 binds to a regulatory element in CD6 intron 4, activating exon 5 splicing and promoting exon 5 inclusion. Concomitant with T cell activation-induced exon 5 skipping, we observed a downregulation of SRSF1. Using RNA immunoprecipitation, we showed that in activated T cells, SRSF1 recruitment to the CD6 transcript is impaired by increased chromatin acetylation levels. We propose that upon T cell activation, SRSF1 becomes limiting, and its function in CD6 exon 5 splicing is countered by an increase in CD6 transcription, dependent on chromatin acetylation.

  11. Quantitative Protein and mRNA Profiling Shows Selective Post-Transcriptional Control of Protein Expression by Vasopressin in Kidney Cells*

    PubMed Central

    Khositseth, Sookkasem; Pisitkun, Trairak; Slentz, Dane H.; Wang, Guanghui; Hoffert, Jason D.; Knepper, Mark A.; Yu, Ming-Jiun

    2011-01-01

    Previous studies in yeast have supported the view that post-transcriptional regulation of protein abundances may be more important than previously believed. Here we ask the question: “In a physiological regulatory process (the response of mammalian kidney cells to the hormone vasopressin), what fraction of the expressed proteome undergoes a change in abundance and what fraction of the regulated proteins have corresponding changes in mRNA levels?” In humans and other mammals, vasopressin fulfills a vital homeostatic role (viz. regulation of renal water excretion) by regulating the water channel aquaporin-2 in collecting duct cells. To address the question posed, we utilized large-scale quantitative protein mass spectrometry (LC-MS/MS) employing stable isotopic labeling in cultured mpkCCD cells (‘SILAC’) coupled with transcriptomic profiling using oligonucleotide expression arrays (Affymetrix). Preliminary studies analyzing two nominally identical control samples by SILAC LC-MS/MS yielded a relative S.D. of 13% (for ratios), establishing the precision of the SILAC approach in our hands. We quantified nearly 3000 proteins with nontargeted SILAC LC-MS/MS, comparing vasopressin- versus vehicle-treated samples. Of these proteins 786 of them were quantified in each of 3 experiments, allowing statistical analysis and 188 of these showed significant vasopressin-induced changes in abundance, including aquaporin-2 (20-fold increase). Among the proteins with statistically significant abundance changes, a large fraction (at least one-third) was found to lack changes in the corresponding mRNA species (despite sufficient statistical power), indicating that post-transcriptional regulation of protein abundance plays an important role in the vasopressin response. Bioinformatic analysis of the regulated proteins (versus all transcripts) shows enrichment of glutathione S-transferase isoforms as well as proteins involved in organization of the actin cytoskeleton. The latter

  12. Selection and Evaluation of Reference Genes for Reverse Transcription-Quantitative PCR Expression Studies in a Thermophilic Bacterium Grown under Different Culture Conditions

    PubMed Central

    Cusick, Kathleen D.; Fitzgerald, Lisa A.; Cockrell, Allison L.; Biffinger, Justin C.

    2015-01-01

    The phylum Deinococcus-Thermus is a deeply-branching lineage of bacteria widely recognized as one of the most extremophilic. Members of the Thermus genus are of major interest due to both their bioremediation and biotechnology potentials. However, the molecular mechanisms associated with these key metabolic pathways remain unknown. Reverse-transcription quantitative PCR (RT-qPCR) is a high-throughput means of studying the expression of a large suite of genes over time and under different conditions. The selection of a stably-expressed reference gene is critical when using relative quantification methods, as target gene expression is normalized to expression of the reference gene. However, little information exists as to reference gene selection in extremophiles. This study evaluated 11 candidate reference genes for use with the thermophile Thermus scotoductus when grown under different culture conditions. Based on the combined stability values from BestKeeper and NormFinder software packages, the following are the most appropriate reference genes when comparing: (1) aerobic and anaerobic growth: TSC_c19900, polA2, gyrA, gyrB; (2) anaerobic growth with varied electron acceptors: TSC_c19900, infA, pfk, gyrA, gyrB; (3) aerobic growth with different heating methods: gyrA, gap, gyrB; (4) all conditions mentioned above: gap, gyrA, gyrB. The commonly-employed rpoC does not serve as a reliable reference gene in thermophiles, due to its expression instability across all culture conditions tested here. As extremophiles exhibit a tendency for polyploidy, absolute quantification was employed to determine the ratio of transcript to gene copy number in a subset of the genes. A strong negative correlation was found to exist between ratio and threshold cycle (CT) values, demonstrating that CT changes reflect transcript copy number, and not gene copy number, fluctuations. Even with the potential for polyploidy in extremophiles, the results obtained via absolute quantification

  13. Single-Step RNA Extraction from Different Hydrogel-Embedded Mesenchymal Stem Cells for Quantitative Reverse Transcription-Polymerase Chain Reaction Analysis.

    PubMed

    Köster, Natascha; Schmiermund, Alexandra; Grubelnig, Stefan; Leber, Jasmin; Ehlicke, Franziska; Czermak, Peter; Salzig, Denise

    2016-06-01

    For many tissue engineering applications, cells such as human mesenchymal stem cells (hMSCs) must be embedded in hydrogels. The analysis of embedded hMSCs requires RNA extraction, but common extraction procedures often produce low yields and/or poor quality RNA. We systematically investigated four homogenization methods combined with eight RNA extraction protocols for hMSCs embedded in three common hydrogel types (alginate, agarose, and gelatin). We found for all three hydrogel types that using liquid nitrogen or a rotor-stator produced low RNA yields, whereas using a microhomogenizer or enzymatic/chemical hydrogel digestion achieved better yields regardless of which extraction protocol was subsequently applied. The hot phenol extraction protocol generally achieved the highest A260 values (representing up to 40.8 μg RNA per 10(6) cells), but the cetyltrimethylammonium bromide (CTAB) method produced RNA of better quality, with A260/A280 and A260/A230 ratios and UV spectra similar to the pure RNA control. The RNA produced by this method was also suitable as a template for endpoint and quantitative reverse transcription-PCR (qRT-PCR), achieving low Ct values of ∼20. The prudent choice of hydrogel homogenization and RNA extraction methods can ensure the preparation of high-quality RNA that generates reliable endpoint and quantitative RT-PCR data. We therefore propose a universal method that is suitable for the extraction of RNA from cells embedded in all three hydrogel types commonly used for tissue engineering. PMID:27094052

  14. Selection and evaluation of potential reference genes for gene expression analysis in the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae) using reverse-transcription quantitative PCR.

    PubMed

    Yuan, Miao; Lu, Yanhui; Zhu, Xun; Wan, Hu; Shakeel, Muhammad; Zhan, Sha; Jin, Byung-Rae; Li, Jianhong

    2014-01-01

    The brown planthopper (BPH), Nilaparvata lugens (Hemiptera, Delphacidae), is one of the most important rice pests. Abundant genetic studies on BPH have been conducted using reverse-transcription quantitative real-time PCR (qRT-PCR). Using qRT-PCR, the expression levels of target genes are calculated on the basis of endogenous controls. These genes need to be appropriately selected by experimentally assessing whether they are stably expressed under different conditions. However, such studies on potential reference genes in N. lugens are lacking. In this paper, we presented a systematic exploration of eight candidate reference genes in N. lugens, namely, actin 1 (ACT), muscle actin (MACT), ribosomal protein S11 (RPS11), ribosomal protein S15e (RPS15), alpha 2-tubulin (TUB), elongation factor 1 delta (EF), 18S ribosomal RNA (18S), and arginine kinase (AK) and used four alternative methods (BestKeeper, geNorm, NormFinder, and the delta Ct method) to evaluate the suitability of these genes as endogenous controls. We examined their expression levels among different experimental factors (developmental stage, body part, geographic population, temperature variation, pesticide exposure, diet change, and starvation) following the MIQE (Minimum Information for publication of Quantitative real time PCR Experiments) guidelines. Based on the results of RefFinder, which integrates four currently available major software programs to compare and rank the tested candidate reference genes, RPS15, RPS11, and TUB were found to be the most suitable reference genes in different developmental stages, body parts, and geographic populations, respectively. RPS15 was the most suitable gene under different temperature and diet conditions, while RPS11 was the most suitable gene under different pesticide exposure and starvation conditions. This work sheds light on establishing a standardized qRT-PCR procedure in N. lugens, and serves as a starting point for screening for reference genes for

  15. Single-Step RNA Extraction from Different Hydrogel-Embedded Mesenchymal Stem Cells for Quantitative Reverse Transcription-Polymerase Chain Reaction Analysis.

    PubMed

    Köster, Natascha; Schmiermund, Alexandra; Grubelnig, Stefan; Leber, Jasmin; Ehlicke, Franziska; Czermak, Peter; Salzig, Denise

    2016-06-01

    For many tissue engineering applications, cells such as human mesenchymal stem cells (hMSCs) must be embedded in hydrogels. The analysis of embedded hMSCs requires RNA extraction, but common extraction procedures often produce low yields and/or poor quality RNA. We systematically investigated four homogenization methods combined with eight RNA extraction protocols for hMSCs embedded in three common hydrogel types (alginate, agarose, and gelatin). We found for all three hydrogel types that using liquid nitrogen or a rotor-stator produced low RNA yields, whereas using a microhomogenizer or enzymatic/chemical hydrogel digestion achieved better yields regardless of which extraction protocol was subsequently applied. The hot phenol extraction protocol generally achieved the highest A260 values (representing up to 40.8 μg RNA per 10(6) cells), but the cetyltrimethylammonium bromide (CTAB) method produced RNA of better quality, with A260/A280 and A260/A230 ratios and UV spectra similar to the pure RNA control. The RNA produced by this method was also suitable as a template for endpoint and quantitative reverse transcription-PCR (qRT-PCR), achieving low Ct values of ∼20. The prudent choice of hydrogel homogenization and RNA extraction methods can ensure the preparation of high-quality RNA that generates reliable endpoint and quantitative RT-PCR data. We therefore propose a universal method that is suitable for the extraction of RNA from cells embedded in all three hydrogel types commonly used for tissue engineering.

  16. Development of a combined immunomagnetic separation and quantitative reverse transcription-PCR assay for sensitive detection of infectious rotavirus in water samples.

    PubMed

    Yang, Wan; Gu, April Z; Zeng, Si-yu; Li, Dan; He, Miao; Shi, Han-chang

    2011-03-01

    A quantitative and rapid detection method for rotavirus in water samples was developed using immunomagnetic separation combined with quantitative reverse transcription-polymerase chain reaction (IMS-RT-qPCR). Magnetic beads coated with antibodies against representative group A rotavirus were used to capture and purify intact rotavirus particles in both artificial and real environmental water sample matrix. Compared to extracting RNA using commercial kits and RT-qPCR assay, the developed IMS-RT-qPCR method increased the detection sensitivity by about one order of magnitude when applied in clean water, with a detection limit of 3.16 50% tissue culture infectious dose (TCID(50))/mL within 5h. This method was compatible with various commonly used virus eluants, including beef extract (BE), beef extract with 0.05M glycine (BEG) and urea arginine phosphate buffer (UAPB). The recovery efficiencies from various eluants using IMS-RT-qPCR are higher than that using direct RT-qPCR method, demonstrating the effectiveness of the IMS step for eliminating inhibitors in the eluant matrix. This method was also successfully applied to purify and detect rotavirus particles seeded in 10(3)-fold concentrated wastewater influent samples. It seemed to reduce the interference from complex sample background and increase the qPCR product reliability comparing to RT-qPCR method without the IMS step. The results indicated that IMS-RT-qPCR is a rapid, sensitive and reliable tool for detecting rotaviruses in complex water environments. PMID:21256895

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

    NASA Astrophysics Data System (ADS)

    Ryan, C. G.; Laird, J. S.; Fisher, L. A.; Kirkham, R.; Moorhead, G. F.

    2015-11-01

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

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

    PubMed

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

    2016-08-01

    Cell theory has officially reached 350 years of age as the first use of the word 'cell' in a biological context can be traced to a description of plant material by Robert Hooke in his historic publication 'Micrographia: or some physiological definitions of minute bodies'. The 2015 Royal Microscopical Society Botanical Microscopy meeting was a celebration of the streams of investigation initiated by Hooke to understand at the subcellular scale how plant cell function and form arises. Much of the work presented, and Honorary Fellowships awarded, reflected the advanced application of bioimaging informatics to extract quantitative data from micrographs that reveal dynamic molecular processes driving cell growth and physiology. The field has progressed from collecting many pixels in multiple modes to associating these measurements with objects or features that are meaningful biologically. The additional complexity involves object identification that draws on a different type of expertise from computer science and statistics that is often impenetrable to biologists. There are many useful tools and approaches being developed, but we now need more interdisciplinary exchange to use them effectively. In this review we show how this quiet revolution has provided tools available to any personal computer user. We also discuss the oft-neglected issue of quantifying algorithm robustness and the exciting possibilities offered through the integration of physiological information generated by biosensors with object detection and tracking. PMID:27145353

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

    PubMed

    Shimp, Charles P

    2004-06-30

    Research on categorization has changed over time, and some of these changes resemble how Wittgenstein's views changed from his Tractatus Logico-Philosophicus to his Philosophical Investigations. Wittgenstein initially focused on unambiguous, abstract, parsimonious, logical propositions and rules, and on independent, static, "atomic facts." This approach subsequently influenced the development of logical positivism and thereby may have indirectly influenced method and theory in research on categorization: much animal research on categorization has focused on learning simple, static, logical rules unambiguously interrelating small numbers of independent features. He later rejected logical simplicity and rigor and focused instead on Gestalt ideas about figure-ground reversals and context, the ambiguity of family resemblance, and the function of details of everyday language. Contemporary contextualism has been influenced by this latter position, some features of which appear in contemporary empirical research on categorization. These developmental changes are illustrated by research on avian local and global levels of visual perceptual analysis, categorization of rectangles and moving objects, and artificial grammar learning. Implications are described for peer review of quantitative theory in which ambiguity, logical rigor, simplicity, or dynamics are designed to play important roles. PMID:15157980

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

    PubMed

    Sobkowicz, Pawel

    2016-01-01

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

  1. Quantitative law describing market dynamics before and after interest-rate change

    NASA Astrophysics Data System (ADS)

    Petersen, Alexander M.; Wang, Fengzhong; Havlin, Shlomo; Stanley, H. Eugene

    2010-06-01

    We study the behavior of U.S. markets both before and after U.S. Federal Open Market Commission meetings and show that the announcement of a U.S. Federal Reserve rate change causes a financial shock, where the dynamics after the announcement is described by an analog of the Omori earthquake law. We quantify the rate n(t) of aftershocks following an interest-rate change at time T and find power-law decay which scales as n(t-T)˜(t-T)-Ω , with Ω positive. Surprisingly, we find that the same law describes the rate n'(|t-T|) of “preshocks” before the interest-rate change at time T . This study quantitatively relates the size of the market response to the news which caused the shock and uncovers the presence of quantifiable preshocks. We demonstrate that the news associated with interest-rate change is responsible for causing both the anticipation before the announcement and the surprise after the announcement. We estimate the magnitude of financial news using the relative difference between the U.S. Treasury Bill and the Federal Funds effective rate. Our results are consistent with the “sign effect,” in which “bad news” has a larger impact than “good news.” Furthermore, we observe significant volatility aftershocks, confirming a “market under-reaction” that lasts at least one trading day.

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

    PubMed

    Shimp, Charles P

    2004-06-30

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

  3. Micro/Nano-Computed Tomography Technology for Quantitative Dynamic, Multi-scale Imaging of Morphogenesis

    PubMed Central

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

    2015-01-01

    Tissue morphogenesis and embryonic development are dynamic events challenging to quantify, especially considering the intricate events that happen simultaneously in different locations and time. Micro-, and more recently nano-computed tomography (micro/nanoCT), has been used for the past 15 years to characterize large 3D fields of tortuous geometries at high spatial resolution. We and others have advanced micro/nanoCT imaging strategies for quantifying tissue and organ level fate changes throughout morphogenesis. Exogenous soft tissue contrast media enables visualization of vascular lumens and tissues via extravasation. Furthermore, the emergence of antigen specific tissue contrast enables direct quantitative visualization of protein and mRNA expression. Micro-CT X-ray doses appear to be non-embryotoxic, enabling longitudinal imaging studies in live embryos. In this paper we present established soft tissue contrast protocols for obtaining high quality micro/nanoCT images and the image processing techniques useful for quantifying anatomical and physiological information from the datasets. PMID:25245686

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

    PubMed

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

    2015-01-01

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

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

    PubMed

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

    2016-08-01

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

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

    PubMed Central

    Sobkowicz, Pawel

    2016-01-01

    We present results of an abstract, agent based model of opinion dynamics simulations based on the emotion/information/opinion (E/I/O) approach, applied to a strongly polarized society, corresponding to the Polish political scene between 2005 and 2015. Under certain conditions the model leads to metastable coexistence of two subcommunities of comparable size (supporting the corresponding opinions)—which corresponds to the bipartisan split found in Poland. Spurred by the recent breakdown of this political duopoly, which occurred in 2015, we present a model extension that describes both the long term coexistence of the two opposing opinions and a rapid, transitory change due to the appearance of a third party alternative. We provide quantitative comparison of the model with the results of polls and elections in Poland, testing the assumptions related to the modeled processes and the parameters used in the simulations. It is shown, that when the propaganda messages of the two incumbent parties differ in emotional tone, the political status quo may be unstable. The asymmetry of the emotions within the support bases of the two parties allows one of them to be ‘invaded’ by a newcomer third party very quickly, while the second remains immune to such invasion. PMID:27171226

  7. Chromatin landscape and circadian dynamics: Spatial and temporal organization of clock transcription

    PubMed Central

    Aguilar-Arnal, Lorena; Sassone-Corsi, Paolo

    2015-01-01

    Circadian rhythms drive the temporal organization of a wide variety of physiological and behavioral functions in ∼24-h cycles. This control is achieved through a complex program of gene expression. In mammals, the molecular clock machinery consists of interconnected transcriptional–translational feedback loops that ultimately ensure the proper oscillation of thousands of genes in a tissue-specific manner. To achieve circadian transcriptional control, chromatin remodelers serve the clock machinery by providing appropriate oscillations to the epigenome. Recent findings have revealed the presence of circadian interactomes, nuclear “hubs” of genome topology where coordinately expressed circadian genes physically interact in a spatial and temporal-specific manner. Thus, a circadian nuclear landscape seems to exist, whose interplay with metabolic pathways and clock regulators translates into specific transcriptional programs. Deciphering the molecular mechanisms that connect the circadian clock machinery with the nuclear landscape will reveal yet unexplored pathways that link cellular metabolism to epigenetic control. PMID:25378702

  8. The Dynamics of Transcript Abundance during Cellularization of Developing Barley Endosperm1[OPEN

    PubMed Central

    Zhang, Runxuan; Burton, Rachel A; Shirley, Neil J.; Little, Alan; Morris, Jenny; Milne, Linda

    2016-01-01

    Within the cereal grain, the endosperm and its nutrient reserves are critical for successful germination and in the context of grain utilization. The identification of molecular determinants of early endosperm development, particularly regulators of cell division and cell wall deposition, would help predict end-use properties such as yield, quality, and nutritional value. Custom microarray data have been generated using RNA isolated from developing barley grain endosperm 3 d to 8 d after pollination (DAP). Comparisons of transcript abundance over time revealed 47 gene expression modules that can be clustered into 10 broad groups. Superimposing these modules upon cytological data allowed patterns of transcript abundance to be linked with key stages of early grain development. Here, attention was focused on how the datasets could be mined to explore and define the processes of cell wall biosynthesis, remodeling, and degradation. Using a combination of spatial molecular network and gene ontology enrichment analyses, it is shown that genes involved in cell wall metabolism are found in multiple modules, but cluster into two main groups that exhibit peak expression at 3 DAP to 4 DAP and 5 DAP to 8 DAP. The presence of transcription factor genes in these modules allowed candidate genes for the control of wall metabolism during early barley grain development to be identified. The data are publicly available through a dedicated web interface (https://ics.hutton.ac.uk/barseed/), where they can be used to interrogate co- and differential expression for any other genes, groups of genes, or transcription factors expressed during early endosperm development. PMID:26754666

  9. Transcription factor expression dynamics of early T-lymphocyte specification and commitment

    PubMed Central

    David-Fung, Elizabeth-Sharon; Butler, Robert; Buzi, Gentian; Yui, Mary A.; Diamond, Rochelle A.; Anderson, Michele K.; Rowen, Lee; Rothenberg, Ellen V.

    2009-01-01

    Summary Mammalian T lymphocytes are a prototype for development from adult pluripotent stem cells. While T-cell specification is driven by Notch signaling, T-lineage commitment is only finalized after prolonged Notch activation. However, no T-lineage specific regulatory factor has been reported that mediates commitment. We used a gene-discovery approach to identify additional candidate T-lineage transcription factors and characterized expression of >100 regulatory genes in early T-cell precursors using realtime RT-PCR. These regulatory genes were also monitored in multilineage precursors as they entered T-cell or non-T-cell pathways in vitro; in non-T cells ex vivo; and in later T-cell developmental stages after lineage commitment. At least three major expression patterns were observed. Transcription factors in the largest group are expressed at relatively stable levels throughout T-lineage specification as a legacy from prethymic precursors, with some continuing while others are downregulated after commitment. Another group is highly expressed in the earliest stages only, and is downregulated before or during commitment. Genes in a third group undergo upregulation at one of three distinct transitions, suggesting a positive regulatory cascade. However, the transcription factors induced during commitment are not T-lineage specific. Different members of the same transcription factor family can follow opposite trajectories during specification and commitment, while factors co-expressed early can be expressed in divergent patterns in later T-cell development. Some factors reveal new regulatory distinctions between αβ and γδ T-lineage differentiation. These results show that T-cell identity has an essentially complex regulatory basis and provide a detailed framework for regulatory network modeling of T-cell specification. PMID:19013443

  10. Evaluation of Appropriate Reference Genes for Reverse Transcription-Quantitative PCR Studies in Different Tissues of a Desert Poplar via Comparision of Different Algorithms.

    PubMed

    Wang, Hou-Ling; Li, Lan; Tang, Sha; Yuan, Chao; Tian, Qianqian; Su, Yanyan; Li, Hui-Guang; Zhao, Lin; Yin, Weilun; Zhao, Rui; Xia, Xinli

    2015-01-01

    Despite the unshakable status of reverse transcription-quantitative PCR in gene expression analysis, it has certain disadvantages, including that the results are highly dependent on the reference genes selected for data normalization. Since inappropriate endogenous control genes will lead to inaccurate target gene expression profiles, the validation of suitable internal reference genes is essential. Given the increasing interest in functional genes and genomics of Populus euphratica, a desert poplar showing extraordinary adaptation to salt stress, we evaluated the expression stability of ten candidate reference genes in P. euphratica roots, stems, and leaves under salt stress conditions. We used five algorithms, namely, ΔCt, NormFinder, geNorm, GrayNorm, and a rank aggregation method (RankAggreg) to identify suitable normalizers. To support the suitability of the identified reference genes and to compare the relative merits of these different algorithms, we analyzed and compared the relative expression levels of nine P. euphratica functional genes in different tissues. Our results indicate that a combination of multiple reference genes recommended by GrayNorm algorithm (e.g., a combination of Actin, EF1α, GAPDH, RP, UBQ in root) should be used instead of a single reference gene. These results are valuable for research of gene identification in different P. euphratica tissues.

  11. Selection of internal reference genes for normalization of quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis in the canine brain and other organs.

    PubMed

    Park, Sang-Je; Huh, Jae-Won; Kim, Young-Hyun; Lee, Sang-Rae; Kim, Sang-Hyun; Kim, Sun-Uk; Kim, Heui-Soo; Kim, Min Kyu; Chang, Kyu-Tae

    2013-05-01

    Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is a specific and sensitive technique for quantifying gene expression. To analyze qRT-PCR data accurately, suitable reference genes that show consistent expression patterns across different tissues and experimental conditions should be selected. The objective of this study was to obtain the most stable reference genes in dogs, using samples from 13 different brain tissues and 10 other organs. 16 well-known candidate reference genes were analyzed by the geNorm, NormFinder, and BestKeeper programs. Brain tissues were derived from several different anatomical regions, including the forebrain, cerebrum, diencephalon, hindbrain, and metencephalon, and grouped accordingly. Combination of the three different analyses clearly indicated that the ideal reference genes are ribosomal protien S5 (RPS5) in whole brain, RPL8 and RPS5 in whole body tissues, RPS5 and RPS19 in the forebrain and cerebrum, RPL32 and RPS19 in the diencephalon, GAPDH and RPS19 in the hindbrain, and MRPS7 and RPL13A in the metencephalon. These genes were identified as ideal for the normalization of qRT-PCR results in the respective tissues. These findings indicate more suitable and stable reference genes for future studies of canine gene expression.

  12. Detection of Cardamom mosaic virus and Banana bract mosaic virus in cardamom using SYBR Green based reverse transcription-quantitative PCR.

    PubMed

    Siljo, A; Bhat, A I; Biju, C N

    2014-01-01

    Cardamom being perennial, propagated vegetatively, detecting viruses in planting material is important to check the spread of viruses through infected material. Thus development of effective and sensitive assay for detection of viruses is need of the time. In this view, assay for the detection of Cardamom mosaic virus (CdMV) and Banana bract mosaic virus (BBrMV), infecting cardamom was developed using SYBR Green one step reverse transcription-quantitative PCR (RT-qPCR). The RT-qPCR assay amplified all isolates of CdMV and BBrMV tested but no amplification was obtained with RNA of healthy plants. Recombinant plasmids carrying target virus regions corresponding to both viruses were quantified, serially diluted and used as standards in qPCR to develop standard curve to enable quantification. When tenfold serial dilutions of the total RNAs from infected plants were tested through RT-qPCR, the detection limit of the assay was estimated to be 16 copies for CdMV and 10 copies for BBrMV, which was approximately 1,000-fold higher than the conventional RT-PCR. The RT-qPCR assay was validated by testing field samples collected from different cardamom growing regions of India. This is the first report of RT-qPCR assay for the detection of CdMV and BBrMV in cardamom. PMID:24426323

  13. Comparison of Automated Quantitative Reverse Transcription-PCR and Direct Fluorescent-Antibody Detection for Routine Rabies Diagnosis in the United States

    PubMed Central

    Dupuis, Michelle; Brunt, Scott; Appler, Kim; Rudd, Robert

    2015-01-01

    Rabies virus found worldwide and prevalent throughout the United States continues to be a public health concern. Direct-fluorescent antibody (DFA) detection remains the gold standard for rabies virus diagnostics. Assessing the utility of a high-throughput molecular platform such as the QIAsymphony SP/AS, in conjunction with quantitative reverse transcription-PCR (qRT-PCR), to augment or potentially replace the DFA test, was the focus of this project. Here we describe a triplex qRT-PCR assay, including assembly and evaluation for sensitivity, specificity, and ability to detect variants. Additionally, we compared the qRT-PCR assay to the gold standard direct fluorescent-antibody test. More than 1,000 specimens submitted for routine rabies diagnosis were tested to directly compare the two methods. All results were in agreement between the two methods, with one additional specimen detected by qRT-PCR below the limits of the DFA sensitivity. With the proper continued validation for variant detection, molecular methods have a place in routine rabies diagnostics within the United States. PMID:26179300

  14. SYBR(®) Green-based real-time quantitative reverse-transcription PCR for detection and discrimination of grapevine viruses.

    PubMed

    Poojari, Sudarsana; Alabi, Olufemi J; Okubara, Patricia A; Naidu, Rayapati A

    2016-09-01

    A SYBR(®) Green-based real-time quantitative reverse transcription PCR (qRT-PCR) assay in combination with melt-curve analysis (MCA) was optimized for the detection of nine grapevine viruses. The detection limits for simplex qRT-PCR for all nine grapevine viruses were estimated to be in the range of 214-1112 copies of the virus genome. Amplicons with melting temperatures (Tm) separated by at least 2°C in the MCA could differentiate two viruses in the same reaction. Therefore, eight of the nine viruses could be co-diagnosed in five different combinations of duplex assays. Of 305 grape leaf samples from the field or greenhouse, 162 were positive for at least one of the nine grapevine viruses using the duplex qRT-PCR assays. In contrast, only 127 samples were positive using endpoint RT-PCR and PCR assays, indicating the enhanced sensitivity of duplex real-time PCR. In addition, the duplex qRT-PCR assays were be used to detect Grapevine leafroll associated virus 3 (GLRaV-3) in its vector, the grape mealybug (Pseudococcus maritimus Ehrhorn), and Grapevine red blotch-associated virus (GRBaV) in Virginia creeper leafhopper (Erythroneura ziczac Walsh). The simplex and duplex real-time PCR assays developed in this study can be used to examine transmission of co-occruing viruses by insect vectors as well as for rapid and sensitive detection of viruses in infected grapevines.

