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Sample records for quantitative transcription dynamic

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

    PubMed

    Ma, Menggen; Liu, Lewis Z

    2010-06-10

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

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

    PubMed Central

    2010-01-01

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

  3. 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

  4. From DNA sequence to transcriptional behaviour: a quantitative approach.

    PubMed

    Segal, Eran; Widom, Jonathan

    2009-07-01

    Complex transcriptional behaviours are encoded in the DNA sequences of gene regulatory regions. Advances in our understanding of these behaviours have been recently gained through quantitative models that describe how molecules such as transcription factors and nucleosomes interact with genomic sequences. An emerging view is that every regulatory sequence is associated with a unique binding affinity landscape for each molecule and, consequently, with a unique set of molecule-binding configurations and transcriptional outputs. We present a quantitative framework based on existing methods that unifies these ideas. This framework explains many experimental observations regarding the binding patterns of factors and nucleosomes and the dynamics of transcriptional activation. It can also be used to model more complex phenomena such as transcriptional noise and the evolution of transcriptional regulation.

  5. 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

  6. 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

  7. Promoter-mediated transcriptional dynamics.

    PubMed

    Zhang, Jiajun; Zhou, Tianshou

    2014-01-21

    Genes in eukaryotic cells are typically regulated by complex promoters containing multiple binding sites for a variety of transcription factors, but how promoter dynamics affect transcriptional dynamics has remained poorly understood. In this study, we analyze gene models at the transcriptional regulation level, which incorporate the complexity of promoter structure (PS) defined as transcriptional exits (i.e., ON states of the promoter) and the transition pattern (described by a matrix consisting of transition rates among promoter activity states). We show that multiple exits of transcription are the essential origin of generating multimodal distributions of mRNA, but promoters with the same transition pattern can lead to multimodality of different modes, depending on the regulation of transcriptional factors. In turn, for similar mRNA distributions in the models, the mean ON or OFF time distributions may exhibit different characteristics, thus providing the supplemental information on PS. In addition, we demonstrate that the transcriptional noise can be characterized by a nonlinear function of mean ON and OFF times. These results not only reveal essential characteristics of promoter-mediated transcriptional dynamics but also provide signatures useful for inferring PS based on characteristics of transcriptional outputs. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  8. 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)

  9. Transcript quantitation in total yeast cellular RNA using kinetic PCR

    PubMed Central

    Kang, John J.; Watson, Robert M.; Fisher, Mary E.; Higuchi, Russell; Gelfand, David H.; Holland, Michael J.

    2000-01-01

    Kinetically monitored, reverse transcriptase-initiated PCR (kinetic RT–PCR, kRT–PCR) is a novel application of kinetic PCR for high throughput transcript quantitation in total cellular RNA. The assay offers the simplicity and flexibility of an enzyme assay with distinct advantages over DNA microarray hybridization and SAGE technologies for certain applications. The reproducibility, sensitivity and accuracy of the kRT–PCR were assessed for yeast transcripts previously quantitated by a variety of methods including SAGE analysis. Changes in transcript levels between different genetic or physiological cell states were reproducibly quantitated with an accuracy of ±20%. The assay was sufficiently sensitive to quantitate yeast transcripts over a range of more than five orders of magnitude, including low abundance transcripts encoding cell cycle and transcriptional regulators. PMID:10606670

  10. Non-transcriptional regulatory processes shape transcriptional network dynamics.

    PubMed

    Ray, J Christian J; Tabor, Jeffrey J; Igoshin, Oleg A

    2011-10-11

    Information about the extra- or intracellular environment is often captured as biochemical signals that propagate through regulatory networks. These signals eventually drive phenotypic changes, typically by altering gene expression programmes in the cell. Reconstruction of transcriptional regulatory networks has given a compelling picture of bacterial physiology, but transcriptional network maps alone often fail to describe phenotypes. Cellular response dynamics are ultimately determined by interactions between transcriptional and non-transcriptional networks, with dramatic implications for physiology and evolution. Here, we provide an overview of non-transcriptional interactions that can affect the performance of natural and synthetic bacterial regulatory networks.

  11. Spatially coordinated dynamic gene transcription in living pituitary tissue.

    PubMed

    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 R H; Davis, Julian R E

    2016-02-01

    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.

  12. Effects of elongation delay in transcription dynamics.

    PubMed

    Zhang, Xuan; Jin, Huiqin; Yang, Zhuoqin; Lei, Jinzhi

    2014-12-01

    In the transcription process, elongation delay is induced by the movement of RNA polymerases (RNAP) along the DNA sequence, and can result in changes in the transcription dynamics. This paper studies the transcription dynamics that involved the elongation delay and effects of cell division and DNA replication. The stochastic process of gene expression is modeled with delay chemical master equation with periodic coefficients, and is studied numerically through the stochastic simulation algorithm with delay. We show that the average transcription level approaches to a periodic dynamics over cell cycles at homeostasis, and the elongation delay can reduce the transcription level and increase the transcription noise. Moreover, the transcription elongation can induce bimodal distribution of mRNA levels that can be measured by the techniques of flow cytometry.

  13. A Quantitative Model of Expert Transcription Typing

    DTIC Science & Technology

    1993-03-08

    monitoring the accuracy of the typing...the deterioration of typing rate that occurs as the text is modified from normal prose to non -language or random...letters...for] non -alphabetical keys. (p. 6) Rumelhart and Norman also do not attempt to make zero-parameter quantitative predictions of typing...Salthouse’s two-choice reaction time task was somewhat non - standard: Stimuli were uppercase and lowercase versions of the letters L and R, and responses

  14. The transcription analysis of duck enteritis virus UL49.5 gene using real-time quantitative reverse transcription PCR.

    PubMed

    Lin, Meng; Jia, Renyong; Wang, Mingshu; Gao, Xinghong; Zhu, Dekang; Chen, Shun; Yin, Zhongqiong; Wang, Yin; Chen, Xiaoyue; Cheng, Anchun

    2013-10-01

    Duck enteritis virus (DEV) UL49.5 encoding glycoprotein N was a conserved gene. The transcription dynamic process of UL49.5 homologous genes in herpesviruses was reported. However, the transcription dynamic process of DEV UL49.5 gene has not yet been established. In this study, a real-time quantitative reverse transcription PCR (real-time qRT-PCR) assay was established to test the transcription dynamic process of DEV UL49.5 gene, and the recombinant plasmid pUCm-T/UL49.5 was constructed as the standard DNA. The samples prepared from DEV-infected (at different time points) and uninfected cell were detected and calculated. The results demonstrated that the real-time qRT-PCR assay was successfully established. The transcription product of DEV UL49.5 gene was first detected at 0.5 h post infection (p.i.), increased at 8 h p.i. and reached a peak at 60 h p.i. Our results illustrated that DEV UL49.5 gene could be regarded as a late gene. The transcription dynamic process of DEV UL49.5 gene may provide a significant clue for further studies of DEV UL49.5 gene.

  15. Quantitatively predictable control of Drosophila transcriptional enhancers in vivo with engineered transcription factors.

    PubMed

    Crocker, Justin; Ilsley, Garth R; Stern, David L

    2016-03-01

    Genes are regulated by transcription factors that bind to regions of genomic DNA called enhancers. Considerable effort is focused on identifying transcription factor binding sites, with the goal of predicting gene expression from DNA sequence. Despite this effort, general, predictive models of enhancer function are currently lacking. Here we combine quantitative models of enhancer function with manipulations using engineered transcription factors to examine the extent to which enhancer function can be controlled in a quantitatively predictable manner. Our models, which incorporate few free parameters, can accurately predict the contributions of ectopic transcription factor inputs. These models allow the predictable 'tuning' of enhancers, providing a framework for the quantitative control of enhancers with engineered transcription factors.

  16. 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

  17. Evolutionary dynamics of prokaryotic transcriptional regulatory networks.

    PubMed

    Madan Babu, M; Teichmann, Sarah A; Aravind, L

    2006-04-28

    The structure of complex transcriptional regulatory networks has been studied extensively in certain model organisms. However, the evolutionary dynamics of these networks across organisms, which would reveal important principles of adaptive regulatory changes, are poorly understood. We use the known transcriptional regulatory network of Escherichia coli to analyse the conservation patterns of this network across 175 prokaryotic genomes, and predict components of the regulatory networks for these organisms. We observe that transcription factors are typically less conserved than their target genes and evolve independently of them, with different organisms evolving distinct repertoires of transcription factors responding to specific signals. We show that prokaryotic transcriptional regulatory networks have evolved principally through widespread tinkering of transcriptional interactions at the local level by embedding orthologous genes in different types of regulatory motifs. Different transcription factors have emerged independently as dominant regulatory hubs in various organisms, suggesting that they have convergently acquired similar network structures approximating a scale-free topology. We note that organisms with similar lifestyles across a wide phylogenetic range tend to conserve equivalent interactions and network motifs. Thus, organism-specific optimal network designs appear to have evolved due to selection for specific transcription factors and transcriptional interactions, allowing responses to prevalent environmental stimuli. The methods for biological network analysis introduced here can be applied generally to study other networks, and these predictions can be used to guide specific experiments.

  18. DNA dynamically directs its own transcription initiation

    SciTech Connect

    Rasmussen, K. O.; Kalosakas, G.; Bishop, A. R.; Choi, C. H.; Usheva, A.

    2004-01-01

    Initiation of DNA gene transcription requires a transient opening in the double helix at the transcriptional start site. It is generally assumed that the location of this 'transcriptional bubble' is determined by sequence-specific protein binding, and that the energy required for unwinding the double helix comes from torsional strain. Physical twisting should cause DNA to open consistently in weakly bonded A/T rich stretches, however, simple base-pairing energetics alone can not account for the variety of observed transcriptional start sites. Applying the Peyrard-Bishop nonlinear cooperativity model to DNA, we are able to predict that thermally-induced DNA bubbles, similar in size to transcription bubbles, form at specific locations on DNA promoters. These predicted openings agree remarkably well with experiment, and that they correlate exactly with known transcription start sites and important regulatory sites on three different promoters. We propose that the sequence-specific location of the transcriptional start site is predetermined by the inherent opening patterns of specific DNA sequences. As DNA bubble formation is independent of protein binding, it appears that DNA is not only a passive carrier of information, but its dynamics plays an important role in directing the transcription and regulation of the genes it contains.

  19. 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

  20. 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

  1. Protein Synthesis Driven by Dynamical Stochastic Transcription.

    PubMed

    Innocentini, Guilherme C P; Forger, Michael; Radulescu, Ovidiu; Antoneli, Fernando

    2016-01-01

    In this manuscript, we propose a mathematical framework to couple transcription and translation in which mRNA production is described by a set of master equations, while the dynamics of protein density is governed by a random differential equation. The coupling between the two processes is given by a stochastic perturbation whose statistics satisfies the master equations. In this approach, from the knowledge of the analytical time-dependent distribution of mRNA number, we are able to calculate the dynamics of the probability density of the protein population.

  2. Identifying genes associated with a quantitative trait or quantitative trait locus via selective transcriptional profiling.

    PubMed

    Wang, Dong; Nettleton, Dan

    2006-06-01

    Genetical genomics is an approach that blends the mapping of quantitative trait loci (QTL) with microarray analysis. The approach can be used to identify associations between the allelic state of a genomic region and a gene's transcript abundance. However, the large number of microarrays required for adequate power results in high material and labor costs that prevent wide adoption of the genetical genomics strategy outside of some well-funded laboratories. We present a method called selective transcriptional profiling that involves selecting an optimal subset of individuals to microarray from a larger set of individuals for which relatively inexpensive quantitative trait and molecular marker data are available. We show how to use microarray data from the selected individuals, along with the trait and marker data from all individuals, to identify genes whose transcript abundance is associated with a quantitative trait of interest through linkage to a trait QTL or correlation with the trait. Our methods for selection and analysis are derived within a missing data framework.

  3. 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.

  4. 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

  5. Quantitative changes in gene transcription during induction of differentiation in porcine neural progenitor cells

    PubMed Central

    Yang, Jing; Gu, Ping; Menges, Steven

    2012-01-01

    Purpose Differentiation of neural stem/progenitor cells involves changes in the gene expression of these cells. Less clear is the extent to which incremental changes occur and the time course of such changes, particularly in non-rodents. Methods Using porcine genome microarrays, we analyzed changes in the expression of 23,256 genes in porcine neural progenitor cells (pNPCs) subject to two established differentiation protocols. In addition, we performed sequential quantitative assessment of a defined transcription profile consisting of 15 progenitor- and lineage-associated genes following exposure to the same treatment protocols, to examine the temporal dynamics of phenotypic changes following induction of differentiation. Immunocytochemistry was also used to examine the expression of seven of these phenotypically important genes at the protein level. Initial primary isolates were passaged four times in proliferation medium containing 20 ng/ml epidermal growth factor (EGF) and 20 ng/ml basic fibroblast growth factor (bFGF) before differentiation was induced. Differentiation was induced by medium without EGF or bFGF and containing either 10 ng/ml ciliary neurotrophic factor or 10% fetal bovine serum (FBS). Cultures were fed every two days and harvested on days 0, 1, 3, and 5 for quantitative real-time PCR. Results The microarray results illustrated and contrasted the global shifts in the porcine transcriptome associated with both treatment conditions. PCR confirmed dramatic upregulation of transcripts for myelin basic protein (up to 88 fold), claudin 11 (up to 32 fold), glial fibrillary acidic protein (GFAP; up to 26 fold), together with notable (>twofold) increases in message for microtubule associated protein 2 (MAP2) and C-X-C chemokine receptor type 4 (CXCR4), Janus kinase 1 (Jak1), signal transducer and activator of transcription 1 (STAT1), and signal transducer and activator of transcription 3 (STAT3). Transcripts for nestin and Krüppel-like factor 4 (KLF4

  6. Quantitative characterization of gene regulation by Rho dependent transcription termination.

    PubMed

    Hussein, Razika; Lee, Tiffany Y; Lim, Han N

    2015-08-01

    Rho factor dependent transcription termination (RTT) is common within the coding sequences of bacterial genes and it acts to couple transcription and translation levels. Despite the importance of RTT for gene regulation, its effects on mRNA and protein concentrations have not been quantitatively characterized. Here we demonstrate that the exogenous cfp gene encoding the cyan fluorescent protein can serve as a model for gene regulation by RTT. This was confirmed by showing that Psu and bicyclomycin decrease RTT and increase full length cfp mRNAs (but remarkably they have little effect on protein production). We then use cfp to characterize the relationship between its protein and full length mRNA concentrations when the translation initiation rate is varied by sequence modifications of the translation initiation region (TIR). These experiments reveal that the fold change in protein concentration (RP) and the fold change in full length mRNA concentration (Rm) have the relationship RP≈Rm(b), where b is a constant. The average value of b was determined from three separate data sets to be ~3.6. We demonstrate that the above power law function can predict how altering the translation initiation rate of a gene in an operon will affect the mRNA concentrations of downstream genes and specify a lower bound for the associated changes in protein concentrations. In summary, this study defines a simple phenomenological model to help program expression from single genes and operons that are regulated by RTT, and to guide molecular models of RTT.

  7. Co-transcriptional nuclear actin dynamics

    PubMed Central

    Percipalle, Piergiorgio

    2013-01-01

    Actin is a key player for nuclear structure and function regulating both chromosome organization and gene activity. In the cell nucleus actin interacts with many different proteins. Among these proteins several studies have identified classical nuclear factors involved in chromatin structure and function, transcription and RNA processing as well as proteins that are normally involved in controlling the actin cytoskeleton. These discoveries have raised the possibility that nuclear actin performs its multi task activities through tight interactions with different sets of proteins. This high degree of promiscuity in the spectrum of protein-to-protein interactions correlates well with the conformational plasticity of actin and the ability to undergo regulated changes in its polymerization states. Several of the factors involved in controlling head-to-tail actin polymerization have been shown to be in the nucleus where they seem to regulate gene activity. By focusing on the multiple tasks performed by actin and actin-binding proteins, possible models of how actin dynamics controls the different phases of the RNA polymerase II transcription cycle are being identified. PMID:23138849

  8. Modulation of RNA polymerase assembly dynamics in transcriptional regulation

    PubMed Central

    Gorski, Stanislaw A.; Snyder, Sara K.; John, Sam; Grummt, Ingrid; Misteli, Tom

    2008-01-01

    The interaction of transcription factors with target genes is highly dynamic. Whether the dynamic nature of these interactions is merely an intrinsic property of transcriptions factors or serves a regulatory role is unknown. Here, we have used single cell fluorescence imaging combined with computational modeling and chromatin immunoprecipitation to analyze transcription complex dynamics in gene regulation during the cell cycle in living cells. We demonstrate a link between the dynamics of RNA polymerase I (RNA pol I) assembly and transcriptional output. We show that transcriptional upregulation is accompanied by prolonged retention of RNA pol I components at the promoter, resulting in longer promoter dwell time, and an increase in the steady state population of assembling polymerase. As a consequence, polymerase assembly efficiency, and ultimately, an rate of entry into processive elongation are elevated. Our results show that regulation of rDNA transcription in vivo occurs via modulation of the efficiency of transcription complex subunit capture and assembly. PMID:18498750

  9. Design and optimization of reverse-transcription quantitative PCR experiments.

    PubMed

    Tichopad, Ales; Kitchen, Rob; Riedmaier, Irmgard; Becker, Christiane; Ståhlberg, Anders; Kubista, Mikael

    2009-10-01

    Quantitative PCR (qPCR) is a valuable technique for accurately and reliably profiling and quantifying gene expression. Typically, samples obtained from the organism of study have to be processed via several preparative steps before qPCR. We estimated the errors of sample withdrawal and extraction, reverse transcription (RT), and qPCR that are introduced into measurements of mRNA concentrations. We performed hierarchically arranged experiments with 3 animals, 3 samples, 3 RT reactions, and 3 qPCRs and quantified the expression of several genes in solid tissue, blood, cell culture, and single cells. A nested ANOVA design was used to model the experiments, and relative and absolute errors were calculated with this model for each processing level in the hierarchical design. We found that intersubject differences became easily confounded by sample heterogeneity for single cells and solid tissue. In cell cultures and blood, the noise from the RT and qPCR steps contributed substantially to the overall error because the sampling noise was less pronounced. We recommend the use of sample replicates preferentially to any other replicates when working with solid tissue, cell cultures, and single cells, and we recommend the use of RT replicates when working with blood. We show how an optimal sampling plan can be calculated for a limited budget. .

  10. 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.

  11. Nonlinear dynamics and quantitative EEG analysis.

    PubMed

    Jansen, B H

    1996-01-01

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

  12. Interplay of dynamic transcription and chromatin remodeling: lessons from yeast.

    PubMed

    Niederacher, Gerhard; Klopf, Eva; Schüller, Christoph

    2011-01-01

    Regulation of transcription involves dynamic rearrangements of chromatin structure. The budding yeast Saccharomyces cerevisiae has a variety of highly conserved factors necessary for these reconstructions. Chromatin remodelers, histone modifiers and histone chaperones directly associate to promoters and open reading frames of exposed genes and facilitate activation and repression of transcription. We compare two distinct patterns of induced transcription: Sustained transcribed genes switch to an activated state where they remain as long as the induction signal is present. In contrast, single pulsed transcribed genes show a quick and strong induction pulse resulting in high transcript levels followed by adaptation and repression to basal levels. We discuss intensively studied promoters and coding regions from both groups for their co-factor requirements during transcription. Interplay between chromatin restructuring factors and dynamic transcription is highly variable and locus dependent.

  13. 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.

  14. Systematic Discovery of Archaeal Transcription Factor Functions in Regulatory Networks through Quantitative Phenotyping Analysis.

    PubMed

    Darnell, Cynthia L; Tonner, Peter D; Gulli, Jordan G; Schmidler, Scott C; Schmid, Amy K

    2017-01-01

    Gene regulatory networks (GRNs) are critical for dynamic transcriptional responses to environmental stress. However, the mechanisms by which GRN regulation adjusts physiology to enable stress survival remain unclear. Here we investigate the functions of transcription factors (TFs) within the global GRN of the stress-tolerant archaeal microorganism Halobacterium salinarum. We measured growth phenotypes of a panel of TF deletion mutants in high temporal resolution under heat shock, oxidative stress, and low-salinity conditions. To quantitate the noncanonical functional forms of the growth trajectories observed for these mutants, we developed a novel modeling framework based on Gaussian process regression and functional analysis of variance (FANOVA). We employ unique statistical tests to determine the significance of differential growth relative to the growth of the control strain. This analysis recapitulated known TF functions, revealed novel functions, and identified surprising secondary functions for characterized TFs. Strikingly, we observed that the majority of the TFs studied were required for growth under multiple stress conditions, pinpointing regulatory connections between the conditions tested. Correlations between quantitative phenotype trajectories of mutants are predictive of TF-TF connections within the GRN. These phenotypes are strongly concordant with predictions from statistical GRN models inferred from gene expression data alone. With genome-wide and targeted data sets, we provide detailed functional validation of novel TFs required for extreme oxidative stress and heat shock survival. Together, results presented in this study suggest that many TFs function under multiple conditions, thereby revealing high interconnectivity within the GRN and identifying the specific TFs required for communication between networks responding to disparate stressors. IMPORTANCE To ensure survival in the face of stress, microorganisms employ inducible damage repair

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

    PubMed

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

    2010-04-01

    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.

  16. Dynamic usage of transcription start sites within core promoters

    PubMed Central

    Kawaji, Hideya; Frith, Martin C; Katayama, Shintaro; Sandelin, Albin; Kai, Chikatoshi; Kawai, Jun; Carninci, Piero; Hayashizaki, Yoshihide

    2006-01-01

    Background Mammalian promoters do not initiate transcription at single, well defined base pairs, but rather at multiple, alternative start sites spread across a region. We previously characterized the static structures of transcription start site usage within promoters at the base pair level, based on large-scale sequencing of transcript 5' ends. Results In the present study we begin to explore the internal dynamics of mammalian promoters, and demonstrate that start site selection within many mouse core promoters varies among tissues. We also show that this dynamic usage of start sites is associated with CpG islands, broad and multimodal promoter structures, and imprinting. Conclusion Our results reveal a new level of biologic complexity within promoters - fine-scale regulation of transcription starting events at the base pair level. These events are likely to be related to epigenetic transcriptional regulation. PMID:17156492

  17. RNA Pol II Dynamics Modulate Co-transcriptional Chromatin Modification, CTD Phosphorylation, and Transcriptional Direction.

    PubMed

    Fong, Nova; Saldi, Tassa; Sheridan, Ryan M; Cortazar, Michael A; Bentley, David L

    2017-05-18

    Eukaryotic genes are marked by conserved post-translational modifications on the RNA pol II C-terminal domain (CTD) and the chromatin template. How the 5'-3' profiles of these marks are established is poorly understood. Using pol II mutants in human cells, we found that slow transcription repositioned specific co-transcriptionally deposited chromatin modifications; histone H3 lysine 36 trimethyl (H3K36me3) shifted within genes toward 5' ends, and histone H3 lysine 4 dimethyl (H3K4me2) extended farther upstream of start sites. Slow transcription also evoked a hyperphosphorylation of CTD Ser2 residues at 5' ends of genes that is conserved in yeast. We propose a "dwell time in the target zone" model to explain the effects of transcriptional dynamics on the establishment of co-transcriptionally deposited protein modifications. Promoter-proximal Ser2 phosphorylation is associated with a longer pol II dwell time at start sites and reduced transcriptional polarity because of strongly enhanced divergent antisense transcription at promoters. These results demonstrate that pol II dynamics help govern the decision between sense and divergent antisense transcription. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. 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.

  19. Transcription factor binding dynamics during human ESC differentiation

    PubMed Central

    Tsankov, Alexander M.; Gu, Hongcang; Akopian, Veronika; Ziller, Michael J.; Donaghey, Julie; Amit, Ido; Gnirke, Andreas; Meissner, Alexander

    2015-01-01

    Summary Pluripotent stem cells provide a powerful system to dissect the underlying molecular dynamics that regulate cell fate changes during mammalian development. Here we report the integrative analysis of genome wide binding data for 38 transcription factors with extensive epigenome and transcriptional data across the differentiation of human embryonic stem cells to the three germ layers. We describe core regulatory dynamics and show the lineage specific behavior of selected factors. In addition to the orchestrated remodeling of the chromatin landscape, we find that the binding of several transcription factors is strongly associated with specific loss of DNA methylation in one germ layer and in many cases a reciprocal gain in the other layers. Taken together, our work shows context-dependent rewiring of transcription factor binding, downstream signaling effectors, and the epigenome during human embryonic stem cell differentiation. PMID:25693565

  20. Deciphering modular and dynamic behaviors of transcriptional networks.

    PubMed

    Zhan, Ming

    2007-01-01

    The coordinated and dynamic modulation or interaction of genes or proteins acts as an important mechanism used by a cell in functional regulation. Recent studies have shown that many transcriptional networks exhibit a scale-free topology and hierarchical modular architecture. It has also been shown that transcriptional networks or pathways are dynamic and behave only in certain ways and controlled manners in response to disease development, changing cellular conditions, and different environmental factors. Moreover, evolutionarily conserved and divergent transcriptional modules underline fundamental and species-specific molecular mechanisms controlling disease development or cellular phenotypes. Various computational algorithms have been developed to explore transcriptional networks and modules from gene expression data. In silico studies have also been made to mimic the dynamic behavior of regulatory networks, analyzing how disease or cellular phenotypes arise from the connectivity or networks of genes and their products. Here, we review the recent development in computational biology research on deciphering modular and dynamic behaviors of transcriptional networks, highlighting important findings. We also demonstrate how these computational algorithms can be applied in systems biology studies as on disease, stem cells, and drug discovery.

  1. 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 Central

    Novinscak, A.; Filion, M.

    2011-01-01

    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. PMID:21724880

  2. 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.

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

    PubMed

    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) τ(L)≫max(τ(R),τ(E)), (b) τ(LT)≫τ(T), and (c) τ(I)≥(τ(EL)+τ(TR)) where τ(L) 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, (τ(R),τ(E)) 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, τ(LT) is the time associated with the relaxation of the looped-out segment of DNA that connects the cis-acting site and promoter, τ(T) is the time required to generate a complete transcript, τ(I) is the transcription initiation time, τ(EL) is the elongation time, and τ(TR) 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.

  4. A sandwich assay for quantitative detection of transcription factors in cell lysate.

    PubMed

    Fang, Zhiyuan; Zhang, Wenjuan; Ge, Chenchen; Liu, Jie; Lie, Puchang; Zeng, Lingwen

    2012-09-21

    A double-stranded DNA (dsDNA) mediated sandwich assay was developed for quantitative detection of transcription factors. The detection limit for human recombinant c-jun protein is 2.5 ng, and for c-jun protein the limit is as low as 0.625 μg of cell lysate.

  5. A dynamic mode of mitotic bookmarking by transcription factors

    PubMed Central

    Teves, Sheila S; An, Luye; Hansen, Anders S; Xie, Liangqi; Darzacq, Xavier; Tjian, Robert

    2016-01-01

    During mitosis, transcription is shut off, chromatin condenses, and most transcription factors (TFs) are reported to be excluded from chromosomes. How do daughter cells re-establish the original transcription program? Recent discoveries that a select set of TFs remain bound on mitotic chromosomes suggest a potential mechanism for maintaining transcriptional programs through the cell cycle termed mitotic bookmarking. Here we report instead that many TFs remain associated with chromosomes in mouse embryonic stem cells, and that the exclusion previously described is largely a fixation artifact. In particular, most TFs we tested are significantly enriched on mitotic chromosomes. Studies with Sox2 reveal that this mitotic interaction is more dynamic than in interphase and is facilitated by both DNA binding and nuclear import. Furthermore, this dynamic mode results from lack of transcriptional activation rather than decreased accessibility of underlying DNA sequences in mitosis. The nature of the cross-linking artifact prompts careful re-examination of the role of TFs in mitotic bookmarking. DOI: http://dx.doi.org/10.7554/eLife.22280.001 PMID:27855781

  6. Sequential Logic Model Deciphers Dynamic Transcriptional Control of Gene Expressions

    PubMed Central

    Yeo, Zhen Xuan; Wong, Sum Thai; Arjunan, Satya Nanda Vel; Piras, Vincent; Tomita, Masaru; Selvarajoo, Kumar; Giuliani, Alessandro; Tsuchiya, Masa

    2007-01-01

    Background Cellular signaling involves a sequence of events from ligand binding to membrane receptors through transcription factors activation and the induction of mRNA expression. The transcriptional-regulatory system plays a pivotal role in the control of gene expression. A novel computational approach to the study of gene regulation circuits is presented here. Methodology Based on the concept of finite state machine, which provides a discrete view of gene regulation, a novel sequential logic model (SLM) is developed to decipher control mechanisms of dynamic transcriptional regulation of gene expressions. The SLM technique is also used to systematically analyze the dynamic function of transcriptional inputs, the dependency and cooperativity, such as synergy effect, among the binding sites with respect to when, how much and how fast the gene of interest is expressed. Principal Findings SLM is verified by a set of well studied expression data on endo16 of Strongylocentrotus purpuratus (sea urchin) during the embryonic midgut development. A dynamic regulatory mechanism for endo16 expression controlled by three binding sites, UI, R and Otx is identified and demonstrated to be consistent with experimental findings. Furthermore, we show that during transition from specification to differentiation in wild type endo16 expression profile, SLM reveals three binary activities are not sufficient to explain the transcriptional regulation of endo16 expression and additional activities of binding sites are required. Further analyses suggest detailed mechanism of R switch activity where indirect dependency occurs in between UI activity and R switch during specification to differentiation stage. Conclusions/Significance The sequential logic formalism allows for a simplification of regulation network dynamics going from a continuous to a discrete representation of gene activation in time. In effect our SLM is non-parametric and model-independent, yet providing rich biological

  7. A quantitative assay for assessing the effects of DNA lesions on transcription.

    PubMed

    You, Changjun; Dai, Xiaoxia; Yuan, Bifeng; Wang, Jin; Wang, Jianshuang; Brooks, Philip J; Niedernhofer, Laura J; Wang, Yinsheng

    2012-10-01

    Most mammalian cells in nature are quiescent but actively transcribing mRNA for normal physiological processes; thus, it is important to investigate how endogenous and exogenous DNA damage compromises transcription in cells. Here we describe a new competitive transcription and adduct bypass (CTAB) assay to determine the effects of DNA lesions on the fidelity and efficiency of transcription. Using this strategy, we demonstrate that the oxidatively induced lesions 8,5'-cyclo-2'-deoxyadenosine (cdA) and 8,5'-cyclo-2'-deoxyguanosine (cdG) and the methylglyoxal-induced lesion N(2)-(1-carboxyethyl)-2'-deoxyguanosine (N(2)-CEdG) strongly inhibited transcription in vitro and in mammalian cells. In addition, cdA and cdG, but not N(2)-CEdG, induced transcriptional mutagenesis in vitro and in vivo. Furthermore, when located on the template DNA strand, all examined lesions were primarily repaired by transcription-coupled nucleotide excision repair in mammalian cells. This newly developed CTAB assay should be generally applicable for quantitatively assessing how other DNA lesions affect DNA transcription in vitro and in cells.

  8. Dynamic equilibrium on DNA defines transcriptional regulation of a multidrug binding transcriptional repressor, LmrR.

    PubMed

    Takeuchi, Koh; Imai, Misaki; Shimada, Ichio

    2017-03-21

    LmrR is a multidrug binding transcriptional repressor that controls the expression of a major multidrug transporter, LmrCD, in Lactococcus lactis. Promiscuous compound ligations reduce the affinity of LmrR for the lmrCD operator by several fold to release the transcriptional repression; however, the affinity reduction is orders of magnitude smaller than that of typical transcriptional repressors. Here, we found that the transcriptional regulation of LmrR is achieved through an equilibrium between the operator-bound and non-specific DNA-adsorption states in vivo. The effective dissociation constant of LmrR for the lmrCD operator under the equilibrium is close to the endogenous concentration of LmrR, which allows a substantial reduction of LmrR occupancy upon compound ligations. Therefore, LmrR represents a dynamic type of transcriptional regulation of prokaryotic multidrug resistance systems, where the small affinity reduction induced by compounds is coupled to the functional relocalization of the repressor on the genomic DNA via nonspecific DNA adsorption.

  9. 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)

  10. Dynamic Post-Transcriptional Regulation of HIV-1 Gene Expression

    PubMed Central

    Kula, Anna; Marcello, Alessandro

    2012-01-01

    Gene expression of the human immunodeficiency virus type 1 (HIV-1) is a highly regulated process. Basal transcription of the integrated provirus generates early transcripts that encode for the viral products Tat and Rev. Tat promotes the elongation of RNA polymerase while Rev mediates the nuclear export of viral RNAs that contain the Rev-responsive RNA element (RRE). These RNAs are exported from the nucleus to allow expression of Gag-Pol and Env proteins and for the production of full-length genomic RNAs. A balance exists between completely processed mRNAs and RRE-containing RNAs. Rev functions as an adaptor that recruits cellular factors to re-direct singly spliced and unspliced viral RNAs to nuclear export. The aim of this review is to address the dynamic regulation of this post-transcriptional pathway in light of recent findings that implicate several novel cellular cofactors of Rev function. PMID:24832221

  11. Quantitative assessment of BAX transcript and flow cytometric expression in acute myeloid leukemia: a prospective study.

    PubMed

    Sharawat, Surender Kumar; Raina, Vinod; Kumar, Lalit; Sharma, Atul; Bakhshi, Radhika; Vishnubhatla, Sreenivas; Gupta, Ritu; Bakhshi, Sameer

    2014-10-01

    Quantitative assessment of BAX transcripts and protein in acute myeloid leukemia (AML). We quantitatively evaluated BAX gene transcripts by real-time polymerase chain reaction (TaqMan probe chemistry) and protein expression by flow cytometry. Consecutive 112 AML patients with a median age of 16 (1-59) years were recruited in the study. By flow cytometry, the percentage expression was in linear correlation with relative median fluorescent intensity (RMFI; R = 0.4425; P < 0.001). However, there was no linear relationship between the transcript copies of the BAX with its RMFI (R = -0.0559; P = 0.586). The expression of the BAX at both protein and transcript level was significantly higher in AML patients as compared with normal control. RMFI of the BAX were higher in the cohort with lower white blood cell count (P = 0.029). None of the other baseline characteristics correlated with either the BAX transcript or the RMFI. BAX expression did not correlate with complete remission rate, event free, disease free, and overall survival. BAX gene expression in AML was evaluated first time with two different methods but did not correlate with the survival outcome.

  12. 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.

  13. 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

  14. 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.

  15. Binding Site Turnover Produces Pervasive Quantitative Changes in Transcription Factor Binding between Closely Related Drosophila Species

    PubMed Central

    Trapnell, Cole; Davidson, Stuart; Pachter, Lior; Chu, Hou Cheng; Tonkin, Leath A.; Biggin, Mark D.; Eisen, Michael B.