  15. Evaluation of Appropriate Reference Genes for Reverse Transcription-Quantitative PCR Studies in Different Tissues of a Desert Poplar via Comparision of Different Algorithms

    PubMed Central

    Wang, Hou-Ling; Li, Lan; Tang, Sha; Yuan, Chao; Tian, Qianqian; Su, Yanyan; Li, Hui-Guang; Zhao, Lin; Yin, Weilun; Zhao, Rui; Xia, Xinli

    2015-01-01

    Despite the unshakable status of reverse transcription-quantitative PCR in gene expression analysis, it has certain disadvantages, including that the results are highly dependent on the reference genes selected for data normalization. Since inappropriate endogenous control genes will lead to inaccurate target gene expression profiles, the validation of suitable internal reference genes is essential. Given the increasing interest in functional genes and genomics of Populus euphratica, a desert poplar showing extraordinary adaptation to salt stress, we evaluated the expression stability of ten candidate reference genes in P. euphratica roots, stems, and leaves under salt stress conditions. We used five algorithms, namely, ΔCt, NormFinder, geNorm, GrayNorm, and a rank aggregation method (RankAggreg) to identify suitable normalizers. To support the suitability of the identified reference genes and to compare the relative merits of these different algorithms, we analyzed and compared the relative expression levels of nine P. euphratica functional genes in different tissues. Our results indicate that a combination of multiple reference genes recommended by GrayNorm algorithm (e.g., a combination of Actin, EF1α, GAPDH, RP, UBQ in root) should be used instead of a single reference gene. These results are valuable for research of gene identification in different P. euphratica tissues. PMID:26343648

  16. Detection of Cardamom mosaic virus and Banana bract mosaic virus in cardamom using SYBR Green based reverse transcription-quantitative PCR.

    PubMed

    Siljo, A; Bhat, A I; Biju, C N

    2014-01-01

    Cardamom being perennial, propagated vegetatively, detecting viruses in planting material is important to check the spread of viruses through infected material. Thus development of effective and sensitive assay for detection of viruses is need of the time. In this view, assay for the detection of Cardamom mosaic virus (CdMV) and Banana bract mosaic virus (BBrMV), infecting cardamom was developed using SYBR Green one step reverse transcription-quantitative PCR (RT-qPCR). The RT-qPCR assay amplified all isolates of CdMV and BBrMV tested but no amplification was obtained with RNA of healthy plants. Recombinant plasmids carrying target virus regions corresponding to both viruses were quantified, serially diluted and used as standards in qPCR to develop standard curve to enable quantification. When tenfold serial dilutions of the total RNAs from infected plants were tested through RT-qPCR, the detection limit of the assay was estimated to be 16 copies for CdMV and 10 copies for BBrMV, which was approximately 1,000-fold higher than the conventional RT-PCR. The RT-qPCR assay was validated by testing field samples collected from different cardamom growing regions of India. This is the first report of RT-qPCR assay for the detection of CdMV and BBrMV in cardamom.

  17. SYBR(®) Green-based real-time quantitative reverse-transcription PCR for detection and discrimination of grapevine viruses.

    PubMed

    Poojari, Sudarsana; Alabi, Olufemi J; Okubara, Patricia A; Naidu, Rayapati A

    2016-09-01

    A SYBR(®) Green-based real-time quantitative reverse transcription PCR (qRT-PCR) assay in combination with melt-curve analysis (MCA) was optimized for the detection of nine grapevine viruses. The detection limits for simplex qRT-PCR for all nine grapevine viruses were estimated to be in the range of 214-1112 copies of the virus genome. Amplicons with melting temperatures (Tm) separated by at least 2°C in the MCA could differentiate two viruses in the same reaction. Therefore, eight of the nine viruses could be co-diagnosed in five different combinations of duplex assays. Of 305 grape leaf samples from the field or greenhouse, 162 were positive for at least one of the nine grapevine viruses using the duplex qRT-PCR assays. In contrast, only 127 samples were positive using endpoint RT-PCR and PCR assays, indicating the enhanced sensitivity of duplex real-time PCR. In addition, the duplex qRT-PCR assays were be used to detect Grapevine leafroll associated virus 3 (GLRaV-3) in its vector, the grape mealybug (Pseudococcus maritimus Ehrhorn), and Grapevine red blotch-associated virus (GRBaV) in Virginia creeper leafhopper (Erythroneura ziczac Walsh). The simplex and duplex real-time PCR assays developed in this study can be used to examine transmission of co-occruing viruses by insect vectors as well as for rapid and sensitive detection of viruses in infected grapevines. PMID:27246908

  18. Two distinct promoter architectures centered on dynamic nucleosomes control ribosomal protein gene transcription

    PubMed Central

    Knight, Britta; Kubik, Slawomir; Ghosh, Bhaswar; Bruzzone, Maria Jessica; Geertz, Marcel; Martin, Victoria; Dénervaud, Nicolas; Jacquet, Philippe; Ozkan, Burak; Rougemont, Jacques; Maerkl, Sebastian J.; Naef, Félix

    2014-01-01

    In yeast, ribosome production is controlled transcriptionally by tight coregulation of the 138 ribosomal protein genes (RPGs). RPG promoters display limited sequence homology, and the molecular basis for their coregulation remains largely unknown. Here we identify two prevalent RPG promoter types, both characterized by upstream binding of the general transcription factor (TF) Rap1 followed by the RPG-specific Fhl1/Ifh1 pair, with one type also binding the HMG-B protein Hmo1. We show that the regulatory properties of the two promoter types are remarkably similar, suggesting that they are determined to a large extent by Rap1 and the Fhl1/Ifh1 pair. Rapid depletion experiments allowed us to define a hierarchy of TF binding in which Rap1 acts as a pioneer factor required for binding of all other TFs. We also uncovered unexpected features underlying recruitment of Fhl1, whose forkhead DNA-binding domain is not required for binding at most promoters, and Hmo1, whose binding is supported by repeated motifs. Finally, we describe unusually micrococcal nuclease (MNase)-sensitive nucleosomes at all RPG promoters, located between the canonical +1 and −1 nucleosomes, which coincide with sites of Fhl1/Ifh1 and Hmo1 binding. We speculate that these “fragile” nucleosomes play an important role in regulating RPG transcriptional output. PMID:25085421

  19. Acetylation of histone H3 at lysine 64 regulates nucleosome dynamics and facilitates transcription.

    PubMed

    Di Cerbo, Vincenzo; Mohn, Fabio; Ryan, Daniel P; Montellier, Emilie; Kacem, Salim; Tropberger, Philipp; Kallis, Eleni; Holzner, Monika; Hoerner, Leslie; Feldmann, Angelika; Richter, Florian Martin; Bannister, Andrew J; Mittler, Gerhard; Michaelis, Jens; Khochbin, Saadi; Feil, Robert; Schuebeler, Dirk; Owen-Hughes, Tom; Daujat, Sylvain; Schneider, Robert

    2014-03-25

    Post-translational modifications of proteins have emerged as a major mechanism for regulating gene expression. However, our understanding of how histone modifications directly affect chromatin function remains limited. In this study, we investigate acetylation of histone H3 at lysine 64 (H3K64ac), a previously uncharacterized acetylation on the lateral surface of the histone octamer. We show that H3K64ac regulates nucleosome stability and facilitates nucleosome eviction and hence gene expression in vivo. In line with this, we demonstrate that H3K64ac is enriched in vivo at the transcriptional start sites of active genes and it defines transcriptionally active chromatin. Moreover, we find that the p300 co-activator acetylates H3K64, and consistent with a transcriptional activation function, H3K64ac opposes its repressive counterpart H3K64me3. Our findings reveal an important role for a histone modification within the nucleosome core as a regulator of chromatin function and they demonstrate that lateral surface modifications can define functionally opposing chromatin states. DOI: http://dx.doi.org/10.7554/eLife.01632.001.

  20. Acetylation of histone H3 at lysine 64 regulates nucleosome dynamics and facilitates transcription

    PubMed Central

    Di Cerbo, Vincenzo; Mohn, Fabio; Ryan, Daniel P; Montellier, Emilie; Kacem, Salim; Tropberger, Philipp; Kallis, Eleni; Holzner, Monika; Hoerner, Leslie; Feldmann, Angelika; Richter, Florian Martin; Bannister, Andrew J; Mittler, Gerhard; Michaelis, Jens; Khochbin, Saadi; Feil, Robert; Schuebeler, Dirk; Owen-Hughes, Tom; Daujat, Sylvain; Schneider, Robert

    2014-01-01

    Post-translational modifications of proteins have emerged as a major mechanism for regulating gene expression. However, our understanding of how histone modifications directly affect chromatin function remains limited. In this study, we investigate acetylation of histone H3 at lysine 64 (H3K64ac), a previously uncharacterized acetylation on the lateral surface of the histone octamer. We show that H3K64ac regulates nucleosome stability and facilitates nucleosome eviction and hence gene expression in vivo. In line with this, we demonstrate that H3K64ac is enriched in vivo at the transcriptional start sites of active genes and it defines transcriptionally active chromatin. Moreover, we find that the p300 co-activator acetylates H3K64, and consistent with a transcriptional activation function, H3K64ac opposes its repressive counterpart H3K64me3. Our findings reveal an important role for a histone modification within the nucleosome core as a regulator of chromatin function and they demonstrate that lateral surface modifications can define functionally opposing chromatin states. DOI: http://dx.doi.org/10.7554/eLife.01632.001 PMID:24668167

  1. Dynamic transcriptional changes in response to rehydration in Anabaena sp. PCC 7120.

    PubMed

    Higo, Akiyoshi; Suzuki, Takayuki; Ikeuchi, Masahiko; Ohmori, Masayuki

    2007-11-01

    Global transcriptional responses to dehydration and rehydration were determined in Anabaena sp. PCC 7120. Nearly 300 genes were up- or downregulated during both dehydration and rehydration. While as many as 133 genes showed dehydration-specific downregulation, only 29 genes showed dehydration-specific upregulation. In contrast, while only 13 genes showed rehydration-specific downregulation, as many as 259 genes showed rehydration-specific upregulation. The genes upregulated during rehydration responded rapidly and transiently, whereas those upregulated during dehydration did so gradually and persistently. The expression of various genes involved in DNA repair, protein folding and NAD synthesis, as well as genes responding to nitrogen depletion and CO2 limitation, was upregulated during rehydration. Although no genes for transcriptional regulators showed dehydration-specific upregulation, eight showed rehydration-specific upregulation. Among them, two genes, ancrpB and alr0618, encode putative transcriptional activators of the cAMP receptor protein (CRP) family. DNA microarray analysis using gene disruptants revealed that AnCrpB and Alr0618 regulate the genes induced by nitrogen depletion and by CO2 limitation, respectively. We conclude that rehydration is a complex process in which the expression of certain genes, particularly those for metabolism, is dramatically induced. PMID:17975076

  2. Increasing the dynamic control space of mammalian transcription devices by combinatorial assembly of homologous regulatory elements from different bacterial species.

    PubMed

    Bacchus, William; Weber, Wilfried; Fussenegger, Martin

    2013-01-01

    Prokaryotic transcriptional regulatory elements are widely utilized building blocks for constructing regulatory genetic circuits adapted for mammalian cells and have found their way into a broad range of biotechnological applications. Prokaryotic transcriptional repressors, fused to eukaryotic transactivation or repression domains, compose the transcription factor, which binds and adjusts transcription from chimeric promoters containing the repressor-specific operator sequence. Escherichia coli and Chlamydia trachomatis share common features in the regulatory mechanism of the biosynthesis of l-tryptophan. The repressor protein TrpR of C. trachomatis regulates the trpRBA operon and the TrpR of E. coli regulates the trpEDCBA operon, both requiring l-tryptophan as a co-repressor. Fusion of these bacterial repressors to the VP16 transactivation domain of Herpes simplex virus creates synthetic transactivators that could bind and activate chimeric promoters, assembled by placing repressor-specific operator modules adjacent to a minimal promoter, in an l-tryptophan-adjustable manner. Combinations of different transactivator and promoter variants from the same or different bacterial species resulted in a multitude of regulatory systems where l-tryptophan regulation properties, background noise, and maximal gene expression levels were significantly diverse. Different l-tryptophan analogues showed diverse regulatory capacity depending on the promoter/transactivator combination. We believe the systems approach to rationally choose promoters, transactivators and inducer molecules, to obtain desired and predefined genetic expression dynamics and control profiles, will significantly advance the design of new regulatory circuits as well as improving already existing ones. PMID:23178502

  3. Conformational Dynamics and the Binding of Specific and Nonspecific DNA by the Autoinhibited Transcription Factor Ets-1.

    PubMed

    Desjardins, Geneviève; Okon, Mark; Graves, Barbara J; McIntosh, Lawrence P

    2016-07-26

    The affinity of the Ets-1 transcription factor for DNA is autoinhibited by an intrinsically disordered serine-rich region (SRR) and a helical inhibitory module (IM) appended to its winged helix-turn-helix ETS domain. Using NMR spectroscopy, we investigated how Ets-1 recognizes specific versus nonspecific DNA, with a focus on the roles of protein dynamics and autoinhibition in these processes. Upon binding either DNA, the two marginally stable N-terminal helices of the IM predominantly unfold, but still sample partially ordered conformations. Also, on the basis of amide chemical shift perturbation mapping, Ets-1 associates with both specific and nonspecific DNA through the same canonical ETS domain interface. These interactions are structurally independent of the SRR, and thus autoinhibition does not impart DNA-binding specificity. However, relative to the pronounced NMR spectroscopic changes in Ets-1 resulting from specific DNA binding, the spectra of the nonspecific DNA complexes showed conformational exchange broadening and lacked several diagnostic amide and indole signals attributable to hydrogen bonding interactions seen in reported X-ray crystallographic structures of this transcription factor with its cognate DNA sequences. Such differences are highlighted by the chemical shift and relaxation properties of several interfacial lysine and arginine side chains. Collectively, these data support a general model in which Ets-1 interacts with nonspecific DNA via dynamic electrostatic interactions, whereas hydrogen bonding drives the formation of well-ordered complexes with specific DNA. PMID:27362745

  4. Conformational Dynamics and the Binding of Specific and Nonspecific DNA by the Autoinhibited Transcription Factor Ets-1.

    PubMed

    Desjardins, Geneviève; Okon, Mark; Graves, Barbara J; McIntosh, Lawrence P

    2016-07-26

    The affinity of the Ets-1 transcription factor for DNA is autoinhibited by an intrinsically disordered serine-rich region (SRR) and a helical inhibitory module (IM) appended to its winged helix-turn-helix ETS domain. Using NMR spectroscopy, we investigated how Ets-1 recognizes specific versus nonspecific DNA, with a focus on the roles of protein dynamics and autoinhibition in these processes. Upon binding either DNA, the two marginally stable N-terminal helices of the IM predominantly unfold, but still sample partially ordered conformations. Also, on the basis of amide chemical shift perturbation mapping, Ets-1 associates with both specific and nonspecific DNA through the same canonical ETS domain interface. These interactions are structurally independent of the SRR, and thus autoinhibition does not impart DNA-binding specificity. However, relative to the pronounced NMR spectroscopic changes in Ets-1 resulting from specific DNA binding, the spectra of the nonspecific DNA complexes showed conformational exchange broadening and lacked several diagnostic amide and indole signals attributable to hydrogen bonding interactions seen in reported X-ray crystallographic structures of this transcription factor with its cognate DNA sequences. Such differences are highlighted by the chemical shift and relaxation properties of several interfacial lysine and arginine side chains. Collectively, these data support a general model in which Ets-1 interacts with nonspecific DNA via dynamic electrostatic interactions, whereas hydrogen bonding drives the formation of well-ordered complexes with specific DNA.

  5. How Intrinsic Molecular Dynamics Control Intramolecular Communication in Signal Transducers and Activators of Transcription Factor STAT5

    PubMed Central

    Langenfeld, Florent; Guarracino, Yann; Arock, Michel; Trouvé, Alain; Tchertanov, Luba

    2015-01-01

    Signal Transducer and Activator of Transcription STAT5 is a key mediator of cell proliferation, differentiation and survival. While STAT5 activity is tightly regulated in normal cells, its constitutive activation directly contributes to oncogenesis and is associated with a broad range of hematological and solid tumor cancers. Therefore the development of compounds able to modulate pathogenic activation of this protein is a very challenging endeavor. A crucial step of drug design is the understanding of the protein conformational features and the definition of putative binding site(s) for such modulators. Currently, there is no structural data available for human STAT5 and our study is the first footprint towards the description of structure and dynamics of this protein. We investigated structural and dynamical features of the two STAT5 isoforms, STAT5a and STAT5b, taken into account their phosphorylation status. The study was based on the exploration of molecular dynamics simulations by different analytical methods. Despite the overall folding similarity of STAT5 proteins, the MD conformations display specific structural and dynamical features for each protein, indicating first, sequence-encoded structural properties and second, phosphorylation-induced effects which contribute to local and long-distance structural rearrangements interpreted as allosteric event. Further examination of the dynamical coupling between distant sites provides evidence for alternative profiles of the communication pathways inside and between the STAT5 domains. These results add a new insight to the understanding of the crucial role of intrinsic molecular dynamics in mediating intramolecular signaling in STAT5. Two pockets, localized in close proximity to the phosphotyrosine-binding site and adjacent to the channel for communication pathways across STAT5, may constitute valid targets to develop inhibitors able to modulate the function-related communication properties of this signaling

  6. The Bias Associated with Amplicon Sequencing Does Not Affect the Quantitative Assessment of Bacterial Community Dynamics

    PubMed Central

    Figuerola, Eva L. M.; Erijman, Leonardo

    2014-01-01

    The performance of two sets of primers targeting variable regions of the 16S rRNA gene V1–V3 and V4 was compared in their ability to describe changes of bacterial diversity and temporal turnover in full-scale activated sludge. Duplicate sets of high-throughput amplicon sequencing data of the two 16S rRNA regions shared a collection of core taxa that were observed across a series of twelve monthly samples, although the relative abundance of each taxon was substantially different between regions. A case in point was the changes in the relative abundance of filamentous bacteria Thiothrix, which caused a large effect on diversity indices, but only in the V1–V3 data set. Yet the relative abundance of Thiothrix in the amplicon sequencing data from both regions correlated with the estimation of its abundance determined using fluorescence in situ hybridization. In nonmetric multidimensional analysis samples were distributed along the first ordination axis according to the sequenced region rather than according to sample identities. The dynamics of microbial communities indicated that V1–V3 and the V4 regions of the 16S rRNA gene yielded comparable patterns of: 1) the changes occurring within the communities along fixed time intervals, 2) the slow turnover of activated sludge communities and 3) the rate of species replacement calculated from the taxa–time relationships. The temperature was the only operational variable that showed significant correlation with the composition of bacterial communities over time for the sets of data obtained with both pairs of primers. In conclusion, we show that despite the bias introduced by amplicon sequencing, the variable regions V1–V3 and V4 can be confidently used for the quantitative assessment of bacterial community dynamics, and provide a proper qualitative account of general taxa in the community, especially when the data are obtained over a convenient time window rather than at a single time point. PMID:24923665

  7. Evaluation of internal reference genes for quantitative expression analysis by real-time reverse transcription-PCR in somatic cells from goat milk.

    PubMed

    Modesto, P; Peletto, S; Pisoni, G; Cremonesi, P; Castiglioni, B; Colussi, S; Caramelli, M; Bronzo, V; Moroni, P; Acutis, P L

    2013-01-01

    Reverse transcription (RT) quantitative real-time PCR (qPCR) is the most accurate and easy-to-perform technique to measure the expression level of a selected gene of interest by quantifying mRNA transcripts. The use of reference genes is commonly accepted as the most reliable approach to normalize RT-qPCR data and reduce possible errors generated in the quantification of gene expression. The optimal number and choice of reference genes are experimentally validated for specific tissues or cell types and experimental designs. To date, data on qPCR normalization in goats are scarce and the most suitable reference genes in this species have been identified for only a limited number of tissues. The aim of this study was to determine an optimal combination of stably expressed reference genes in caprine milk somatic cells (MSC) from healthy and infected mammary glands. For the purpose, we performed RT-qPCR for 10 commonly used reference genes from various functional classes and then determined their expression level in MSC from goats intramammary challenged with Staphylococcus aureus and in MSC from healthy controls, with a view to select genes whose stability would be unaffected under infection conditions. The geNorm and NormFinder algorithms were used for validating the reference genes. Furthermore, to demonstrate the importance of normalization of gene expression with appropriate reference genes, we tested the effect of using a combination of the least stable genes for expression analysis evaluation. On the basis of our evaluation, we recommend the use of a panel of reference genes that should include G6PD, YWHAZ, and ACTB for caprine MSC gene expression profiling. The expression of the 2 genes of interest, pentraxin-related protein (PTX3) and secreted phosphoprotein 1 (SPP1), was evaluated by RT-qPCR in all samples collected pre- and postinfection, and the recommended reference genes were used to normalize the data. Our study provides a validated panel of optimal

  8. Identification and validation of quantitative real-time reverse transcription PCR reference genes for gene expression analysis in teak (Tectona grandis L.f.)

    PubMed Central

    2014-01-01

    Background Teak (Tectona grandis L.f.) is currently the preferred choice of the timber trade for fabrication of woody products due to its extraordinary qualities and is widely grown around the world. Gene expression studies are essential to explore wood formation of vascular plants, and quantitative real-time reverse transcription PCR (qRT-PCR) is a sensitive technique employed for quantifying gene expression levels. One or more appropriate reference genes are crucial to accurately compare mRNA transcripts through different tissues/organs and experimental conditions. Despite being the focus of some genetic studies, a lack of molecular information has hindered genetic exploration of teak. To date, qRT-PCR reference genes have not been identified and validated for teak. Results Identification and cloning of nine commonly used qRT-PCR reference genes from teak, including ribosomal protein 60s (rp60s), clathrin adaptor complexes medium subunit family (Cac), actin (Act), histone 3 (His3), sand family (Sand), β-Tubulin (Β-Tub), ubiquitin (Ubq), elongation factor 1-α (Ef-1α), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Expression profiles of these genes were evaluated by qRT-PCR in six tissue and organ samples (leaf, flower, seedling, root, stem and branch secondary xylem) of teak. Appropriate gene cloning and sequencing, primer specificity and amplification efficiency was verified for each gene. Their stability as reference genes was validated by NormFinder, BestKeeper, geNorm and Delta Ct programs. Results obtained from all programs showed that TgUbq and TgEf-1α are the most stable genes to use as qRT-PCR reference genes and TgAct is the most unstable gene in teak. The relative expression of the teak cinnamyl alcohol dehydrogenase (TgCAD) gene in lignified tissues at different ages was assessed by qRT-PCR, using TgUbq and TgEf-1α as internal controls. These analyses exposed a consistent expression pattern with both reference genes. Conclusion This study

  9. Dynamic contrast-enhanced quantitative susceptibility mapping with ultrashort echo time MRI for evaluating renal function.

    PubMed

    Xie, Luke; Layton, Anita T; Wang, Nian; Larson, Peder E Z; Zhang, Jeff L; Lee, Vivian S; Liu, Chunlei; Johnson, G Allan

    2016-01-15

    Dynamic contrast-enhanced (DCE) MRI can provide key insight into renal function. DCE MRI is typically achieved through an injection of a gadolinium (Gd)-based contrast agent, which has desirable T1 quenching and tracer kinetics. However, significant T2* blooming effects and signal voids can arise when Gd becomes very concentrated, especially in the renal medulla and pelvis. One MRI sequence designed to alleviate T2* effects is the ultrashort echo time (UTE) sequence. In the present study, we observed T2* blooming in the inner medulla of the mouse kidney, despite using UTE at an echo time of 20 microseconds and a low dose of 0.03 mmol/kg Gd. We applied quantitative susceptibility mapping (QSM) and resolved the signal void into a positive susceptibility signal. The susceptibility values [in parts per million (ppm)] were converted into molar concentrations of Gd using a calibration curve. We determined the concentrating mechanism (referred to as the concentrating index) as a ratio of maximum Gd concentration in the inner medulla to the renal artery. The concentrating index was assessed longitudinally over a 17-wk course (3, 5, 7, 9, 13, 17 wk of age). We conclude that the UTE-based DCE method is limited in resolving extreme T2* content caused by the kidney's strong concentrating mechanism. QSM was able to resolve and confirm the source of the blooming effect to be the large positive susceptibility of concentrated Gd. UTE with QSM can complement traditional magnitude UTE and offer a powerful tool to study renal pathophysiology. PMID:26447222

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

    NASA Astrophysics Data System (ADS)

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

    2016-04-01

    Over the past decade, energy-dependent ambient pressure X-ray photoelectron spectroscopy (XPS) has emerged as a powerful analytical probe of the ion spatial distributions at the vapor (vacuum)-aqueous electrolyte interface. These experiments are often paired with complementary molecular dynamics (MD) simulations in an attempt to provide a complete description of the liquid interface. There is, however, no systematic protocol that permits a straightforward comparison of the two sets of results. XPS is an integrated technique that averages signals from multiple layers in a solution even at the lowest photoelectron kinetic energies routinely employed, whereas MD simulations provide a microscopic layer-by-layer description of the solution composition near the interface. Here, we use the National Institute of Standards and Technology database for the Simulation of Electron Spectra for Surface Analysis (SESSA) to quantitatively interpret atom-density profiles from MD simulations for XPS signal intensities using sodium and potassium iodide solutions as examples. We show that electron inelastic mean free paths calculated from a semi-empirical formula depend strongly on solution composition, varying by up to 30% between pure water and concentrated NaI. The XPS signal thus arises from different information depths in different solutions for a fixed photoelectron kinetic energy. XPS signal intensities are calculated using SESSA as a function of photoelectron kinetic energy (probe depth) and compared with a widely employed ad hoc method. SESSA simulations illustrate the importance of accounting for elastic-scattering events at low photoelectron kinetic energies (<300 eV) where the ad hoc method systematically underestimates the preferential enhancement of anions over cations. Finally, some technical aspects of applying SESSA to liquid interfaces are discussed.

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

    PubMed

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

    2016-04-21

    Over the past decade, energy-dependent ambient pressure X-ray photoelectron spectroscopy(XPS) has emerged as a powerful analytical probe of the ion spatial distributions at the vapor (vacuum)-aqueous electrolyteinterface. These experiments are often paired with complementary molecular dynamics (MD) simulations in an attempt to provide a complete description of the liquidinterface. There is, however, no systematic protocol that permits a straightforward comparison of the two sets of results. XPS is an integrated technique that averages signals from multiple layers in a solution even at the lowest photoelectron kinetic energies routinely employed, whereas MD simulations provide a microscopic layer-by-layer description of the solution composition near the interface. Here, we use the National Institute of Standards and Technology database for the Simulation of Electron Spectra for Surface Analysis (SESSA) to quantitatively interpret atom-density profiles from MD simulations for XPS signal intensities using sodium and potassium iodide solutions as examples. We show that electron inelastic mean free paths calculated from a semi-empirical formula depend strongly on solution composition, varying by up to 30% between pure water and concentrated NaI. The XPS signal thus arises from different information depths in different solutions for a fixed photoelectron kinetic energy. XPS signal intensities are calculated using SESSA as a function of photoelectron kinetic energy (probe depth) and compared with a widely employed ad hoc method. SESSA simulations illustrate the importance of accounting for elastic-scattering events at low photoelectron kinetic energies (<300 eV) where the ad hoc method systematically underestimates the preferential enhancement of anions over cations. Finally, some technical aspects of applying SESSA to liquidinterfaces are discussed. PMID:27389231

  12. New technique to quantitate regional pulmonary microvascular transit times from dynamic x-ray CT images

    NASA Astrophysics Data System (ADS)

    Tajik, Jehangir K.; Tran, Binh Q.; Hoffman, Eric A.