    2010-01-01

    Changes in gene expression play an important role in evolution, yet the molecular mechanisms underlying regulatory evolution are poorly understood. Here we compare genome-wide binding of the six transcription factors that initiate segmentation along the anterior-posterior axis in embryos of two closely related species: Drosophila melanogaster and Drosophila yakuba. Where we observe binding by a factor in one species, we almost always observe binding by that factor to the orthologous sequence in the other species. Levels of binding, however, vary considerably. The magnitude and direction of the interspecies differences in binding levels of all six factors are strongly correlated, suggesting a role for chromatin or other factor-independent forces in mediating the divergence of transcription factor binding. Nonetheless, factor-specific quantitative variation in binding is common, and we show that it is driven to a large extent by the gain and loss of cognate recognition sequences for the given factor. We find only a weak correlation between binding variation and regulatory function. These data provide the first genome-wide picture of how modest levels of sequence divergence between highly morphologically similar species affect a system of coordinately acting transcription factors during animal development, and highlight the dominant role of quantitative variation in transcription factor binding over short evolutionary distances. PMID:20351773

  16. Quantitative studies of ribosome conformational dynamics.

    PubMed

    Fraser, Christopher S; Doudna, Jennifer A

    2007-05-01

    The ribosome is a dynamic machine that undergoes many conformational rearrangements during the initiation of protein synthesis. Significant differences exist between the process of protein synthesis initiation in eubacteria and eukaryotes. In particular, the initiation of eukaryotic protein synthesis requires roughly an order of magnitude more initiation factors to promote efficient mRNA recruitment and ribosomal recognition of the start codon than are needed for eubacterial initiation. The mechanisms by which these initiation factors promote ribosome conformational changes during stages of initiation have been studied using cross-linking, footprinting, site-directed probing, cryo-electron microscopy, X-ray crystallography, fluorescence spectroscopy and single-molecule techniques. Here, we review how the results of these different approaches have begun to converge to yield a detailed molecular understanding of the dynamic motions that the eukaryotic ribosome cycles through during the initiation of protein synthesis.

  17. 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. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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

    PubMed Central

    Castanera, Raúl; López-Varas, Leticia; Pisabarro, Antonio G.

    2015-01-01

    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

  19. 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

  20. Quantitative biomolecular imaging by dynamic nanomechanical mapping.

    PubMed

    Zhang, Shuai; Aslan, Hüsnü; Besenbacher, Flemming; Dong, Mingdong

    2014-11-07

    The ability to 'see' down to nanoscale has always been one of the most challenging obstacles for researchers to address fundamental questions. For many years, researchers have been developing scanning probe microscopy techniques to improve imaging capability at nanoscale. Among them, atomic force microscopy (AFM) has received considerable attention, which allows probing topography of biological species at real space under physiological environment. Importantly, force measurements in AFM enable researchers to reveal not only the topography but also the relevant physical-chemical properties. AFM-based dynamic nanomechanical mapping (DNM) provides insights into the functions of biological systems by the interpretation of 'force', which are inaccessible by most of the other analytic techniques. This review is aiming to shed light on these recently developed AFM-based DNM techniques for biomolecular imaging, and discuss the relative applications in biological research from the nanomechanical point of view.

  1. Advanced fluorescence microscopy methods for the real-time study of transcription and chromatin dynamics

    PubMed Central

    Annibale, Paolo; Gratton, Enrico

    2014-01-01

    In this contribution we provide an overview of the recent advances allowed by the use of fluorescence microscopy methods in the study of transcriptional processes and their interplay with the chromatin architecture in living cells. Although the use of fluorophores to label nucleic acids dates back at least to about half a century ago,1 two recent breakthroughs have effectively opened the way to use fluorescence routinely for specific and quantitative probing of chromatin organization and transcriptional activity in living cells: namely, the possibility of labeling first the chromatin loci and then the mRNA synthesized from a gene using fluorescent proteins. In this contribution we focus on methods that can probe rapid dynamic processes by analyzing fast fluorescence fluctuations. PMID:25764219

  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.

  3. Energy-dependent fitness: a quantitative model for the evolution of yeast transcription factor binding sites.

    PubMed

    Mustonen, Ville; Kinney, Justin; Callan, Curtis G; Lässig, Michael

    2008-08-26

    We present a genomewide cross-species analysis of regulation for broad-acting transcription factors in yeast. Our model for binding site evolution is founded on biophysics: the binding energy between transcription factor and site is a quantitative phenotype of regulatory function, and selection is given by a fitness landscape that depends on this phenotype. The model quantifies conservation, as well as loss and gain, of functional binding sites in a coherent way. Its predictions are supported by direct cross-species comparison between four yeast species. We find ubiquitous compensatory mutations within functional sites, such that the energy phenotype and the function of a site evolve in a significantly more constrained way than does its sequence. We also find evidence for substantial evolution of regulatory function involving point mutations as well as sequence insertions and deletions within binding sites. Genes lose their regulatory link to a given transcription factor at a rate similar to the neutral point mutation rate, from which we infer a moderate average fitness advantage of functional over nonfunctional sites. In a wider context, this study provides an example of inference of selection acting on a quantitative molecular trait.

  4. 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.

  5. 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.

  6. 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.

  7. Merging Absolute and Relative Quantitative PCR Data to Quantify STAT3 Splice Variant Transcripts

    PubMed Central

    Turton, Keren B.; Esnault, Stephane; Delain, Larissa P.; Mosher, Deane F.

    2016-01-01

    Human signal transducer and activator of transcription 3 (STAT3) is one of many genes containing a tandem splicing site. Alternative donor splice sites 3 nucleotides apart result in either the inclusion (S) or exclusion (ΔS) of a single residue, Serine-701. Further downstream, splicing at a pair of alternative acceptor splice sites result in transcripts encoding either the 55 terminal residues of the transactivation domain (α) or a truncated transactivation domain with 7 unique residues (β). As outlined in this manuscript, measuring the proportions of STAT3's four spliced transcripts (Sα, Sβ, ΔSα and ΔSβ) was possible using absolute qPCR (quantitative polymerase chain reaction). The protocol therefore distinguishes and measures highly similar splice variants. Absolute qPCR makes use of calibrator plasmids and thus specificity of detection is not compromised for the sake of efficiency. The protocol necessitates primer validation and optimization of cycling parameters. A combination of absolute qPCR and efficiency-dependent relative qPCR of total STAT3 transcripts allowed a description of the fluctuations of STAT3 splice variants' levels in eosinophils treated with cytokines. The protocol also provided evidence of a co-splicing interdependence between the two STAT3 splicing events. The strategy based on a combination of the two qPCR techniques should be readily adaptable to investigation of co-splicing at other tandem splicing sites. PMID:27768061

  8. Qualitative and quantitative change in the dynamics of motor learning.

    PubMed

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

    2006-04-01

    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 attractor dynamic. Results showed 3 different learning patterns to the changes in the dynamics as a function of practice that were mediated by the initial ball speed. Only participants who learned the task showed a bifurcation in coordination mode that was preceded by enhanced performance variability. The observed multiple time scales to motor learning are interpreted as the products of the dynamical stability and instability realized from (a) the continually evolving landscape dynamics due to bifurcations between attractor organization and (b) the transient phenomena associated with moving toward and away from fixed-point dynamics.

  9. Nucleosomal arrangement affects single-molecule transcription dynamics

    PubMed Central

    Fitz, Veronika; Shin, Jaeoh; Ehrlich, Christoph; Farnung, Lucas; Cramer, Patrick; Zaburdaev, Vasily; Grill, Stephan W.

    2016-01-01

    In eukaryotes, gene expression depends on chromatin organization. However, how chromatin affects the transcription dynamics of individual RNA polymerases has remained elusive. Here, we use dual trap optical tweezers to study single yeast RNA polymerase II (Pol II) molecules transcribing along a DNA template with two nucleosomes. The slowdown and the changes in pausing behavior within the nucleosomal region allow us to determine a drift coefficient, χ, which characterizes the ability of the enzyme to recover from a nucleosomal backtrack. Notably, χ can be used to predict the probability to pass the first nucleosome. Importantly, the presence of a second nucleosome changes χ in a manner that depends on the spacing between the two nucleosomes, as well as on their rotational arrangement on the helical DNA molecule. Our results indicate that the ability of Pol II to pass the first nucleosome is increased when the next nucleosome is turned away from the first one to face the opposite side of the DNA template. These findings help to rationalize how chromatin arrangement affects Pol II transcription dynamics. PMID:27791062

  10. Expression and epigenetic dynamics of transcription regulator Lhx8 during mouse oogenesis.

    PubMed

    Zhang, Lian-Jun; Pan, Bo; Chen, Bo; Zhang, Xi-Feng; Liang, Gui-Jin; Feng, Yan-Ni; Wang, Lin-Qing; Ma, Jin-Mei; Li, Lan; Shen, Wei

    2012-09-10

    The spatial and temporal specific activation and inhibition of numerous genes are required for successful oogenesis which is precisely regulated by germ cell-related transcription factors, and appropriate epigenetic modifications, including DNA methylation, histone modification and other mechanisms that closely regulate the functional exertion of these transcription factors. In this study, we characterized the correlation between the expression and epigenetic dynamics of Lhx8, a germ cell specific transcription factor during mouse oogenesis. Immunohistochemistry, quantitative PCR and western blots were performed to localize and quantify the expressional characteristics of Lhx8 in oocytes of 13.5 dpc (day post coitum), 17.5 dpc, 0 dpp (day post partum), 3 dpp, 7 dpp and 14 dpp. The results showed that LHX8 protein was located in the nucleus of oocytes, and increasingly expressed during primordial follicle activation. Sequencing of bisulfite-converted genomic DNAs revealed that the methylation dynamics of Lhx8-3' was highly changeable but almost no change occurred in Lhx8-5'. ChIP-QPCR analysis showed that histone H3 acetylation of Lhx8 was also increased during primordial follicle assembly and activation. In conclusion, Lhx8 expression is related with the activation of primordial follicles, which is highly correlated with the demethylation of Lhx8-3' untranslated region and the high acetylation of histone H3.

  11. 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…

  12. Quantitative Reverse Transcription-qPCR-Based Gene Expression Analysis in Plants.

    PubMed

    Abdallah, Heithem Ben; Bauer, Petra

    2016-01-01

    The investigation of gene expression is an initial and essential step to understand the function of a gene in a physiological context. Reverse transcription-quantitative real-time PCR (RT-qPCR) assays are reproducible, quantitative, and fast. They can be adapted to study model and non-model plant species without the need to have whole genome or transcriptome sequence data available. Here, we provide a protocol for a reliable RT-qPCR assay, which can be easily adapted to any plant species of interest. We describe the design of the qPCR strategy and primer design, considerations for plant material generation, RNA preparation and cDNA synthesis, qPCR setup and run, and qPCR data analysis, interpretation, and final presentation.

  13. Complementary quantitative proteomics reveals that transcription factor AP-4 mediates E-box-dependent complex formation for transcriptional repression of HDM2.

    PubMed

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

    2009-09-01

    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 a

  14. 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.

  15. 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.

  16. Characterizing dynamic changes in the human blood transcriptional network.

    PubMed

    Zhu, Jun; Chen, Yanqing; Leonardson, Amy S; Wang, Kai; Lamb, John R; Emilsson, Valur; Schadt, Eric E

    2010-02-12

    Gene expression data generated systematically in a given system over multiple time points provides a source of perturbation that can be leveraged to infer causal relationships among genes explaining network changes. Previously, we showed that food intake has a large impact on blood gene expression patterns and that these responses, either in terms of gene expression level or gene-gene connectivity, are strongly associated with metabolic diseases. In this study, we explored which genes drive the changes of gene expression patterns in response to time and food intake. We applied the Granger causality test and the dynamic Bayesian network to gene expression data generated from blood samples collected at multiple time points during the course of a day. The simulation result shows that combining many short time series together is as powerful to infer Granger causality as using a single long time series. Using the Granger causality test, we identified genes that were supported as the most likely causal candidates for the coordinated temporal changes in the network. These results show that PER1 is a key regulator of the blood transcriptional network, in which multiple biological processes are under circadian rhythm regulation. The fasted and fed dynamic Bayesian networks showed that over 72% of dynamic connections are self links. Finally, we show that different processes such as inflammation and lipid metabolism, which are disconnected in the static network, become dynamically linked in response to food intake, which would suggest that increasing nutritional load leads to coordinate regulation of these biological processes. In conclusion, our results suggest that food intake has a profound impact on the dynamic co-regulation of multiple biological processes, such as metabolism, immune response, apoptosis and circadian rhythm. The results could have broader implications for the design of studies of disease association and drug response in clinical trials.

  17. Meloidogyne javanica Chorismate Mutase Transcript Expression Profile Using Real-Time Quantitative RT-PCR.

    PubMed

    Painter, Janet E; Lambert, Kris N

    2003-03-01

    A developmental expression profile of the Meloidodgyne javanica esophageal gland gene chorismate mutase-1 (Mj-cm-1) could suggest when in the lifecycle of the nematode the Mj-cm-1 product is functional. This study used real-time quantitative RT-PCR to examine the variation in Mj-cm-1 transcript levels over six timepoints in the nematode lifecycle: egg, infective second-stage juveniles (Inf-J2), 2-day post-inoculation (pi), 7-day pi, 14-day pi, and adult. The Mj-cm-1 mRNA levels peaked at 2-day pi, about 100-fold above levels expressed at the egg and Inf-J2 stages. Some expression of Mj-cm-1 remained during the 7-day pi, 14-day pi, and adult stages. High transcript levels of the beta-actin control gene M. javanica Beta-actin-1 (Mj-ba-1) demonstrated the presence of cDNA at all timepoints. The peak in Mj-cm-1 transcript expression at 2-day pi as well as the previously shown esophageal gland localization of Mj-cm-1 mRNA suggest that the product of this gene may be involved early in the establishment of parasitism.

  18. 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.

  19. Transcription dependent dynamic supercoiling is a short-range genomic force

    PubMed Central

    Kouzine, Fedor; Gupta, Ashutosh; Baranello, Laura; Wojtowicz, Damian; Benaissa, Khadija; Liu, Juhong; Przytycka, Teresa M.; Levens, David

    2013-01-01

    Transcription has the capacity to modify mechanically DNA topology, DNA structure, and nucleosome arrangement. Resulting from ongoing transcription, these modifications in turn, may provide instant feedback to the transcription machinery. To substantiate the connection between transcription and DNA dynamics, we charted an ENCODE map of transcription-dependent dynamic supercoiling in human Burkitt lymphoma cells using psoralen photobinding to probe DNA topology in vivo. Dynamic supercoils spread ~1.5 kb upstream of the start sites of active genes. Low and high output promoters handle this torsional stress differently as shown using inhibitors of transcription and topoisomerases, and by chromatin immunoprecipation of RNA polymerase and topoisomerases I and II. Whereas lower outputs are managed adequately by topoisomerase I, high output promoters additionally require topoisomerase II. The genome-wide coupling between transcription and DNA topology emphasizes the importance of dynamic supercoiling for gene regulation. PMID:23416947

  20. 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

  1. The developmental expression dynamics of Drosophila melanogaster transcription factors.

    PubMed

    Adryan, Boris; Teichmann, Sarah A

    2010-01-01

    Site-specific transcription factors (TFs) are coordinators of developmental and physiological gene expression programs. Their binding to cis-regulatory modules of target genes mediates the precise cell- and context-specific activation and repression of genes. The expression of TFs should therefore reflect the core expression program of each cell. We studied the expression dynamics of about 750 TFs using the available genomics resources in Drosophila melanogaster. We find that 95% of these TFs are expressed at some point during embryonic development, with a peak roughly between 10 and 12 hours after egg laying, the core stages of organogenesis. We address the differential utilization of DNA-binding domains in different developmental programs systematically in a spatio-temporal context, and show that the zinc finger class of TFs is predominantly early expressed, while Homeobox TFs exhibit later expression in embryogenesis. Previous work, dissecting cis-regulatory modules during Drosophila development, suggests that TFs are deployed in groups acting in a cooperative manner. In contrast, we find that there is rapid exchange of co-expressed partners amongst the fly TFs, at rates similar to the genome-wide dynamics of co-expression clusters. This suggests there may also be a high level of combinatorial complexity of TFs at cis-regulatory modules.