    1998-07-01

    Microvascular red blood cell mean transit time is a crucial parameter underlying basic pulmonary physiology. Dynamic x-ray CT imaging during bolus radiopaque tracer injection offers the ability to make functional measurements throughout the lungs, but is not able to resolve individual microvascular beds. We have implemented a model-free Fast Fourier Transform deconvolution algorithm to extract the microvascular transport characteristics from the acquired time-intensity data. The deconvolved feeding arterial bolus input curves and corresponding regional pulmonary parenchymal 'response' functions provide measures of regional pulmonary tracer residence times, allowing calculation of microvascular transit times for different spatial regions of the pulmonary system. The acquired feeding (main) pulmonary artery and regional pulmonary parenchyma time-intensity curves were fit to gamma variate functions which were then sampled with a temporal resolution of 0.1 seconds. Deconvolution of the feeding arterial and regional parenchymal curves consistently results in bimodal regional residue functions. The two modes consist of a relatively large, sharp, narrow peak approximating a delta function followed by a smaller more dispersed curve. The sharp, narrow peak appears to be due to small artery inclusion in the sampled parenchymal region (partial volume effects). The magnitude of the dominant arterial peak decreases as sampling locations are moved from the less expanded dependent to the more expanded non-dependent lung regions of supine dogs. Mathematical separation of the two modes allowed isolation of the arterial and microvascular components. The shape and transit times of the putative microvascular components agree well with results from similar measurements via microfocal angiography and in vivo microscopy. Reconvolving the microvascular component with the input curve results in a corrected parenchymal curve representing the regional microvascular transport characteristics

  13. Dynamic dialog between cytokeratin 18 and annexin A1 in breast cancer: a transcriptional disequilibrium.

    PubMed

    Araujo, Thaise G; Marangoni, Karina; Rocha, Rafael M; Maia, Yara C P; Araujo, Galber R; Alcântar, Tânia M; Alves, Patrícia T; Calábria, Luanda; Neves, Adriana F; Soares, Fernando A; Goulart, Luiz R

    2014-09-01

    Cytokeratins (CKs) constitute the cytoskeletal network and are regulated by post-translational modifications, acting not only as a mechanical support, but also in cell signaling and regulatory processes. Signaling is mediated by CK-associated proteins, such as Annexin A1 (ANXA1), a ligand of the CK18/CK8 complex. ANXA1 has a pivotal role in cellular and immunological responses, and together with CK18 have been implicated in several processes related to malignant transformation in breast cancer (BC). Our aim was to demonstrate how their interaction might be linked to BC development. We investigated transcript levels, protein expression and distribution for both targets in breast tissues of 92 patients (42 BCs and 50 benign diseases) using qPCR and immunohistochemistry, respectively. ANXA1 and CK18 mRNAs were inversely correlated, and their ratio in each TNM stage significantly differentiated BC from benign diseases (OR=5.62). These differences did not mirror tissue protein levels, but a significant dichotomous protein distribution in tumor tissues was observed, differing from the expected co-localization observed during cell homeostasis. The disequilibrium of transcriptional levels between ANXA1/CK18 and alterations in their tissue distribution are present either in initial events or tumor progression, which suggest a critical event in BC. The broken dialog between ANXA1 and CK18 in normal breast tissues may play a critical role in BC development, and together may be used as combined targets for BC diagnostics. PMID:25028131

  14. Dynamic Sumoylation of a Conserved Transcription Corepressor Prevents Persistent Inclusion Formation during Hyperosmotic Stress

    PubMed Central

    Oeser, Michelle L.; Amen, Triana; Nadel, Cory M.; Bradley, Amanda I.; Reed, Benjamin J.; Jones, Ramon D.; Gopalan, Janani; Kaganovich, Daniel; Gardner, Richard G.

    2016-01-01

    Cells are often exposed to physical or chemical stresses that can damage the structures of essential biomolecules. Stress-induced cellular damage can become deleterious if not managed appropriately. Rapid and adaptive responses to stresses are therefore crucial for cell survival. In eukaryotic cells, different stresses trigger post-translational modification of proteins with the small ubiquitin-like modifier SUMO. However, the specific regulatory roles of sumoylation in each stress response are not well understood. Here, we examined the sumoylation events that occur in budding yeast after exposure to hyperosmotic stress. We discovered by proteomic and biochemical analyses that hyperosmotic stress incurs the rapid and transient sumoylation of Cyc8 and Tup1, which together form a conserved transcription corepressor complex that regulates hundreds of genes. Gene expression and cell biological analyses revealed that sumoylation of each protein directs distinct outcomes. In particular, we discovered that Cyc8 sumoylation prevents the persistence of hyperosmotic stress-induced Cyc8-Tup1 inclusions, which involves a glutamine-rich prion domain in Cyc8. We propose that sumoylation protects against persistent inclusion formation during hyperosmotic stress, allowing optimal transcriptional function of the Cyc8-Tup1 complex. PMID:26800527

  15. Evaluation of Reference Genes for Reverse Transcription Quantitative PCR Studies of Physiological Responses in the Ghost Moth, Thitarodes armoricanus (Lepidoptera, Hepialidae)

    PubMed Central

    Liu, Guiqing; Qiu, Xuehong; Cao, Li; Zhang, Yi; Zhan, Zubing; Han, Richou

    2016-01-01

    Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is the sensitive method to quantify the expression levels of target genes on the basis of endogenous control. An appropriate reference gene set for normalization is essential for reliable results. The ghost moth, Thitarodes armoricanus, a host species of a medicinal fungus, Ophiocordyceps sinensis, is an economically important member of the Lepidoptera. Recent studies have focused on the mechanism of adaptation of this species to its high-altitude environment and host immune response to O. sinensis infection and RT-qPCR is commonly used in these studies to decipher the genetic basis of physiological functions. However, a thorough assessment of candidate reference genes in the genus Thitarodes is lacking. Here, the expression levels of eight candidate reference genes (ACT, EF, EIF4A, GAPDH, G6PDH, RPL13A, TUB and 18S) in T. armoricanus at different developmental stages and in different body parts of the seventh instar larvae were analyzed, along with larvae kept under low temperatures, larvae exposed to two fungal infections and larvae fed different diets. Three established software programs–Bestkeeper, geNorm and NormFinder–were employed to calculate variation among the treatments. The results revealed that the best-suited reference genes differed across the treatments, with EF, EIF4A and GAPDH found to be the best suited for the different developmental stages and larvae body parts; EF, EIF4A and RPL13A found to be the best suited for low-temperature challenge; and EF, EIF4A and TUB found to be the best suited for the fungal infections and dietary treatments. This study thus further contributes to the establishment of an accurate method for normalizing RT-qPCR results for T. armoricanus and serves as a reference for gene expression studies of related insect species. PMID:27392023

  16. Real-time quantitative reverse transcription-polymerase chain reaction to detect propionibacterial ribosomal RNA in the lymph nodes of Chinese patients with sarcoidosis.

    PubMed

    Zhou, Y; Wei, Y-R; Zhang, Y; Du, S-S; Baughman, R P; Li, H-P

    2015-09-01

    The aim of this study was to investigate the diagnostic value of using the copy number of propionibacterial rRNA as a biomarker for sarcoidosis. Ribosomal RNA of Propionibacterium acnes and P. granulosum was measured by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) using formalin-fixed and paraffin-embedded tissue of lymph node biopsy from 65 Chinese patients with sarcoidosis, 45 with tuberculosis and 50 controls with other diseases (23 with non-specific lymphadenitis and 27 with mediastinal lymph node metastasis from lung cancer). The receiver operating characteristic (ROC) curve was analysed to determine an optimal cut-off value for diagnosis, and the diagnostic accuracy of the cut-off value was evaluated in additional tissue samples [24 patients with sarcoidosis and 22 with tuberculosis (TB)]. P. acnes or P. granulosum rRNA was detected in 48 of the 65 sarcoidosis samples but only in four of the 45 TB samples and three of the 50 control samples. Analysis of the ROC curve revealed that an optimal cut-off value of the copy number of propionibacterial rRNA for diagnosis of sarcoidosis was 50·5 copies/ml with a sensitivity and specificity of 73·8 and 92·6%, respectively. Based on the cut-off value, 19 of the 24 additional sarcoidosis samples exhibited positive P. acnes or P. granulosum, whereas only one of the 22 additional TB samples was positive, resulting in a sensitivity and specificity of 79·2 and 95·5%, respectively. These findings suggest that propionibacteria might be associated with sarcoidosis granulomatous inflammation. Detection of propionibacterial rRNA by RT-PCR might possibly distinguish sarcoidosis from TB. PMID:25959360

  17. Real-time quantitative reverse transcription-polymerase chain reaction to detect propionibacterial ribosomal RNA in the lymph nodes of Chinese patients with sarcoidosis.

    PubMed

    Zhou, Y; Wei, Y-R; Zhang, Y; Du, S-S; Baughman, R P; Li, H-P

    2015-09-01

    The aim of this study was to investigate the diagnostic value of using the copy number of propionibacterial rRNA as a biomarker for sarcoidosis. Ribosomal RNA of Propionibacterium acnes and P. granulosum was measured by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) using formalin-fixed and paraffin-embedded tissue of lymph node biopsy from 65 Chinese patients with sarcoidosis, 45 with tuberculosis and 50 controls with other diseases (23 with non-specific lymphadenitis and 27 with mediastinal lymph node metastasis from lung cancer). The receiver operating characteristic (ROC) curve was analysed to determine an optimal cut-off value for diagnosis, and the diagnostic accuracy of the cut-off value was evaluated in additional tissue samples [24 patients with sarcoidosis and 22 with tuberculosis (TB)]. P. acnes or P. granulosum rRNA was detected in 48 of the 65 sarcoidosis samples but only in four of the 45 TB samples and three of the 50 control samples. Analysis of the ROC curve revealed that an optimal cut-off value of the copy number of propionibacterial rRNA for diagnosis of sarcoidosis was 50·5 copies/ml with a sensitivity and specificity of 73·8 and 92·6%, respectively. Based on the cut-off value, 19 of the 24 additional sarcoidosis samples exhibited positive P. acnes or P. granulosum, whereas only one of the 22 additional TB samples was positive, resulting in a sensitivity and specificity of 79·2 and 95·5%, respectively. These findings suggest that propionibacteria might be associated with sarcoidosis granulomatous inflammation. Detection of propionibacterial rRNA by RT-PCR might possibly distinguish sarcoidosis from TB.

  18. Detection of Viral Hemorrhagic Septicemia Virus by Quantitative Reverse Transcription Polymerase Chain Reaction from Two Fish Species at Two Sites in Lake Superior

    USGS Publications Warehouse

    Cornwell, Emily R.; Eckerlin, Geofrey E.; Getchell, Rodman G.; Groocock, Geoffrey H.; Thompson, Tarin M.; Batts, William N.; Casey, Rufina N.; Kurath, Gael; Winton, James R.; Bowser, Paul R.; Bain, Mark B.; Casey, James W.

    2011-01-01

    Viral hemorrhagic septicemia virus (VHSV) was first detected in the Laurentian Great Lakes in 2005 during a mortality event in the Bay of Quinte, Lake Ontario. Subsequent analysis of archived samples determined that the first known isolation of VHSV in the Laurentian Great Lakes was from a muskellunge Esox masquinongy collected in Lake St. Clair in 2003. By the end of 2008, mortality events and viral isolations had occurred in all of the Laurentian Great Lakes except Lake Superior. In 2009, a focused disease surveillance program was designed to determine whether VHSV was also present in Lake Superior. In this survey, 874 fish from 7 sites along the U.S. shoreline of Lake Superior were collected during June 2009. Collections were focused on nearshore species known to be susceptible to VHSV. All fish were dissected individually by using aseptic techniques and were tested for the presence of VHSV genetic material by use of a quantitative reverse transcription (qRT) polymerase chain reaction (PCR) targeting the viral nucleoprotein gene. Seventeen fish from two host species at two different sites tested positive at low levels for VHSV. All attempts to isolate virus in cell culture were unsuccessful. However, the presence of viral RNA was confirmed independently in five fish by using a nested PCR that targeted the glycoprotein (G) gene. Partial G gene sequences obtained from three fish were identical to the corresponding sequence from the original 2003 VHSV isolate (MI03) from muskellunge. These detections represent the earliest evidence for the presence of VHSV in Lake Superior and illustrate the utility of the highly sensitive qRT-PCR assay for disease surveillance in aquatic animals.

  19. Evaluation of Reference Genes for Reverse Transcription Quantitative PCR Studies of Physiological Responses in the Ghost Moth, Thitarodes armoricanus (Lepidoptera, Hepialidae).

    PubMed

    Liu, Guiqing; Qiu, Xuehong; Cao, Li; Zhang, Yi; Zhan, Zubing; Han, Richou

    2016-01-01

    Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is the sensitive method to quantify the expression levels of target genes on the basis of endogenous control. An appropriate reference gene set for normalization is essential for reliable results. The ghost moth, Thitarodes armoricanus, a host species of a medicinal fungus, Ophiocordyceps sinensis, is an economically important member of the Lepidoptera. Recent studies have focused on the mechanism of adaptation of this species to its high-altitude environment and host immune response to O. sinensis infection and RT-qPCR is commonly used in these studies to decipher the genetic basis of physiological functions. However, a thorough assessment of candidate reference genes in the genus Thitarodes is lacking. Here, the expression levels of eight candidate reference genes (ACT, EF, EIF4A, GAPDH, G6PDH, RPL13A, TUB and 18S) in T. armoricanus at different developmental stages and in different body parts of the seventh instar larvae were analyzed, along with larvae kept under low temperatures, larvae exposed to two fungal infections and larvae fed different diets. Three established software programs-Bestkeeper, geNorm and NormFinder-were employed to calculate variation among the treatments. The results revealed that the best-suited reference genes differed across the treatments, with EF, EIF4A and GAPDH found to be the best suited for the different developmental stages and larvae body parts; EF, EIF4A and RPL13A found to be the best suited for low-temperature challenge; and EF, EIF4A and TUB found to be the best suited for the fungal infections and dietary treatments. This study thus further contributes to the establishment of an accurate method for normalizing RT-qPCR results for T. armoricanus and serves as a reference for gene expression studies of related insect species. PMID:27392023

  20. Quantitative vibrational dynamics of the metal site in a tin porphyrin: an IR, NRVS, and DFT study.

    PubMed

    Leu, Bogdan M; Zgierski, Marek Z; Bischoff, Christian; Li, Ming; Hu, Michael Y; Zhao, Jiyong; Martin, Steve W; Alp, Esen Ercan; Scheidt, W Robert

    2013-09-01

    We used a newer, synchrotron-based, spectroscopic technique (nuclear resonance vibrational spectroscopy, NRVS) in combination with a more traditional one (infrared absorption, IR) to obtain a complete, quantitative picture of the metal center vibrational dynamics in a six-coordinated tin porphyrin. From the NRVS (119)Sn site-selectivity and the sensitivity of the IR signal to (112)Sn/(119)Sn isotope substitution, we identified the frequency of the antisymmetric stretching of the axial bonds (290 cm(-1)) and all the other vibrations involving Sn. Experimentally authenticated density functional theory (DFT) calculations aid the data interpretation by providing detailed normal mode descriptions for each observed vibration. These results may represent a starting point toward the characterization of the local vibrational dynamics of the metallic site in tin porphyrins and compounds with related structures. The quantitative complementariness between IR, NRVS, and DFT is emphasized.

  1. Dynamics of transcriptional start site selection during nitrogen stress-induced cell differentiation in Anabaena sp. PCC7120

    PubMed Central

    Mitschke, Jan; Vioque, Agustín; Haas, Fabian; Hess, Wolfgang R.; Muro-Pastor, Alicia M.

    2011-01-01

    The fixation of atmospheric N2 by cyanobacteria is a major source of nitrogen in the biosphere. In Nostocales, such as Anabaena, this process is spatially separated from oxygenic photosynthesis and occurs in heterocysts. Upon nitrogen step-down, these specialized cells differentiate from vegetative cells in a process controlled by two major regulators: NtcA and HetR. However, the regulon controlled by these two factors is only partially defined, and several aspects of the differentiation process have remained enigmatic. Using differential RNA-seq, we experimentally define a genome-wide map of >10,000 transcriptional start sites (TSS) of Anabaena sp. PCC7120, a model organism for the study of prokaryotic cell differentiation and N2 fixation. By analyzing the adaptation to nitrogen stress, our global TSS map provides insight into the dynamic changes that modify the transcriptional organization at a critical step of the differentiation process. We identify >900 TSS with minimum fold change in response to nitrogen deficiency of eight. From these TSS, at least 209 were under control of HetR, whereas at least 158 other TSS were potentially directly controlled by NtcA. Our analysis of the promoters activated during the switch to N2 fixation adds hundreds of protein-coding genes and noncoding transcripts to the list of potentially involved factors. These data experimentally define the NtcA regulon and the DIF+ motif, a palindrome at or close to position −35 that seems essential for heterocyst-specific expression of certain genes. PMID:22135468

  2. Dissecting mechanisms of mouse embryonic stem cells heterogeneity through a model-based analysis of transcription factor dynamics.

    PubMed

    Herberg, Maria; Glauche, Ingmar; Zerjatke, Thomas; Winzi, Maria; Buchholz, Frank; Roeder, Ingo

    2016-04-01

    Pluripotent mouse embryonic stem cells (mESCs) show heterogeneous expression levels of transcription factors (TFs) involved in pluripotency regulation, among them Nanog and Rex1. The expression of both TFs can change dynamically between states of high and low activity, correlating with the cells' capacity for self-renewal. Stochastic fluctuations as well as sustained oscillations in gene expression are possible mechanisms to explain this behaviour, but the lack of suitable data hampered their clear distinction. Here, we present a systems biology approach in which novel experimental data on TF heterogeneity is complemented by an agent-based model of mESC self-renewal. Because the model accounts for intracellular interactions, cell divisions and heredity structures, it allows for evaluating the consistency of the proposed mechanisms with data on population growth and on TF dynamics after cell sorting. Our model-based analysis revealed that a bistable, noise-driven network model fulfils the minimal requirements to consistently explain Nanog and Rex1 expression dynamics in heterogeneous and sorted mESC populations. Moreover, we studied the impact of TF-related proliferation capacities on the frequency of state transitions and demonstrate that cellular genealogies can provide insights into the heredity structures of mESCs.

  3. DNA-Directed Assembly of Nanogold Dimers: A Unique Dynamic Light Scattering Sensing Probe for Transcription Factor Detection

    PubMed Central

    Seow, Nianjia; Tan, Yen Nee; Yung, Lin-Yue Lanry; Su, Xiaodi

    2015-01-01

    We have developed a unique DNA-assembled gold nanoparticles (AuNPs) dimer for dynamic light scattering (DLS) sensing of transcription factors, exemplified by estrogen receptor (ER) that binds specifically to a double-stranded (ds) DNA sequence containing estrogen response element (ERE). Here, ERE sequence is incorporated into the DNA linkers to bridge the AuNPs dimer for ER binding. Coupled with DLS, this AuNP dimer-based DLS detection system gave distinct readout of a single ‘complex peak’ in the presence of the target molecule (i.e., ER). This unique signature marked the first time that such nanostructures can be used to study transcription factor-DNA interactions, which DLS alone cannot do. This was also unlike previously reported AuNP-DLS assays that gave random and broad distribution of particles size upon target binding. In addition, the ERE-containing AuNP dimers could also suppress the light-scattering signal from the unbound proteins and other interfering factors (e.g., buffer background), and has potential for sensitive detection of target proteins in complex biological samples such as cell lysates. In short, the as-developed AuNP dimer probe coupled with DLS is a simple (mix and test), rapid (readout in ~5 min) and sensitive (low nM levels of ER) platform to detect sequence-specific protein-DNA binding event. PMID:26678946

  4. DNA-Directed Assembly of Nanogold Dimers: A Unique Dynamic Light Scattering Sensing Probe for Transcription Factor Detection

    NASA Astrophysics Data System (ADS)

    Seow, Nianjia; Tan, Yen Nee; Yung, Lin-Yue Lanry; Su, Xiaodi

    2015-12-01

    We have developed a unique DNA-assembled gold nanoparticles (AuNPs) dimer for dynamic light scattering (DLS) sensing of transcription factors, exemplified by estrogen receptor (ER) that binds specifically to a double-stranded (ds) DNA sequence containing estrogen response element (ERE). Here, ERE sequence is incorporated into the DNA linkers to bridge the AuNPs dimer for ER binding. Coupled with DLS, this AuNP dimer-based DLS detection system gave distinct readout of a single ‘complex peak’ in the presence of the target molecule (i.e., ER). This unique signature marked the first time that such nanostructures can be used to study transcription factor-DNA interactions, which DLS alone cannot do. This was also unlike previously reported AuNP-DLS assays that gave random and broad distribution of particles size upon target binding. In addition, the ERE-containing AuNP dimers could also suppress the light-scattering signal from the unbound proteins and other interfering factors (e.g., buffer background), and has potential for sensitive detection of target proteins in complex biological samples such as cell lysates. In short, the as-developed AuNP dimer probe coupled with DLS is a simple (mix and test), rapid (readout in ~5 min) and sensitive (low nM levels of ER) platform to detect sequence-specific protein-DNA binding event.

  5. DNA-Directed Assembly of Nanogold Dimers: A Unique Dynamic Light Scattering Sensing Probe for Transcription Factor Detection.

    PubMed

    Seow, Nianjia; Tan, Yen Nee; Yung, Lin-Yue Lanry; Su, Xiaodi

    2015-12-18

    We have developed a unique DNA-assembled gold nanoparticles (AuNPs) dimer for dynamic light scattering (DLS) sensing of transcription factors, exemplified by estrogen receptor (ER) that binds specifically to a double-stranded (ds) DNA sequence containing estrogen response element (ERE). Here, ERE sequence is incorporated into the DNA linkers to bridge the AuNPs dimer for ER binding. Coupled with DLS, this AuNP dimer-based DLS detection system gave distinct readout of a single 'complex peak' in the presence of the target molecule (i.e., ER). This unique signature marked the first time that such nanostructures can be used to study transcription factor-DNA interactions, which DLS alone cannot do. This was also unlike previously reported AuNP-DLS assays that gave random and broad distribution of particles size upon target binding. In addition, the ERE-containing AuNP dimers could also suppress the light-scattering signal from the unbound proteins and other interfering factors (e.g., buffer background), and has potential for sensitive detection of target proteins in complex biological samples such as cell lysates. In short, the as-developed AuNP dimer probe coupled with DLS is a simple (mix and test), rapid (readout in ~5 min) and sensitive (low nM levels of ER) platform to detect sequence-specific protein-DNA binding event.

  6. A living biosensor model to dynamically trace glucocorticoid transcriptional activity during development and adult life in zebrafish.

    PubMed

    Benato, Francesca; Colletti, Elisa; Skobo, Tatjana; Moro, Enrico; Colombo, Lorenzo; Argenton, Francesco; Dalla Valle, Luisa

    2014-07-01

    Glucocorticoids (GCs) modulate many cellular processes through the binding of the glucocorticoid receptor (GR) to specific responsive elements located upstream of the transcription starting site or within an intron of GC target genes. Here we describe a transgenic fish line harboring a construct with nine GC-responsive elements (GREs) upstream of a reporter (EGFP) coding sequence. Transgenic fish exhibit strong fluorescence in many known GC-responsive organs. Moreover, its enhanced sensitivity allowed the discovery of novel GC-responsive tissue compartments, such as fin, eyes, and otic vesicles. Long-term persistence of transgene expression is seen during adult stages in several organs. Pharmacological and genetic analysis demonstrates that the transgenic line is highly responsive to drug administration and molecular manipulation. Moreover, reporter expression is sensitively and dynamically modulated by the photoperiod, thus proving that these fish are an in vivo valuable platform to explore GC responsiveness to both endogenous and exogenous stimuli.

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

    PubMed

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

    2016-02-01

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

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

    PubMed

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

    2016-02-01

    Based on the fine root samples of the first and second generations of poplar (Populus x euramericana ' Neva'), this study examined the response of quantitative and morphological traits of fine roots of different orders and the difference between generations. The results showed that, the quantitative traits of fine roots, such as root length, root surface area and root biomass, presented obvious seasonal variation, and the fine root traits had obvious difference among root orders. The quantitative traits of lower-order fine roots showed significant seasonal difference, and the fine root biomass increased in the growing season and then decreased significantly. The specific root length (SRL) of higher-order roots also showed significant change with season, while the root length density (RLD) and root tissue density (RTD) changed a little. The successive rotation resulted in the significant increase of root length, root biomass, SRL and RLD of 1-2 orders in the growing season. The quantitative traits of first order root significantly positively correlated with soil temperature and moisture, and significantly negatively correlated with the soil organic matter and soil available nitrogen content. However, the quantitative traits of second order root only showed significant correlation with soil nutrient content. The seasonal dynamics of poplar fine roots and the difference between successive rotation plantations implied carbon investment change of poplar to roots. Soil nutrient deficiency induced more carbon investment into roots, and this carbon allocation pattern might affect the aboveground productivity of poplar plantation. PMID:27396110

  9. Dynamic transcriptional profiling provides insights into tuberous root development in Rehmannia glutinosa

    PubMed Central

    Sun, Peng; Xiao, Xingguo; Duan, Liusheng; Guo, Yuhai; Qi, Jianjun; Liao, Dengqun; Zhao, Chunli; Liu, Yan; Zhou, Lili; Li, Xianen

    2015-01-01

    Rehmannia glutinosa, an herb of the Scrophulariaceae family, is widely cultivated in the Northern part of China. The tuberous root has well-known medicinal properties; however, yield and quality are threatened by abiotic and biotic stresses. Understanding the molecular process of tuberous root development may help identify novel targets for its control. In the present study, we used Illumina sequencing and de novo assembly strategies to obtain a reference transcriptome that is relevant to tuberous root development. We then conducted RNA-seq quantification analysis to determine gene expression profiles of the adventitious root (AR), thickening adventitious root (TAR), and the developing tuberous root (DTR). Expression profiling identified a total of 6794 differentially expressed unigenes during root development. Bioinformatics analysis and gene expression profiling revealed changes in phenylpropanoid biosynthesis, starch and sucrose metabolism, and plant hormone biosynthesis during root development. Moreover, we identified and allocated putative functions to the genes involved in tuberous root development, including genes related to major carbohydrate metabolism, hormone metabolism, and transcription regulation. The present study provides the initial description of gene expression profiles of AR, TAR, and DTR, which facilitates identification of genes of interest. Moreover, our work provides insights into the molecular mechanisms underlying tuberous root development and may assist in the design and development of improved breeding schemes for different R. glutinosa varieties through genetic manipulation. PMID:26113849

  10. A continuum model of transcriptional bursting

    PubMed Central

    Corrigan, Adam M; Tunnacliffe, Edward; Cannon, Danielle; Chubb, Jonathan R

    2016-01-01

    Transcription occurs in stochastic bursts. Early models based upon RNA hybridisation studies suggest bursting dynamics arise from alternating inactive and permissive states. Here we investigate bursting mechanism in live cells by quantitative imaging of actin gene transcription, combined with molecular genetics, stochastic simulation and probabilistic modelling. In contrast to early models, our data indicate a continuum of transcriptional states, with a slowly fluctuating initiation rate converting the gene between different levels of activity, interspersed with extended periods of inactivity. We place an upper limit of 40 s on the lifetime of fluctuations in elongation rate, with initiation rate variations persisting an order of magnitude longer. TATA mutations reduce the accessibility of high activity states, leaving the lifetime of on- and off-states unchanged. A continuum or spectrum of gene states potentially enables a wide dynamic range for cell responses to stimuli. DOI: http://dx.doi.org/10.7554/eLife.13051.001 PMID:26896676

  11. Evolutionary Dynamics of Floral Homeotic Transcription Factor Protein-Protein Interactions.

    PubMed

    Bartlett, Madelaine; Thompson, Beth; Brabazon, Holly; Del Gizzi, Robert; Zhang, Thompson; Whipple, Clinton

    2016-06-01

    Protein-protein interactions (PPIs) have widely acknowledged roles in the regulation of development, but few studies have addressed the timing and mechanism of shifting PPIs over evolutionary history. The B-class MADS-box transcription factors, PISTILLATA (PI) and APETALA3 (AP3) are key regulators of floral development. PI-like (PI(L)) and AP3-like (AP3(L)) proteins from a number of plants, including Arabidopsis thaliana (Arabidopsis) and the grass Zea mays (maize), bind DNA as obligate heterodimers. However, a PI(L) protein from the grass relative Joinvillea can bind DNA as a homodimer. To ascertain whether Joinvillea PI(L) homodimerization is an anomaly or indicative of broader trends, we characterized PI(L) dimerization across the Poales and uncovered unexpected evolutionary lability. Both obligate B-class heterodimerization and PI(L) homodimerization have evolved multiple times in the order, by distinct molecular mechanisms. For example, obligate B-class heterodimerization in maize evolved very recently from PI(L) homodimerization. A single amino acid change, fixed during domestication, is sufficient to toggle one maize PI(L) protein between homodimerization and obligate heterodimerization. We detected a signature of positive selection acting on residues preferentially clustered in predicted sites of contact between MADS-box monomers and dimers, and in motifs that mediate MADS PPI specificity in Arabidopsis. Changing one positively selected residue can alter PI(L) dimerization activity. Furthermore, ectopic expression of a Joinvillea PI(L) homodimer in Arabidopsis can homeotically transform sepals into petals. Our results provide a window into the evolutionary remodeling of PPIs, and show that novel interactions have the potential to alter plant form in a context-dependent manner. PMID:26908583

  12. Evolutionary Dynamics of Floral Homeotic Transcription Factor Protein–Protein Interactions

    PubMed Central

    Bartlett, Madelaine; Thompson, Beth; Brabazon, Holly; Del Gizzi, Robert; Zhang, Thompson; Whipple, Clinton

    2016-01-01

    Protein–protein interactions (PPIs) have widely acknowledged roles in the regulation of development, but few studies have addressed the timing and mechanism of shifting PPIs over evolutionary history. The B-class MADS-box transcription factors, PISTILLATA (PI) and APETALA3 (AP3) are key regulators of floral development. PI-like (PIL) and AP3-like (AP3L) proteins from a number of plants, including Arabidopsis thaliana (Arabidopsis) and the grass Zea mays (maize), bind DNA as obligate heterodimers. However, a PIL protein from the grass relative Joinvillea can bind DNA as a homodimer. To ascertain whether Joinvillea PIL homodimerization is an anomaly or indicative of broader trends, we characterized PIL dimerization across the Poales and uncovered unexpected evolutionary lability. Both obligate B-class heterodimerization and PIL homodimerization have evolved multiple times in the order, by distinct molecular mechanisms. For example, obligate B-class heterodimerization in maize evolved very recently from PIL homodimerization. A single amino acid change, fixed during domestication, is sufficient to toggle one maize PIL protein between homodimerization and obligate heterodimerization. We detected a signature of positive selection acting on residues preferentially clustered in predicted sites of contact between MADS-box monomers and dimers, and in motifs that mediate MADS PPI specificity in Arabidopsis. Changing one positively selected residue can alter PIL dimerization activity. Furthermore, ectopic expression of a Joinvillea PIL homodimer in Arabidopsis can homeotically transform sepals into petals. Our results provide a window into the evolutionary remodeling of PPIs, and show that novel interactions have the potential to alter plant form in a context-dependent manner. PMID:26908583

  13. New in situ capture quantitative (real-time) reverse transcription-PCR method as an alternative approach for determining inactivation of Tulane virus.