  2. The developmental expression dynamics of Drosophila melanogaster transcription factors

    PubMed Central

    2010-01-01

    Background Site-specific transcription factors (TFs) are coordinators of developmental and physiological gene expression programs. Their binding to cis-regulatory modules of target genes mediates the precise cell- and context-specific activation and repression of genes. The expression of TFs should therefore reflect the core expression program of each cell. Results We studied the expression dynamics of about 750 TFs using the available genomics resources in Drosophila melanogaster. We find that 95% of these TFs are expressed at some point during embryonic development, with a peak roughly between 10 and 12 hours after egg laying, the core stages of organogenesis. We address the differential utilization of DNA-binding domains in different developmental programs systematically in a spatio-temporal context, and show that the zinc finger class of TFs is predominantly early expressed, while Homeobox TFs exhibit later expression in embryogenesis. Conclusions Previous work, dissecting cis-regulatory modules during Drosophila development, suggests that TFs are deployed in groups acting in a cooperative manner. In contrast, we find that there is rapid exchange of co-expressed partners amongst the fly TFs, at rates similar to the genome-wide dynamics of co-expression clusters. This suggests there may also be a high level of combinatorial complexity of TFs at cis-regulatory modules. PMID:20384991

  3. 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

  4. Global Regulation of Transcription by a Small RNA: A Quantitative View

    PubMed Central

    Nitzan, Mor; Wassarman, Karen M.; Biham, Ofer; Margalit, Hanah

    2014-01-01

    Small RNAs are integral regulators of bacterial gene expression, the majority of which act posttranscriptionally by basepairing with target mRNAs, altering translation or mRNA stability. 6S RNA, however, is a small RNA that is a transcriptional regulator, acting by binding directly to σ70-RNA polymerase (σ70-RNAP) and preventing its binding to gene promoters. At the transition from exponential to stationary phase, 6S RNA accumulates and globally downregulates the transcription of hundreds of genes. At the transition from stationary to exponential phase (outgrowth), 6S RNA is released from σ70-RNAP, resulting in a fast increase in free σ70-RNAP and transcription of many genes. The transition from stationary to exponential phase is sharp, and is thus accessible for experimental study. However, the transition from exponential to stationary phase is gradual and complicated by changes in other factors, making it more difficult to isolate 6S RNA effects experimentally at this transition. Here, we use mathematical modeling and simulation to study the dynamics of 6S RNA-dependent regulation, focusing on transitions in growth mediated by altered nutrient availability. We first show that our model reproduces the sharp increase in σ70-RNAP at outgrowth, as well as the behavior of two experimentally tested mutants, thus justifying its use for characterizing the less accessible dynamics of the transition from exponential to stationary phase. We characterize the dynamics of the two transitions for Escherichia coli wild-type, as well as for mutants with various 6S RNA-RNAP affinities, demonstrating that the 6S RNA regulation mechanism is generally robust to a wide range of such mutations, although the level of regulation at single promoters and their resulting expression fold change will be altered with changes in affinity. Our results provide insight into the potential advantage of transcription regulation by 6S RNA, as it enables storage and efficient release of σ70-RNAP

  5. 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

  6. Relative transcript quantification by Quantitative PCR: Roughly right or precisely wrong?

    PubMed Central

    Skern, Rasmus; Frost, Petter; Nilsen, Frank

    2005-01-01

    Background When estimating relative transcript abundances by quantitative real-time PCR (Q-PCR) we found that the results can vary dramatically depending on the method chosen for data analysis. Results Analyses of Q-PCR results from a salmon louse starvation experiment show that, even with apparently good raw data, different analytical approaches [1,2] may lead to opposing biological conclusions. Conclusion The results emphasise the importance of being cautious when analysing Q-PCR data and indicate that uncritical routine application of an analytical method will eventually result in incorrect conclusions. We do not know the extent of, or have a universal solution to this problem. However, we strongly recommend caution when analysing Q-PCR results e.g. by using two or more analytical approaches to validate conclusions. In our view a common effort should be made to standardise methods for analysis and validation of Q-PCR results. PMID:15854230

  7. 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.

  8. GETPrime: a gene- or transcript-specific primer database for quantitative real-time PCR

    PubMed Central

    Gubelmann, Carine; Gattiker, Alexandre; Massouras, Andreas; Hens, Korneel; David, Fabrice; Decouttere, Frederik; Rougemont, Jacques; Deplancke, Bart

    2011-01-01

    The vast majority of genes in humans and other organisms undergo alternative splicing, yet the biological function of splice variants is still very poorly understood in large part because of the lack of simple tools that can map the expression profiles and patterns of these variants with high sensitivity. High-throughput quantitative real-time polymerase chain reaction (qPCR) is an ideal technique to accurately quantify nucleic acid sequences including splice variants. However, currently available primer design programs do not distinguish between splice variants and also differ substantially in overall quality, functionality or throughput mode. Here, we present GETPrime, a primer database supported by a novel platform that uniquely combines and automates several features critical for optimal qPCR primer design. These include the consideration of all gene splice variants to enable either gene-specific (covering the majority of splice variants) or transcript-specific (covering one splice variant) expression profiling, primer specificity validation, automated best primer pair selection according to strict criteria and graphical visualization of the latter primer pairs within their genomic context. GETPrime primers have been extensively validated experimentally, demonstrating high transcript specificity in complex samples. Thus, the free-access, user-friendly GETPrime database allows fast primer retrieval and visualization for genes or groups of genes of most common model organisms, and is available at http://updepla1srv1.epfl.ch/getprime/. Database URL: http://deplanckelab.epfl.ch. PMID:21917859

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

    PubMed

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

    2016-09-29

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

  10. 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

  11. Quantitative phase retrieval in dynamic laser speckle interferometry

    NASA Astrophysics Data System (ADS)

    Huang, Y. H.; Hung, S. Y.; Janabi-Sharifi, Farrokh; Wang, W.; Liu, Y. S.

    2012-04-01

    The rapid progress of modern manufacturing and inspection technologies has posed stringent requirements on optical techniques for vibration characterization and dynamic testing. Due to its simplicity, accuracy and whole-field characters, laser speckle interferometry has served as one of the major techniques for dynamic measurement. In this paper, a two-step phase shifting method is developed for quantitative speckle phase measurement, which helps to eliminate the specklegrams needed for phase evaluation and facilitate dynamic measurement. Unlike previously reported two-step methods using fringe patterns with known phase shift of π/2, a small unknown phase shift is employed instead in the proposed method, which eliminates the need for phase shifting devices. Further investigation shows that small phase shifts are preferable over large phase shifts in this method. Shearographic experiments conducted have demonstrated the effectiveness of the proposed technique.

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

    NASA Astrophysics Data System (ADS)

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

    2016-09-01

    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.

  13. 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.

  14. Minimum Fuel Trajectory Design in Multiple Dynamical Environments Utilizing Direct Transcription Methods and Particle Swarm Optimization

    DTIC Science & Technology

    2016-03-01

    MINIMUM-FUEL TRAJECTORY DESIGN IN MULTIPLE DYNAMICAL ENVIRONMENTS UTILIZING DIRECT TRANSCRIPTION METHODS AND PARTICLE SWARM OPTIMIZATION THESIS...250 MINIMUM-FUEL TRAJECTORY DESIGN IN MULTIPLE DYNAMICAL ENVIRONMENTS UTILIZING DIRECT TRANSCRIPTION METHODS AND PARTICLE SWARM OPTIMIZATION THESIS... Education and Training Command in Partial Fulfillment of the Requirements for the Degree of Master of Science in Astronautical Engineering Alfredo G

  15. 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.

  16. Imaging red blood cell dynamics by quantitative phase microscopy.

    PubMed

    Popescu, Gabriel; Park, YoungKeun; Choi, Wonshik; Dasari, Ramachandra R; Feld, Michael S; Badizadegan, Kamran

    2008-01-01

    Red blood cells (RBCs) play a crucial role in health and disease, and structural and mechanical abnormalities of these cells have been associated with important disorders such as Sickle cell disease and hereditary cytoskeletal abnormalities. Although several experimental methods exist for analysis of RBC mechanical properties, optical methods stand out as they enable collecting mechanical and dynamic data from live cells without physical contact and without the need for exogenous contrast agents. In this report, we present quantitative phase microscopy techniques that enable imaging RBC membrane fluctuations with nanometer sensitivity at arbitrary time scales from milliseconds to hours. We further provide a theoretical framework for extraction of membrane mechanical and dynamical properties using time series of quantitative phase images. Finally, we present an experimental approach to extend quantitative phase imaging to 3-dimensional space using tomographic methods. By providing non-invasive methods for imaging mechanics of live cells, these novel techniques provide an opportunity for high-throughput analysis and study of RBC mechanical properties in health and disease.

  17. Newly developed quantitative transactivation system shows difference in activation by Vitis CBF transcription factors on DRE/CRT elements.

    PubMed

    Nassuth, Annette; Siddiqua, Mahbuba; Xiao, Huogen; Moody, Michelle A; Carlow, Chevonne E

    2014-01-01

    Agroinfiltration-based transactivation systems can determine if a protein functions as a transcription factor, and via which promoter element. However, this activation is not always a yes or no proposition. Normalization for variation in plasmid delivery into plant cells, sample collection and protein extraction is desired to allow for a quantitative comparison between transcription factors or promoter elements. We developed new effector and reporter plasmids which carry additional reporter genes, as well as a procedure to assay all three reporter enzymes from a single extract. The applicability of these plasmids was demonstrated with the analysis of CBF transcription factors and their target promoter sequence, DRE/CRT. Changes in the core DRE/CRT sequence abolished activation by Vitis CBF1 or Vitis CBF4, whereas changes in the surrounding sequence lowered activation by Vitis CBF1 but much less so for Vitis CBF4. The system also detected a reduction in activation due to one amino acid change in Vitis CBF1. The newly developed effector and reporter plasmids improve the ability to quantitatively compare the activation on two different promoter elements by the same transcription factor, or between two different transcription factors on the same promoter element. The quantitative difference in activation by VrCBF1 and VrCBF4 on various DRE/CRT elements support the hypothesis that these transcription factors have unique roles in the cold acclimation process.

  18. Thermal plasticity in Drosophila melanogaster populations from eastern Australia: quantitative traits to transcripts.

    PubMed

    Clemson, A S; Sgrò, C M; Telonis-Scott, M

    2016-12-01

    The flexibility afforded to genotypes in different environments by phenotypic plasticity is of interest to biologists studying thermal adaptation because of the thermal lability of many traits. Differences in thermal performance and reaction norms can provide insight into the evolution of thermal adaptation to explore broader questions such as species distributions and persistence under climate change. One approach is to study the effects of temperature on fitness, morphological and more recently gene expression traits in populations from different climatic origins. The diverse climatic conditions experienced by Drosophila melanogaster along the eastern Australian temperate-tropical gradient are ideal given the high degree of continuous trait differentiation, but reaction norm variation has not been well studied in this system. Here, we reared a tropical and temperate population from the ends of the gradient over six developmental temperatures and examined reaction norm variation for five quantitative traits including thermal performance for fecundity, and reaction norms for thermotolerance, body size, viability and 23 transcript-level traits. Despite genetic variation for some quantitative traits, we found no differentiation between the populations for fecundity thermal optima and breadth, and the reaction norms for the other traits were largely parallel, supporting previous work suggesting that thermal evolution occurs by changes in trait means rather than by reaction norm shifts. We examined reaction norm variation in our expanded thermal regime for a gene set shown to previously exhibit GxE for expression plasticity in east Australian flies, as well as key heat-shock genes. Although there were differences in curvature between the populations suggesting a higher degree of thermal plasticity in expression patterns than for the quantitative traits, we found little evidence to support a role for genetic variation in maintaining expression plasticity. © 2016

  19. The primary transcription unit of the human alpha 2 globin gene defined by quantitative RT/PCR.

    PubMed Central

    Owczarek, C M; Enriquez-Harris, P; Proudfoot, N J

    1992-01-01

    We have set up an experimental system to map the primary transcription unit of the human alpha 2 globin gene. The duplicated human alpha globin genes (alpha 2-alpha 1) were linked to the alpha globin locus Positive Regulatory Element (PRE) and stably transfected into murine erythroleukaemia cells. We then developed a quantitative reverse transcriptase, polymerase chain reaction assay to map alpha 2 primary transcripts using primer pairs derived from different parts of the alpha 2 globin gene and its 3' flanking region. This approach has revealed the presence of steady state nuclear RNA past the poly(A) site of the alpha 2 globin gene at approximately 40% of the level of unspliced intron transcript. Furthermore, these 3' flanking transcripts diminish 500 bp into the 3' flanking region, identifying this part of the alpha 2 globin gene as the principal region of termination of transcription. Images PMID:1371868

  20. 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

  1. Quantitative Fluorescent Speckle Microscopy (QFSM) to Measure Actin Dynamics

    PubMed Central

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

    2012-01-01

    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 meotic/mitotic spindle. Here, we focus 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 microinjection of fluorophore-labeled actin into cells, which generate a low ratio of fluorescently-labeled:endogenous unlabeled actin monomers. Spinning disk confocal or wide-field imaging then visualizes fluorophore clusters (2–8 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

  2. Quantitative assessment of actin transcript number in eggs, embryos, and tube feet of the sea star Pisaster ochraceus.

    PubMed Central

    Kovesdi, I; Smith, M J

    1985-01-01

    Actin coding sequence cDNA probes were used to quantitate the number of transcripts in RNA from eggs, embryos, and tube feet of the sea star Pisaster ochraceus. Transcript concentrations were measured in both total RNA and in poly(A)+ RNA by titration and hybridization kinetic methods. Surprisingly, the actin transcript number in sea star eggs is two orders of magnitude greater than in sea urchin eggs. There are at least 2.9 X 10(5) actin transcripts per sea star egg, 1.2 X 10(5) per 48-h gastrula and 1.9 X 10(5) per 72-h gastrula. The number of actin transcripts per unit mass of extracted tube foot RNA is lower than in developmental stages. The relative abundance and size of actin transcripts was determined by Northern and dot blot analyses using probes containing actin coding DNA or 3'-untranslated-region sequences. The actin transcript in eggs and embryos is 2,300 nucleotides (nt) long and originates from the Cy (cytoplasmic) gene class. In tube feet, the most abundant actin transcript is 2,200 nt long and originates from the M (muscle) gene class. Tube feet also contain, at lower abundance, 2,300-nt transcripts of the Cy gene type expressed in eggs and embryos. Images PMID:3018493

  3. 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

  4. 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.

  5. Network component analysis provides quantitative insights on an Arabidopsis transcription factor-gene regulatory network

    PubMed Central

    2013-01-01

    Background Gene regulatory networks (GRNs) are models of molecule-gene interactions instrumental in the coordination of gene expression. Transcription factor (TF)-GRNs are an important subset of GRNs that characterize gene expression as the effect of TFs acting on their target genes. Although such networks can qualitatively summarize TF-gene interactions, it is highly desirable to quantitatively determine the strengths of the interactions in a TF-GRN as well as the magnitudes of TF activities. To our knowledge, such analysis is rare in plant biology. A computational methodology developed for this purpose is network component analysis (NCA), which has been used for studying large-scale microbial TF-GRNs to obtain nontrivial, mechanistic insights. In this work, we employed NCA to quantitatively analyze a plant TF-GRN important in floral development using available regulatory information from AGRIS, by processing previously reported gene expression data from four shoot apical meristem cell types. Results The NCA model satisfactorily accounted for gene expression measurements in a TF-GRN of seven TFs (LFY, AG, SEPALLATA3 [SEP3], AP2, AGL15, HY5 and AP3/PI) and 55 genes. NCA found strong interactions between certain TF-gene pairs including LFY → MYB17, AG → CRC, AP2 → RD20, AGL15 → RAV2 and HY5 → HLH1, and the direction of the interaction (activation or repression) for some AGL15 targets for which this information was not previously available. The activity trends of four TFs - LFY, AG, HY5 and AP3/PI as deduced by NCA correlated well with the changes in expression levels of the genes encoding these TFs across all four cell types; such a correlation was not observed for SEP3, AP2 and AGL15. Conclusions For the first time, we have reported the use of NCA to quantitatively analyze a plant TF-GRN important in floral development for obtaining nontrivial information about connectivity strengths between TFs and their target genes as well as TF

  6. Quantitative genomics of voluntary exercise in mice: transcriptional analysis and mapping of expression QTL in muscle.

    PubMed

    Kelly, Scott A; Nehrenberg, Derrick L; Hua, Kunjie; Garland, Theodore; Pomp, Daniel

    2014-08-15

    Motivation and ability both underlie voluntary exercise, each with a potentially unique genetic architecture. Muscle structure and function are one of many morphological and physiological systems acting to simultaneously determine exercise ability. We generated a large (n = 815) advanced intercross line of mice (G4) derived from a line selectively bred for increased wheel running (high runner) and the C57BL/6J inbred strain. We previously mapped quantitative trait loci (QTL) contributing to voluntary exercise, body composition, and changes in body composition as a result of exercise. Using brain tissue in a subset of the G4 (n = 244), we have also previously reported expression QTL (eQTL) colocalizing with the QTL for the higher-level phenotypes. Here, we examined the transcriptional landscape of hind limb muscle tissue via global mRNA expression profiles. Correlations revealed an ∼1,168% increase in significant relationships between muscle transcript expression levels and the same exercise and body composition phenotypes examined previously in the brain. The exercise trait most often significantly correlated with gene expression in the brain was running duration while in the muscle it was maximum running speed. This difference may indicate that time spent engaging in exercise behavior may be more influenced by central (neurobiological) mechanisms, while intensity of exercise may be largely controlled by peripheral mechanisms. Additionally, we used subsets of cis-acting eQTL, colocalizing with QTL, to identify candidate genes based on both positional and functional evidence. We discuss three plausible candidate genes (Insig2, Prcp, Sparc) and their potential regulatory role.

  7. 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.

  8. 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

  9. Assessment of viable periodontal pathogens by reverse transcription quantitative polymerase chain reaction.

    PubMed

    Polonyi, M; Prenninger, N; Arweiler, N B; Haririan, H; Winklehner, P; Kierstein, S

    2013-10-01

    Molecular biological methods for the detection of periodontitis-associated bacteria based on DNA amplification have many advantages over classical culture techniques. However, when it comes to assessing immediate therapeutic success, e.g. reduction of viable bacteria, DNA-based polymerase chain reaction is unsuitable because it does not distinguish between live and dead bacteria. Our objective was to establish a simple RNA-based method that is easily set up and allows reliable assessment of the live bacterial load. We compared conventional quantitative real-time PCR (qPCR), propidium monoazide-qPCR and reverse transcription qPCR (RT-qPCR) for the detection of periodontal pathogens after antibiotic treatment in vitro. Applicability was tested using clinical samples of subgingival plaque obtained from patients at different treatment stages. The bacterial load was remarkably stable over prolonged periods when assessed by conventional qPCR, while both propidium monoazide intercalation as well as cDNA quantitation showed a decline according to decreasing numbers of viable bacteria after antibiotic treatment. Clinical samples of subgingival plaque were directly subjected to DNase I treatment and RT without previous extraction or purification steps. While the results of the DNA- and RNA-based methods are comparable in untreated patients, the classical qPCR frequently detected substantial bacterial load in treated patients where RT-qPCR no longer indicates the presence of those pathogens. The disagreement rates ranged between 4 and 20% in first visit patients and 8-50% in the group of currently treated patients. We propose to use RNA-based detection methods to verify the successful eradication of periodontal pathogens. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  10. 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.

  11. 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.

  12. Organic Substances Interfere with Reverse Transcription-Quantitative PCR-Based Virus Detection in Water Samples

    PubMed Central

    Katayama, Hiroyuki; Furumai, Hiroaki

    2014-01-01

    Reverse transcription (RT)-PCR-based virus detection from water samples is occasionally hampered by organic substances that are coconcentrated during virus concentration procedures. To characterize these organic substances, samples containing commercially available humic acid, which is known to inhibit RT-PCR, and river water samples were subjected to adsorption-elution-based virus concentration using an electronegative membrane. In this study, the samples before, during, and after the concentration were analyzed in terms of organic properties and virus detection efficiencies. Two out of the three humic acid solutions resulted in RT-quantitative PCR (qPCR) inhibition that caused >3-log10-unit underestimation of spiked poliovirus. Over 60% of the organics contained in the two solutions were recovered in the concentrate, while over 60% of the organics in the uninhibited solution were lost during the concentration process. River water concentrates also caused inhibition of RT-qPCR. Organic concentrations in the river water samples increased by 2.3 to 3.9 times after the virus concentration procedure. The inhibitory samples contained organic fractions in the 10- to 100-kDa size range, which are suspected to be RT-PCR inhibitors. According to excitation-emission matrices, humic acid-like and protein-like fractions were also recovered from river water concentrates, but these fractions did not seem to affect virus detection. Our findings reveal that detailed organic analyses are effective in characterizing inhibitory substances. PMID:25527552

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

    PubMed

    Shatzkes, Kenneth; Teferedegne, Belete; Murata, Haruhiko

    2014-04-11

    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.

  14. 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

  15. Evaluation of various real-time reverse transcription quantitative PCR assays for norovirus detection.

    PubMed

    Yoo, Ju Eun; Lee, Cheonghoon; Park, SungJun; Ko, GwangPyo

    2017-02-01

    Human noroviruses are widespread and contagious viruses causing nonbacterial gastroenteritis. Real-time reverse transcription quantitative PCR (real-time RT-qPCR) is currently the gold standard for sensitive and accurate detection for these pathogens and serves as a critical tool in outbreak prevention and control. Different surveillance teams, however, may use different assays and variability in specimen conditions may lead to disagreement in results. Furthermore, the norovirus genome is highly variable and continuously evolving. These issues necessitate the re-examination of the real-time RT-qPCR's robustness in the context of accurate detection as well as the investigation of practical strategies to enhance assay performance. Four widely referenced real-time RT-qPCR assays (Assay A-D) were simultaneously performed to evaluate characteristics such as PCR efficiency, detection limit, as well as sensitivity and specificity with RT-PCR, and to assess the most accurate method for detecting norovirus genogroups I and II. Overall, Assay D was evaluated to be the most precise and accurate assay in this study. A Zen internal quencher, which decreases nonspecific fluorescence during the PCR reaction, was added to Assay D's probe which further improved assay performance. This study compared several detection assays for noroviruses and an improvement strategy based on such comparisons provided useful characterizations of a highly optimized real-time RT-qPCR assay for norovirus detection.

  16. 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...

  17. 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...

  18. Quantitative Analysis of Transcription Elongation by RNA Polymerase I In Vitro

    PubMed Central

    Schneider, David Alan

    2016-01-01

    The elongation step in transcription has gained attention for its roles in regulation of eukaryotic gene expression and for its influence on RNA processing. Sophisticated genetic analyses have identified factors and/or conditions that may affect transcription elongation rate or processivity; however, differentiation of direct and indirect effects on transcription is difficult using in vivo strategies. Therefore, effective, reproducible in vitro assays have been developed to test whether a given factor or condition can have a direct effect on the kinetics of transcription elongation. We have adapted a fully reconstituted transcription system for RNA polymerase I (Pol I) for kinetic analysis of transcription elongation rate in vitro. The assay described here has proven to be effective in the characterization of defects or enhancement of wild-type transcription elongation by RNA Pol I. Since transcription elongation by RNA Pol I has only recently gained significant attention, this assay will be a valuable resource for years to come. PMID:22113301

  19. Bayesian B-spline mapping for dynamic quantitative traits.

    PubMed

    Xing, Jun; Li, Jiahan; Yang, Runqing; Zhou, Xiaojing; Xu, Shizhong

    2012-04-01

    Owing to their ability and flexibility to describe individual gene expression at different time points, random regression (RR) analyses have become a popular procedure for the genetic analysis of dynamic traits whose phenotypes are collected over time. Specifically, when modelling the dynamic patterns of gene expressions in the RR framework, B-splines have been proved successful as an alternative to orthogonal polynomials. In the so-called Bayesian B-spline quantitative trait locus (QTL) mapping, B-splines are used to characterize the patterns of QTL effects and individual-specific time-dependent environmental errors over time, and the Bayesian shrinkage estimation method is employed to estimate model parameters. Extensive simulations demonstrate that (1) in terms of statistical power, Bayesian B-spline mapping outperforms the interval mapping based on the maximum likelihood; (2) for the simulated dataset with complicated growth curve simulated by B-splines, Legendre polynomial-based Bayesian mapping is not capable of identifying the designed QTLs accurately, even when higher-order Legendre polynomials are considered and (3) for the simulated dataset using Legendre polynomials, the Bayesian B-spline mapping can find the same QTLs as those identified by Legendre polynomial analysis. All simulation results support the necessity and flexibility of B-spline in Bayesian mapping of dynamic traits. The proposed method is also applied to a real dataset, where QTLs controlling the growth trajectory of stem diameters in Populus are located.

  20. Evidence for a Common Mode of Transcription Factor Interaction with Chromatin as Revealed by Improved Quantitative Fluorescence Recovery after Photobleaching

    PubMed Central

    Mueller, Florian; Wach, Paul; McNally, James G.

    2008-01-01

    How site-specific transcription factors scan the genome to locate their target sites is a fundamental question in gene regulation. The in vivo binding interactions of several different transcription factors with chromatin have been investigated recently using quantitative fluorescence recovery after photobleaching (FRAP). These analyses have yielded significantly different estimates of both the binding rates and the number of predicted binding states of the respective transcription factors. We show here that these discrepancies are not due to fundamental differences among the site-specific transcription factors, but rather arise from errors in FRAP modeling. The two principal errors are a neglect of diffusion's role and an oversimplified approximation of the photobleach profile. Accounting for these errors by developing a revised FRAP protocol eliminates most of the previous discrepancies in the binding estimates for the three different transcription factors analyzed here. The new estimates predict that for each of the three transcription factors, ∼75% of the molecules are freely diffusing within the nucleus, whereas the remainder is bound with an average residence time of ∼2.5 s to a single type of chromatin binding site. Such consistent predictions for three different molecules suggest that many site-specific transcription factors may exhibit similar in vivo interactions with native chromatin. PMID:18199661

  1. Uncovering a Dynamic Feature of the Transcriptional Regulatory Network for Anterior-Posterior Patterning in the Drosophila Embryo

    PubMed Central

    Liu, Junbo; Ma, Jun

    2013-01-01

    Anterior-posterior (AP) patterning in the Drosophila embryo is dependent on the Bicoid (Bcd) morphogen gradient. However, most target genes of Bcd also require additional inputs to establish their expression domains, reflective of the operation of a cross-regulatory network and contributions of other maternal signals. This is in contrast to hunchback (hb), which has an anterior expression domain driven by an enhancer that appears to respond primarily to the Bcd input. To gain a better understanding of the regulatory logic of the AP patterning network, we perform quantitative studies that specifically investigate the dynamics of hb transcription during development. We show that Bcd-dependent hb transcription, monitored by the intron-containing nascent transcripts near the P2 promoter, is turned off quickly–on the order of a few minutes–upon entering the interphase of nuclear cycle 14A. This shutdown contrasts with earlier cycles during which active hb transcription can persist until the moment when the nucleus enters mitosis. The shutdown takes place at a time when the nuclear Bcd gradient profile in the embryo remains largely intact, suggesting that this is a process likely subject to control of a currently unknown regulatory mechanism. We suggest that this dynamic feature offers a window of opportunity for hb to faithfully interpret, and directly benefit from, Bcd gradient properties, including its scaling properties, to help craft a robust AP patterning outcome. PMID:23646132

  2. 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

  3. [Prognostic value of dynamic monitoring of RUNX1-RUNX1T1 transcript in pediatric acute myeloid leukemia].

    PubMed

    Gao, H T; Zhang, Y; Sun, K; Guo, J M; Chen, Y Q; Chen, X L; Shi, J; Niu, X N; Wang, F; Huo, L

    2017-03-14

    Objective: To investigate the prognostic value of dynamic monitoring of RUNX1-RUNX1T1 transcript in pediatric patients with t (8;21) acute myeloid leukemia (AML) . Methods: The clinical features and RUNX1-RUNX1T1 transcript levels of 55 pediatric t (8;21) AML patients, newly diagnosed from Jan. 2010 to Apr. 2016, were analyzed retrospectively. The relationship between the minimal residual disease (MRD) and prognosis was analysed by dynamic monitoring of RUNX1-RUNX1T1 transcript levels using real-time quantitative PCR (RQ-PCR) technology. Results: The RUNX1-RUNX1T1 transcript levels in bone marrow cells at diagnosis was not related to relapse. After one course of induction therapy, patients with a more than 2 Log reduction of RUNX1-RUNX1T1 transcript levels (>2 Log) had lower 5 years cumulative incidence of relapse (CIR) [ (24.3±8.4) % vs (52.6±9.7) %, χ(2)=9.046, P=0.003], relapse-free survival (RFS) [ (71.6±12.7) % vs (48.1±13.2) %, χ(2)=5.814, P=0.016], and better overall survival (OS) [ (76.9±12.5) % vs (48.9±14.7) %, χ(2)=6.346, P=0.012], compared to patients with a less than 2 Log reduction (a<2 Log) . Multivariate Cox survival analysis suggested that a>2 Log reduction in RUNX1-RUNX1T1 transcript levels after a course of induction therapy was an independent prognostic factor for RFS (HR=0.263, 95%CI 0.081-0.851, P=0.026) and OS (HR=0.214, 95% CI 0.057-0.808, P=0.023) . During consolidation therapy and follow-up period, molecular relapse of 16 cases and hematologic relapse of 13 cases were identified by continuous dynamic monitoring of RUNX1-RUNX1T1 transcript levels, with a median interval of 4.0 (1.5-5.8) months from the molecular relapse to hematologic relapse. 2 cases of molecular relapse who received timely allogeneic hematopoietic stem cell transplantation did not experience hematologic relapse. Conclusion: Dynamic monitoring RUNX1-RUNX1T1 transcript levels by RQ-PCR technique can subdivide patients into relatively low and high risk group, early

  4. 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.

  5. Fractional Dynamics of Globally Slow Transcription and Its Impact on Deterministic Genetic Oscillation

    PubMed Central

    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. PMID:22679500

  6. Mapping of HPV transcripts in four human cervical lesions using RNAseq suggests quantitative rearrangements during carcinogenic progression.

    PubMed

    Chen, Jinmiao; Xue, Yuezhen; Poidinger, Michael; Lim, Thimothy; Chew, Sung Hock; Pang, Chai Ling; Abastado, Jean-Pierre; Thierry, Françoise

    2014-08-01

    Two classes of Human papillomaviruses (HPV) infect the anogenital track: high risk viruses that are associated with risk of cervical cancer and low risk types that drive development of benign lesions, such as condylomas. In the present study, we established quantitative transcriptional maps of the viral genome in clinical lesions associated with high risk HPV16 or low risk HPV6b. Marked qualitative and quantitative changes in the HPV16 transcriptome were associated with progression from low to high grade lesions. Specific transcripts encoding essential regulatory proteins such as E7, E2, E1^E4 and E5 were identified. We also identified intrinsic differences between the HPV6b-associated condyloma transcript map and that of the HPV16-associated low grade CIN specifically regarding promoter usage. Characterization and quantification of HPV transcripts in patient samples thus establish the impact of viral transcriptional regulation on the status of HPV-associated lesions and may therefore help in defining new biologically-relevant prognosis markers. Copyright © 2014 Elsevier Inc. All rights reserved.

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

    PubMed Central

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

    2015-01-01

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

  8. 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

  9. 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)

  10. Tracing the dynamics of gene transcripts after organismal death

    PubMed Central

    2017-01-01

    In life, genetic and epigenetic networks precisely coordinate the expression of genes—but in death, it is not known if gene expression diminishes gradually or abruptly stops or if specific genes and pathways are involved. We studied this by identifying mRNA transcripts that apparently increase in relative abundance after death, assessing their functions, and comparing their abundance profiles through postmortem time in two species, mouse and zebrafish. We found mRNA transcript profiles of 1063 genes became significantly more abundant after death of healthy adult animals in a time series spanning up to 96 h postmortem. Ordination plots revealed non-random patterns in the profiles by time. While most of these transcript levels increased within 0.5 h postmortem, some increased only at 24 and 48 h postmortem. Functional characterization of the most abundant transcripts revealed the following categories: stress, immunity, inflammation, apoptosis, transport, development, epigenetic regulation and cancer. The data suggest a step-wise shutdown occurs in organismal death that is manifested by the apparent increase of certain transcripts with various abundance maxima and durations. PMID:28123054

  11. Relative neurotoxin gene expression in clostridium botulinum type B, determined using quantitative reverse transcription-PCR.

    PubMed

    Lövenklev, Maria; Holst, Elisabet; Borch, Elisabeth; Rådström, Peter

    2004-05-01

    A quantitative reverse transcription-PCR (qRT-PCR) method was developed to monitor the relative expression of the type B botulinum neurotoxin (BoNT/B) gene (cntB) in Clostridium botulinum. The levels of cntB mRNA in five type B strains were accurately monitored by using primers specific for cntB and for the reference gene encoding the 16S rRNA. The patterns and relative expression of cntB were different in the different strains. Except for one of the strains investigated, an increase in cntB expression was observed when the bacteria entered the early stationary growth phase. In the proteolytic strain C. botulinum ATCC 7949, the level of cntB mRNA was four- to fivefold higher than the corresponding levels in the other strains. This was confirmed when we quantified the production of extracellular BoNT/B by an enzyme-linked immunosorbent assay and measured the toxicity of BoNT/B by a mouse bioassay. When the effect of exposure to air on cntB expression was investigated, no decline in the relative expression was observed in spite of an 83% reduction in the viable count based on the initial cell number. Instead, the level of cntB mRNA remained the same. When there was an increase in the sodium nitrite concentration, the bacteria needed a longer adjustment time in the medium before exponential growth occurred. In addition, there was a reduction in the expression of cntB compared to the expression of the 16S rRNA gene at higher sodium nitrite concentrations. This was most obvious in the late exponential growth phase, but at the highest sodium nitrite concentration investigated, 45 ppm, a one- to threefold decline in the cntB mRNA level was observed in all growth phases.

  12. Transcriptional dynamics with time-dependent reaction rates

    NASA Astrophysics Data System (ADS)

    Nandi, Shubhendu; Ghosh, Anandamohan

    2015-02-01

    Transcription is the first step in the process of gene regulation that controls cell response to varying environmental conditions. Transcription is a stochastic process, involving synthesis and degradation of mRNAs, that can be modeled as a birth-death process. We consider a generic stochastic model, where the fluctuating environment is encoded in the time-dependent reaction rates. We obtain an exact analytical expression for the mRNA probability distribution and are able to analyze the response for arbitrary time-dependent protocols. Our analytical results and stochastic simulations confirm that the transcriptional machinery primarily act as a low-pass filter. We also show that depending on the system parameters, the mRNA levels in a cell population can show synchronous/asynchronous fluctuations and can deviate from Poisson statistics.

  13. Quantitative analysis of gene expression by reverse transcription polymerase chain reaction and capillary electrophoresis with laser-induced fluorescence detection.

    PubMed

    Richards, Mark P; Poch, Stephen M

    2002-05-01

    There has been a dramatic expansion of DNA sequence information compiled over the past several years for a variety of eukaryotic and prokaryotic genomes. Accompanying this increase in knowledge of genomic structure and organization has been a growing interest in studying the function of individual genes including regulation of their expression. A number of methods such as Northern blotting, ribonuclease protection assay, and hybridization arrays have been developed to analyze gene expression at the transcriptional (mRNA) level. Although quantitative estimates of mRNA transcripts can be obtained from each of these methods, oftentimes they lack sufficient sensitivity or the methodology is too costly or too labor-intensive to be applied to the analysis of a large number of samples. The most sensitive method for analyzing gene expression at the mRNA level involves the combination of reverse transcription and polymerase chain reaction (RT-PCR). However, in order to provide accurate quantitative estimates of gene expression, a rapid and efficient method is required for separation and detection of the double-stranded DNA (dsDNA) products of RT-PCR. Recent advances in capillary electrophoresis with laser-induced fluorescence detection (CE/LIF) have made this method suitable for the automated analysis of large numbers of RT-PCR samples. An overview of the application of CE/LIF to quantitative analysis of gene expression by RT-PCR is presented along with selected protocols and examples. Both relative-quantitative (RQ) and quantitative-competitive (QC) approaches to RT-PCR are discussed in conjunction with the use of CE/LIF for rapid and accurate quantitative analysis of PCR products.

  14. Analyzing transcription dynamics during the budding yeast cell cycle.

    PubMed

    Leman, Adam R; Bristow, Sara L; Haase, Steven B

    2014-01-01

    Assaying global cell cycle-regulated transcription in budding yeast involves extracting RNA from a synchronous population and proper normalization of detected transcript levels. Here, we describe synchronization of Saccharomyces cerevisiae cell populations by centrifugal elutriation, followed by the isolation of RNA for microarray analysis. Further, we outline the computational methods required to directly compare RNA abundance from individual time points within an experiment and to compare independent experiments. Together, these methods describe the complete workflow necessary to observe RNA abundance during the cell cycle.