    PubMed

    Wang, Dapeng; Xu, Shuxia; Yang, David; Young, Glenn M; Tian, Peng

    2014-04-01

    Human noroviruses (HuNoVs) are the major cause of epidemic nonbacterial gastroenteritis. Although quantitative (real-time) reverse transcription-PCR (qRT-PCR) is widely used for detecting HuNoVs, it only detects the presence of viral RNA and does not indicate viral infectivity. Human blood group antigens (HBGAs) have been identified as receptors/co-receptors for both HuNoVs and Tulane virus (TV) and are crucial for viral infection. We propose that viral infectivity can be evaluated with a molecular assay based on receptor-captured viruses. In this study, we employed TV as an HuNoV surrogate to validate the HBGA-based capture qRT-PCR method against the 50% tissue culture infectious dose (TCID50) method. We employed type B HBGA on an immuno-well module to concentrate TV, followed by amplification of the captured viral genome by in situ qRT-PCR. We first demonstrated that this in situ capture qRT-PCR (ISC-qRT-PCR) method could effectively concentrate and detect TV. We then treated TV under either partial or full inactivation conditions and measured the remaining infectivity by ISC-qRT-PCR and a tissue culture-based amplification method (TCID50). We found that the ISC-qRT-PCR method could be used to evaluate virus inactivation deriving from damage to the capsid and study interactions between the capsid and viral receptor. Heat, chlorine, and ethanol treatment primarily affect the capsid structure, which in turns affects the ability of the capsid to bind to viral receptors. Inactivation of the virus by these methods could be reflected by the ISC-qRT-PCR method and confirmed by TCID50 assay. However, the loss of the infectivity caused by damage to the viral genome (such as that from UV irradiation) could not be effectively reflected by this method. Despite this limitation, the ISC-qRT-PCR provides an alternative approach to determine inactivation of Tulane virus. A particular advantage of the ISC-qRT-PCR method is that it is also a faster and easier method to effectively

  14. Identification of Suitable Reference Genes for Investigating Gene Expression in Anterior Cruciate Ligament Injury by Using Reverse Transcription-Quantitative PCR

    PubMed Central

    Leal, Mariana Ferreira; Astur, Diego Costa; Debieux, Pedro; Arliani, Gustavo Gonçalves; Franciozi, Carlos Eduardo Silveira; Loyola, Leonor Casilla; Andreoli, Carlos Vicente; Smith, Marília Cardoso; Pochini, Alberto de Castro; Ejnisman, Benno; Cohen, Moises

    2015-01-01

    The anterior cruciate ligament (ACL) is one of the most frequently injured structures during high-impact sporting activities. Gene expression analysis may be a useful tool for understanding ACL tears and healing failure. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) has emerged as an effective method for such studies. However, this technique requires the use of suitable reference genes for data normalization. Here, we evaluated the suitability of six reference genes (18S, ACTB, B2M, GAPDH, HPRT1, and TBP) by using ACL samples of 39 individuals with ACL tears (20 with isolated ACL tears and 19 with ACL tear and combined meniscal injury) and of 13 controls. The stability of the candidate reference genes was determined by using the NormFinder, geNorm, BestKeeper DataAssist, and RefFinder software packages and the comparative ΔCt method. ACTB was the best single reference gene and ACTB+TBP was the best gene pair. The GenEx software showed that the accumulated standard deviation is reduced when a larger number of reference genes is used for gene expression normalization. However, the use of a single reference gene may not be suitable. To identify the optimal combination of reference genes, we evaluated the expression of FN1 and PLOD1. We observed that at least 3 reference genes should be used. ACTB+HPRT1+18S is the best trio for the analyses involving isolated ACL tears and controls. Conversely, ACTB+TBP+18S is the best trio for the analyses involving (1) injured ACL tears and controls, and (2) ACL tears of patients with meniscal tears and controls. Therefore, if the gene expression study aims to compare non-injured ACL, isolated ACL tears and ACL tears from patients with meniscal tear as three independent groups ACTB+TBP+18S+HPRT1 should be used. In conclusion, 3 or more genes should be used as reference genes for analysis of ACL samples of individuals with and without ACL tears. PMID:26192306

  15. Identification of Suitable Reference Genes for Investigating Gene Expression in Anterior Cruciate Ligament Injury by Using Reverse Transcription-Quantitative PCR.

    PubMed

    Leal, Mariana Ferreira; Astur, Diego Costa; Debieux, Pedro; Arliani, Gustavo Gonçalves; Silveira Franciozi, Carlos Eduardo; Loyola, Leonor Casilla; Andreoli, Carlos Vicente; Smith, Marília Cardoso; Pochini, Alberto de Castro; Ejnisman, Benno; Cohen, Moises

    2015-01-01

    The anterior cruciate ligament (ACL) is one of the most frequently injured structures during high-impact sporting activities. Gene expression analysis may be a useful tool for understanding ACL tears and healing failure. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) has emerged as an effective method for such studies. However, this technique requires the use of suitable reference genes for data normalization. Here, we evaluated the suitability of six reference genes (18S, ACTB, B2M, GAPDH, HPRT1, and TBP) by using ACL samples of 39 individuals with ACL tears (20 with isolated ACL tears and 19 with ACL tear and combined meniscal injury) and of 13 controls. The stability of the candidate reference genes was determined by using the NormFinder, geNorm, BestKeeper DataAssist, and RefFinder software packages and the comparative ΔCt method. ACTB was the best single reference gene and ACTB+TBP was the best gene pair. The GenEx software showed that the accumulated standard deviation is reduced when a larger number of reference genes is used for gene expression normalization. However, the use of a single reference gene may not be suitable. To identify the optimal combination of reference genes, we evaluated the expression of FN1 and PLOD1. We observed that at least 3 reference genes should be used. ACTB+HPRT1+18S is the best trio for the analyses involving isolated ACL tears and controls. Conversely, ACTB+TBP+18S is the best trio for the analyses involving (1) injured ACL tears and controls, and (2) ACL tears of patients with meniscal tears and controls. Therefore, if the gene expression study aims to compare non-injured ACL, isolated ACL tears and ACL tears from patients with meniscal tear as three independent groups ACTB+TBP+18S+HPRT1 should be used. In conclusion, 3 or more genes should be used as reference genes for analysis of ACL samples of individuals with and without ACL tears.

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

    NASA Astrophysics Data System (ADS)

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

    2016-03-01

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

  17. Effect of the Concentration Difference between Magnesium Ions and Total Ribonucleotide Triphosphates in Governing the Specificity of T7 RNA Polymerase-Based Rolling Circle Transcription for Quantitative Detection.

    PubMed

    Li, Zhiyan; Lau, Choiwan; Lu, Jianzhong

    2016-06-01

    T7 RNA polymerase-based rolling circle transcription (RCT) is a more powerful tool than universal runoff transcription and traditional DNA polymerase-based rolling circle amplification (RCA). However, RCT is rarely employed in quantitative detection due to its poor specificity for small single-stranded DNA (ssDNA), which can be transcribed efficiently by T7 RNA polymerase even without a promoter. Herein we show that the concentration difference between Mg(2+) and total ribonucleotide triphosphates (rNTPs) radically governs the specificity of T7 RNA polymerase. Only when the total rNTP concentration is 9 mM greater than the Mg(2+) concentration can T7 RNA polymerase transcribe ssDNA specifically and efficiently. This knowledge improves our traditional understanding of T7 RNA polymerase and makes convenient application of RCT in quantitative detection possible. Subsequently, an RCT-based label-free chemiluminescence method for microRNA detection was designed to test the capability of this sensing platform. Using this simple method, microRNA as low as 20 amol could be quantitatively detected. The results reveal that the developed sensing platform holds great potential for further applications in the quantitative detection of a variety of targets. PMID:27167591

  18. Quantitative recurrence statistics and convergence to an extreme value distribution for non-uniformly hyperbolic dynamical systems

    NASA Astrophysics Data System (ADS)

    Holland, M. P.; Rabassa, P.; Sterk, A. E.

    2016-08-01

    For non-uniformly hyperbolic dynamical systems we consider the time series of maxima along typical orbits. Using ideas based upon quantitative recurrence time statistics we prove convergence of the maxima (under suitable normalization) to an extreme value distribution, and obtain estimates on the rate of convergence. We show that our results are applicable to a range of examples, and include new results for Lorenz maps, certain partially hyperbolic systems, and non-uniformly expanding systems with sub-exponential decay of correlations. For applications where analytic results are not readily available we show how to estimate the rate of convergence to an extreme value distribution based upon numerical information of the quantitative recurrence statistics. We envisage that such information will lead to more efficient statistical parameter estimation schemes based upon the block-maxima method.

  19. Quantitative analysis of dynamic behavior of osteoblasts during in vitro formation of micro-mass cell cultures.

    PubMed

    Schäfer, Susanne; Dekiff, Markus; Plate, Ulrich; Szuwart, Thomas; Denz, Cornelia; Dirksen, Dieter

    2013-08-01

    Improvements in bone tissue engineering require an understanding of cellular and tissue level behavior of osteoblast-like cells. Experiments indicate that in the absence of an anchoring material, intercellular adhesion may be based on signals that promote cell activity resulting in the formation of a spheroid cell-matrix. The aim of the present study is to investigate the formation of scaffold-free three-dimensional micro-mass cell spheroids in vitro, and to characterize quantitatively the cell movement. A new correlation based automated tracking method is evaluated in order to optimize the processing parameters and to identify statistical parameters that characterize the cell behavior. Results suggest that the temporal development of the mean distance of the cells to the center of gravity may be described by an exponential function, thus providing a characteristic time constant as a quantitative measure of cell dynamics. (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim).

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

    PubMed

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

    2015-10-01

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

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

    PubMed Central

    Mutukula, Naresh; Edri, Reuven; Elkabetz, Yechiel

    2015-01-01

    Neural stem cells (NSCs) are progenitor cells for brain development, where cellular spatial composition (cytoarchitecture) and dynamics are hypothesized to be linked to critical NSC capabilities. However, understanding cytoarchitectural dynamics of this process has been limited by the difficulty to quantitatively image brain development in vivo. Here, we study NSC dynamics within Neural Rosettes—highly organized multicellular structures derived from human pluripotent stem cells. Neural rosettes contain NSCs with strong epithelial polarity and are expected to perform apical-basal interkinetic nuclear migration (INM)—a hallmark of cortical radial glial cell development. We developed a quantitative live imaging framework to characterize INM dynamics within rosettes. We first show that the tendency of cells to follow the INM orientation—a phenomenon we referred to as radial organization, is associated with rosette size, presumably via mechanical constraints of the confining structure. Second, early forming rosettes, which are abundant with founder NSCs and correspond to the early proliferative developing cortex, show fast motions and enhanced radial organization. In contrast, later derived rosettes, which are characterized by reduced NSC capacity and elevated numbers of differentiated neurons, and thus correspond to neurogenesis mode in the developing cortex, exhibit slower motions and decreased radial organization. Third, later derived rosettes are characterized by temporal instability in INM measures, in agreement with progressive loss in rosette integrity at later developmental stages. Finally, molecular perturbations of INM by inhibition of ACTIN or NON-MUSCLE MYOSIN-II (NMII) reduced INM measures. Our framework enables quantification of cytoarchitecture NSC dynamics and may have implications in functional molecular studies, drug screening, and iPS cell-based platforms for disease modeling. PMID:26473351

  2. Quantitative analysis of interferon alpha receptor subunit 1 and suppressor of cytokine signaling 1 gene transcription in blood cells of patients with chronic hepatitis C

    PubMed Central

    2010-01-01

    Background Interferon (IFN)-α receptor 1 (ifnar1) and suppressor of cytokine signaling 1 (socs1) transcription levels were quantified in peripheral blood mononuclear cells (PBMC) of 59 patients infected with hepatitis C virus (HCV) and 17 non-infected individuals. Samples were obtained from patients infected with HCV that were either untreated or treated with IFN-α2 plus ribavirin for 1 year and divided into responders and non-responders based on viral load reduction 6 months after treatment. Ifnar1 and socs1 transcription was quantified by real-time RT-PCR, and the fold difference (2-ΔΔCT) with respect to hprt housekeeping gene was calculated. Results Ifnar1 transcription increased significantly in HCV-infected patients either untreated (3.26 ± 0.31), responders (3.1 ± 0.23) and non-responders (2.18 ± 0.23) with respect to non-infected individuals (1 ± 0.34; P = 0.005). Ifnar1 transcription increased significantly (P = 0.003) in patients infected with HCV genotypes 1a (4.74 ± 0.25) and 1b (2.81 ± 0.25) but not in 1a1b (1.58 ± 0.21). No association was found of Ifnar1 transcription with disease progress, initial viral load or other clinical factors. With respect to socs1 transcription, values were similar for non-infected individuals (1 ± 0.28) and untreated patients (0.99 ± 0.41) but increased in responders (2.81 ± 0.17) and non-responder patients (1.67 ± 0.41). Difference between responder and non-responder patients was not statistically significant. Socs1 transcription increased in patients infected with HCV genotypes 1a and 1b (2.87 ± 0.45 and 2.22 ± 0.17, respectively) but not in 1a1b (1.28 ± 0.40). Socs1 transcript was absent in three patients infected with HCV genotype 1b. A weak correlation between ifnar1 and socs1 transcription was found, when Spearman's correlation coefficient was calculated. Conclusion Our results suggest that HCV infection may up-regulate ifnar1 transcription. HCV genotypes differ in their capacity to affect ifnar1 and

  3. Transcription and imprinting dynamics in developing postnatal male germline stem cells.

    PubMed

    Hammoud, Saher Sue; Low, Diana H P; Yi, Chongil; Lee, Chee Leng; Oatley, Jon M; Payne, Christopher J; Carrell, Douglas T; Guccione, Ernesto; Cairns, Bradley R

    2015-11-01

    Postnatal spermatogonial stem cells (SSCs) progress through proliferative and developmental stages to populate the testicular niche prior to productive spermatogenesis. To better understand, we conducted extensive genomic profiling at multiple postnatal stages on subpopulations enriched for particular markers (THY1, KIT, OCT4, ID4, or GFRa1). Overall, our profiles suggest three broad populations of spermatogonia in juveniles: (1) epithelial-like spermatogonia (THY1(+); high OCT4, ID4, and GFRa1), (2) more abundant mesenchymal-like spermatogonia (THY1(+); moderate OCT4 and ID4; high mesenchymal markers), and (3) (in older juveniles) abundant spermatogonia committing to gametogenesis (high KIT(+)). Epithelial-like spermatogonia displayed the expected imprinting patterns, but, surprisingly, mesenchymal-like spermatogonia lacked imprinting specifically at paternally imprinted loci but fully restored imprinting prior to puberty. Furthermore, mesenchymal-like spermatogonia also displayed developmentally linked DNA demethylation at meiotic genes and also at certain monoallelic neural genes (e.g., protocadherins and olfactory receptors). We also reveal novel candidate receptor-ligand networks involving SSCs and the developing niche. Taken together, neonates/juveniles contain heterogeneous epithelial-like or mesenchymal-like spermatogonial populations, with the latter displaying extensive DNA methylation/chromatin dynamics. We speculate that this plasticity helps SSCs proliferate and migrate within the developing seminiferous tubule, with proper niche interaction and membrane attachment reverting mesenchymal-like spermatogonial subtype cells back to an epithelial-like state with normal imprinting profiles. PMID:26545815

  4. Transcription and imprinting dynamics in developing postnatal male germline stem cells

    PubMed Central

    Hammoud, Saher Sue; Low, Diana H.P.; Yi, Chongil; Lee, Chee Leng; Oatley, Jon M.; Payne, Christopher J.; Carrell, Douglas T.; Guccione, Ernesto; Cairns, Bradley R.

    2015-01-01

    Postnatal spermatogonial stem cells (SSCs) progress through proliferative and developmental stages to populate the testicular niche prior to productive spermatogenesis. To better understand, we conducted extensive genomic profiling at multiple postnatal stages on subpopulations enriched for particular markers (THY1, KIT, OCT4, ID4, or GFRa1). Overall, our profiles suggest three broad populations of spermatogonia in juveniles: (1) epithelial-like spermatogonia (THY1+; high OCT4, ID4, and GFRa1), (2) more abundant mesenchymal-like spermatogonia (THY1+; moderate OCT4 and ID4; high mesenchymal markers), and (3) (in older juveniles) abundant spermatogonia committing to gametogenesis (high KIT+). Epithelial-like spermatogonia displayed the expected imprinting patterns, but, surprisingly, mesenchymal-like spermatogonia lacked imprinting specifically at paternally imprinted loci but fully restored imprinting prior to puberty. Furthermore, mesenchymal-like spermatogonia also displayed developmentally linked DNA demethylation at meiotic genes and also at certain monoallelic neural genes (e.g., protocadherins and olfactory receptors). We also reveal novel candidate receptor–ligand networks involving SSCs and the developing niche. Taken together, neonates/juveniles contain heterogeneous epithelial-like or mesenchymal-like spermatogonial populations, with the latter displaying extensive DNA methylation/chromatin dynamics. We speculate that this plasticity helps SSCs proliferate and migrate within the developing seminiferous tubule, with proper niche interaction and membrane attachment reverting mesenchymal-like spermatogonial subtype cells back to an epithelial-like state with normal imprinting profiles. PMID:26545815

  5. Nucleo-cytoplasmic shuttling dynamics of the transcriptional regulators XYR1 and CRE1 under conditions of cellulase and xylanase gene expression in Trichoderma reesei

    PubMed Central

    Lichius, Alexander; Seidl-Seiboth, Verena; Seiboth, Bernhard; Kubicek, Christian P

    2014-01-01

    Trichoderma reesei is a model for investigating the regulation of (hemi-)cellulase gene expression. Cellulases are formed adaptively, and the transcriptional activator XYR1 and the carbon catabolite repressor CRE1 are main regulators of their expression. We quantified the nucleo-cytoplasmic shuttling dynamics of GFP-fusion proteins of both transcription factors under cellulase and xylanase inducing conditions, and correlated their nuclear presence/absence with transcriptional changes. We also compared their subcellular localization in conidial germlings and mature hyphae. We show that cellulase gene expression requires de novo biosynthesis of XYR1 and its simultaneous nuclear import, whereas carbon catabolite repression is regulated through preformed CRE1 imported from the cytoplasmic pool. Termination of induction immediately stopped cellulase gene transcription and was accompanied by rapid nuclear degradation of XYR1. In contrast, nuclear CRE1 rapidly decreased upon glucose depletion, and became recycled into the cytoplasm. In mature hyphae, nuclei containing activated XYR1 were concentrated in the colony center, indicating that this is the main region of XYR1 synthesis and cellulase transcription. CRE1 was found to be evenly distributed throughout the entire mycelium. Taken together, our data revealed novel aspects of the dynamic shuttling and spatial bias of the major regulator of (hemi-)cellulase gene expression, XYR1, in T. reesei. PMID:25302561

  6. High speed optical tomography system for quantitative measurement and visualization of dynamic features in a round jet

    NASA Astrophysics Data System (ADS)

    McMackin, L.; Hugo, R. J.; Bishop, K. P.; Chen, E. Y.; Pierson, R. E.; Truman, C. R.

    An optical tomography system that is capable of operating at frame rates of up to 5 kHz has been used to obtain spatially resolved cross-sectional temperature images of a heated round jet. These tomographic images show dynamic details in the evolving vortical flow structures found in the near field of the jet that are consistent with previous studies of low speed jet flow. Reconstructions produced by the system are quantitative temperature distributions of a planar cross section of the jet measuring temperature differences with a spatial resolution of 1.4 mm.

  7. Biophysical models of transcription in cells

    NASA Astrophysics Data System (ADS)

    Choubey, Sandeep

    Cells constantly face environmental challenges and deal with them by changing their gene expression patterns. They make decisions regarding which genes to express and which genes not to express based on intra-cellular and environmental cues. These decisions are often made by regulating the process of transcription. While the identities of the different molecules that take part in regulating transcription have been determined for a number of different genes, their dynamics inside the cell are still poorly understood. One key feature of these regulatory dynamics is that the numbers of the bio-molecules involved is typically small, resulting in large temporal fluctuations in transcriptional outputs (mRNA and protein). In this thesis I show that measurements of the cell-to-cell variability of the distribution of transcribing RNA polymerases along a gene provide a previously unexplored method for deciphering the mechanism of its transcription in vivo. First, I propose a simple kinetic model of transcription initiation and elongation from which I calculate transcribing RNA polymerase copy-number fluctuations. I test my theory against published data obtained for yeast genes and propose a novel mechanism of transcription. Rather than transcription being initiated through a single rate-limiting step, as was previously proposed, my single-cell analysis reveals the presence of at least two rate limiting steps. Second, I compute the distribution of inter-polymerase distance distribution along a gene and propose a method for analyzing inter-polymerase distance distributions acquired in experiments. By applying this method to images of polymerases transcribing ribosomal genes in E.coli I show that one model of regulation of these genes is consistent with inter-polymerase distance data while a number of other models are not. The analytical framework described in this thesis can be used to extract quantitative information about the dynamics of transcription from single

  8. Quantitative measurement of intracellular protein dynamics using photobleaching or photoactivation of fluorescent proteins.

    PubMed

    Matsuda, Tomoki; Nagai, Takeharu

    2014-12-01

    Unlike in vitro protein dynamics, intracellular protein dynamics are intricately regulated by protein-protein interactions or interactions between proteins and other cellular components, including nucleic acids, the plasma membrane and the cytoskeleton. Alteration of these dynamics plays a crucial role in physiological phenomena such as gene expression and cell division. Live-cell imaging via microscopy with the inherent properties of fluorescent proteins, i.e. photobleaching and photoconversion, or fluorescence correlation spectroscopy, provides insight into the movement of proteins and their interactions with cellular components. This article reviews techniques based on photo-induced changes in the physicochemical properties of fluorescent proteins to measure protein dynamics inside living cells, and it also discusses the strengths and weaknesses of these techniques.

  9. Static and dynamic systems in Rickettsia slovaca life cycle evaluated by quantitative real-time polymerase chain reaction.

    PubMed

    Spitalská, E; Sparagano, O; Boldis, V

    2010-04-01

    Quantitative real-time polymerase chain reaction was used to characterize the growth of Rickettsia slovaca, a tick-borne pathogen transmitted by Dermacentor reticulatus and D. marginatus ticks, in static (L929 and Vero cells) and dynamic (D. marginatus and Ixodes ricinus ticks) cultivation systems. The highest points of bacterial multiplication and the time-spans between the inoculum and the maximum of rickettsial copies were increased in consecutive order from eukaryotic cells, I. ricinus to D. marginatus systems. In dynamic system, multiplication maximum of R. slovaca was achieved 9 days earlier in I. ricinus; however, the number of rickettsial DNA copies was approximately 3.6 x 10(6) more in D. marginatus. PMID:20537110

  10. Dynamic quantitative photothermal monitoring of cell death of individual human red blood cells upon glucose depletion

    NASA Astrophysics Data System (ADS)

    Vasudevan, Srivathsan; Chen, George Chung Kit; Andika, Marta; Agarwal, Shuchi; Chen, Peng; Olivo, Malini

    2010-09-01

    Red blood cells (RBCs) have been found to undergo ``programmed cell death,'' or eryptosis, and understanding this process can provide more information about apoptosis of nucleated cells. Photothermal (PT) response, a label-free photothermal noninvasive technique, is proposed as a tool to monitor the cell death process of living human RBCs upon glucose depletion. Since the physiological status of the dying cells is highly sensitive to photothermal parameters (e.g., thermal diffusivity, absorption, etc.), we applied linear PT response to continuously monitor the death mechanism of RBC when depleted of glucose. The kinetics of the assay where the cell's PT response transforms from linear to nonlinear regime is reported. In addition, quantitative monitoring was performed by extracting the relevant photothermal parameters from the PT response. Twofold increases in thermal diffusivity and size reduction were found in the linear PT response during cell death. Our results reveal that photothermal parameters change earlier than phosphatidylserine externalization (used for fluorescent studies), allowing us to detect the initial stage of eryptosis in a quantitative manner. Hence, the proposed tool, in addition to detection of eryptosis earlier than fluorescence, could also reveal physiological status of the cells through quantitative photothermal parameter extraction.

  11. Transcriptional regulation of cell cycle genes in response to abiotic stresses correlates with dynamic changes in histone modifications in maize.

    PubMed

    Zhao, Lin; Wang, Pu; Hou, Haoli; Zhang, Hao; Wang, Yapei; Yan, Shihan; Huang, Yan; Li, Hui; Tan, Junjun; Hu, Ao; Gao, Fei; Zhang, Qi; Li, Yingnan; Zhou, Hong; Zhang, Wei; Li, Lijia

    2014-01-01

    The histone modification level has been shown to be related with gene activation and repression in stress-responsive process, but there is little information on the relationship between histone modification and cell cycle gene expression responsive to environmental cues. In this study, the function of histone modifications in mediating the transcriptional regulation of cell cycle genes under various types of stress was investigated in maize (Zea mays L.). Abiotic stresses all inhibit the growth of maize seedlings, and induce total acetylation level increase compared with the control group in maize roots. The positive and negative regulation of the expression of some cell cycle genes leads to perturbation of cell cycle progression in response to abiotic stresses. Chromatin immunoprecipitation analysis reveals that dynamic histone acetylation change in the promoter region of cell cycle genes is involved in the control of gene expression in response to external stress and different cell cycle genes have their own characteristic patterns for histone acetylation. The data also showed that the combinations of hyperacetylation and hypoacetylation states of specific lysine sites on the H3 and H4 tails on the promoter regions of cell cycle genes regulate specific cell cycle gene expression under abiotic stress conditions, thus resulting in prolonged cell cycle duration and an inhibitory effect on growth and development in maize seedlings. PMID:25171199

  12. Elucidating the evolutionary conserved DNA-binding specificities of WRKY transcription factors by molecular dynamics and in vitro binding assays

    PubMed Central

    Brand, Luise H.; Fischer, Nina M.; Harter, Klaus; Kohlbacher, Oliver; Wanke, Dierk

    2013-01-01

    WRKY transcription factors constitute a large protein family in plants that is involved in the regulation of developmental processes and responses to biotic or abiotic stimuli. The question arises how stimulus-specific responses are mediated given that the highly conserved WRKY DNA-binding domain (DBD) exclusively recognizes the ‘TTGACY’ W-box consensus. We speculated that the W-box consensus might be more degenerate and yet undetected differences in the W-box consensus of WRKYs of different evolutionary descent exist. The phylogenetic analysis of WRKY DBDs suggests that they evolved from an ancestral group IIc-like WRKY early in the eukaryote lineage. A direct descent of group IIc WRKYs supports a monophyletic origin of all other group II and III WRKYs from group I by loss of an N-terminal DBD. Group I WRKYs are of paraphyletic descent and evolved multiple times independently. By homology modeling, molecular dynamics simulations and in vitro DNA–protein interaction-enzyme-linked immunosorbent assay with AtWRKY50 (IIc), AtWRKY33 (I) and AtWRKY11 (IId) DBDs, we revealed differences in DNA-binding specificities. Our data imply that other components are essentially required besides the W-box-specific binding to DNA to facilitate a stimulus-specific WRKY function. PMID:23975197

  13. Interaction of human mitochondrial transcription factor A in mitochondria: its involvement in the dynamics of mitochondrial DNA nucleoids.

    PubMed

    Kasashima, Katsumi; Endo, Hitoshi

    2015-12-01

    Mitochondrial transcription factor A (TFAM) is a key regulator of mitochondrial DNA (mtDNA). TFAM interacts with itself and forms dimers; however, the precise interaction domain in vivo has not yet been determined. We herein showed that human TFAM formed oligomers in mitochondria by in situ chemical cross-linking. We used the separated fluorescent protein, monomeric Kusabira-Green, as a reporter to monitor their self-association in mitochondria. This reporter successfully detected the TFAM-TFAM interaction in cells as fluorescent signals on mitochondria. We also found that the N-terminal high-mobility group box domain was sufficient for this interaction. The expression of the dimer-defective mutant induced enlarged mtDNA nucleoids, suggesting the importance of dimerization in the distribution of mtDNA. The reporter system also supported the association and mixture between independent nucleoids through TFAM by a cell fusion assay using hemagglutinating virus of Japan. We here, for the first time, visualized the interaction of TFAM molecules in mitochondria and proposed its implications for the dynamics of mtDNA nucleoids.