  15. Signal-dependent dynamics of transcription factor translocation controls gene expression

    PubMed Central

    Hao, Nan; O'Shea, Erin K.

    2014-01-01

    Summary Information about environmental stimuli is often transmitted using common signalling molecules, but the mechanisms that ensure signalling specificity are not entirely known. Here we show that the identities and intensities of different stresses are transmitted by modulation of the amplitude, duration or frequency of nuclear translocation of the budding yeast general stress responsive transcription factor Msn2. Through artificial control of the dynamics of Msn2 translocation, we reveal how distinct dynamical schemes differentially affect reporter gene expression. Using a simple model, we predict stress-induced reporter gene expression from single-cell translocation dynamics. We then demonstrate that the response of natural target genes to dynamical modulation of Msn2 translocation is influenced by differences in the kinetics of promoter transitions and transcription factor binding properties. Thus, multiple environmental signals can trigger qualitatively different dynamics of a single transcription factor, and influence gene expression patterns. PMID:22179789

  16. Genesis of chromatin and transcription dynamics in the origin of species.

    PubMed

    Koster, Maria J E; Snel, Berend; Timmers, H Th Marc

    2015-05-07

    Histone proteins compact and stabilize the genomes of Eukarya and Archaea. By forming nucleosome(-like) structures they restrict access of DNA-binding transcription regulators to cis-regulatory DNA elements. Dynamic competition between histones and transcription factors is facilitated by different classes of proteins including ATP-dependent remodeling enzymes that control assembly, access, and editing of chromatin. Here, we summarize the knowledge on dynamics underlying transcriptional regulation across the domains of life with a focus on ATP-dependent enzymes in chromatin structure or in TATA-binding protein activity. These insights suggest directions for future studies on the evolution of transcription regulation and chromatin dynamics. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Measuring dynamic changes in histone modifications and nucleosome density during activated transcription in budding yeast.

    PubMed

    Govind, Chhabi K; Ginsburg, Daniel; Hinnebusch, Alan G

    2012-01-01

    Chromatin immunoprecipitation is widely utilized to determine the in vivo binding of factors that regulate transcription. This procedure entails formaldehyde-mediated cross-linking of proteins and isolation of soluble chromatin followed by shearing. The fragmented chromatin is subjected to immunoprecipitation using antibodies against the protein of interest and the associated DNA is identified using quantitative PCR. Since histones are posttranslationally modified during transcription, this technique can be effectively used to determine the changes in histone modifications that occur during transcription. In this paper, we describe a detailed methodology to determine changes in histone modifications in budding yeast that takes into account reductions in nucleosome.

  18. Blurring the line between the DNA damage response and transcription: the importance of chromatin dynamics.

    PubMed

    Adam, Salomé; Polo, Sophie E

    2014-11-15

    DNA damage interferes with the progression of transcription machineries. A tight coordination of transcription with signaling and repair of DNA damage is thus critical for safeguarding genome function. This coordination involves modulations of chromatin organization. Here, we focus on the central role of chromatin dynamics, in conjunction with DNA Damage Response (DDR) factors, in controlling transcription inhibition and restart at sites of DNA damage in mammalian cells. Recent work has identified chromatin modifiers and histone chaperones as key regulators of transcriptional activity in damaged chromatin regions. Conversely, the transcriptional state of chromatin before DNA damage influences both DNA damage signaling and repair. We discuss the importance of chromatin plasticity in coordinating the interplay between the DDR and transcription, with major implications for cell fate maintenance. Copyright © 2014 Elsevier Inc. All rights reserved.

  19. Transcriptome discovery in non-model wild fish species for the development of quantitative transcript abundance assays

    USGS Publications Warehouse

    Hahn, Cassidy M.; Iwanowicz, Luke R.; Cornman, Robert S.; Mazik, Patricia M.; Blazer, Vicki S.

    2016-01-01

    Environmental studies increasingly identify the presence of both contaminants of emerging concern (CECs) and legacy contaminants in aquatic environments; however, the biological effects of these compounds on resident fishes remain largely unknown. High throughput methodologies were employed to establish partial transcriptomes for three wild-caught, non-model fish species; smallmouth bass (Micropterus dolomieu), white sucker (Catostomus commersonii) and brown bullhead (Ameiurus nebulosus). Sequences from these transcriptome databases were utilized in the development of a custom nCounter CodeSet that allowed for direct multiplexed measurement of 50 transcript abundance endpoints in liver tissue. Sequence information was also utilized in the development of quantitative real-time PCR (qPCR) primers. Cross-species hybridization allowed the smallmouth bass nCounter CodeSet to be used for quantitative transcript abundance analysis of an additional non-model species, largemouth bass (Micropterus salmoides). We validated the nCounter analysis data system with qPCR for a subset of genes and confirmed concordant results. Changes in transcript abundance biomarkers between sexes and seasons were evaluated to provide baseline data on transcript modulation for each species of interest.

  20. Molecular dynamics simulation study of conformational changes of transcription factor TFIIS during RNA polymerase II transcriptional arrest and reactivation.

    PubMed

    Eun, Changsun; Ortiz-Sánchez, Juan Manuel; Da, Lintai; Wang, Dong; McCammon, J Andrew

    2014-01-01

    Transcription factor IIS (TFIIS) is a protein known for catalyzing the cleavage reaction of the 3'-end of backtracked RNA transcript, allowing RNA polymerase II (Pol II) to reactivate the transcription process from the arrested state. Recent structural studies have provided a molecular basis of protein-protein interaction between TFIIS and Pol II. However, the detailed dynamic conformational changes of TFIIS upon binding to Pol II and the related thermodynamic information are largely unknown. Here we use computational approaches to investigate the conformational space of TFIIS in the Pol II-bound and Pol II-free (unbound) states. Our results reveal two distinct conformations of TFIIS: the closed and the open forms. The closed form is dominant in the Pol II-free (unbound) state of TFIIS, whereas the open form is favorable in the Pol II-bound state. Furthermore, we discuss the free energy difference involved in the conformational changes between the two forms in the presence or absence of Pol II. Additionally, our analysis indicates that hydrophobic interactions and the protein-protein interactions between TFIIS and Pol II are crucial for inducing the conformational changes of TFIIS. Our results provide novel insights into the functional interplay between Pol II and TFIIS as well as mechanism of reactivation of Pol II transcription by TFIIS.

  1. Real-time RT-qPCR assay for the analysis of human influenza A virus transcription and replication dynamics.

    PubMed

    Vester, Diana; Lagoda, Antje; Hoffmann, Diana; Seitz, Claudius; Heldt, Stefan; Bettenbrock, Katja; Genzel, Yvonne; Reichl, Udo

    2010-09-01

    A quantitative real-time reverse transcriptase PCR (RT-qPCR) assay was developed for the analysis of influenza A virus transcription and replication dynamics in mammalian cell culture. The assay is based on a polarity- and sequence-specific reverse transcription used to distinguish specifically between viral genomes (vRNA(-)), replicative intermediates (cRNA(+)) and viral messenger RNAs (vmRNA(+)) of segments 4 (HA), 6 (NA), 7 (M) and 8 (NS) during the life cycle of influenza virus. Synthetic viral RNAs used as reference standards for validation and quantitation were prepared for each viral RNA type and segment. Assay validation demonstrated linearity over five orders of magnitude, sensitivity of 1.0 x 10(3) to 8.9 x 10(3) of viral RNA molecules, repeatability and reproducibility of less than 0.8-3.1% CV (coefficient of variation). Dynamics of influenza A virus infection in adherent MDCK cells, a substrate considered for human influenza vaccine manufacturing, were analyzed. In general, mainly vmRNA(+) were synthesized during early phases of infection at about 0.6 hpi, followed immediately by cRNA(+) synthesis and after a short delay of about 1.9 hpi viral genome replication could be detected. The vRNA(-)s were synthesized in equimolar amounts and similar dynamics whereas preferential synthesis of NS1 vmRNA(+) in early transcription phases and a delay for M1 vmRNA(+) was found. Copyright 2010 Elsevier B.V. All rights reserved.

  2. Contribution of cell culture, RNA extraction, and reverse transcription to the measurement error in quantitative reverse transcription polymerase chain reaction-based gene expression quantification.

    PubMed

    Combes, Jean-Damien; Grelier, Gaël; Laversanne, Matthieu; Voirin, Nicolas; Chabaud, Sylvie; Ecochard, René; Lasset, Christine; Moyret-Lalle, Caroline

    2009-10-01

    Quantitative polymerase chain reaction (qPCR) instruments are known to be reliable. However, many authors have underlined the poor reliability of the procedures that precede the measurement of gene expression--cell culture, RNA extraction, and reverse transcription. Here we quantified the measurement errors due to each step and estimated the correction that would accrue from replicating any of those steps. We measured the relative expression of the APC-11 gene (the catalytic anaphase-promoting complex/cyclosome subunit suspected to be involved in breast cancer) with step replication in 18 breast cancer cell lines. The final qPCR step was found to be reproducible (standard deviation [SD]=0.26). In comparison with the between-cell-line variability (SD=1.7), the variability due to the previous steps (cell culture, RNA extraction, and reverse transcription) was on the same order of magnitude (SD=1.2-2.0). Misclassification rates were used to assess the impact of replicating each manual procedure. The misclassification rates improved with replication of cell culture, RNA extraction, and reverse transcription (90.0, 60.9, and 61.1% decreases, respectively). The results point out a high error level in the quantification of gene expression, and these errors may stem from all steps of the procedure. The best correction would accrue from replicating cell culture.

  3. 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.

  4. Quantitative and Dynamic Imaging of ATM Kinase Activity.

    PubMed

    Nyati, Shyam; Young, Grant; Ross, Brian Dale; Rehemtulla, Alnawaz

    2017-01-01

    Ataxia telangiectasia mutated (ATM) is a serine/threonine kinase critical to the cellular DNA-damage response, including DNA double-strand breaks (DSBs). ATM activation results in the initiation of a complex cascade of events facilitating DNA damage repair, cell cycle checkpoint control, and survival. Traditionally, protein kinases have been analyzed in vitro using biochemical methods (kinase assays using purified proteins or immunological assays) requiring a large number of cells and cell lysis. Genetically encoded biosensors based on optical molecular imaging such as fluorescence or bioluminescence have been developed to enable interrogation of kinase activities in live cells with a high signal to background. We have genetically engineered a hybrid protein whose bioluminescent activity is dependent on the ATM-mediated phosphorylation of a substrate. The engineered protein consists of the split luciferase-based protein complementation pair with a CHK2 (a substrate for ATM kinase activity) target sequence and a phospho-serine/threonine-binding domain, FHA2, derived from yeast Rad53. Phosphorylation of the serine residue within the target sequence by ATM would lead to its interaction with the phospho-serine-binding domain, thereby preventing complementation of the split luciferase pair and loss of reporter activity. Bioluminescence imaging of reporter expressing cells in cultured plates or as mouse xenografts provides a quantitative surrogate for ATM kinase activity and therefore the cellular DNA damage response in a noninvasive, dynamic fashion.

  5. Bruchid egg induced transcript dynamics in developing seeds of black gram (Vigna mungo).

    PubMed

    Baruah, Indrani K; Panda, Debashis; M V, Jagadale; Das, Deba Jit; Acharjee, Sumita; Sen, Priyabrata; Sarmah, Bidyut Kumar

    2017-01-01

    Black gram (Vigna mungo) seeds are a rich source of digestible proteins, however, during storage these seeds are severely damaged by bruchids (Callosobruchus spp.), reducing seed quality and yield losses. Most of the cultivated genotypes of black gram are susceptible to bruchids, however, few tolerant genotypes have also been identified but the mechanism of tolerance is poorly understood. We employed Suppression Subtractive Hybridization (SSH) to identify specifically, but rarely expressed bruchid egg induced genes in black gram. In this study, Suppression Subtractive Hybridization (SSH) library was constructed to study the genes involved in defense response in black gram against bruchid infestation. An EST library of 277 clones was obtained for further analyses. Based on CAP3 assembly, 134 unigenes were computationally annotated using Blast2GOPRO software. In all, 20 defense related genes were subject to quantitative PCR analysis (qPCR) out of which 12 genes showed up-regulation in developing seeds of the pods oviposited by bruchids. Few major defense genes like defensin, pathogenesis related protein (PR), lipoxygenase (LOX) showed high expression levels in the oviposited population when compared with the non-oviposited plants. This is the first report on defense related gene transcript dynamics during the bruchid-black gram interaction using SSH library. This library would be useful to clone defense related gene(s) such as defensin as represented in our library for crop improvement.

  6. Bruchid egg induced transcript dynamics in developing seeds of black gram (Vigna mungo)

    PubMed Central

    Baruah, Indrani K.; Panda, Debashis; M.V, Jagadale; Das, Deba Jit; Acharjee, Sumita; Sen, Priyabrata; Sarmah, Bidyut Kumar

    2017-01-01

    Black gram (Vigna mungo) seeds are a rich source of digestible proteins, however, during storage these seeds are severely damaged by bruchids (Callosobruchus spp.), reducing seed quality and yield losses. Most of the cultivated genotypes of black gram are susceptible to bruchids, however, few tolerant genotypes have also been identified but the mechanism of tolerance is poorly understood. We employed Suppression Subtractive Hybridization (SSH) to identify specifically, but rarely expressed bruchid egg induced genes in black gram. In this study, Suppression Subtractive Hybridization (SSH) library was constructed to study the genes involved in defense response in black gram against bruchid infestation. An EST library of 277 clones was obtained for further analyses. Based on CAP3 assembly, 134 unigenes were computationally annotated using Blast2GOPRO software. In all, 20 defense related genes were subject to quantitative PCR analysis (qPCR) out of which 12 genes showed up-regulation in developing seeds of the pods oviposited by bruchids. Few major defense genes like defensin, pathogenesis related protein (PR), lipoxygenase (LOX) showed high expression levels in the oviposited population when compared with the non-oviposited plants. This is the first report on defense related gene transcript dynamics during the bruchid-black gram interaction using SSH library. This library would be useful to clone defense related gene(s) such as defensin as represented in our library for crop improvement. PMID:28448540

  7. Temporal Hierarchy of Gene Expression Mediated by Transcription Factor Binding Affinity and Activation Dynamics

    PubMed Central

    Gao, Rong

    2015-01-01

    ABSTRACT Understanding cellular responses to environmental stimuli requires not only the knowledge of specific regulatory components but also the quantitative characterization of the magnitude and timing of regulatory events. The two-component system is one of the major prokaryotic signaling schemes and is the focus of extensive interest in quantitative modeling and investigation of signaling dynamics. Here we report how the binding affinity of the PhoB two-component response regulator (RR) to target promoters impacts the level and timing of expression of PhoB-regulated genes. Information content has often been used to assess the degree of conservation for transcription factor (TF)-binding sites. We show that increasing the information content of PhoB-binding sites in designed phoA promoters increased the binding affinity and that the binding affinity and concentration of phosphorylated PhoB (PhoB~P) together dictate the level and timing of expression of phoA promoter variants. For various PhoB-regulated promoters with distinct promoter architectures, expression levels appear not to be correlated with TF-binding affinities, in contrast to the intuitive and oversimplified assumption that promoters with higher affinity for a TF tend to have higher expression levels. However, the expression timing of the core set of PhoB-regulated genes correlates well with the binding affinity of PhoB~P to individual promoters and the temporal hierarchy of gene expression appears to be related to the function of gene products during the phosphate starvation response. Modulation of the information content and binding affinity of TF-binding sites may be a common strategy for temporal programming of the expression profile of RR-regulated genes. PMID:26015501

  8. Is transcription the dominant force during dynamic changes in gene expression?

    PubMed

    Turner, Martin

    2011-01-01

    Dynamic changes in gene expression punctuate lymphocyte development and are a characteristic of lymphocyte activation. A prevailing view has been that these changes are driven by DNA transcription factors, which are the dominant force in gene expression. Accumulating evidence is challenging this DNA centric view and has highlighted the prevalence and dynamic nature of RNA handling mechanisms. Alternative splicing and differential polyadenylation appear to be more widespread than first thought. Changes in mRNA decay rates also affect the abundance of transcripts and this mechanism may contribute significantly to gene expression. Additional RNA handling mechanisms that control the intracellular localization of mRNA and association with translating ribosomes are also important. Thus, gene expression is regulated through the coordination of transcriptional and post-transcriptional mechanisms. Developing a more "RNA centric" view of gene expression will allow a more systematic understanding of how gene expression and cell function are integrated.

  9. Dynamics of transcriptional (re)-programming of syncytial nuclei in developing muscles.

    PubMed

    Bataillé, Laetitia; Boukhatmi, Hadi; Frendo, Jean-Louis; Vincent, Alain

    2017-06-09

    A stereotyped array of body wall muscles enables precision and stereotypy of animal movements. In Drosophila, each syncytial muscle forms via fusion of one founder cell (FC) with multiple fusion competent myoblasts (FCMs). The specific morphology of each muscle, i.e. distinctive shape, orientation, size and skeletal attachment sites, reflects the specific combination of identity transcription factors (iTFs) expressed by its FC. Here, we addressed three questions: Are FCM nuclei naive? What is the selectivity and temporal sequence of transcriptional reprogramming of FCMs recruited into growing syncytium? Is transcription of generic myogenic and identity realisation genes coordinated during muscle differentiation? The tracking of nuclei in developing muscles shows that FCM nuclei are competent to be transcriptionally reprogrammed to a given muscle identity, post fusion. In situ hybridisation to nascent transcripts for FCM, FC-generic and iTF genes shows that this reprogramming is progressive, beginning by repression of FCM-specific genes in fused nuclei, with some evidence that FC nuclei retain specific characteristics. Transcription of identity realisation genes is linked to iTF activation and regulated at levels of both transcription initiation rate and period of transcription. The generic muscle differentiation programme is activated independently. Transcription reprogramming of fused myoblast nuclei is progressive, such that nuclei within a syncytial fibre at a given time point during muscle development are heterogeneous with regards to specific gene transcription. This comprehensive view of the dynamics of transcriptional (re)programming of post-mitotic nuclei within syncytial cells provides a new framework for understanding the transcriptional control of the lineage diversity of multinucleated cells.