  14. Imaging Transcription: Past, Present, and Future

    PubMed Central

    Coleman, Robert A.; Liu, Zhe; Darzacq, Xavier; Tjian, Robert; Singer, Robert H.; Lionnet, Timothée

    2016-01-01

    Transcription, the first step of gene expression, is exquisitely regulated in higher eukaryotes to ensure correct development and homeostasis. Traditional biochemical, genetic, and genomic approaches have proved successful at identifying factors, regulatory sequences, and potential pathways that modulate transcription. However, they typically only provide snapshots or population averages of the highly dynamic, stochastic biochemical processes involved in transcriptional regulation. Single molecule live-cell imaging has, therefore, emerged as a complementary approach capable of circumventing these limitations. By observing sequences of molecular events in real time as they occur in their native context, imaging has the power to derive cause-and-effect relationships and quantitative kinetics to build predictive models of transcription. Ongoing progress in fluorescence imaging technology has brought new microscopes and labeling technologies that now make it possible to visualize and quantify the transcription process with single-molecule resolution in living cells and animals. Here we provide an overview of the evolution and current state of transcription imaging technologies. We discuss some of the important concepts they uncovered and present possible future developments that might solve long-standing questions in transcriptional regulation. PMID:26763984

  15. Monitoring the dynamics of syntrophic β-oxidizing bacteria during anaerobic degradation of oleic acid by quantitative PCR.

    PubMed

    Ziels, Ryan M; Beck, David A C; Martí, Magalí; Gough, Heidi L; Stensel, H David; Svensson, Bo H

    2015-04-01

    The ecophysiology of long-chain fatty acid-degrading syntrophic β-oxidizing bacteria has been poorly understood due to a lack of quantitative abundance data. Here, TaqMan quantitative PCR (qPCR) assays targeting the 16S rRNA gene of the known mesophilic syntrophic β-oxidizing bacterial genera Syntrophomonas and Syntrophus were developed and validated. Microbial community dynamics were followed using qPCR and Illumina-based high-throughput amplicon sequencing in triplicate methanogenic bioreactors subjected to five consecutive batch feedings of oleic acid. With repeated oleic acid feeding, the initial specific methane production rate significantly increased along with the relative abundances of Syntrophomonas and methanogenic archaea in the bioreactor communities. The novel qPCR assays showed that Syntrophomonas increased from 7 to 31% of the bacterial community 16S rRNA gene concentration, whereas that of Syntrophus decreased from 0.02 to less than 0.005%. High-throughput amplicon sequencing also revealed that Syntrophomonas became the dominant genus within the bioreactor microbiomes. These results suggest that increased specific mineralization rates of oleic acid were attributed to quantitative shifts within the microbial communities toward higher abundances of syntrophic β-oxidizing bacteria and methanogenic archaea. The novel qPCR assays targeting syntrophic β-oxidizing bacteria may thus serve as monitoring tools to indicate the fatty acid β-oxidization potential of anaerobic digester communities.

  16. Quantitative Analysis of Dynamic Softening Behaviors Induced by Dynamic Recrystallization for Ti-10V-2Fe-2Al Alloy

    NASA Astrophysics Data System (ADS)

    Quan, Guozheng; Pu, Shiao; Wen, Hairong; Zou, Zhenyu; Zhou, Jie

    2015-10-01

    In order to investigate the effect of dynamic recrystallization (DRX) behavior on dynamic softening behavior of wrought Ti-10V-2Fe-3Al titanium alloy, a series of laboratory scale isothermal hot compression tests with a height reduction of 60% were performed in a temperature range of 948 K 1023 K in the (σ + β) phase field, and a strain rate range of 0.01 10 s-1 on a Gleeble-3500 thermo-mechanical simulator. The flow curves show a continuous softening at all strain rate after peak stress. The constitutive equation and the DRX kinetic mold were established to study the dynamic softening based on the flow curves. By the regression analysis for conventional hyperbolic sine equation, the activation energy was determined as Q = 479.4169 kJ·mol-1, According to the strain hardening rate curves (dσ/dɛ versus σ), two characteristic parameters including the critical strain for DRX initiation (ɛc) and the strain for peak stress (ɛp) were identified, and the linear dependence of the critical strain (ɛc) for DRX initiation on the strain for peak stress (ɛp) can be specified by the equation: ɛc = 0.5667ɛp. A modified Avrami type equation X_{DRX} = 1 - exp[-β_{d}(\\varepsilon - \\varepsilon_c over \\varepsilon_{0.5})k_d] was introduced to characterize the evolution of DRX volume fraction. The evolution of DRX volume was described as the following: for a fixed strain rate, the strain required for the same amount of DRX volume fraction increases with decreasing deformation temperature, in contrast, for a fixed temperature, it increases with increasing strain rate. Finally, the impact of dynamic recrystallized behavior on degree of dynamic softening became weaker and weaker with the increasing of temperature for the strain rate of 0.01 s-1, 0.1 s-1, 1 s-1 and 10 s-1, due to the volume of α phase decreased with the increasing of temperature.

  17. A new dynamic myocardial phantom for evaluation of SPECT and PET quantitation in systolic and diastolic conditions

    SciTech Connect

    Dreuille, O. de; Bendriem, B.; Riddell, C.

    1996-12-31

    We present a new dynamic myocardial phantom designed to evaluate SPECT and PET imaging in systolic and diastolic conditions. The phantom includes a thoracic attenuating media and the myocardial wall thickness varying during the scan can be performed. In this study the phantom was used with three different wall thickness characteristic of a systolic, end-diastolic and pathologic end-diastolic condition. The myocardium was filled with {sup 99m}Tc, {sup 18}F and Gd and imaged by SPECT, PET and MRI. SPECT attenuation correction was performed using a modified PET transmission. A bull`s eyes image was obtained for all data and wall ROI were then drawn for analysis. Using MRI as a reference, error from PET, SPECT and attenuation corrected SPECT were calculated. Systolic PET performances agree with MRI. Quantitation loss due to wall thickness reduction compared to the systole. Attenuation correction in SPECT leads to significant decrease of the error both in systole (from 29% to 14%) and diastole (35% to 22%). This is particularly sensitive for septum and inferior walls. SPECT residual errors (14% in systole and 22% in pathologic end-diastole) are likely caused by scatter, noise and depth dependent resolution effect. The results obtained with this dynamical phantom demonstrate the quantitation improvement achieved in SPECT with attenuation correction and also reinforce the need for variable resolution correction in addition to attenuation correction.

  18. Quantitation of Cellular Dynamics in Growing Arabidopsis Roots with Light Sheet Microscopy

    PubMed Central

    Birnbaum, Kenneth D.; Leibler, Stanislas

    2011-01-01

    To understand dynamic developmental processes, living tissues have to be imaged frequently and for extended periods of time. Root development is extensively studied at cellular resolution to understand basic mechanisms underlying pattern formation and maintenance in plants. Unfortunately, ensuring continuous specimen access, while preserving physiological conditions and preventing photo-damage, poses major barriers to measurements of cellular dynamics in growing organs such as plant roots. We present a system that integrates optical sectioning through light sheet fluorescence microscopy with hydroponic culture that enables us to image, at cellular resolution, a vertically growing Arabidopsis root every few minutes and for several consecutive days. We describe novel automated routines to track the root tip as it grows, to track cellular nuclei and to identify cell divisions. We demonstrate the system's capabilities by collecting data on divisions and nuclear dynamics. PMID:21731697

  19. Quantitation of cellular dynamics in growing Arabidopsis roots with light sheet microscopy.

    PubMed

    Sena, Giovanni; Frentz, Zak; Birnbaum, Kenneth D; Leibler, Stanislas

    2011-01-01

    To understand dynamic developmental processes, living tissues have to be imaged frequently and for extended periods of time. Root development is extensively studied at cellular resolution to understand basic mechanisms underlying pattern formation and maintenance in plants. Unfortunately, ensuring continuous specimen access, while preserving physiological conditions and preventing photo-damage, poses major barriers to measurements of cellular dynamics in growing organs such as plant roots. We present a system that integrates optical sectioning through light sheet fluorescence microscopy with hydroponic culture that enables us to image, at cellular resolution, a vertically growing Arabidopsis root every few minutes and for several consecutive days. We describe novel automated routines to track the root tip as it grows, to track cellular nuclei and to identify cell divisions. We demonstrate the system's capabilities by collecting data on divisions and nuclear dynamics.

  20. A high-throughput imaging system to quantitatively analyze the growth dynamics of plant seedlings.

    PubMed

    Men, Yongfan; Yu, Qiang; Chen, Zitian; Wang, Jianbin; Huang, Yanyi; Guo, Hongwei

    2012-08-01

    Most current methods for analyzing the growth rate of plant seedlings are limited to low-throughput experimental configurations. We have developed an automatic system to investigate the dynamics of the growth of hypocotyls using Arabidopsis as model. This system is able to capture time-lapse infrared images of 24 seedlings automatically, with a spatial resolution of 2 μm per pixel and temporal interval of 5 min. Seedling length is rapidly calculated using automated geometric image-processing algorithms. With this high-throughput platform, we have investigated the genotype dependent difference of growth patterns, as well as the response to plant hormone - ethylene. Our analyses suggest that cytoskeleton function is not required in ethylene-induced hypocotyl inhibition. This novel integrative method can be applied to large-scale dynamic screening of plants, as well as any other image-based biological studies related to dynamic growth.

  1. Semiquantitative and Quantitative Dynamic Contrast-Enhanced Magnetic Resonance Imaging Measurements Predict Radiation Response in Cervix Cancer

    SciTech Connect

    Zahra, Mark A. Tan, Li Tee; Priest, Andrew N.; Graves, Martin J.; Arends, Mark; Crawford, Robin A.F.; Brenton, James D.; Lomas, David J.; Sala, Evis

    2009-07-01

    Purpose: To evaluate semiquantitative and quantitative dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) measurements in predicting the response to radiotherapy in cervix cancer. Methods and Materials: Patients with cervix cancer treated radically with chemoradiotherapy had DCE-MRI at three time points: before starting treatment, after 2 weeks of radiotherapy, and in the 5th week of radiotherapy. Semiquantitative measurements obtained from the signal intensity vs. time plots included arrival time of contrast, the slope and maximum slope of contrast uptake, time for peak enhancement, and the contrast enhancement ratio (CER). Pharmacokinetic modeling with a modeled vascular input function was used for the quantitative measurements volume transfer constant (K{sup trans}), rate constant (k{sub ep}), fraction plasma volume (fPV), and the initial area under gadolinium-time curve. The correlation of these measurements at each of the three time points with radiologic tumor response was investigated. Results: Thirteen patients had a total of 38 scans. There was no correlation between the DCE-MRI measurements and the corresponding tumor volumes. A statistically significant correlation with percentage tumor regression was shown with the pretreatment DCE-MRI semiquantitative parameters of peak time (p = 0.046), slope (p = 0.025), maximum slope (p = 0.046), and CER (p = 0.025) and the quantitative parameters K{sup trans} (p = 0.043) and k{sub ep} (p = 0.022). Second and third scan measurements did not show any correlation. Conclusions: This is the first study to show that pretreatment DCE-MRI quantitative parameters predict the radiation response in cervix cancer. These measurements may allow a more meaningful comparison of DCE-MRI studies from different centers.

  2. Static and dynamic crystalline lens accommodation evaluated using quantitative 3-D OCT

    PubMed Central

    Gambra, Enrique; Ortiz, Sergio; Perez-Merino, Pablo; Gora, Michalina; Wojtkowski, Maciej; Marcos, Susana

    2013-01-01

    Custom high-resolution high-speed anterior segment spectral domain Optical Coherence Tomography (OCT) provided with automatic quantification and distortion correction algorithms was used to characterize three-dimensionally (3-D) the human crystalline lens in vivo in four subjects, for accommodative demands between 0 to 6 D in 1 D steps. Anterior and posterior lens radii of curvature decreased with accommodative demand at rates of 0.73 and 0.20 mm/D, resulting in an increase of the estimated optical power of the eye of 0.62 D per diopter of accommodative demand. Dynamic fluctuations in crystalline lens radii of curvature, anterior chamber depth and lens thickness were also estimated from dynamic 2-D OCT images (14 Hz), acquired during 5-s of steady fixation, for different accommodative demands. Estimates of the eye power from dynamical geometrical measurements revealed an increase of the fluctuations of the accommodative response from 0.07 D to 0.47 D between 0 and 6 D (0.044 D per D of accommodative demand). A sensitivity analysis showed that the fluctuations of accommodation were driven by dynamic changes in the lens surfaces, particularly in the posterior lens surface. PMID:24049680

  3. Quantitative high-throughput population dynamics in continuous-culture by automated microscopy.

    PubMed

    Merritt, Jason; Kuehn, Seppe

    2016-01-01

    We present a high-throughput method to measure abundance dynamics in microbial communities sustained in continuous-culture. Our method uses custom epi-fluorescence microscopes to automatically image single cells drawn from a continuously-cultured population while precisely controlling culture conditions. For clonal populations of Escherichia coli our instrument reveals history-dependent resilience and growth rate dependent aggregation. PMID:27616752

  4. Quantitative high-throughput population dynamics in continuous-culture by automated microscopy

    PubMed Central

    Merritt, Jason; Kuehn, Seppe

    2016-01-01

    We present a high-throughput method to measure abundance dynamics in microbial communities sustained in continuous-culture. Our method uses custom epi-fluorescence microscopes to automatically image single cells drawn from a continuously-cultured population while precisely controlling culture conditions. For clonal populations of Escherichia coli our instrument reveals history-dependent resilience and growth rate dependent aggregation. PMID:27616752

  5. Quantitative 3D analysis of shape dynamics of the left ventricle

    NASA Astrophysics Data System (ADS)

    Scowen, Barry C.; Smith, Stephen L.; Vannan, Mani A.; Arsenault, Marie

    1998-07-01

    There is an established link between Left Ventricular (LV) geometry and its performance. As a consequence of ischemic heart disease and the attempt to relieve myocardial tissue stress, ventricle shape begins to distort from a conical to spherical geometry with a reduction in pumping efficiency of the chamber. If untreated, premature heart failure will result. To increase the changes of successful treatment it is obviously important for the benefit of the patient to detect these abnormalities as soon as possible. It is the development of a technique to characterize and quantify the shape of the left ventricle that is described here. The system described in this paper uses a novel helix model which combines the advantages of current two dimensional (2D) quantitative measures which provide limited information, with 3D qualitative methods which provide accurate reconstructions of the LV using computationally expensive rendering schemes. A phantom object and dog ventricle (normal/abnormal) were imaged and helical models constructed. The result are encouraging with differences between normal and abnormal ventricles in both diastole and systole able to be determined. Further work entails building a library of subjects in order to determine the relationship between ventricle geometry and quantitative measurements.

  6. A Quantitative Dynamic Simulation of Bremia lactucae Airborne Conidia Concentration above a Lettuce Canopy

    PubMed Central

    Fall, Mamadou Lamine; Van der Heyden, Hervé; Carisse, Odile

    2016-01-01

    Lettuce downy mildew, caused by the oomycete Bremia lactucae Regel, is a major threat to lettuce production worldwide. Lettuce downy mildew is a polycyclic disease driven by airborne spores. A weather-based dynamic simulation model for B. lactucae airborne spores was developed to simulate the aerobiological characteristics of the pathogen. The model was built using the STELLA platform by following the system dynamics methodology. The model was developed using published equations describing disease subprocesses (e.g., sporulation) and assembled knowledge of the interactions among pathogen, host, and weather. The model was evaluated with four years of independent data by comparing model simulations with observations of hourly and daily airborne spore concentrations. The results show an accurate simulation of the trend and shape of B. lactucae temporal dynamics of airborne spore concentration. The model simulated hourly and daily peaks in airborne spore concentrations. More than 95% of the simulation runs, the daily-simulated airborne conidia concentration was 0 when airborne conidia were not observed. Also, the relationship between the simulated and the observed airborne spores was linear. In more than 94% of the simulation runs, the proportion of the linear variation in the hourly-observed values explained by the variation in the hourly-simulated values was greater than 0.7 in all years except one. Most of the errors came from the deviation from the 1:1 line, and the proportion of errors due to the model bias was low. This model is the only dynamic model developed to mimic the dynamics of airborne inoculum and represents an initial step towards improved lettuce downy mildew understanding, forecasting and management. PMID:26953691

  7. A Quantitative Dynamic Simulation of Bremia lactucae Airborne Conidia Concentration above a Lettuce Canopy.

    PubMed

    Fall, Mamadou Lamine; Van der Heyden, Hervé; Carisse, Odile

    2016-01-01

    Lettuce downy mildew, caused by the oomycete Bremia lactucae Regel, is a major threat to lettuce production worldwide. Lettuce downy mildew is a polycyclic disease driven by airborne spores. A weather-based dynamic simulation model for B. lactucae airborne spores was developed to simulate the aerobiological characteristics of the pathogen. The model was built using the STELLA platform by following the system dynamics methodology. The model was developed using published equations describing disease subprocesses (e.g., sporulation) and assembled knowledge of the interactions among pathogen, host, and weather. The model was evaluated with four years of independent data by comparing model simulations with observations of hourly and daily airborne spore concentrations. The results show an accurate simulation of the trend and shape of B. lactucae temporal dynamics of airborne spore concentration. The model simulated hourly and daily peaks in airborne spore concentrations. More than 95% of the simulation runs, the daily-simulated airborne conidia concentration was 0 when airborne conidia were not observed. Also, the relationship between the simulated and the observed airborne spores was linear. In more than 94% of the simulation runs, the proportion of the linear variation in the hourly-observed values explained by the variation in the hourly-simulated values was greater than 0.7 in all years except one. Most of the errors came from the deviation from the 1:1 line, and the proportion of errors due to the model bias was low. This model is the only dynamic model developed to mimic the dynamics of airborne inoculum and represents an initial step towards improved lettuce downy mildew understanding, forecasting and management. PMID:26953691

  8. Dynamic contrast optical coherence tomography: quantitative measurement of microvascular transit-time distributions in vivo (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Merkle, Conrad W.; Srinivasan, Vivek J.

    2016-03-01

    Transit time is a fundamental microcirculatory parameter that is critical in determining oxygen delivery from capillaries to surrounding tissue. Recently, it was demonstrated theoretically that capillary transit-time heterogeneity potentially leads to non-uniform oxygen extraction in micro-domains. However, in spite of its importance, capillary transit-time distribution has been challenging to quantify comprehensively and efficiently at the microscopic level. Here, we introduce a method, called Dynamic Contrast Optical Coherence Tomography (DyC-OCT), based on dynamic cross-sectional OCT imaging of the kinetics of an intravascular tracer during its passage through the field-of-view. DyC-OCT is used to quantitatively measure the transit-time distribution in microvascular networks in cross-section at the single-capillary level. Transit-time metrics are derived from analysis of the temporal characteristics of the dynamic scattering signal, related to tracer concentration, using indicator-dilution theory. Since DyC-OCT does not require calibration of the optical focus, quantitative accuracy is achieved even deep in highly scattering brain tissue where the focal spot degrades. After direct validation of DyC-OCT against the dilution curves measured using a fluorescent plasma label in the surface pial vessels of a mouse brain, imaged through a thinned-skull, glass coverslip-reinforced cranial window, the laminar transit-time distribution was investigated in microvasculature across the entire depth of the mouse somatosensory cortex. Laminar trends were identified, with the earliest transit times in the middle cortical layers, and the lowest heterogeneity in cortical layer 4. The new DyC-OCT technique affords a novel perspective of microvascular networks, with the unique capability of performing simultaneous measurements of transit-time distributions across cortical laminae.

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

    PubMed

    Bernitt, Erik; Koh, Cheng Gee; Gov, Nir; Döbereiner, Hans-Günther

    2015-01-01

    Circular Dorsal Ruffles (CDRs) have been known for decades, but the mechanism that organizes these actin waves remains unclear. In this article we systematically analyze the dynamics of CDRs on fibroblasts with respect to characteristics of current models of actin waves. We studied CDRs on heterogeneously shaped cells and on cells that we forced into disk-like morphology. We show that CDRs exhibit phenomena such as periodic cycles of formation, spiral patterns, and mutual wave annihilations that are in accord with an active medium description of CDRs. On cells of controlled morphologies, CDRs exhibit extremely regular patterns of repeated wave formation and propagation, whereas on random-shaped cells the dynamics seem to be dominated by the limited availability of a reactive species. We show that theoretical models of reaction-diffusion type incorporating conserved species capture partially the behavior we observe in our data. PMID:25574668

  10. Heteronuclear Adiabatic Relaxation Dispersion (HARD) for quantitative analysis of conformational dynamics in proteins

    NASA Astrophysics Data System (ADS)

    Traaseth, Nathaniel J.; Chao, Fa-An; Masterson, Larry R.; Mangia, Silvia; Garwood, Michael; Michaeli, Shalom; Seelig, Burckhard; Veglia, Gianluigi

    2012-06-01

    NMR relaxation methods probe biomolecular motions over a wide range of timescales. In particular, the rotating frame spin-lock R1ρ and Carr-Purcell-Meiboom-Gill (CPMG) R2 experiments are commonly used to characterize μs to ms dynamics, which play a critical role in enzyme folding and catalysis. In an effort to complement these approaches, we introduced the Heteronuclear Adiabatic Relaxation Dispersion (HARD) method, where dispersion in rotating frame relaxation rate constants (longitudinal R1ρ and transverse R2ρ) is created by modulating the shape and duration of adiabatic full passage (AFP) pulses. Previously, we showed the ability of the HARD method to detect chemical exchange dynamics in the fast exchange regime (kex ˜ 104-105 s-1). In this article, we show the sensitivity of the HARD method to slower exchange processes by measuring R1ρ and R2ρ relaxation rates for two soluble proteins (ubiquitin and 10C RNA ligase). One advantage of the HARD method is its nominal dependence on the applied radio frequency field, which can be leveraged to modulate the dispersion in the relaxation rate constants. In addition, we also include product operator simulations to define the dynamic range of adiabatic R1ρ and R2ρ that is valid under all exchange regimes. We conclude from both experimental observations and simulations that this method is complementary to CPMG-based and rotating frame spin-lock R1ρ experiments to probe conformational exchange dynamics for biomolecules. Finally, this approach is germane to several NMR-active nuclei, where relaxation rates are frequency-offset independent.

  11. Evolutionary dynamics of a quantitative trait in a finite asexual population.

    PubMed

    Débarre, Florence; Otto, Sarah P

    2016-04-01

    In finite populations, mutation limitation and genetic drift can hinder evolutionary diversification. We consider the evolution of a quantitative trait in an asexual population whose size can vary and depends explicitly on the trait. Previous work showed that evolutionary branching is certain ("deterministic branching") above a threshold population size, but uncertain ("stochastic branching") below it. Using the stationary distribution of the population's trait variance, we identify three qualitatively different sub-domains of "stochastic branching" and illustrate our results using a model of social evolution. We find that in very small populations, branching will almost never be observed; in intermediate populations, branching is intermittent, arising and disappearing over time; in larger populations, finally, branching is expected to occur and persist for substantial periods of time. Our study provides a clearer picture of the ecological conditions that facilitate the appearance and persistence of novel evolutionary lineages in the face of genetic drift.

  12. Quantitative scanning thermal microscopy based on determination of thermal probe dynamic resistance.

    PubMed

    Bodzenta, J; Juszczyk, J; Chirtoc, M

    2013-09-01

    Resistive thermal probes used in scanning thermal microscopy provide high spatial resolution of measurement accompanied with high sensitivity to temperature changes. At the same time their sensitivity to variations of thermal conductivity of a sample is relatively low. In typical dc operation mode the static resistance of the thermal probe is measured. It is shown both analytically and experimentally that the sensitivity of measurement can be improved by a factor of three by measuring the dynamic resistance of a dc biased probe superimposed with small ac current. The dynamic resistance can be treated as a complex value. Its amplitude represents the slope of the static voltage-current U-I characteristic for a given I while its phase describes the delay between the measured ac voltage and applied ac current component in the probe. The phase signal also reveals dependence on the sample thermal conductivity. Signal changes are relatively small but very repeatable. In contrast, the difference between dynamic and static resistance has higher sensitivity (the same maximum value as that of the 2nd and 3rd harmonics), and also much higher amplitude than higher harmonics. The proposed dc + ac excitation scheme combines the benefits of dc excitation (mechanical stability of probe-sample contact, average temperature control) with those of ac excitation (base-line stability, rejection of ambient temperature influence, high sensitivity, lock-in signal processing), when the experimental conditions prohibit large ac excitation.

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

    PubMed Central

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

    2014-01-01

    Understanding stem cell (SC) population dynamics is essential for developing models that can be used in basic science and medicine, to aid in predicting cells fate. These models can be used as tools e.g. in studying patho-physiological events at the cellular and tissue level, predicting (mal)functions along the developmental course, and personalized regenerative medicine. Using time-lapsed imaging and statistical tools, we show that the dynamics of SC populations involve a heterogeneous structure consisting of multiple sub-population behaviors. Using non-Gaussian statistical approaches, we identify the co-existence of fast and slow dividing subpopulations, and quiescent cells, in stem cells from three species. The mathematical analysis also shows that, instead of developing independently, SCs exhibit a time-dependent fractal behavior as they interact with each other through molecular and tactile signals. These findings suggest that more sophisticated models of SC dynamics should view SC populations as a collective and avoid the simplifying homogeneity assumption by accounting for the presence of more than one dividing sub-population, and their multi-fractal characteristics. PMID:24769917

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

    NASA Astrophysics Data System (ADS)

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

    2014-04-01

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

  15. Dynamics of Nuclear Receptor Helix-12 Switch of Transcription Activation by Modeling Time-Resolved Fluorescence Anisotropy Decays

    PubMed Central

    Batista, Mariana R.B.; Martínez, Leandro

    2013-01-01

    Nuclear hormone receptors (NRs) are major targets for pharmaceutical development. Many experiments demonstrate that their C-terminal Helix (H12) is more flexible in the ligand-binding domains (LBDs) without ligand, this increased mobility being correlated with transcription repression and human diseases. Crystal structures have been obtained in which the H12 is extended, suggesting the possibility of large amplitude H12 motions in solution. However, these structures were interpreted as possible crystallographic artifacts, and thus the microscopic nature of H12 movements is not well known. To bridge the gap between experiments and molecular models and provide a definitive picture of H12 motions in solution, extensive molecular dynamics simulations of the peroxisome proliferator-activated receptor-γ LBD, in which the H12 was bound to a fluorescent probe, were performed. A direct comparison of the modeled anisotropy decays to time-resolved fluorescence anisotropy experiments was obtained. It is shown that the decay rates are dependent on the interactions of the probe with the surface of the protein, and display little correlation with the flexibility of the H12. Nevertheless, for the probe to interact with the surface of the LBD, the H12 must be folded over the body of the LBD. Therefore, the molecular mobility of the H12 should preserve the globularity of the LBD, so that ligand binding and dissociation occur by diffusion through the surface of a compact receptor. These results advance the comprehension of both ligand-bound and ligand-free receptor structures in solution, and also guide the interpretation of time-resolved anisotropy decays from a molecular perspective, particularly by the use of simulations. PMID:24094408

  16. Comparison of Myocardial Perfusion Estimates From Dynamic Contrast-Enhanced Magnetic Resonance Imaging With Four Quantitative Analysis Methods

    PubMed Central

    Pack, Nathan A.; DiBella, Edward V. R.

    2012-01-01

    Dynamic contrast-enhanced MRI has been used to quantify myocardial perfusion in recent years. Published results have varied widely, possibly depending on the method used to analyze the dynamic perfusion data. Here, four quantitative analysis methods (two-compartment modeling, Fermi function modeling, model-independent analysis, and Patlak plot analysis) were implemented and compared for quantifying myocardial perfusion. Dynamic contrast-enhanced MRI data were acquired in 20 human subjects at rest with low-dose (0.019 ± 0.005 mmol/kg) bolus injections of gadolinium. Fourteen of these subjects were also imaged at adenosine stress (0.021 ± 0.005 mmol/kg). Aggregate rest perfusion estimates were not significantly different between all four analysis methods. At stress, perfusion estimates were not significantly different between two-compartment modeling, model-independent analysis, and Patlak plot analysis. Stress estimates from the Fermi model were significantly higher (~20%) than the other three methods. Myocardial perfusion reserve values were not significantly different between all four methods. Model-independent analysis resulted in the lowest model curve-fit errors. When more than just the first pass of data was analyzed, perfusion estimates from two-compartment modeling and model-independent analysis did not change significantly, unlike results from Fermi function modeling. PMID:20577976

  17. Quantitative analysis of the angular dynamics of a single spheroid in simple shear flow at moderate Reynolds numbers

    NASA Astrophysics Data System (ADS)

    Rosén, Tomas; Nordmark, Arne; Aidun, Cyrus K.; Do-Quang, Minh; Lundell, Fredrik

    2016-08-01

    A spheroidal particle in simple shear flow shows surprisingly complicated angular dynamics; caused by effects of fluid inertia (characterized by the particle Reynolds number Rep) and particle inertia (characterized by the Stokes number St). Understanding this behavior can provide important fundamental knowledge of suspension flows with spheroidal particles. Up to now only qualitative analysis has been available at moderate Rep. Rigorous analytical methods apply only to very small Rep and numerical results lack accuracy due to the difficulty in treating the moving boundary of the particle. Here we show that the dynamics of the rotational motion of a prolate spheroidal particle in a linear shear flow can be quantitatively analyzed through the eigenvalues of the log-rolling particle (particle aligned with vorticity). This analysis provides an accurate description of stable rotational states in terms of Rep,St, and particle aspect ratio (rp). Furthermore we find that the effect on the orientational dynamics from fluid inertia can be modeled with a Duffing-Van der Pol oscillator. This opens up the possibility of developing a reduced-order model that takes into account effects from both fluid and particle inertia.