  10. Quantitative dynamic imaging of immune cell signalling using lentiviral gene transfer.

    PubMed

    Bagnall, J; Boddington, C; Boyd, J; Brignall, R; Rowe, W; Jones, N A; Schmidt, L; Spiller, D G; White, M R H; Paszek, P

    2015-06-01

    Live-cell imaging of fluorescent fusion proteins has transformed our understanding of mammalian cell signalling and function. However, some cellular systems such as immune cells are unsuitable or refractory to many existing transgene delivery methods thus limiting systematic analyses. Here, a flexible lentiviral gene transfer platform for dynamic time-lapse imaging has been developed and validated with single-molecule spectroscopy, mathematical modelling and transcriptomics and used for analysis of a set of inflammation-related signalling networks. Time-lapse imaging of nuclear factor kappa B (NF-κB), signal transducer and activator of transcription (STATs) and nuclear factor of activated T-cells (NFAT) in mammalian immune cell lines provided evidence for heterogeneous temporal encoding of inflammatory signals. In particular, the absolute quantification of single-cell responses over time via fluorescent correlation spectroscopy (FCS) showed that NF-κB p65 activation in response to tumour necrosis factor α (TNFα) was differentially encoded in variable amplitude of nuclear translocation between immune and non-immune cells. The absolute number of activated molecules was dictated in part by the cell size, suggesting a morphology-dependent regulatory mechanism. The developed platform will enable further absolute quantitative analyses of the dynamic interactions between signalling networks, in and between individual cells, allowing better integration with mathematical models of signalling networks.

  11. 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

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

    PubMed

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

    2015-04-01

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

  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. Quantitative Assessment of Molecular Dynamics Sampling for Flexible Systems.

    PubMed

    Nemec, Mike; Hoffmann, Daniel

    2017-02-14

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

  15. 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

  16. A quantitative proteomics approach identifies ETV6 and IKZF1 as new regulators of an ERG-driven transcriptional network

    PubMed Central

    Unnikrishnan, Ashwin; Guan, Yi F.; Huang, Yizhou; Beck, Dominik; Thoms, Julie A. I.; Peirs, Sofie; Knezevic, Kathy; Ma, Shiyong; de Walle, Inge V.; de Jong, Ineke; Ali, Zara; Zhong, Ling; Raftery, Mark J.; Taghon, Tom; Larsson, Jonas; MacKenzie, Karen L.; Van Vlierberghe, Pieter; Wong, Jason W. H.; Pimanda, John E.

    2016-01-01

    Aberrant stem cell-like gene regulatory networks are a feature of leukaemogenesis. The ETS-related gene (ERG), an important regulator of normal haematopoiesis, is also highly expressed in T-ALL and acute myeloid leukaemia (AML). However, the transcriptional regulation of ERG in leukaemic cells remains poorly understood. In order to discover transcriptional regulators of ERG, we employed a quantitative mass spectrometry-based method to identify factors binding the 321 bp ERG +85 stem cell enhancer region in MOLT-4 T-ALL and KG-1 AML cells. Using this approach, we identified a number of known binders of the +85 enhancer in leukaemic cells along with previously unknown binders, including ETV6 and IKZF1. We confirmed that ETV6 and IKZF1 were also bound at the +85 enhancer in both leukaemic cells and in healthy human CD34+ haematopoietic stem and progenitor cells. Knockdown experiments confirmed that ETV6 and IKZF1 are transcriptional regulators not just of ERG, but also of a number of genes regulated by a densely interconnected network of seven transcription factors. At last, we show that ETV6 and IKZF1 expression levels are positively correlated with expression of a number of heptad genes in AML and high expression of all nine genes confers poorer overall prognosis. PMID:27604872

  17. A quantitative proteomics approach identifies ETV6 and IKZF1 as new regulators of an ERG-driven transcriptional network.

    PubMed

    Unnikrishnan, Ashwin; Guan, Yi F; Huang, Yizhou; Beck, Dominik; Thoms, Julie A I; Peirs, Sofie; Knezevic, Kathy; Ma, Shiyong; de Walle, Inge V; de Jong, Ineke; Ali, Zara; Zhong, Ling; Raftery, Mark J; Taghon, Tom; Larsson, Jonas; MacKenzie, Karen L; Van Vlierberghe, Pieter; Wong, Jason W H; Pimanda, John E

    2016-12-15

    Aberrant stem cell-like gene regulatory networks are a feature of leukaemogenesis. The ETS-related gene (ERG), an important regulator of normal haematopoiesis, is also highly expressed in T-ALL and acute myeloid leukaemia (AML). However, the transcriptional regulation of ERG in leukaemic cells remains poorly understood. In order to discover transcriptional regulators of ERG, we employed a quantitative mass spectrometry-based method to identify factors binding the 321 bp ERG +85 stem cell enhancer region in MOLT-4 T-ALL and KG-1 AML cells. Using this approach, we identified a number of known binders of the +85 enhancer in leukaemic cells along with previously unknown binders, including ETV6 and IKZF1. We confirmed that ETV6 and IKZF1 were also bound at the +85 enhancer in both leukaemic cells and in healthy human CD34(+) haematopoietic stem and progenitor cells. Knockdown experiments confirmed that ETV6 and IKZF1 are transcriptional regulators not just of ERG, but also of a number of genes regulated by a densely interconnected network of seven transcription factors. At last, we show that ETV6 and IKZF1 expression levels are positively correlated with expression of a number of heptad genes in AML and high expression of all nine genes confers poorer overall prognosis.

  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. Characterization and improvement of RNA-Seq precision in quantitative transcript expression profiling

    PubMed Central

    Łabaj, Paweł P.; Leparc, Germán G.; Linggi, Bryan E.; Markillie, Lye Meng; Wiley, H. Steven; Kreil, David P.

    2011-01-01

    Motivation: 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 datasets 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 identification and measurement precision, including their dependence on transcript expression levels, read depth and other parameters. In particular, an impressive recall 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, <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 analysing 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. Contact: rnaseq10@boku.ac.at Supplementary information: Supplementary data are available at Bioinformatics online. PMID:21685096

  20. Deep sequencing approaches for the analysis of prokaryotic transcriptional boundaries and dynamics.

    PubMed

    James, Katherine; Cockell, Simon J; Zenkin, Nikolay

    2017-05-01

    The identification of the protein-coding regions of a genome is straightforward due to the universality of start and stop codons. However, the boundaries of the transcribed regions, conditional operon structures, non-coding RNAs and the dynamics of transcription, such as pausing of elongation, are non-trivial to identify, even in the comparatively simple genomes of prokaryotes. Traditional methods for the study of these areas, such as tiling arrays, are noisy, labour-intensive and lack the resolution required for densely-packed bacterial genomes. Recently, deep sequencing has become increasingly popular for the study of the transcriptome due to its lower costs, higher accuracy and single nucleotide resolution. These methods have revolutionised our understanding of prokaryotic transcriptional dynamics. Here, we review the deep sequencing and data analysis techniques that are available for the study of transcription in prokaryotes, and discuss the bioinformatic considerations of these analyses. Copyright © 2017 Elsevier Inc. All rights reserved.

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

    PubMed Central

    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. PMID:26252791

  2. Phytochrome dependent quantitative control of Hd3a transcription is the basis of the night break effect in rice flowering.

    PubMed

    Ishikawa, Ryo; Shinomura, Tomoko; Takano, Makoto; Shimamoto, Ko

    2009-04-01

    A short exposure to light during relative night (night break; NB) delays flowering in the short day plant rice. NB acts by downregulating Heading date 3a (Hd3a) expression. Because phytochrome B mutants do not respond to NB and their flowering time is not affected even under NB conditions, phyB is required for the suppression of Hd3a expression. The effect of NB is quantitatively controlled by light quality and by either light intensity or duration. However, the molecular mechanisms that regulate these interactions are poorly understood. Here, we examine the roles of phytochromes in the regulation of Hd3a transcription under NB conditions using monochromatic red, far-red and blue light. Red and blue light downregulated Hd3a expression, but far-red light NB did not. The effect of red light NB on Hd3a is dependent on photon fluence and is restored by subsequent far-red light irradiation. Our results suggest that quantitative effect of light on flowering in rice NB is mediated by the regulation of Hd3a transcription by phyB.

  3. The flavonoid pathway in tomato seedlings: transcript abundance and the modeling of metabolite dynamics.

    PubMed

    Groenenboom, Marian; Gomez-Roldan, Victoria; Stigter, Hans; Astola, Laura; van Daelen, Raymond; Beekwilder, Jules; Bovy, Arnaud; Hall, Robert; Molenaar, Jaap

    2013-01-01

    Flavonoids are secondary metabolites present in all terrestrial plants. The flavonoid pathway has been extensively studied, and many of the involved genes and metabolites have been described in the literature. Despite this extensive knowledge, the functioning of the pathway in vivo is still poorly understood. Here, we study the flavonoid pathway using both experiments and mathematical models. We measured flavonoid metabolite dynamics in two tissues, hypocotyls and cotyledons, during tomato seedling development. Interestingly, the same backbone of interactions leads to very different accumulation patterns in the different tissues. Initially, we developed a mathematical model with constant enzyme concentrations that described the metabolic networks separately in both tissues. This model was unable to fit the measured flavonoid dynamics in the hypocotyls, even if we allowed unrealistic parameter values. This suggested us to investigate the effect of transcript abundance on flavonoid accumulation. We found that the expression of candidate flavonoid genes varies considerably with time. Variation in transcript abundance results in enzymatic variation, which could have a large effect on metabolite accumulation. Candidate transcript abundance was included in the mathematical model as representative for enzyme concentration. We fitted the resulting model to the flavonoid dynamics in the cotyledons, and tested it by applying it to the data from hypocotyls. When transcript abundance is included, we are indeed able to explain flavonoid dynamics in both tissues. Importantly, this is possible under the biologically relevant restriction that the enzymatic properties estimated by the model are conserved between the tissues.

  4. Dynamic Changes in Nucleosome Occupancy Are Not Predictive of Gene Expression Dynamics but Are Linked to Transcription and Chromatin Regulators

    PubMed Central

    Huebert, Dana J.; Kuan, Pei-Fen; Keleş, Sündüz

    2012-01-01

    The response to stressful stimuli requires rapid, precise, and dynamic gene expression changes that must be coordinated across the genome. To gain insight into the temporal ordering of genome reorganization, we investigated dynamic relationships between changing nucleosome occupancy, transcription factor binding, and gene expression in Saccharomyces cerevisiae yeast responding to oxidative stress. We applied deep sequencing to nucleosomal DNA at six time points before and after hydrogen peroxide treatment and revealed many distinct dynamic patterns of nucleosome gain and loss. The timing of nucleosome repositioning was not predictive of the dynamics of downstream gene expression change but instead was linked to nucleosome position relative to transcription start sites and specific cis-regulatory elements. We measured genome-wide binding of the stress-activated transcription factor Msn2p over time and found that Msn2p binds different loci with different dynamics. Nucleosome eviction from Msn2p binding sites was common across the genome; however, we show that, contrary to expectation, nucleosome loss occurred after Msn2p binding and in fact required Msn2p. This negates the prevailing model that nucleosomes obscuring Msn2p sites regulate DNA access and must be lost before Msn2p can bind DNA. Together, these results highlight the complexities of stress-dependent chromatin changes and their effects on gene expression. PMID:22354995

  5. Prognostic value of quantitative analysis of WT1 gene transcripts in adult acute lymphoblastic leukemia.

    PubMed

    Chiusa, Luigi; Francia di Celle, Paola; Campisi, Paola; Ceretto, Cristina; Marmont, Filippo; Pich, Achille

    2006-02-01

    We quantified Wilm's tumor gene (WT1) using a real time quantitative polymerase chain reaction in 20 adult patients with acute lymphoblastic leukemia at presentation. A WT1 level greater than 906 (median value for the whole series) was a significant predictor of a poor disease-free and overall survival in uni- and multivariate analyses.

  6. 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.

  7. Quantitative Proteomics Demonstrates That the RNA Polymerase II Subunits Rpb4 and Rpb7 Dissociate during Transcriptional Elongation*

    PubMed Central

    Mosley, Amber L.; Hunter, Gerald O.; Sardiu, Mihaela E.; Smolle, Michaela; Workman, Jerry L.; Florens, Laurence; Washburn, Michael P.

    2013-01-01

    Eukaryotic RNA polymerase II (RNAPII) is a 12-subunit enzyme that is responsible for the transcription of messenger RNA. Two of the subunits of RNA polymerase II, Rpb4 and Rpb7, have been shown to dissociate from the enzyme under a number of specific laboratory conditions. However, a biological context for the dissociation of Rpb4 and Rpb7 has not been identified. We have found that Rpb4/7 dissociate from RNAPII upon interaction with specific transcriptional elongation-associated proteins that are recruited to the hyperphosphorylated form of the C-terminal domain. However, the dissociation of Rpb4/7 is likely short lived because a significant level of free Rpb4/7 was not detected by quantitative proteomic analyses. In addition, we have found that RNAPII that is isolated through Rpb7 is depleted in serine 2 C-terminal domain phosphorylation. In contrast to previous reports, these data indicate that Rpb4/7 are dispensable during specific stages of transcriptional elongation in Saccharomyces cerevisiae. PMID:23418395

  8. Charting Latency Transcripts in Kaposi's Sarcoma-Associated Herpesvirus by Whole-Genome Real-Time Quantitative PCR

    PubMed Central

    Fakhari, Farnaz D.; Dittmer, Dirk P.

    2002-01-01

    The division into a latent or lytic life cycle is fundamental to all herpesviridae. In the case of Kaposi's sarcoma-associated herpesvirus (KSHV) (human herpesvirus 8), latent genes have been implicated in cell autonomous transformation, while certain lytic genes procure a tumor friendly milieu through paracrine mechanism. To query KSHV transcription, we devised and validated a high-throughput, high-specificity, high-sensitivity, real-time quantitative reverse transcription-PCR array. This novel methodology is applicable to many human pathogens. Its first use demonstrated that the mRNA levels for KSHV LANA, v-cyclin, and v-FLIP do not increase at any time after viral reactivation. The mRNA for LANA-2/vIRF-3 is similarly resistant to viral reactivation. In contrast, every other latent or lytic message was induced. Hence, LANA, v-FLIP, v-cyclin, and LANA-2 constitute a group of uniquely regulated transcripts in the KSHV genome. PMID:12021355

  9. The transcription bubble of the RNA polymerase-promoter open complex exhibits conformational heterogeneity and millisecond-scale dynamics: implications for transcription start-site selection.

    PubMed

    Robb, Nicole C; Cordes, Thorben; Hwang, Ling Chin; Gryte, Kristofer; Duchi, Diego; Craggs, Timothy D; Santoso, Yusdi; Weiss, Shimon; Ebright, Richard H; Kapanidis, Achillefs N

    2013-03-11

    Bacterial transcription is initiated after RNA polymerase (RNAP) binds to promoter DNA, melts ~14 bp around the transcription start site and forms a single-stranded "transcription bubble" within a catalytically active RNAP-DNA open complex (RP(o)). There is significant flexibility in the transcription start site, which causes variable spacing between the promoter elements and the start site; this in turn causes differences in the length and sequence at the 5' end of RNA transcripts and can be important for gene regulation. The start-site variability also implies the presence of some flexibility in the positioning of the DNA relative to the RNAP active site in RP(o). The flexibility may occur in the positioning of the transcription bubble prior to RNA synthesis and may reflect bubble expansion ("scrunching") or bubble contraction ("unscrunching"). Here, we assess the presence of dynamic flexibility in RP(o) with single-molecule FRET (Förster resonance energy transfer). We obtain experimental evidence for dynamic flexibility in RP(o) using different FRET rulers and labeling positions. An analysis of FRET distributions of RP(o) using burst variance analysis reveals conformational fluctuations in RP(o) in the millisecond timescale. Further experiments using subsets of nucleotides and DNA mutations allowed us to reprogram the transcription start sites, in a way that can be described by repositioning of the single-stranded transcription bubble relative to the RNAP active site within RP(o). Our study marks the first experimental observation of conformational dynamics in the transcription bubble of RP(o) and indicates that DNA dynamics within the bubble affect the search for transcription start sites. Copyright © 2013 Elsevier Ltd. All rights reserved.

  10. 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. © 2013.