  18. Quantitative Analysis of Mechanisms That Govern Red Blood Cell Age Structure and Dynamics during Anaemia

    PubMed Central

    Savill, Nicholas J.; Chadwick, William; Reece, Sarah E.

    2009-01-01

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

  19. Quantitative NMR characterization of long-range chain dynamics prior to reptation: polyethylene-oxide

    PubMed

    Cohen Addad JP; Guillermo

    2000-10-16

    The thorough analysis of the transverse magnetic relaxation of protons, attached to highly entangled polyethylene-oxide chains in the melt, reveals two striking chain-length dependent properties; these are interpreted from the description (reminiscent of the Rouse model) of the long-range chain dynamics supposed to occur prior to the reptation motion. Experimental results are well matched by this specific NMR approach which accounts for the novel properties and provides the monomeric friction coefficient and the terminal relaxation time, over the molecular weight range 65K to 760K.

  20. Quantitative NMR Characterization of Long-Range Chain Dynamics prior to Reptation: Polyethylene-Oxide

    NASA Astrophysics Data System (ADS)

    Cohen Addad, Jean-Pierre; Guillermo, Armel

    2000-10-01

    The thorough analysis of the transverse magnetic relaxation of protons, attached to highly entangled polyethylene-oxide chains in the melt, reveals two striking chain-length dependent properties; these are interpreted from the description (reminiscent of the Rouse model) of the long-range chain dynamics supposed to occur prior to the reptation motion. Experimental results are well matched by this specific NMR approach which accounts for the novel properties and provides the monomeric friction coefficient and the terminal relaxation time, over the molecular weight range 65K to 760K.

  1. WRKY transcription factors.

    PubMed

    Rushton, Paul J; Somssich, Imre E; Ringler, Patricia; Shen, Qingxi J

    2010-05-01

    WRKY transcription factors are one of the largest families of transcriptional regulators in plants and form integral parts of signalling webs that modulate many plant processes. Here, we review recent significant progress in WRKY transcription factor research. New findings illustrate that WRKY proteins often act as repressors as well as activators, and that members of the family play roles in both the repression and de-repression of important plant processes. Furthermore, it is becoming clear that a single WRKY transcription factor might be involved in regulating several seemingly disparate processes. Mechanisms of signalling and transcriptional regulation are being dissected, uncovering WRKY protein functions via interactions with a diverse array of protein partners, including MAP kinases, MAP kinase kinases, 14-3-3 proteins, calmodulin, histone deacetylases, resistance proteins and other WRKY transcription factors. WRKY genes exhibit extensive autoregulation and cross-regulation that facilitates transcriptional reprogramming in a dynamic web with built-in redundancy.

  2. A Quantitative Model of Glucose Signaling in Yeast Reveals an Incoherent Feed Forward Loop Leading to a Specific, Transient Pulse of Transcription

    NASA Astrophysics Data System (ADS)

    Kuttykrishnan, Sooraj; Sabina, Jeffrey; Langton, Laura; Johnston, Mark; Brent, Michael R.

    The ability to design and engineer organisms demands the ability to predict kinetic responses of novel regulatory networks built from well-characterized biological components. Surprisingly, few validated kinetic models of complex regulatory networks have been derived by combining models of the network components. A major bottleneck in producing such models is the difficulty of measuring in vivo rate constants for components of complex networks. We demonstrate that a simple, genetic approach to measuring rate constants in vivo produces an accurate kinetic model of the complex network that Saccharomyces cerevisiae employs to regulate the expression of genes encoding glucose transporters. The model predicts a transient pulse of transcription of HXT4 (but not HXT2 or HXT3) in response to addition of a small amount of glucose to cells, an outcome we observed experimentally. Our model also provides a mechanistic explanation for this result: HXT24 are governed by a type 2, incoherent feed forward regulatory loop involving the Rgt1 and Mig2 transcriptional repressors. The efficiency with which Rgt1 and Mig2 repress expression of each HXT gene determines which of them have a pulse of transcription in response to glucose. Finally, the model correctly predicts how lesions in the feed forward loop change the kinetics of induction of HXT4 expression.

  3. Quantitative assessment of ratiometric bimolecular beacons as a tool for imaging single engineered RNA transcripts and measuring gene expression in living cells.

    PubMed

    Zhang, Xuemei; Song, Yang; Shah, Akash Y; Lekova, Virzhiniya; Raj, Arjun; Huang, Ling; Behlke, Mark A; Tsourkas, Andrew

    2013-08-01

    Recently, we developed an oligonucleotide-based probe, ratiometric bimolecular beacon (RBMB), which generates a detectable fluorescent signal in living cells that express the target RNA. Here, we show that RBMBs can also be used to image single RNA transcripts in living cells, when the target RNA is engineered to contain as few as four hybridization sites. Moreover, comparison with single-molecule fluorescence in situ hybridization confirmed that RBMBs could be used to accurately quantify the number of RNA transcripts within individual cells. Measurements of gene expression could be acquired within 30 min and using a wide range of RBMB concentrations. The ability to acquire accurate measurements of RNA copy number in both HT-1080 cells and CHO cells also suggests that RBMBs can be used to image and quantify single RNA transcripts in a wide range of cell lines. Overall, these findings highlight the robustness and versatility of RBMBs as a tool for imaging RNA in live cells. We envision that the unique capabilities of RBMBs will open up new avenues for RNA research.

  4. Quantitative image analysis identifies pVHL as a key regulator of microtubule dynamic instability.

    PubMed

    Thoma, Claudio R; Matov, Alexandre; Gutbrodt, Katrin L; Hoerner, Christian R; Smole, Zlatko; Krek, Wilhelm; Danuser, Gaudenz

    2010-09-20

    Von Hippel-Lindau (VHL) tumor suppressor gene mutations predispose carriers to kidney cancer. The protein pVHL has been shown to interact with microtubules (MTs), which is critical to cilia maintenance and mitotic spindle orientation. However, the function for pVHL in the regulation of MT dynamics is unknown. We tracked MT growth via the plus end marker EB3 (end-binding protein 3)-GFP and inferred additional parameters of MT dynamics indirectly by spatiotemporal grouping of growth tracks from live cell imaging. Our data establish pVHL as a near-optimal MT-stabilizing protein: it attenuates tubulin turnover, both during MT growth and shrinkage, inhibits catastrophe, and enhances rescue frequencies. These functions are mediated, in part, by inhibition of tubulin guanosine triphosphatase activity in vitro and at MT plus ends and along the MT lattice in vivo. Mutants connected to the VHL cancer syndrome are differentially compromised in these activities. Thus, single cell-level analysis of pVHL MT regulatory function allows new predictions for genotype to phenotype associations that deviate from the coarser clinically defined mutant classifications. PMID:20855504

  5. Quantitative Attribution of Major Driving Forces on Soil Organic Carbon Dynamics

    NASA Astrophysics Data System (ADS)

    WU, Y.; Liu, S.; Tan, Z.

    2014-12-01

    Soil organic carbon (SOC) storage plays a major role in the global carbon cycle and is affected by many factors including land use/management changes. However, the contributions of various factors to SOC changes are not well understood and quantified. This study was to investigate the impacts of changing farming practices, initial SOC levels, and biological enhancement of grain production on SOC dynamics and to attribute the relative contributions of major driving forces (CO2 enrichment and farming practices) using a fractional factorial modeling design. The case study using a crop site in Iowa in the United States demonstrated that the traditional corn-soybean (CS) rotation had the potential of accumulating SOC over this century under the current condition; whereas the continuous-corn (CC) system might have a higher SOC sequestration potential than CS. In either case, however, straw removal could reverse the sink potential to carbon neutral or a weak sink/source. Our results also suggested that the equilibrium SOC level may vary greatly depending on cropping systems and management practices. Importantly, the factorial design analysis indicated that residue management had the most significant impact on SOC changes, followed by CO2 enrichment. In brief, this study is valuable for understanding the major forces driving SOC dynamics of agro-ecosystems and informative for decision-makers when seeking the enhancement of SOC sequestration potential and sustainability of biofuel production, especially in the Corn Belt region.

  6. Quantitative adaptation analytics for assessing dynamic systems of systems: LDRD Final Report

    SciTech Connect

    Gauthier, John H.; Miner, Nadine E.; Wilson, Michael L.; Le, Hai D.; Kao, Gio K.; Melander, Darryl J.; Longsine, Dennis Earl; Vander Meer, Jr., Robert C.

    2015-01-01

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

  7. 3D quantitative visualization of altered LV wall thickening dynamics caused by coronary microembolization

    NASA Astrophysics Data System (ADS)

    Eusemann, Christian D.; Mohlenkamp, Stefan; Ritman, Erik L.; Robb, Richard A.

    2001-05-01

    Regional heart wall dynamics has been shown to be a sensitive indicator of LV wall ischemia. Rates of local LV wall thickening during a cardiac cycle can be measured and illustrated using functional parametric mappings. This display conveys the spatial distribution of dynamic strain in the myocardium and thereby provides a rapid qualitative appreciation of the severity and extent of the ischemic region. 3D reconstructions were obtained in an anesthetized pig from 8 adjacent, shortaxis, slices of the left ventricle imaged with an Electron Beam Computer Tomograph at 11 time points through one complete cardiac cycle. The 3D reconstructions were obtained before and after injection of 100 micrometer microspheres into the Left Anterior Descending (LAD) coronary artery. This injection causes microembolization of LAD artery branches within the heart wall. The image processing involved radially dividing the tomographic images of the myocardium into small subdivisions with color encoding of the local magnitude of regional thickness or regional velocities of LV wall thickening throughout the cardiac cycle. We compared the effectiveness of animation of wall thickness encoded in color versus a static image of computed rate of wall thickness change in color. The location, extent and severity of regional wall akinesis or dyskinesis, as determined from these displays, can then be compared to the region of embolization as indicated by the distribution of altered LV wall perfusion.

  8. Dynamic quantitative phase images of pond life, insect wings, and in vitro cell cultures.

    PubMed

    Creath, Katherine

    2010-08-01

    This paper presents images and data of live biological samples taken with a novel Linnik interference microscope. The specially designed optical system enables instantaneous and 3D video measurements of dynamic motions within and among live cells without the need for contrast agents. This "label-free", vibration insensitive imaging system enables measurement of biological objects in reflection using harmless light levels with current magnifications of 10X (NA 0.3) and 20X (NA 0.5) and wavelengths of 660 nm and 785 nm over fields of view from several hundred microns up to a millimeter. At the core of the instrument is a phase-measurement camera (PMC) enabling simultaneous measurement of multiple interference patterns utilizing a pixelated phase mask taking advantage of the polarization properties of light. Utilizing this technology enables the creation of phase image movies in real time at video rates so that dynamic motions and volumetric changes can be tracked. Objects are placed on a reflective surface in liquid under a coverslip. Phase values are converted to optical thickness data enabling volumetric, motion and morphological studies. Data from a number of different mud puddle organisms such as paramecium, flagellates and rotifers will be presented, as will measurements of flying ant wings and cultures of human breast cancer cells. These data highlight examples of monitoring different biological processes and motions. The live presentation features 4D phase movies of these examples. PMID:24357900

  9. Dynamic quantitative phase images of pond life, insect wings, and in vitro cell cultures

    NASA Astrophysics Data System (ADS)

    Creath, Katherine

    2010-08-01

    This paper presents images and data of live biological samples taken with a novel Linnik interference microscope. The specially designed optical system enables instantaneous and 3D video measurements of dynamic motions within and among live cells without the need for contrast agents. This "label-free", vibration insensitive imaging system enables measurement of biological objects in reflection using harmless light levels with current magnifications of 10X (NA 0.3) and 20X (NA 0.5) and wavelengths of 660 nm and 785 nm over fields of view from several hundred microns up to a millimeter. At the core of the instrument is a phasemeasurement camera (PMC) enabling simultaneous measurement of multiple interference patterns utilizing a pixelated phase mask taking advantage of the polarization properties of light. Utilizing this technology enables the creation of phase image movies in real time at video rates so that dynamic motions and volumetric changes can be tracked. Objects are placed on a reflective surface in liquid under a coverslip. Phase values are converted to optical thickness data enabling volumetric, motion and morphological studies. Data from a number of different mud puddle organisms such as paramecium, flagellates and rotifers will be presented, as will measurements of flying ant wings and cultures of human breast cancer cells. These data highlight examples of monitoring different biological processes and motions. The live presentation features 4D phase movies of these examples.

  10. Quantitative assessment of myocardial perfusion using dynamic three-dimensional x-ray computed angiography

    SciTech Connect

    Teslow, T.N.

    1985-01-01

    Using computed tomogram time series, myocardial perfusion was angiographically measured as distributions of x-ray circulatory indicators in three dimensions. By separating the dynamic function from the cardiac structure, these separate components were tested using region-of-interest (ROI) mensuration in simulation, phantom, and in vivo experiments. Statistical criteria were used to evaluate the dynamic component which was represented by analytic mathematical models of indicator dilution. The spatial component was represented by three-dimensional (3-D) and two-dimensional (2-D) geometric models of the heart. Each of these components were determined in individual ROI's and globally integrated to manifest the perfusion heterogeneities. A physical heart phantom with controllable regional perfusion characteristics was also developed and studied. Experiments conducted on dogs compared the accuracy of 2-D and 3-D perfusion measurements by imaging to those using gamma-radioactive microspheres. Accurate reproducible localization of the heart was found to be important for obtaining accurate measures of regional perfusion in 3-D volume images exhibiting high noise.

  11. Impact of dynamical scattering on quantitative contrast for aberration-corrected transmission electron microscope images.

    PubMed

    Wen, C; Smith, David J

    2016-10-01

    Aberration-corrected transmission electron microscope images taken under optimum-defocus conditions or processed offline can correctly reflect the projected crystal structure with atomic resolution. However, dynamical scattering, which will seriously influence image contrast, is still unavoidable. Here, the multislice image simulation approach was used to quantify the impact of dynamical scattering on the contrast of aberration-corrected images for a 3C-SiC specimen with changes in atomic occupancy and thickness. Optimum-defocus images with different spherical aberration (CS) coefficients, and structure images restored by deconvolution processing, were studied. The results show that atomic-column positions and the atomic occupancy for SiC 'dumbbells' can be determined by analysis of image contrast profiles only below a certain thickness limit. This limit is larger for optimum-defocus and restored structure images with negative CS coefficient than those with positive CS coefficient. The image contrast of C (or Si) atomic columns with specific atomic occupancy changes differently with increasing crystal thickness. Furthermore, contrast peaks for C atomic columns overlapping with neighboring peaks of Si atomic columns with varied Si atomic occupancy, which is enhanced with increasing crystal thickness, can be neglected in restored structure images, but the effect is substantial in optimum-defocus images.

  12. Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells.

    PubMed

    Zuck, Meghan; Feng, Caroline; Hybiske, Kevin

    2015-10-13

    The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C. trachomatis is the leading bacterial cause of sexually transmitted infection and also the primary cause of preventable blindness in the world. An essential determinant for successful infection of host cells by Chlamydia is the bacterium's ability to manipulate host cell signaling from within a novel, vacuolar compartment called the inclusion. From within the inclusion, Chlamydia acquire nutrients required for their 2-3 day developmental growth, and they additionally secrete a panel of effector proteins onto the cytosolic face of the vacuole membrane and into the host cytosol. Gaps in our understanding of Chlamydia biology, however, present significant challenges for visualizing and analyzing this intracellular compartment. Recently, a reverse-imaging strategy for visualizing the inclusion using GFP expressing host cells was described. This approach rationally exploits the intrinsic impermeability of the inclusion membrane to large molecules such as GFP. In this work, we describe how GFP- or mCherry-expressing host cells are generated for subsequent visualization of chlamydial inclusions. Furthermore, this method is shown to effectively substitute for costly antibody-based enumeration methods, can be used in tandem with other fluorescent labels, such as GFP-expressing Chlamydia, and can be exploited to derive key quantitative data about inclusion membrane growth from a range of Chlamydia species and strains.

  13. Streaking artifact reduction for quantitative susceptibility mapping of sources with large dynamic range.

    PubMed

    Wei, Hongjiang; Dibb, Russell; Zhou, Yan; Sun, Yawen; Xu, Jianrong; Wang, Nian; Liu, Chunlei

    2015-10-01

    Quantitative susceptibility mapping (QSM) is a novel MRI technique for the measurement of tissue magnetic susceptibility in three dimensions. Although numerous algorithms have been developed to solve this ill-posed inverse problem, the estimation of susceptibility maps with a wide range of values is still problematic. In cases such as large veins, contrast agent uptake and intracranial hemorrhages, extreme susceptibility values in focal areas cause severe streaking artifacts. To enable the reduction of these artifacts, whilst preserving subtle susceptibility contrast, a two-level QSM reconstruction algorithm (streaking artifact reduction for QSM, STAR-QSM) was developed in this study by tuning a regularization parameter to automatically reconstruct both large and small susceptibility values. Compared with current state-of-the-art QSM methods, such as the improved sparse linear equation and least-squares (iLSQR) algorithm, STAR-QSM significantly reduced the streaking artifacts, whilst preserving the sharp boundaries for blood vessels of mouse brains in vivo and fine anatomical details of high-resolution mouse brains ex vivo. Brain image data from patients with cerebral hematoma and multiple sclerosis further illustrated the superiority of this method in reducing streaking artifacts caused by large susceptibility sources, whilst maintaining sharp anatomical details. STAR-QSM is implemented in STI Suite, a comprehensive shareware for susceptibility imaging and quantification.

  14. Measuring the dynamics of E. coli ribosome biogenesis using pulse-labeling and quantitative mass spectrometry

    PubMed Central

    Chen, Stephen S.; Sperling, Edit; Silverman, Joshua M.; Davis, Joseph H.; Williamson, James R.

    2012-01-01

    The ribosome is an essential organelle responsible for cellular protein synthesis. Until recently, the study of ribosome assembly has been largely limited to in vitro assays, with few attempts to reconcile these results with the more complex in vivo ribosome biogenesis process. Here, we characterize the ribosome synthesis and assembly pathway for each E. coli ribosomal protein (r-protein) in vivo using a stable isotope pulse-labeling timecourse. Isotope incorporation into assembled ribosomes was measured by quantitative mass spectrometry (qMS) and fit using steady-state flux models. Most r-proteins exhibit precursor pools ranging in size from 0% to 7% of completed ribosomes, and that the sizes of these individual r-protein pools correlate well with the order of r-protein binding in vitro. Additionally, we observe anomalously large precursor pools for specific r-proteins with known extra-ribosomal functions and we have detected three r-proteins with significant turnover during steady-state growth. Taken together, this highly precise, time-dependent proteomic qMS approach should prove useful in future studies of ribosome biogenesis and could be easily extended to explore other complex biological processes in a cellular context. PMID:23090316

  15. Technical note: development of a quantitative PCR method for monitoring strain dynamics during yogurt manufacture.

    PubMed

    Miller, D M; Dudley, E G; Roberts, R F

    2012-09-01

    Yogurt starter cultures may consist of multiple strains of Lactobacillus delbrueckii ssp. bulgaricus (LB) and Streptococcus thermophilus (ST). Conventional plating methods for monitoring LB and ST levels during yogurt manufacture do not allow for quantification of individual strains. The objective of the present work was to develop a quantitative PCR method for quantification of individual strains in a commercial yogurt starter culture. Strain-specific primers were designed for 2 ST strains (ST DGCC7796 and ST DGCC7710), 1 LB strain (DGCC4078), and 1 Lactobacillus delbrueckii ssp. lactis strain (LL; DGCC4550). Primers for the individual ST and LB strains were designed to target unique DNA sequences in clustered regularly interspersed short palindromic repeats. Primers for LL were designed to target a putative mannitol-specific IIbC component of the phosphotransferase system. Following evaluation of primer specificity, standard curves relating cell number to cycle threshold were prepared for each strain individually and in combination in yogurt mix, and no significant differences in the slopes were observed. Strain balance data was collected for yogurt prepared at 41 and 43°C to demonstrate the potential application of this method.

  16. Qualitative and Quantitative Assessment of Adenosine Triphosphate Stress Whole-Heart Dynamic Myocardial Perfusion Imaging Using 256-Slice Computed Tomography

    PubMed Central

    Kurata, Akira; Kawaguchi, Naoto; Kido, Teruhito; Inoue, Katsuji; Suzuki, Jun; Ogimoto, Akiyoshi; Funada, Jun-ichi; Higaki, Jitsuo; Miyagawa, Masao; Vembar, Mani; Mochizuki, Teruhito

    2013-01-01

    Background The aim of this study was to investigate the correlation of the qualitative transmural extent of hypoperfusion areas (HPA) using stress dynamic whole-heart computed tomography perfusion (CTP) imaging by 256-slice CT with CTP-derived myocardial blood flow (MBF) for the estimation of the severity of coronary artery stenosis. Methods and Results Eleven patients underwent adenosine triphosphate (0.16 mg/kg/min, 5 min) stress dynamic CTP by 256-slice CT (coverage: 8 cm, 0.27 s/rotation), and 9 of the 11 patients underwent coronary angiography (CAG). Stress dynamic CTP (whole–heart datasets over 30 consecutive heart beats in systole without spatial and temporal gaps) was acquired with prospective ECG gating (effective radiation dose: 10.4 mSv). The extent of HPAs was visually graded using a 3-point score (normal, subendocardial, transmural). MBF (ml/100g/min) was measured by deconvolution. Differences in MBF (mean ± standard error) according to HPA and CAG results were evaluated. In 27 regions (3 major coronary territories in 9 patients), 11 coronary stenoses (> 50% reduction in diameter) were observed. In 353 myocardial segments, HPA was significantly related to MBF (P < 0.05; normal 295 ± 94; subendocardial 186 ± 67; and transmural 80 ± 53). Coronary territory analysis revealed a significant relationship between coronary stenosis severity and MBF (P < 0.05; non-significant stenosis [< 50%], 284 ± 97; moderate stenosis [50–70%], 184 ± 74; and severe stenosis [> 70%], 119 ± 69). Conclusion The qualitative transmural extent of HPA using stress whole-heart dynamic CTP imaging by 256-slice CT exhibits a good correlation with quantitative CTP-derived MBF and may aid in assessing the hemodynamic significance of coronary artery disease. PMID:24376774

  17. Automated continuous quantitative measurement of proximal airways on dynamic ventilation CT: initial experience using an ex vivo porcine lung phantom

    PubMed Central

    Yamashiro, Tsuneo; Tsubakimoto, Maho; Nagatani, Yukihiro; Moriya, Hiroshi; Sakuma, Kotaro; Tsukagoshi, Shinsuke; Inokawa, Hiroyasu; Kimoto, Tatsuya; Teramoto, Ryuichi; Murayama, Sadayuki

    2015-01-01

    Background The purpose of this study was to evaluate the feasibility of continuous quantitative measurement of the proximal airways, using dynamic ventilation computed tomography (CT) and our research software. Methods A porcine lung that was removed during meat processing was ventilated inside a chest phantom by a negative pressure cylinder (eight times per minute). This chest phantom with imitated respiratory movement was scanned by a 320-row area-detector CT scanner for approximately 9 seconds as dynamic ventilatory scanning. Obtained volume data were reconstructed every 0.35 seconds (total 8.4 seconds with 24 frames) as three-dimensional images and stored in our research software. The software automatically traced a designated airway point in all frames and measured the cross-sectional luminal area and wall area percent (WA%). The cross-sectional luminal area and WA% of the trachea and right main bronchus (RMB) were measured for this study. Two radiologists evaluated the traceability of all measurable airway points of the trachea and RMB using a three-point scale. Results It was judged that the software satisfactorily traced airway points throughout the dynamic ventilation CT (mean score, 2.64 at the trachea and 2.84 at the RMB). From the maximum inspiratory frame to the maximum expiratory frame, the cross-sectional luminal area of the trachea decreased 17.7% and that of the RMB 29.0%, whereas the WA% of the trachea increased 6.6% and that of the RMB 11.1%. Conclusion It is feasible to measure airway dimensions automatically at designated points on dynamic ventilation CT using research software. This technique can be applied to various airway and obstructive diseases. PMID:26445535

  18. Quantitative measurement of the medial surface dynamics of the vocal folds using high-speed digital imaging

    NASA Astrophysics Data System (ADS)

    Doellinger, Michael; Neubauer, Juergen; Berry, David A.

    2003-10-01

    To increase our understanding of pathological and healthy voice production, the quantitative measurement of the medial surface dynamics of the vocal folds is significant, albeit rarely performed because of the inaccessibility of the vocal folds. Hence, an excised hemilarynx procedure is applied, Berry et al. recently reported such quantitative measurements along one coronal plane of the left vocal fold of a canine [J. Acoust. Soc. Am. 110, 2539-2547 (2001)]. The present work extends previous studies by capturing vibrations along the entire medial surface of the left vocal fold. The number of vertical rows of sutures used to demarcate fleshpoints was increased from one to five. An automatic algorithm for tracking vocal fold fleshpoints will be reported, along with calibration techniques, and error estimation. Preliminary results will be reported for both periodic and aperiodic vocal fold vibrations. High-speed digital imaging was performed using a Photron APX machine with a sampling frequency of 4000 Hz, a spatial resolution of 1024×512 pixels, and 256 levels of grayscale.

  19. In situ flash x-ray high-speed computed tomography for the quantitative analysis of highly dynamic processes

    NASA Astrophysics Data System (ADS)

    Moser, Stefan; Nau, Siegfried; Salk, Manfred; Thoma, Klaus

    2014-02-01

    The in situ investigation of dynamic events, ranging from car crash to ballistics, often is key to the understanding of dynamic material behavior. In many cases the important processes and interactions happen on the scale of milli- to microseconds at speeds of 1000 m s-1 or more. Often, 3D information is necessary to fully capture and analyze all relevant effects. High-speed 3D-visualization techniques are thus required for the in situ analysis. 3D-capable optical high-speed methods often are impaired by luminous effects and dust, while flash x-ray based methods usually deliver only 2D data. In this paper, a novel 3D-capable flash x-ray based method, in situ flash x-ray high-speed computed tomography is presented. The method is capable of producing 3D reconstructions of high-speed processes based on an undersampled dataset consisting of only a few (typically 3 to 6) x-ray projections. The major challenges are identified, discussed and the chosen solution outlined. The application is illustrated with an exemplary application of a 1000 m s-1 high-speed impact event on the scale of microseconds. A quantitative analysis of the in situ measurement of the material fragments with a 3D reconstruction with 1 mm voxel size is presented and the results are discussed. The results show that the HSCT method allows gaining valuable visual and quantitative mechanical information for the understanding and interpretation of high-speed events.

  20. Real-Time Quantitative RT-PCR of Defense-Associated Gene Transcripts of Rhizoctonia solani-Infected Bean Seedlings in Response to Inoculation with a Nonpathogenic Binucleate Rhizoctonia Isolate.

    PubMed

    Wen, Kui; Seguin, Philippe; St-Arnaud, Marc; Jabaji-Hare, Suha

    2005-04-01

    ABSTRACT Certain isolates of nonpathogenic binucleate Rhizoctonia spp. (np-BNR) are effective biocontrol agents against seedling root rot and damping-off. Inoculation of bean seed with np-BNR strain 232-CG at sowing reduced disease symptoms in bean (Phaseolus vulgaris) seedlings caused by R. solani. Molecular analyses of the spatial expression of three defense-associated genes were carried out using real-time quantitative reverse transcription-polymerase chain reaction (QRT-PCR) assays. This method allowed accurate quantitative evaluation of transcript levels of pG101 encoding for 1,3-beta-D-glucanase, gPAL1 encoding for phenylalanine ammonia lyase, and CHS17 encoding for chalcone synthase in 1- and 2-week-old bean seedlings that were inoculated simultaneously with np-BNR and infected with R. solani, and in seedlings that were singly inoculated with either fungi or not inoculated. In the seedlings that were infected with R. solani only, results revealed that, following infection, activation of all defense-associated gene transcripts was achieved with significant increases ranging from 7- to 40-fold greater than the control, depending on the defense gene and tissue analyzed. Seedlings that were treated with np-BNR and infected with R. solani had expression similar to those that were treated with np-BNR only, but the levels were significantly down-regulated compared with those that were infected with R. solani only. These findings indicate that disease suppression by np-BNR isolate is not correlated to pG101, gPAL1, and CHS17 gene activation.

  1. Quantitative real-time imaging of molecular dynamics during cancer cell invasion and metastasis in vivo

    PubMed Central

    Serrels, Alan; Canel, Marta; Frame, Margaret C; Brunton, Valerie G; Anderson, Kurt I

    2009-01-01

    Despite our advanced understanding of primary cancer development and progression, metastasis and the systemic spread of the disease to secondary sites remains the leading cause of cancer-associated death. The metastatic process is therefore a major potential therapeutic target area for cancer researchers and elucidating the key steps that are susceptible to therapeutic intervention will be critical to improve our treatment strategies. Recent advances in intravital imaging are rapidly improving our insight into this process and are helping in the design of stage-specific drug regimes for the treatment of metastatic cancer. Here we discuss current developments in intravital imaging and our recent use of photobleaching and photoactivation in the analysis of dynamic biomarkers in living animals to assess the efficacy of therapeutic intervention on early stages of tumor cell metastasis. PMID:19690469

  2. A Quantitative Analysis of Aqueous Nanofilm Rupture by Molecular Dynamic Simulation

    SciTech Connect

    Peng, Tiefeng; Nguyen, Anh V.; Peng, Hong; Dang, Liem X.

    2012-01-26

    In this study, we used molecular dynamic (MD) simulations of the rupture process for a water film to define and determine the critical rupture time (CRT). This new approach could be an important method for authentically defining and determining the rupture point of a water film and associated phenomena. We were able to generically predict the CRT and the critical thickness of the water film. Then, we studied the effect of ions on the film rupture process. Our results showed that addition of sodium chloride did not significantly affect on the stability of the water film. Results from MD simulations, when compared with results from experimental measurements, can provide insights into the film rupture process.

  3. Quantitative analysis of antimicrobial effect kinetics in an in vitro dynamic model.

    PubMed Central

    Firsov, A A; Chernykh, V M; Navashin, S M

    1990-01-01

    Variants of the available methods for estimating antimicrobial effect kinetics in an in vitro dynamic model were analyzed. Two integral parameters characterizing antimicrobial effect duration (TE) and intensity (IE) are suggested to define and analyze the concentration-effect relationships in these models, irrespective of the method of recording. TE is defined by the time from the moment of antibiotic administration to the movement when the bacterial count again reaches its initial level. IE is defined by the area between the microbial growth curves in the presence and absence of an antibiotic. TE and IE were used to quantify the antimicrobial effects of sisomicin on Pseudomonas aeruginosa 58, Escherichia coli 93, and Klebsiella pneumoniae 5056, simulating the pharmacokinetic profiles of the drugs observed following intramuscular administration in therapeutic doses, including the variability of aminoglycoside concentrations in human blood. PMID:2117416

  4. Quantitative attribution of major driving forces on soil organic carbon dynamics

    NASA Astrophysics Data System (ADS)

    Wu, Yiping; Liu, Shuguang; Tan, Zhengxi

    2015-03-01

    Soil organic carbon (SOC) storage plays a major role in the global carbon cycle and is affected by many factors including land use/management changes (e.g., biofuel production-oriented changes). However, the contributions of various factors to SOC changes are not well understood and quantified. This study was designed to investigate the impacts of changing farming practices, initial SOC levels, and biological enhancement of grain production on SOC dynamics and to attribute the relative contributions of major driving forces (CO2 enrichment and farming practices) using a fractional factorial modeling design. The case study at a crop site in Iowa in the United States demonstrated that the traditional corn-soybean (CS) rotation could still accumulate SOC over this century (from 4.2 to 6.8 kg C/m2) under the current condition; whereas the continuous-corn (CC) system might have a higher SOC sequestration potential than CS. In either case, however, residue removal could reduce the sink potential substantially. Long-term simulation results also suggested that the equilibrium SOC level may vary greatly (˜5.7 to ˜11 kg C/m2) depending on cropping systems and management practices, and projected growth enhancement could make the magnitudes higher (˜7.8 to ˜13 kg C/m2). Importantly, the factorial design analysis indicated that residue management had the most significant impact (contributing 49.4%) on SOC changes, followed by CO2 Enrichment (37%), Tillage (6.2%), the combination of CO2 Enrichment-Residue removal (5.8%), and Fertilization (1.6%). In brief, this study is valuable for understanding the major forces driving SOC dynamics of agroecosystems and informative for decision-makers when seeking the enhancement of SOC sequestration potential and sustainability of biofuel production, especially in the Corn Belt region of the United States.

  5. Transcriptional regulatory dynamics of the hypothalamic-pituitary-gonadal axis and its peripheral pathways as impacted by the 3-beta HSD inhibitor trilostane in zebrafish (Danio rerio).

    PubMed

    Wang, Rong-Lin; Bencic, David; Lazorchak, Jim; Villeneuve, Daniel; Ankley, Gerald T

    2011-09-01

    To study mechanisms underlying generalized effects of 3β hydroxysteroid dehydrogenase (HSD3B) inhibition, reproductively mature zebrafish (Danio rerio) were exposed to trilostane at two dosages for 24, 48, or 96 h and their gonadal RNA samples profiled with Agilent zebrafish microarrays. Trilostane had substantial impact on the transcriptional dynamics of zebrafish, as reflected by a number of differentially expressed genes (DEGs) including transcription factors (TFs), altered TF networks, signaling pathways, and Gene Ontology (GO) biological processes. Changes in gene expression between a treatment and its control were mostly moderate, ranging from 1.3 to 2.0 fold. Expression of genes coding for HSD3B and many of its transcriptional regulators remained unchanged, suggesting transcriptional up-regulation is not a primary compensatory mechanism for HSD3B enzyme inhibition. While some trilostane-responsive TFs appear to share cellular functions linked to endocrine disruption, there are also many other DEGs not directly linked to steroidogenesis. Of the 65 significant TF networks, little similarity, and therefore little cross-talk, existed between them and the hypothalamic-pituitary-gonadal (HPG) axis. The most enriched GO biological processes are regulations of transcription, phosphorylation, and protein kinase activity. Most of the impacted TFs and TF networks are involved in cellular proliferation, differentiation, migration, and apoptosis. While these functions are fairly broad, their underlying TF networks may be useful to development of generalized toxicological screening methods. These findings suggest that trilostane-induced effects on fish endocrine functions are not confined to the HPG-axis alone. Its impact on corticosteroid synthesis could also have contributed to some system wide transcriptional changes in zebrafish observed in this study.

  6. Imaging of Lamb Waves in Plates for Quantitative Determination of Anisotropy using Photorefractive Dynamic Holography

    SciTech Connect

    Telschow, Kenneth Louis; Deason, Vance Albert; Schley, Robert Scott; Watson, Scott Marshall

    1998-06-01

    Anisotropic properties of sheet materials can be determined by measuring the propagation of Lamb waves in different directions. Electromagnetic acoustic transduction and laser ultrasonic methods provide noncontacting approaches that are often desired for application to industrial and processing environments. This paper describes a laser imaging approach utilizing the adaptive property of photorefractive materials to produce a real-time measurement of the antisymmetric Lamb wave mode in all directions simultaneously. Continuous excitation is employed enabling the data to be recorded and displayed by a CCD camera. Analysis of the image produces a direct quantitative determination of the phase velocity in all directions showing plate anisotropy in the plane. Many optical techniques for measuring ultrasonic motion at surfaces have been developed for use in applications such as vibration measurement and laser ultrasonics. Most of these methods have similar sensitivities and are based on time domain processing using homodyne, Fabry-Perot [1], and, more recently, photorefractive interferometry [2]. Generally, the methods described above do not allow measurement at more than one surface point simultaneously, requiring multiple beam movements and scanning in order to produce images of surface ultrasonic motion over a large area. Electronic speckle interferometry, including shearography, does provide images directly of vibrations over large surface areas. This method has proven very durable in the field for large displacement amplitudes of several wavelengths. In addition, a sensitivity of ë/3000 has been demonstrated under laboratory conditions [3]. Full-field imaging of traveling ultrasonic waves using digital shearography has been recently reported with sensitivity in the nanometer range [4]. With this method, optical interference occurs at the photodetector

  7. Quantitative imaging of cell dynamics in mouse embryos using light-sheet microscopy

    PubMed Central

    Udan, Ryan S.; Piazza, Victor G.; Hsu, Chih-wei; Hadjantonakis, Anna-Katerina; Dickinson, Mary E.

    2014-01-01

    Single/selective-plane illumination, or light-sheet, systems offer several advantages over other fluorescence microscopy methods for live, 3D microscopy. These systems are valuable for studying embryonic development in several animal systems, such as Drosophila, C. elegans and zebrafish. The geometry of the light path in this form of microscopy requires the sample to be accessible from multiple sides and fixed in place so that it can be rotated around a single axis. Popular methods for mounting include hanging the specimen from a pin or embedding it in 1-2% agarose. These methods can be particularly problematic for certain samples, such as post-implantation mouse embryos, that expand significantly in size and are very delicate and sensitive to mounting. To overcome the current limitations and to establish a robust strategy for long-term (24 h) time-lapse imaging of E6.5-8.5 mouse embryos with light-sheet microscopy, we developed and tested a method using hollow agarose cylinders designed to accommodate for embryonic growth, yet provide boundaries to minimize tissue drift and enable imaging in multiple orientations. Here, we report the first 24-h time-lapse sequences of post-implantation mouse embryo development with light-sheet microscopy. We demonstrate that light-sheet imaging can provide both quantitative data for tracking changes in morphogenesis and reveal new insights into mouse embryogenesis. Although we have used this approach for imaging mouse embryos, it can be extended to imaging other types of embryos as well as tissue explants. PMID:25344073

  8. Quantitative nuclear magnetic resonance characterization of long-range chain dynamics: Polybutadiene, polyethylene-oxide solution

    NASA Astrophysics Data System (ADS)

    Guillermo, Armel; Cohen Addad, Jean-Pierre

    2002-02-01

    We report two sets of independent nuclear magnetic resonance (NMR) measurements of self-diffusion and proton transverse relaxation in molten cis1,4-polybutadiene (PB) performed in order to investigate chain dynamics properties. Self-diffusion coefficients were measured as a function of temperature and of molecular weight (M) over the range 104 to 6.7×104g/mol. The crossover from the Rouse-type behavior (D≈M-1) to the reptation one was found to occur for MCross≈3×104g/mol; for M>MCross the data were consistent with the scaling dependence: D≈M-2.4±0.05, in agreement with the data analysis recently reported in the literature. The thorough analysis of the transverse relaxation of protons attached to highly entangled PB chains (6.7×104⩽M⩽43×104g/mol) gave evidence for the dynamics partition of one chain into two end-submolecules and one inner part clearly discriminated from one another. The number NEnd of monomeric units in one end-submolecule, independent of M, is shown to be closely related to the monomeric friction coefficient ζ0 measured from short chain diffusion over the temperature range 25 to 85 °C. The interpretation both of diffusion results and of proton relaxation of inner monomeric units lead to the definition of an effective friction coefficient ζ0Eff≈ζ0(M/NEnd)0.4 associated with the curvilinear diffusion of one chain in its tube. The friction coefficient ζLoc associated with local monomeric rotations is discriminated from ζ0 from its weaker temperature dependence. This approach was applied to polyethylene-oxide chains in solution (dimethyl formamide, 0.18⩽c⩽1, w/w) where the segmental size of end-submolecules was found to vary as 1/c. Experimental results are well matched by this specific NMR approach which accounts for the novel properties of the proton relaxation function.

  9. Semi-quantitative differences in gene transcription profiles between sexes of a marine snail by a new variant of cDNA-AFLP analysis.

    PubMed

    Martínez-Fernández, M; Bernatchez, L; Rolán-Alvarez, E; Quesada, H

    2010-03-01

    A variant of the cDNA-AFLP method coupled to an automated sequencer was used to quantify transcripts differentially expressed between sexes of the marine snail Littorina saxatilis. First, we conducted a validation study of the technique using known concentrations of a commercial marker. Second, we analysed six replicates of males and females from a population showing no apparent sexual dimorphism. The results confirm that the method can be properly used within the range of DNA concentrations utilized. In addition, we detected a small percentage of spots (1.8%) differentially expressed between sexes, as expected from a low to moderately sexual dimorphic species. PMID:21565027

  10. Chromatin immunoprecipitation (ChIP) coupled to detection by quantitative real-time PCR to study transcription factor binding to DNA in Caenorhabditis elegans

    PubMed Central

    Mukhopadhyay, Arnab; Deplancke, Bart; Walhout, Albertha J M; Tissenbaum, Heidi A

    2009-01-01

    In order to determine how signaling pathways differentially regulate gene expression, it is necessary to identify the interactions between transcription factors (TFs) and their cognate cis-regulatory DNA elements. Here, we have outlined a chromatin immunoprecipitation (ChIP) protocol for use in whole Caenorhabditis elegans extracts. We discuss optimization of the procedure, including growth and harvesting of the worms, formaldehyde fixation, TF immunoprecipitation and analysis of bound sequences through real-time PCR. It takes ∼10–12 d to obtain the worm culture for ChIP; the ChIP procedure is spaced out over a period of 2.5 d with two overnight incubations. PMID:18388953

  11. Dynamics and quantitative analysis of the synthesis of fermentative aromas by an evolved wine strain of Saccharomyces cerevisiae.

    PubMed

    Mouret, J R; Cadiere, A; Aguera, E; Rollero, S; Ortiz-Julien, A; Sablayrolles, J M; Dequin, S

    2015-01-01

    We performed a dynamic and quantitative analysis of the synthesis of fermentative aromas by an aromatic wine yeast, ECA5, obtained by adaptive evolution. During fermentation at pilot scale on synthetic and natural musts, ECA5 produced volatile compounds (higher alcohols and their acetates, ethyl esters) at higher rates than the ancestral strain, with the exception of propanol. Marked differences in the chronology of synthesis of several compounds were observed between the two strains. Overproduction of phenyl ethanol occurred mainly during the growth phase for ECA5, consistent with its higher flux through the pentose phosphate pathway, which plays a key role in biosynthetic processes. The kinetics of production of isobutanol and isoamyl alcohol were differently affected by different media (synthetic or natural must) and, in particular, according to the nature of the sterols in the media (ergosterol or phytosterols). We also observed differences in the chronology of synthesis of ethyl acetate and isoamyl acetate or ethyl esters, suggesting that the regulation of the synthesis of these compounds in the evolved strain differs from that in the ancestral strain. This study shows that a dynamic analysis of volatile compounds, using high acquisition frequency online gas chromatography, can provide novel insights into the synthesis and regulation of aromas and is thus a potentially powerful tool for strain characterization. PMID:24989462

  12. The Effect of Long Term Calorie Restriction on in Vivo Hepatic Proteostatis: A Novel Combination of Dynamic and Quantitative Proteomics

    PubMed Central

    Price, John C.; Khambatta, Cyrus F.; Li, Kelvin W.; Bruss, Matthew D.; Shankaran, Mahalakshmi; Dalidd, Marcy; Floreani, Nicholas A.; Roberts, Lindsay S.; Turner, Scott M.; Holmes, William E.; Hellerstein, Marc K.

    2012-01-01

    Calorie restriction (CR) promotes longevity. A prevalent mechanistic hypothesis explaining this effect suggests that protein degradation, including mitochondrial autophagy, is increased with CR, removing damaged proteins and improving cellular fitness. At steady state, increased catabolism must be balanced by increasing mitochondrial biogenesis and protein synthesis, resulting in faster protein replacement rates. To test this hypothesis, we measured replacement kinetics and relative concentrations of hundreds of proteins in vivo in long-term CR and ad libitum-fed mice using metabolic 2H2O-labeling combined with the Stable Isotope Labeling in Mammals protocol and LC-MS/MS analysis of mass isotopomer abundances in tryptic peptides. CR reduced absolute synthesis and breakdown rates of almost all measured hepatic proteins and prolonged the half-lives of most (∼80%), particularly mitochondrial proteins (but not ribosomal subunits). Proteins with related functions exhibited coordinated changes in relative concentration and replacement rates. In silico expression pathway interrogation allowed the testing of potential regulators of altered network dynamics (e.g. peroxisome proliferator-activated receptor gamma coactivator 1-alpha). In summary, our combination of dynamic and quantitative proteomics suggests that long-term CR reduces mitochondrial biogenesis and mitophagy. Our findings contradict the theory that CR increases mitochondrial protein turnover and provide compelling evidence that cellular fitness is accompanied by reduced global protein synthetic burden. PMID:22984287

  13. Quantitative Verification of Dynamic Wedge Dose Distribution Using a 2D Ionization Chamber Array.

    PubMed

    Sahnoun, Tarek; Farhat, Leila; Mtibaa, Anis; Besbes, Mounir; Daoud, Jamel

    2015-10-01

    The accuracy of two calculation algorithms of the Eclipse 8.9 treatment planning system (TPS)--the anisotropic analytic algorithm (AAA) and pencil-beam convolution (PBC)--in modeling the enhanced dynamic wedge (EDW) was investigated. Measurements were carried out for 6 and 18 MV photon beams using a 2D ionization chamber array. Accuracy of the TPS was evaluated using a gamma index analysis with the following acceptance criteria for dose differences (DD) and distance to agreement (DTA): 3%/3 mm and 2%/2 mm. The TPS models the dose distribution accurately except for 20×20 cm(2) field size, 60 (°) and 45 (°) wedge angles using PBC at 6 MV photon energy. For these latter fields, the pass rate and the mean value of gamma were less than 90% and more than 0.5, respectively at the (3%/3 mm) acceptance criteria. In addition, an accuracy level of (2%/2 mm) was achieved using AAA with better agreement for 18 MV photon energy.

  14. Toward a quantitative understanding of mantle dynamics beneath western United States

    NASA Astrophysics Data System (ADS)

    Liu, L.; Stegman, D. R.

    2012-12-01

    The western United States have a complex subduction and tectonic history during the Cenozoic, leading to an as complex mantle structure as imaged by recent tomography inversions. A full understanding of this dynamic history requires a simultaneous evaluation of the various components of the 4-dimensional system. We adopt the approach of large-scale geodynamic modeling with data assimilation to address this multi-variable problem, as is the case for western US. Here we report some results of our ongoing study that describes tectonic, magmatic, and tomographic signals of the Farallon plate's influence on the geologic history of the western North America. We simulate the Farallon subduction since the Eocene, using a recent plate reconstruction (Muller et al., G-cubed, 2008) to guide the model on top of the surface. The uncertain mantle properties such as mantle viscosity, slab strength, phase transformations, etc, are inferred during model construction and comparison with the present-day slab seismic image. One model output is slab evolution, when a major tearing event of the Farallon slab beneath eastern Oregon correlates perfect with the enigmatic Steens-Columbia River flood basalt during the mid-Miocene, thus providing a novel mechanism for continental flood basalt formation. Another output is mantle flow, which is being evaluated against the present-day seismic anisotropy measurements.

  15. Quantitative reconstruction of thermal and dynamic characteristics of lava flow from surface thermal measurements

    NASA Astrophysics Data System (ADS)

    Korotkii, Alexander; Kovtunov, Dmitry; Ismail-Zadeh, Alik; Tsepelev, Igor; Melnik, Oleg

    2016-06-01

    We study a model of lava flow to determine its thermal and dynamic characteristics from thermal measurements of the lava at its surface. Mathematically this problem is reduced to solving an inverse boundary problem. Namely, using known conditions at one part of the model boundary we determine the missing condition at the remaining part of the boundary. We develop a numerical approach to the mathematical problem in the case of steady-state flow. Assuming that the temperature and the heat flow are prescribed at the upper surface of the model domain, we determine the flow characteristics in the entire model domain using a variational (adjoint) method. We have performed computations of model examples and showed that in the case of smooth input data the lava temperature and the flow velocity can be reconstructed with a high accuracy. As expected, a noise imposed on the smooth input data results in a less accurate solution, but still acceptable below some noise level. Also we analyse the influence of optimization methods on the solution convergence rate. The proposed method for reconstruction of physical parameters of lava flows can also be applied to other problems in geophysical fluid flows.

  16. Quantitative genetic insights into the coevolutionary dynamics of male and female genitalia.

    PubMed

    Evans, Jonathan P; van Lieshout, Emile; Gasparini, Clelia

    2013-07-22

    The spectacular variability that typically characterizes male genital traits has largely been attributed to the role of sexual selection. Among the evolutionary mechanisms proposed to account for this diversity, two processes in particular have generated considerable interest. On the one hand, females may exploit postcopulatory mechanisms of selection to favour males with preferred genital traits (cryptic female choice; CFC), while on the other hand females may evolve structures or behaviours that mitigate the direct costs imposed by male genitalia (sexual conflict; SC). A critical but rarely explored assumption underlying both processes is that male and female reproductive traits coevolve, either via the classic Fisherian model of preference-trait coevolution (CFC) or through sexually antagonistic selection (SC). Here, we provide evidence for this prediction in the guppy (Poecilia reticulata), a polyandrous livebearing fish in which males transfer sperm internally to females via consensual and forced matings. Our results from a paternal half-sibling breeding design reveal substantial levels of additive genetic variation underlying male genital size and morphology-two traits known to predict mating success during non-consensual matings. Our subsequent finding that physically interacting female genital traits exhibit corresponding levels of genetic (co)variation reveals the potential intersexual coevolutionary dynamics of male and female genitalia, thereby fulfilling a fundamental assumption underlying CFC and SC theory.

  17. Quantitative evaluation of mast cells in cellularly dynamic and adynamic vascular malformations.

    PubMed

    Pasyk, K A; Cherry, G W; Grabb, W C; Sasaki, G H

    1984-01-01

    Mast cells were counted in 78 histologic specimens from 70 patients with various vascular malformations showing cellularly dynamic and cellularly adynamic lesions. In growing stages of strawberry hemangiomas, there was an increased number of mast cells (mean 11.0 cells per high-power field in stage III and 23.7 in stage IV), as well as a high number of mast cells in the initial involution of strawberry hemangiomas (stage V, mean 21.0 cells per high-power field). In later involuting stages (stages VI and VII), the number of mast cells decreased (mean 9.3 in stage VI; mean 4.7 in stage VII). In cellularly adynamic lesions, i.e., port wine stains, the mean number of mast cells was 4.8, and in congenital arteriovenous malformations, it was 3.6. In normal skin, the mean number of mast cells was 3.2. In cellular hemangiomas that showed active growth (stages III to IV), the number of mast cells was strikingly low (mean 1.3). It seems that the mast cells are not responsible for the proliferation of the endothelium or for growth of the hemangioma. The markedly increased number of mast cells in the growing stages and initial involuting stage of strawberry hemangiomas parallels the gradual growth of fibrous connective tissue inside the tumor. Mast cells may thus be a precursor of the beginning of the involution of a strawberry hemangioma. PMID:6691077

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

    PubMed

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

    2015-11-01

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

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

    PubMed

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

    2015-02-01

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

  20. Rapid and quantitative imaging of excitation polarized fluorescence reveals ordered septin dynamics in live yeast.

    PubMed

    DeMay, Bradley S; Noda, Naoki; Gladfelter, Amy S; Oldenbourg, Rudolf

    2011-08-17

    We report an imaging method for fast, sensitive analysis of the orientation of fluorescent molecules by employing a liquid-crystal based universal polarizer in the optical path of a wide-field light microscope. We developed specific acquisition and processing algorithms for measuring the anisotropy and for correcting artifacts caused by fluorescence bleaching, background light, and differential transmission of optical components. We call this approach the Fluorescence LC-PolScope and we used it to analyze the architectural dynamics of septin-green fluorescent protein (septin-GFP) constructs in the neck region of budding yeast. We describe three different states of highly anisotropic septin arrays in which the prevailing orientation of GFP dipoles was either parallel or perpendicular to the mother-bud axis. The transitions between these ordered states were characterized by transient isotropic states. To analyze the patterns of polarized fluorescence, we modeled the alignment of septin-GFP constructs in different stages of septin ring formation. Based on our model, our experimental data are consistent with the formation of paired rather than single filaments and the axis of the α-helical septin terminus linked to a GFP molecule is likely oriented normal to the cell surface. The Fluorescence LC-PolScope combines the molecular specificity of fluorescence tagging with the structural specificity of polarized light analysis.

  1. Quantitative genetic insights into the coevolutionary dynamics of male and female genitalia

    PubMed Central

    Evans, Jonathan P.; van Lieshout, Emile; Gasparini, Clelia

    2013-01-01

    The spectacular variability that typically characterizes male genital traits has largely been attributed to the role of sexual selection. Among the evolutionary mechanisms proposed to account for this diversity, two processes in particular have generated considerable interest. On the one hand, females may exploit postcopulatory mechanisms of selection to favour males with preferred genital traits (cryptic female choice; CFC), while on the other hand females may evolve structures or behaviours that mitigate the direct costs imposed by male genitalia (sexual conflict; SC). A critical but rarely explored assumption underlying both processes is that male and female reproductive traits coevolve, either via the classic Fisherian model of preference-trait coevolution (CFC) or through sexually antagonistic selection (SC). Here, we provide evidence for this prediction in the guppy (Poecilia reticulata), a polyandrous livebearing fish in which males transfer sperm internally to females via consensual and forced matings. Our results from a paternal half-sibling breeding design reveal substantial levels of additive genetic variation underlying male genital size and morphology—two traits known to predict mating success during non-consensual matings. Our subsequent finding that physically interacting female genital traits exhibit corresponding levels of genetic (co)variation reveals the potential intersexual coevolutionary dynamics of male and female genitalia, thereby fulfilling a fundamental assumption underlying CFC and SC theory. PMID:23720546

  2. Shedding light on microbial predator-prey population dynamics using a quantitative bioluminescence assay.

    PubMed

    Im, Hansol; Kim, Dasol; Ghim, Cheol-Min; Mitchell, Robert J

    2014-01-01

    This study assessed the dynamics of predation by Bdellovibrio bacteriovorus HD 100. Predation tests with two different bioluminescent strains of Escherichia coli, one expressing a heat-labile bacterial luciferase and the other a heat-stable form, showed near identical losses from both, indicating that protein expression and stability are not responsible for the "shutting-off" of the prey bioluminescence (BL). Furthermore, it was found that the loss in the prey BL was not proportional with the predator-to-prey ratio (PPR), with significantly greater losses seen as this value was increased. This suggests that other factors also play a role in lowering the prey BL. The loss in BL, however, was very consistent within nine independent experiments to the point that we were able to reliably estimate the predator numbers within only 1 h when present at a PPR of 6 or higher, Using a fluorescent prey, we found that premature lysis of the prey occurs at a significant level and was more prominent as the PPR ratio increased. Based upon the supernatant fluorescent signal, even a relatively low PPR of 10-20 led to approximately 5% of the prey population being prematurely lysed within 1 h, while a PPR of 90 led to nearly 15% lysis. Consequently, we developed a modified Lotka-Volterra predator-prey model that accounted for this lysis and is able to reliably estimate the prey and bdelloplast populations for a wide range of PPRs. PMID:24272279

  3. Quantitative genetic insights into the coevolutionary dynamics of male and female genitalia.

    PubMed

    Evans, Jonathan P; van Lieshout, Emile; Gasparini, Clelia

    2013-07-22

    The spectacular variability that typically characterizes male genital traits has largely been attributed to the role of sexual selection. Among the evolutionary mechanisms proposed to account for this diversity, two processes in particular have generated considerable interest. On the one hand, females may exploit postcopulatory mechanisms of selection to favour males with preferred genital traits (cryptic female choice; CFC), while on the other hand females may evolve structures or behaviours that mitigate the direct costs imposed by male genitalia (sexual conflict; SC). A critical but rarely explored assumption underlying both processes is that male and female reproductive traits coevolve, either via the classic Fisherian model of preference-trait coevolution (CFC) or through sexually antagonistic selection (SC). Here, we provide evidence for this prediction in the guppy (Poecilia reticulata), a polyandrous livebearing fish in which males transfer sperm internally to females via consensual and forced matings. Our results from a paternal half-sibling breeding design reveal substantial levels of additive genetic variation underlying male genital size and morphology-two traits known to predict mating success during non-consensual matings. Our subsequent finding that physically interacting female genital traits exhibit corresponding levels of genetic (co)variation reveals the potential intersexual coevolutionary dynamics of male and female genitalia, thereby fulfilling a fundamental assumption underlying CFC and SC theory. PMID:23720546

  4. The differential expression of alternatively polyadenylated transcripts is a common stress-induced response mechanism that modulates mammalian mRNA expression in a quantitative and qualitative fashion

    PubMed Central

    Hollerer, Ina; Curk, Tomaz; Haase, Bettina; Benes, Vladimir; Hauer, Christian; Neu-Yilik, Gabriele; Bhuvanagiri, Madhuri; Hentze, Matthias W.; Kulozik, Andreas E.

    2016-01-01

    Stress adaptation plays a pivotal role in biological processes and requires tight regulation of gene expression. In this study, we explored the effect of cellular stress on mRNA polyadenylation and investigated the implications of regulated polyadenylation site usage on mammalian gene expression. High-confidence polyadenylation site mapping combined with global pre-mRNA and mRNA expression profiling revealed that stress induces an accumulation of genes with differentially expressed polyadenylated mRNA isoforms in human cells. Specifically, stress provokes a global trend in polyadenylation site usage toward decreased utilization of promoter-proximal poly(A) sites in introns or ORFs and increased utilization of promoter-distal polyadenylation sites in intergenic regions. This extensively affects gene expression beyond regulating mRNA abundance by changing mRNA length and by altering the configuration of open reading frames. Our study highlights the impact of post-transcriptional mechanisms on stress-dependent gene regulation and reveals the differential expression of alternatively polyadenylated transcripts as a common stress-induced mechanism in mammalian cells. PMID:27407180

  5. Deriving quantitative dynamics information for proteins and RNAs using ROTDIF with a graphical user interface.

    PubMed

    Berlin, Konstantin; Longhini, Andrew; Dayie, T Kwaku; Fushman, David

    2013-12-01

    To facilitate rigorous analysis of molecular motions in proteins, DNA, and RNA, we present a new version of ROTDIF, a program for determining the overall rotational diffusion tensor from single- or multiple-field nuclear magnetic resonance relaxation data. We introduce four major features that expand the program's versatility and usability. The first feature is the ability to analyze, separately or together, (13)C and/or (15)N relaxation data collected at a single or multiple fields. A significant improvement in the accuracy compared to direct analysis of R2/R1 ratios, especially critical for analysis of (13)C relaxation data, is achieved by subtracting high-frequency contributions to relaxation rates. The second new feature is an improved method for computing the rotational diffusion tensor in the presence of biased errors, such as large conformational exchange contributions, that significantly enhances the accuracy of the computation. The third new feature is the integration of the domain alignment and docking module for relaxation-based structure determination of multi-domain systems. Finally, to improve accessibility to all the program features, we introduced a graphical user interface that simplifies and speeds up the analysis of the data. Written in Java, the new ROTDIF can run on virtually any computer platform. In addition, the new ROTDIF achieves an order of magnitude speedup over the previous version by implementing a more efficient deterministic minimization algorithm. We not only demonstrate the improvement in accuracy and speed of the new algorithm for synthetic and experimental (13)C and (15)N relaxation data for several proteins and nucleic acids, but also show that careful analysis required especially for characterizing RNA dynamics allowed us to uncover subtle conformational changes in RNA as a function of temperature that were opaque to previous analysis.

  6. Deriving Quantitative Dynamics Information for Proteins and RNAs using ROTDIF with a Graphical User Interface

    PubMed Central

    Berlin, Konstantin; Longhini, Andrew; Dayie, T. Kwaku; Fushman, David

    2013-01-01

    To facilitate rigorous analysis of molecular motions in proteins, DNA, and RNA, we present a new version of ROTDIF, a program for determining the overall rotational diffusion tensor from single-or multiple-field Nuclear Magnetic Resonance (NMR) relaxation data. We introduce four major features that expand the program’s versatility and usability. The first feature is the ability to analyze, separately or together, 13C and/or 15N relaxation data collected at a single or multiple fields. A significant improvement in the accuracy compared to direct analysis of R2/R1 ratios, especially critical for analysis of 13C relaxation data, is achieved by subtracting high-frequency contributions to relaxation rates. The second new feature is an improved method for computing the rotational diffusion tensor in the presence of biased errors, such as large conformational exchange contributions, that significantly enhances the accuracy of the computation. The third new feature is the integration of the domain alignment and docking module for relaxation-based structure determination of multi-domain systems. Finally, to improve accessibility to all the program features, we introduced a graphical user interface (GUI) that simplifies and speeds up the analysis of the data. Written in Java, the new ROTDIF can run on virtually any computer platform. In addition, the new ROTDIF achieves an order of magnitude speedup over the previous version by implementing a more efficient deterministic minimization algorithm. We not only demonstrate the improvement in accuracy and speed of the new algorithm for synthetic and experimental 13C and 15N relaxation data for several proteins and nucleic acids, but also show that careful analysis required especially for characterizing RNA dynamics allowed us to uncover subtle conformational changes in RNA as a function of temperature that were opaque to previous analysis. PMID:24170368

  7. Quantitative Profiling of Chromatome Dynamics Reveals a Novel Role for HP1BP3 in Hypoxia-induced Oncogenesis*

    PubMed Central

    Dutta, Bamaprasad; Yan, Ren; Lim, Sai Kiang; Tam, James P.; Sze, Siu Kwan

    2014-01-01

    In contrast to the intensely studied genetic and epigenetic changes that induce host cell transformation to initiate tumor development, those that promote the malignant progression of cancer remain poorly defined. As emerging evidence suggests that the hypoxic tumor microenvironment could re-model the chromatin-associated proteome (chromatome) to induce epigenetic changes and alter gene expression in cancer cells, we hypothesized that hypoxia-driven evolution of the chromatome promotes malignant changes and the development of therapy resistance in tumor cells. To test this hypothesis, we isolated chromatins from tumor cells treated with varying conditions of normoxia, hypoxia, and re-oxygenation and then partially digested them with DNase I and analyzed them for changes in euchromatin- and heterochromatin-associated proteins using an iTRAQ-based quantitative proteomic approach. We identified a total of 1446 proteins with a high level of confidence, including 819 proteins that were observed to change their chromatin association topology under hypoxic conditions. These hypoxia-sensitive proteins included key mediators of chromatin organization, transcriptional regulation, and DNA repair. Furthermore, our proteomic and functional experiments revealed a novel role for the chromatin organizer protein HP1BP3 in mediating chromatin condensation during hypoxia, leading to increased tumor cell viability, radio-resistance, chemo-resistance, and self-renewal. Taken together, our findings indicate that HP1BP3 is a key mediator of tumor progression and cancer cell acquisition of therapy-resistant traits, and thus might represent a novel therapeutic target in a range of human malignancies. PMID:25100860

  8. Morphology of nuclear transcription.

    PubMed

    Weipoltshammer, Klara; Schöfer, Christian

    2016-04-01

    Gene expression control is a fundamental determinant of cellular life with transcription being the most important step. The spatial nuclear arrangement of the transcription process driven by RNA polymerases II and III is nonrandomly organized in foci, which is believed to add another regulatory layer on gene expression control. RNA polymerase I transcription takes place within a specialized organelle, the nucleolus. Transcription of ribosomal RNA directly responds to metabolic requirements, which in turn is reflected in the architecture of nucleoli. It differs from that of the other polymerases with respect to the gene template organization, transcription rate, and epigenetic expression control, whereas other features are shared like the formation of DNA loops bringing genes and components of the transcription machinery in close proximity. In recent years, significant advances have been made in the understanding of the structural prerequisites of nuclear transcription, of the arrangement in the nuclear volume, and of the dynamics of these entities. Here, we compare ribosomal RNA and mRNA transcription side by side and review the current understanding focusing on structural aspects of transcription foci, of their constituents, and of the dynamical behavior of these components with respect to foci formation, disassembly, and cell cycle. PMID:26847177

  9. Quantitative dynamics of triacylglycerol accumulation in microalgae populations at single-cell resolution revealed by Raman microspectroscopy

    PubMed Central

    2014-01-01

    Background Rapid, real-time and label-free measurement of the cellular contents of biofuel molecules such as triacylglycerol (TAG) in populations at single-cell resolution are important for bioprocess control and understanding of the population heterogeneity. Raman microspectroscopy can directly detect the changes of metabolite profile in a cell and thus can potentially serve these purposes. Results Single-cell Raman spectra (SCRS) of the unicellular oleaginous microalgae Nannochloropsis oceanica from the cultures under nitrogen depletion (TAG-producing condition) and nitrogen repletion (non-TAG-producing condition) were sampled at eight time points during the first 96 hours upon the onset of nitrogen depletion. Single N. oceanica cells were captured by a 532-nm laser and the SCRS were acquired by the same laser within one second per cell. Using chemometric methods, the SCRS were able to discriminate cells between nitrogen-replete and nitrogen-depleted conditions at as early as 6 hours with >93.3% accuracy, and among the eight time points under nitrogen depletion with >90.4% accuracy. Quantitative prediction of TAG content in single cells was achieved and validated via SCRS and liquid chromatography-mass spectrometry (LC-MS) analysis at population level. SCRS revealed the dynamics of heterogeneity in TAG production among cells in each isogenic population. A significant negative correlation between TAG content and lipid unsaturation degree in individual microalgae cells was observed. Conclusions Our results show that SCRS can serve as a label-free and non-invasive proxy for quantitatively tracking and screening cellular TAG content in real-time at single-cell level. Phenotypic comparison of single cells via SCRS should also help investigating the mechanisms of functional heterogeneity within a cellular population. PMID:24716544

  10. Dynamic nature of fibre-type specific expression of myosin heavy chain transcripts in 14 different human skeletal muscles.

    PubMed

    Smerdu, V; Erzen, I

    2001-01-01

    The main goal of this study was to find out, whether the appearance of fibres without evident myosin heavy chain (MyHC) transcript expression (negative fibres) implies the existence of additional MyHC transcripts in human muscle fibres. Fourteen different skeletal muscles were analysed also to verify how MyHC transcript expression matches histochemical phenotypes of fibres. For this purpose, the expression of beta-slow, 2a and 2x MyHC transcripts, demonstrated by in situ hybridisation technique, was analysed within type I, IIC, IIA, IIAX and IIX fibres, determined according to the activity of myofibrillar ATPase. Additionally, MyHC isoform expression was immunohistochemically demonstrated and metabolic profiles of negative fibres were estimated. From a total of 4444 muscle fibres analysed, only 0.8% of fibres were negative, among them type I prevailed, the remainder were type IIA and IIX fibres. The majority of fibres expressed only beta, 2a and 2x MyHC transcripts and they mostly matched type I, IIA and IIX fibres respectively, but two minor hybrid fibre groups (beta/2a and 2ax) exhibited variable histochemical phenotype. The infrequency, the prevailing oxidative-glycolytic metabolic profile of negative type I fibres and frequent co-appearance with transitional type IIC fibres imply that the negative fibres rather result from fibre type transition than express an additional slow or even 2b MyHC transcripts. The appearance of hybrid and mismatched fibres additionally indicates that fibre type transition occurs also in presumably normal skeletal muscles, what enables the muscles to tune even with minimal changes in mechanical demands.

  11. Comparison of droplet digital PCR and quantitative real-time PCR for examining population dynamics of bacteria in soil.

    PubMed

    Kim, Tae Gwan; Jeong, So-Yeon; Cho, Kyung-Suk

    2014-07-01

    The newly developed droplet digital PCR (DD-PCR) has shown promise as a DNA quantification technology in medical diagnostic fields. This study evaluated the applicability of DD-PCR as a quantitative tool for soil DNA using quantitative real-time PCR (qRT-PCR) as a reference technology. Cupriavidus sp. MBT14 and Sphingopyxis sp. MD2 were used, and a primer/TaqMan probe set was designed for each (CupMBT and SphMD2, respectively). Standard curve analyses on tenfold dilution series showed that both qRT-PCR and DD-PCR exhibited excellent linearity (R (2) = 1.00) and PCR efficiency (≥92 %) across their detectable ranges. However, DD-PCR showed a tenfold greater sensitivity than qRT-PCR. MBT14 and MD2 were added to non-sterile soil at 0 ~ 5 × 10(8) and 0 ~ 5 × 10(7) cells per gram of soil, respectively (n = 5). This bacterial load test indicated that DD-PCR was more sensitive and discriminating than qRT-PCR. For instance, DD-PCR showed a gradual DNA increase from 14 to 141,160 MBT14 rDNA copies μL DNA extract(-1) as the bacterial load increased, while qRT-PCR could quantify the DNA (6,432 copies μL DNA(-1)) at ≥5 × 10(5) MBT14 per gram of soil. When temporal DNA changes were monitored for 3 weeks in the amended soils, the two technologies exhibited nearly identical changes over time. Linearity tests (y = a · x) revealed excellent quantitative agreement between the two technologies (a = 0.98, R (2) = 0.97 in the CupMBT set and a = 0.90, R (2) = 0.94 in the SphMD2 set). These results suggest that DD-PCR is a promising tool to examine temporal dynamics of microorganisms in complex environments.

  12. Systems for Lung Volume Standardization during Static and Dynamic MDCT-based Quantitative Assessment of Pulmonary Structure and Function

    PubMed Central

    Fuld, Matthew K.; Grout, Randall; Guo, Junfeng; Morgan, John H.; Hoffman, Eric A.

    2013-01-01

    Rationale and Objectives Multidetector-row Computed Tomography (MDCT) has emerged as a tool for quantitative assessment of parenchymal destruction, air trapping (density metrics) and airway remodeling (metrics relating airway wall and lumen geometry) in chronic obstructive pulmonary disease (COPD) and asthma. Critical to the accuracy and interpretability of these MDCT-derived metrics is the assurance that the lungs are scanned during a breath-hold at a standardized volume. Materials and Methods A computer monitored turbine-based flow meter system was developed to control patient breath-holds and facilitate static imaging at fixed percentages of the vital capacity. Due to calibration challenges with gas density changes during multi-breath xenon-CT an alternative system was required. The design incorporated dual rolling seal pistons. Both systems were tested in a laboratory environment and human subject trials. Results The turbine-based system successfully controlled lung volumes in 32/37 subjects, having a linear relationship for CT measured air volume between repeated scans: for all scans, the mean and confidence interval of the differences (scan1-scan2) was −9 ml (−169, 151); for TLC alone 6 ml (−164, 177); for FRC alone, −23 ml (−172, 126). The dual-piston system successfully controlled lung volume in 31/41 subjects. Study failures related largely to subject non-compliance with verbal instruction and gas leaks around the mouthpiece. Conclusion We demonstrate the successful use of a turbine-based system for static lung volume control and demonstrate its inadequacies for dynamic xenon-CT studies. Implementation of a dual-rolling seal spirometer has been shown to adequately control lung volume for multi-breath wash-in xenon-CT studies. These systems coupled with proper patient coaching provide the tools for the use of CT to quantitate regional lung structure and function. The wash-in xenon-CT method for assessing regional lung function, while not

  13. The HIRA complex that deposits the histone H3.3 is conserved in Arabidopsis and facilitates transcriptional dynamics.

    PubMed

    Nie, Xin; Wang, Haifeng; Li, Jing; Holec, Sarah; Berger, Frédéric

    2014-08-01

    In animals, replication-independent incorporation of nucleosomes containing the histone variant H3.3 enables global reprogramming of histone modifications and transcriptional profiles. H3.3 enrichment over gene bodies correlates with gene transcription in animals and plants. In animals, H3.3 is deposited into chromatin by specific protein complexes, including the HIRA complex. H3.3 variants evolved independently and acquired similar properties in animals and plants, questioning how the H3.3 deposition machinery evolved in plants and what are its biological functions. We performed phylogenetic analyses in the plant kingdom and identified in Arabidopsis all orthologs of human genes encoding members of the HIRA complex. Genetic analyses, biochemical data and protein localisation suggest that these proteins form a complex able to interact with H3.3 in Arabidopsis in a manner similar to that described in mammals. In contrast to animals, where HIRA is required for fertilization and early development, loss of function of HIRA in Arabidopsis causes mild phenotypes in the adult plant and does not perturb sexual reproduction and embryogenesis. Rather, HIRA function is required for transcriptional reprogramming during dedifferentiation of plant cells that precedes vegetative propagation and for the appropriate transcription of genes responsive to biotic and abiotic factors. We conclude that the molecular function of the HIRA complex is conserved between plants and animals. Yet plants diversified HIRA functions to enable asexual reproduction and responsiveness to the environment in response to the plant sessile lifestyle.

  14. Quantitative Resistance to Verticillium Wilt in Medicago truncatula Involves Eradication of the Fungus from Roots and Is Associated with Transcriptional Responses Related to Innate Immunity

    PubMed Central

    Toueni, Maoulida; Ben, Cécile; Le Ru, Aurélie; Gentzbittel, Laurent; Rickauer, Martina

    2016-01-01

    Resistance mechanisms to Verticillium wilt are well-studied in tomato, cotton, and Arabidopsis, but much less in legume plants. Because legume plants establish nitrogen-fixing symbioses in their roots, resistance to root-attacking pathogens merits particular attention. The interaction between the soil-borne pathogen Verticillium alfalfae and the model legume Medicago truncatula was investigated using a resistant (A17) and a susceptible (F83005.5) line. As shown by histological analyses, colonization by the pathogen was initiated similarly in both lines. Later on, the resistant line A17 eliminated the fungus, whereas the susceptible F83005.5 became heavily colonized. Resistance in line A17 does not involve homologs of the well-characterized tomato Ve1 and V. dahliae Ave1 genes. A transcriptomic study of early root responses during initial colonization (i.e., until 24 h post-inoculation) similarly was performed. Compared to the susceptible line, line A17 displayed already a significantly higher basal expression of defense-related genes prior to inoculation, and responded to infection with up-regulation of only a small number of genes. Although fungal colonization was still low at this stage, the susceptible line F83005.5 exhibited a disorganized response involving a large number of genes from different functional classes. The involvement of distinct phytohormone signaling pathways in resistance as suggested by gene expression patterns was supported by experiments with plant hormone pretreatment before fungal inoculation. Gene co-expression network analysis highlighted five main modules in the resistant line, whereas no structured gene expression was found in the susceptible line. One module was particularly associated to the inoculation response in A17. It contains the majority of differentially expressed genes, genes associated with PAMP perception and hormone signaling, and transcription factors. An in silico analysis showed that a high number of these genes also

  15. Quantitative Evaluation of the Dispersion of Graphene Sheets With and Without Functional Groups Using Molecular Dynamics Simulations

    NASA Astrophysics Data System (ADS)

    Cha, JinHyeok; Kyoung, Woomin; Song, Kyonghwa; Park, Sangbaek; Lim, Taewon; Lee, Jongkook; Kang, Hyunmin

    2016-03-01

    Nanofluids with enhanced thermal properties are candidates for thermal management in automotive systems, with scope for improving energy efficiency. In particular, many studies have reported on dispersions of nanoparticles with long-term stability in the base fluid, with qualitative evaluations of the dispersion stability via either the naked eye or optical instruments. Additives such as surfactants can be used to enhance the dispersion of nanoparticles; however, this may diminish their intrinsic thermal properties. Here, we describe molecular dynamics simulations of nanofluids containing graphene sheets dispersed in ethylene glycol and water. We go on to suggest a quantitative evaluation method for the degree of dispersion, based on the ratio of the total number of nanoparticles to the number of clustered nanoparticles. Moreover, we investigate the effects of functional groups on the surface of graphene, which are expected to improve the dispersion without requiring additives such as surfactants due to steric hindrance and chemical affinity for the surrounding fluid. We find that, for pure graphene, the degree of dispersion decreased as the quantity of graphene sheets increased, which is attributed to an increased probability of aggregation at higher loadings; however, the presence of functional groups inhibited the graphene sheets from forming aggregates.

  16. Genome-wide association mapping of growth dynamics detects time-specific and general quantitative trait loci.

    PubMed

    Bac-Molenaar, Johanna A; Vreugdenhil, Dick; Granier, Christine; Keurentjes, Joost J B

    2015-09-01

    Growth is a complex trait determined by the interplay between many genes, some of which play a role at a specific moment during development whereas others play a more general role. To identify the genetic basis of growth, natural variation in Arabidopsis rosette growth was followed in 324 accessions by a combination of top-view imaging, high-throughput image analysis, modelling of growth dynamics, and end-point fresh weight determination. Genome-wide association (GWA) mapping of the temporal growth data resulted in the detection of time-specific quantitative trait loci (QTLs), whereas mapping of model parameters resulted in another set of QTLs related to the whole growth curve. The positive correlation between projected leaf area (PLA) at different time points during the course of the experiment suggested the existence of general growth factors with a function in multiple developmental stages or with prolonged downstream effects. Many QTLs could not be identified when growth was evaluated only at a single time point. Eleven candidate genes were identified, which were annotated to be involved in the determination of cell number and size, seed germination, embryo development, developmental phase transition, or senescence. For eight of these, a mutant or overexpression phenotype related to growth has been reported, supporting the identification of true positives. In addition, the detection of QTLs without obvious candidate genes implies the annotation of novel functions for underlying genes.

  17. Quantitative imaging of cerebral blood flow velocity and intracellular motility using dynamic light scattering–optical coherence tomography

    PubMed Central

    Lee, Jonghwan; Radhakrishnan, Harsha; Wu, Weicheng; Daneshmand, Ali; Climov, Mihail; Ayata, Cenk; Boas, David A

    2013-01-01

    This paper describes a novel optical method for label-free quantitative imaging of cerebral blood flow (CBF) and intracellular motility (IM) in the rodent cerebral cortex. This method is based on a technique that integrates dynamic light scattering (DLS) and optical coherence tomography (OCT), named DLS–OCT. The technique measures both the axial and transverse velocities of CBF, whereas conventional Doppler OCT measures only the axial one. In addition, the technique produces a three-dimensional map of the diffusion coefficient quantifying nontranslational motions. In the DLS–OCT diffusion map, we observed high-diffusion spots, whose locations highly correspond to neuronal cell bodies and whose diffusion coefficient agreed with that of the motion of intracellular organelles reported in vitro in the literature. Therefore, the present method has enabled, for the first time to our knowledge, label-free imaging of the diffusion-like motion of intracellular organelles in vivo. As an example application, we used the method to monitor CBF and IM during a brief ischemic stroke, where we observed an induced persistent reduction in IM despite the recovery of CBF after stroke. This result supports that the IM measured in this study represent the cellular energy metabolism-related active motion of intracellular organelles rather than free diffusion of intracellular macromolecules. PMID:23403378

  18. Quantitative Evaluation of the Dispersion of Graphene Sheets With and Without Functional Groups Using Molecular Dynamics Simulations.

    PubMed

    Cha, JinHyeok; Kyoung, Woomin; Song, Kyonghwa; Park, Sangbaek; Lim, Taewon; Lee, Jongkook; Kang, Hyunmin

    2016-12-01

    Nanofluids with enhanced thermal properties are candidates for thermal management in automotive systems, with scope for improving energy efficiency. In particular, many studies have reported on dispersions of nanoparticles with long-term stability in the base fluid, with qualitative evaluations of the dispersion stability via either the naked eye or optical instruments. Additives such as surfactants can be used to enhance the dispersion of nanoparticles; however, this may diminish their intrinsic thermal properties. Here, we describe molecular dynamics simulations of nanofluids containing graphene sheets dispersed in ethylene glycol and water. We go on to suggest a quantitative evaluation method for the degree of dispersion, based on the ratio of the total number of nanoparticles to the number of clustered nanoparticles. Moreover, we investigate the effects of functional groups on the surface of graphene, which are expected to improve the dispersion without requiring additives such as surfactants due to steric hindrance and chemical affinity for the surrounding fluid. We find that, for pure graphene, the degree of dispersion decreased as the quantity of graphene sheets increased, which is attributed to an increased probability of aggregation at higher loadings; however, the presence of functional groups inhibited the graphene sheets from forming aggregates. PMID:26964558

  19. KymographClear and KymographDirect: two tools for the automated quantitative analysis of molecular and cellular dynamics using kymographs

    PubMed Central

    Mangeol, Pierre; Prevo, Bram; Peterman, Erwin J. G.

    2016-01-01

    Dynamic processes are ubiquitous and essential in living cells. To properly understand these processes, it is imperative to measure them in a time-dependent way and analyze the resulting data quantitatively, preferably with automated tools. Kymographs are single images that represent the motion of dynamic processes and are widely used in live-cell imaging. Although they contain the full range of dynamics, it is not straightforward to extract this quantitative information in a reliable way. Here we present two complementary, publicly available software tools, KymographClear and KymographDirect, that have the power to reveal detailed insight in dynamic processes. KymographClear is a macro toolset for ImageJ to generate kymographs that provides automatic color coding of the different directions of movement. KymographDirect is a stand-alone tool to extract quantitative information from kymographs obtained from a wide range of dynamic processes in an automated way, with high accuracy and reliability. We discuss the concepts behind these software tools, validate them using simulated data, and test them on experimental data. We show that these tools can be used to extract motility parameters from a diverse set of cell-biological experiments in an automated and user-friendly way. PMID:27099372

  20. Rapidly characterizing the fast dynamics of RNA genetic circuitry with cell-free transcription-translation (TX-TL) systems.

    PubMed

    Takahashi, Melissa K; Chappell, James; Hayes, Clarmyra A; Sun, Zachary Z; Kim, Jongmin; Singhal, Vipul; Spring, Kevin J; Al-Khabouri, Shaima; Fall, Christopher P; Noireaux, Vincent; Murray, Richard M; Lucks, Julius B

    2015-05-15

    RNA regulators are emerging as powerful tools to engineer synthetic genetic networks or rewire existing ones. A potential strength of RNA networks is that they may be able to propagate signals on time scales that are set by the fast degradation rates of RNAs. However, a current bottleneck to verifying this potential is the slow design-build-test cycle of evaluating these networks in vivo. Here, we adapt an Escherichia coli-based cell-free transcription-translation (TX-TL) system for rapidly prototyping RNA networks. We used this system to measure the response time of an RNA transcription cascade to be approximately five minutes per step of the cascade. We also show that this response time can be adjusted with temperature and regulator threshold tuning. Finally, we use TX-TL to prototype a new RNA network, an RNA single input module, and show that this network temporally stages the expression of two genes in vivo.

  1. The dynamic transcriptional and translational landscape of the model antibiotic producer Streptomyces coelicolor A3(2)

    PubMed Central

    Jeong, Yujin; Kim, Ji-Nu; Kim, Min Woo; Bucca, Giselda; Cho, Suhyung; Yoon, Yeo Joon; Kim, Byung-Gee; Roe, Jung-Hye; Kim, Sun Chang; Smith, Colin P.; Cho, Byung-Kwan

    2016-01-01

    Individual Streptomyces species have the genetic potential to produce a diverse array of natural products of commercial, medical and veterinary interest. However, these products are often not detectable under laboratory culture conditions. To harness their full biosynthetic potential, it is important to develop a detailed understanding of the regulatory networks that orchestrate their metabolism. Here we integrate nucleotide resolution genome-scale measurements of the transcriptome and translatome of Streptomyces coelicolor, the model antibiotic-producing actinomycete. Our systematic study determines 3,570 transcription start sites and identifies 230 small RNAs and a considerable proportion (∼21%) of leaderless mRNAs; this enables deduction of genome-wide promoter architecture. Ribosome profiling reveals that the translation efficiency of secondary metabolic genes is negatively correlated with transcription and that several key antibiotic regulatory genes are translationally induced at transition growth phase. These findings might facilitate the design of new approaches to antibiotic discovery and development. PMID:27251447

  2. The dynamic transcriptional and translational landscape of the model antibiotic producer Streptomyces coelicolor A3(2).

    PubMed

    Jeong, Yujin; Kim, Ji-Nu; Kim, Min Woo; Bucca, Giselda; Cho, Suhyung; Yoon, Yeo Joon; Kim, Byung-Gee; Roe, Jung-Hye; Kim, Sun Chang; Smith, Colin P; Cho, Byung-Kwan

    2016-01-01

    Individual Streptomyces species have the genetic potential to produce a diverse array of natural products of commercial, medical and veterinary interest. However, these products are often not detectable under laboratory culture conditions. To harness their full biosynthetic potential, it is important to develop a detailed understanding of the regulatory networks that orchestrate their metabolism. Here we integrate nucleotide resolution genome-scale measurements of the transcriptome and translatome of Streptomyces coelicolor, the model antibiotic-producing actinomycete. Our systematic study determines 3,570 transcription start sites and identifies 230 small RNAs and a considerable proportion (∼21%) of leaderless mRNAs; this enables deduction of genome-wide promoter architecture. Ribosome profiling reveals that the translation efficiency of secondary metabolic genes is negatively correlated with transcription and that several key antibiotic regulatory genes are translationally induced at transition growth phase. These findings might facilitate the design of new approaches to antibiotic discovery and development. PMID:27251447

  3. Transcriptional Dynamics of Two Seed Compartments with Opposing Roles in Arabidopsis Seed Germination1[W][OPEN

    PubMed Central

    Dekkers, Bas J.W.; Pearce, Simon; van Bolderen-Veldkamp, R.P.; Marshall, Alex; Widera, Paweł; Gilbert, James; Drost, Hajk-Georg; Bassel, George W.; Müller, Kerstin; King, John R.; Wood, Andrew T.A.; Grosse, Ivo; Quint, Marcel; Krasnogor, Natalio; Leubner-Metzger, Gerhard; Holdsworth, Michael J.; Bentsink, Leónie

    2013-01-01

    Seed germination is a critical stage in the plant life cycle and the first step toward successful plant establishment. Therefore, understanding germination is of important ecological and agronomical relevance. Previous research revealed that different seed compartments (testa, endosperm, and embryo) control germination, but little is known about the underlying spatial and temporal transcriptome changes that lead to seed germination. We analyzed genome-wide expression in germinating Arabidopsis (Arabidopsis thaliana) seeds with both temporal and spatial detail and provide Web-accessible visualizations of the data reported (vseed.nottingham.ac.uk). We show the potential of this high-resolution data set for the construction of meaningful coexpression networks, which provide insight into the genetic control of germination. The data set reveals two transcriptional phases during germination that are separated by testa rupture. The first phase is marked by large transcriptome changes as the seed switches from a dry, quiescent state to a hydrated and active state. At the end of this first transcriptional phase, the number of differentially expressed genes between consecutive time points drops. This increases again at