  11. Quantitative analysis of transient and sustained transforming growth factor-β signaling dynamics

    PubMed Central

    Zi, Zhike; Feng, Zipei; Chapnick, Douglas A; Dahl, Markus; Deng, Difan; Klipp, Edda; Moustakas, Aristidis; Liu, Xuedong

    2011-01-01

    Mammalian cells can decode the concentration of extracellular transforming growth factor-β (TGF-β) and transduce this cue into appropriate cell fate decisions. How variable TGF-β ligand doses quantitatively control intracellular signaling dynamics and how continuous ligand doses are translated into discontinuous cellular fate decisions remain poorly understood. Using a combined experimental and mathematical modeling approach, we discovered that cells respond differently to continuous and pulsating TGF-β stimulation. The TGF-β pathway elicits a transient signaling response to a single pulse of TGF-β stimulation, whereas it is capable of integrating repeated pulses of ligand stimulation at short time interval, resulting in sustained phospho-Smad2 and transcriptional responses. Additionally, the TGF-β pathway displays different sensitivities to ligand doses at different time scales. While ligand-induced short-term Smad2 phosphorylation is graded, long-term Smad2 phosphorylation is switch-like to a small change in TGF-β levels. Correspondingly, the short-term Smad7 gene expression is graded, while long-term PAI-1 gene expression is switch-like, as is the long-term growth inhibitory response. Our results suggest that long-term switch-like signaling responses in the TGF-β pathway might be critical for cell fate determination. PMID:21613981

  12. Temporal hierarchy of gene expression mediated by transcription factor binding affinity and activation dynamics.

    PubMed

    Gao, Rong; Stock, Ann M

    2015-05-26

    Understanding cellular responses to environmental stimuli requires not only the knowledge of specific regulatory components but also the quantitative characterization of the magnitude and timing of regulatory events. The two-component system is one of the major prokaryotic signaling schemes and is the focus of extensive interest in quantitative modeling and investigation of signaling dynamics. Here we report how the binding affinity of the PhoB two-component response regulator (RR) to target promoters impacts the level and timing of expression of PhoB-regulated genes. Information content has often been used to assess the degree of conservation for transcription factor (TF)-binding sites. We show that increasing the information content of PhoB-binding sites in designed phoA promoters increased the binding affinity and that the binding affinity and concentration of phosphorylated PhoB (PhoB~P) together dictate the level and timing of expression of phoA promoter variants. For various PhoB-regulated promoters with distinct promoter architectures, expression levels appear not to be correlated with TF-binding affinities, in contrast to the intuitive and oversimplified assumption that promoters with higher affinity for a TF tend to have higher expression levels. However, the expression timing of the core set of PhoB-regulated genes correlates well with the binding affinity of PhoB~P to individual promoters and the temporal hierarchy of gene expression appears to be related to the function of gene products during the phosphate starvation response. Modulation of the information content and binding affinity of TF-binding sites may be a common strategy for temporal programming of the expression profile of RR-regulated genes. A single TF often orchestrates the expression of multiple genes in response to environmental stimuli. It is not clear how different TF-binding sites within the regulon dictate the expression profile. Our studies of Escherichia coli PhoB, a response

  13. 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

  14. Systems analysis of transcription factor activities in environments with stable and dynamic oxygen concentrations.

    PubMed

    Rolfe, Matthew D; Ocone, Andrea; Stapleton, Melanie R; Hall, Simon; Trotter, Eleanor W; Poole, Robert K; Sanguinetti, Guido; Green, Jeffrey

    2012-07-01

    Understanding gene regulation requires knowledge of changes in transcription factor (TF) activities. Simultaneous direct measurement of numerous TF activities is currently impossible. Nevertheless, statistical approaches to infer TF activities have yielded non-trivial and verifiable predictions for individual TFs. Here, global statistical modelling identifies changes in TF activities from transcript profiles of Escherichia coli growing in stable (fixed oxygen availabilities) and dynamic (changing oxygen availability) environments. A core oxygen-responsive TF network, supplemented by additional TFs acting under specific conditions, was identified. The activities of the cytoplasmic oxygen-responsive TF, FNR, and the membrane-bound terminal oxidases implied that, even on the scale of the bacterial cell, spatial effects significantly influence oxygen-sensing. Several transcripts exhibited asymmetrical patterns of abundance in aerobic to anaerobic and anaerobic to aerobic transitions. One of these transcripts, ndh, encodes a major component of the aerobic respiratory chain and is regulated by oxygen-responsive TFs ArcA and FNR. Kinetic modelling indicated that ArcA and FNR behaviour could not explain the ndh transcript profile, leading to the identification of another TF, PdhR, as the source of the asymmetry. Thus, this approach illustrates how systematic examination of regulatory responses in stable and dynamic environments yields new mechanistic insights into adaptive processes.

  15. The dynamic response of upstream DNA to transcription-generated torsional stress.

    PubMed

    Kouzine, Fedor; Liu, Juhong; Sanford, Suzanne; Chung, Hye-Jung; Levens, David

    2004-11-01

    The torsional stress caused by counter-rotation of the transcription machinery and template generates supercoils in a closed topological domain, but has been presumed to be too short-lived to be significant in an open domain. This report shows that transcribing RNA polymerases dynamically sustain sufficient torsion to perturb DNA structure even on linear templates. Assays to capture and measure transcriptionally generated torque and to trap short-lived perturbations in DNA structure and conformation showed that the transient forces upstream of active promoters are large enough to drive the supercoil-sensitive far upstream element (FUSE) of the human c-myc into single-stranded DNA. An alternative non-B conformation of FUSE found in stably supercoiled DNA is not accessible dynamically. These results demonstrate that dynamic disturbance of DNA structure provides a real-time measure of ongoing genetic activity.

  16. LXIII Cold Spring Harbor Symposium on Quantitative Biology: Mechanisms of Transcription, 3-8 June 1998.

    PubMed

    Emerson, B M; Bagga, R

    1999-01-29

    A new perspective is emerging in the transcription field towards understanding gene regulation not only at its most fundamental level but also in the context of chromatin, nuclear compartmentalization, and physiological processes. This direction is being fueled by several key observations. Among them is the discovery of multi-protein complexes whose components reveal a link between gene activity, nuclear structure, and cellular signaling pathways. This information will no doubt be extended by identifying expanded regulatory circuitry using the microchip oligonucleotide array technology. In addition to elucidating the regulatory consequences of these intricate connections, another frontier will be to analyze gene expression within chromosomes. This requires deciphering the mechanism of action of a variety of DNA elements that create a genetic domain such as locus control regions, distal enhancers, insulators, silencers, and matrix attachment regions. Hopefully, with the development of new assays these elements can be as rigorously defined as promoters have been. We can also look forward to capturing critical transcriptional processes by increasingly refined structural analyses. Thus, the scope of problems being addressed in gene regulation has been greatly expanded and the opportunity exists to answer very sophisticated questions in the future.

  17. Quantitative dissection of the Notch:CSL interaction: insights into the Notch-mediated transcriptional switch.

    PubMed

    Lubman, Olga Y; Ilagan, Ma Xenia G; Kopan, Raphael; Barrick, Doug

    2007-01-19

    Complex formation between the intracellular domain of the Notch receptor (NICD) and the transcription factor CSL is indispensable for transcriptional activation. To understand how NICD displaces CSL-associated co-repressors, we have quantified the binding of different Notch1 ICD regions to a key interaction domain (the beta trefoil domain, or BTD) of human CSL. Electrophoresis, scattering, and titration calorimetry indicate that NICD and BTD combine to form a 1:1 heterodimer. Neither the Notch1 ankyrin domain (ANK) nor C-terminal region contributes binding energy towards BTD. In contrast, binding energy is attributed largely to a short segment including the conserved WFP sequence motif within the RAM region (the approximately 140 residue polypeptide segment N-terminal to the ANK domain); substitution of this motif substantially reduces affinity. Short (< or =25 residues) WFP-containing peptides encoded by the four mammalian Notch genes have similar affinities to BTD; thus, activity differences between paralogues either result from other regions of NICD and CSL or from differences in interaction with downstream components. The importance of RAM was demonstrated by the ability of a short RAM peptides to dissociate NICD:CSL interaction in cellular lysates. These results support an emerging molecular mechanism for the displacement of co-repressors from DNA-bound CSL by NICD.

  18. Quantitative Dissection of the Notch:CSL Interaction: Insights into the Notch Transcriptional Switch

    PubMed Central

    Lubman, Olga Y.; Ilagan, Ma. Xenia G.; Kopan, Raphael; Barrick, Doug

    2007-01-01

    Complex formation between the intracellular domain of the Notch receptor (NICD) and the transcription factor CSL is indispensable for transcriptional activation. To understand how NICD displaces CSL-associated co-repressors, we have quantified the binding of different Notch1 ICD regions to a key interaction domain (the beta trefoil domain, or BTD) of human CSL. Electrophoresis, scattering, and titration calorimetry indicate that NICD and BTD combine to form a 1:1 heterodimer. Neither the Notch1 ankyrin domain (ANK) nor C-terminal region contributes binding energy towards BTD. In contrast, binding energy is attributed largely to a short segment including the conserved WFP sequence motif within the RAM region (the ~140 residue polypeptide segment N-terminal to the ANK domain); substitution of this motif substantially reduces affinity. Short (≤ 25 residues) WFP-containing peptides encoded by the four mammalian Notch genes have similar affinities to BTD; thus, activity differences between paralogues either result from other regions of NICD and CSL or from differences in interaction with downstream components. The importance of RAM was demonstrated by the ability of a short RAM peptides to dissociate NICD:CSL interaction in cellular lysates. These results support an emerging molecular mechanism for the displacement of co-repressors from DNA-bound CSL by NICD. PMID:17070841

  19. Redefining the transcriptional regulatory dynamics of classically and alternatively activated macrophages by deepCAGE transcriptomics

    PubMed Central

    Roy, Sugata; Schmeier, Sebastian; Arner, Erik; Alam, Tanvir; Parihar, Suraj P.; Ozturk, Mumin; Tamgue, Ousman; Kawaji, Hideya; de Hoon, Michiel J. L.; Itoh, Masayoshi; Lassmann, Timo; Carninci, Piero; Hayashizaki, Yoshihide; Forrest, Alistair R. R.; Bajic, Vladimir B.; Guler, Reto; Consortium, FANTOM; Brombacher, Frank; Suzuki, Harukazu

    2015-01-01

    Classically or alternatively activated macrophages (M1 and M2, respectively) play distinct and important roles for microbiocidal activity, regulation of inflammation and tissue homeostasis. Despite this, their transcriptional regulatory dynamics are poorly understood. Using promoter-level expression profiling by non-biased deepCAGE we have studied the transcriptional dynamics of classically and alternatively activated macrophages. Transcription factor (TF) binding motif activity analysis revealed four motifs, NFKB1_REL_RELA, IRF1,2, IRF7 and TBP that are commonly activated but have distinct activity dynamics in M1 and M2 activation. We observe matching changes in the expression profiles of the corresponding TFs and show that only a restricted set of TFs change expression. There is an overall drastic and transient up-regulation in M1 and a weaker and more sustainable up-regulation in M2. Novel TFs, such as Thap6, Maff, (M1) and Hivep1, Nfil3, Prdm1, (M2) among others, were suggested to be involved in the activation processes. Additionally, 52 (M1) and 67 (M2) novel differentially expressed genes and, for the first time, several differentially expressed long non-coding RNA (lncRNA) transcriptome markers were identified. In conclusion, the finding of novel motifs, TFs and protein-coding and lncRNA genes is an important step forward to fully understand the transcriptional machinery of macrophage activation. PMID:26117544

  20. Dynamic transition of transcription and chromatin landscape during fission yeast adaptation to glucose starvation.

    PubMed

    Oda, Arisa; Takemata, Naomichi; Hirata, Yoshito; Miyoshi, Tomoichiro; Suzuki, Yutaka; Sugano, Sumio; Ohta, Kunihiro

    2015-05-01

    Shortage of glucose, the primary energy source for all organisms, is one of the most critical stresses influencing cell viability. Glucose starvation promptly induces changes in mRNA and noncoding RNA (ncRNA) transcription. We previously reported that glucose starvation induces long ncRNA (lncRNA) transcription in the 5' segment of a fission yeast gluconeogenesis gene (fbp1+), which leads to stepwise chromatin alteration around the fbp1+ promoter and to subsequent robust gene activation. Here, we analyzed genomewide transcription by strand-specific RNA sequencing, together with chromatin landscape by immunoprecipitation sequencing (ChIP-seq). Clustering analysis showed that distinct mRNAs and ncRNAs are induced at the early, middle and later stages of cellular response to glucose starvation. The starvation-induced transcription depends substantially on the stress-responsive transcription factor Atf1. Using a new computer program that examines dynamic changes in expression patterns, we identified ncRNAs with similar behavior to the fbp1+ lncRNA. We confirmed that there are continuous lncRNAs associated with local reduction of histone density. Overlapping with the regions for transcription of these lncRNAs, antisense RNAs are antagonistically transcribed under glucose-rich conditions. These results suggest that Atf1-dependent integrated networks of mRNA and lncRNA govern drastic changes in cell physiology in response to glucose starvation.

  1. Dynamics of oscillatory phenotypes in S. cerevisiae reveal a network of genome-wide transcriptional oscillators

    PubMed Central

    Chin, Shwe L.; Marcus, Ian M.; Klevecz, Robert R.; Li, Caroline M.

    2012-01-01

    Genetic and environmental factors are well-studied influences on phenotype; however, time is a variable that is rarely considered when studying changes in cellular phenotype. Time-resolved microarray data revealed genome-wide transcriptional oscillation in a yeast continuous culture system with ~2 and ~4 h periods. We mapped the global patterns of transcriptional oscillations into a 3D map to represent different cellular phenotypes of redox cycles. This map shows the dynamic nature of gene expression in that transcripts are ordered and coupled to each other through time and concentration space. Although cells differed in oscillation periods, transcripts involved in certain processes were conserved in a deterministic way. When oscillation period lengthened, the peak to trough ratio of transcripts increased and the fraction of cells in the unbudded (G0/G1) phase of the cell division cycle increased. Decreasing the glucose level in the culture media was one way to increase the redox cycle, possibly from changes in metabolic flux. The period may be responding to lower glucose levels by increasing the fraction of cells in G1 and reducing S-phase gating so that cells can spend more time in catabolic processes. Our results support that gene transcripts are coordinated with metabolic functions and the cell division cycle. PMID:22289124

  2. Semi-quantitative analysis of transcript accumulation in response to drought stress by Lepidium latifolium seedlings.

    PubMed

    Gupta, Sanjay Mohan; Singh, Sadhana; Pandey, Pankaj; Grover, Atul; Ahmed, Zakwan

    2013-09-01

    Cross-amplification of five Arabidopsis abiotic stress-responsive genes (AtPAP, ZFAN, Vn, LC4 and SNS) in Lepidium has been documented in plants raised out of seeds pre-treated with potassium nitrate (KNO 3) for assessment of enhanced drought stress tolerance. cDNA was synthesized from Lepidium plants pre-treated with KNO 3 (0.1% and 0.3%) and exposed to drought conditions (5% and 15% PEG) at seedling stage for 30 d. Transcript accumulation of all the five genes were found suppressed in set of seedlings, which were pre-treated with 0.1% KNO 3 and were exposed to 15% PEG for 30 d. The present study establishes that different pre-treatments may further enhance the survivability of Lepidium plants under conditions of drought stress to different degrees.

  3. 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

  4. 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

    2016-04-07

    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.

  5. Dynamic regulation of microRNA expression following interferon-γ-induced gene transcription.

    PubMed

    Reinsbach, Susanne; Nazarov, Petr V; Philippidou, Demetra; Schmitt, Martina; Wienecke-Baldacchino, Anke; Muller, Arnaud; Vallar, Laurent; Behrmann, Iris; Kreis, Stephanie

    2012-07-01

    MicroRNAs are major players in post-transcriptional gene regulation. Even small changes in miRNA levels may have profound consequences for the expression levels of target genes. Hence, miRNAs themselves need to be tightly, albeit dynamically, regulated. Here, we investigated the dynamic behavior of miRNAs over a wide time range following stimulation of melanoma cells with interferon-γ (IFN-γ), which activates the transcription factor STAT1. By applying several bioinformatic and statistical software tools for visualization and identification of differentially expressed miRNAs derived from time-series microarray experiments, 8.9% of 1105 miRNAs appeared to be directly or indirectly regulated by STAT1. Focusing on distinct dynamic expression patterns, we found that the majority of robust miRNA expression changes occurred in the intermediate time range (24-48 h). Three miRNAs (miR-27a, miR-30a, miR-34a) had a delayed regulation occurring at 72 h while none showed significant expression changes at early time points between 30 min and 6 h. Expression patterns of individual miRNAs were altered gradually over time or abruptly increased or decreased between two time points. Furthermore, we observed coordinated dynamic transcription of most miRNA clusters while few were found to be regulated independently of their genetic cluster. Most interestingly, several "star" or passenger strand sequences were specifically regulated over time while their "guide" strands were not.

  6. 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

  7. Quantitative multivariate analysis of dynamic multicellular morphogenic trajectories.

    PubMed

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

    2015-07-01

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

  8. Model-driven mapping of transcriptional networks reveals the circuitry and dynamics of virulence regulation.

    PubMed

    Maier, Ezekiel J; Haynes, Brian C; Gish, Stacey R; Wang, Zhuo A; Skowyra, Michael L; Marulli, Alyssa L; Doering, Tamara L; Brent, Michael R

    2015-05-01

    Key steps in understanding a biological process include identifying genes that are involved and determining how they are regulated. We developed a novel method for identifying transcription factors (TFs) involved in a specific process and used it to map regulation of the key virulence factor of a deadly fungus-its capsule. The map, built from expression profiles of 41 TF mutants, includes 20 TFs not previously known to regulate virulence attributes. It also reveals a hierarchy comprising executive, midlevel, and "foreman" TFs. When grouped by temporal expression pattern, these TFs explain much of the transcriptional dynamics of capsule induction. Phenotypic analysis of TF deletion mutants revealed complex relationships among virulence factors and virulence in mice. These resources and analyses provide the first integrated, systems-level view of capsule regulation and biosynthesis. Our methods dramatically improve the efficiency with which transcriptional networks can be analyzed, making genomic approaches accessible to laboratories focused on specific physiological processes.

  9. Evaluation of quantitative assays for the identification of direct signal transducer and activator of transcription 3 (STAT3) inhibitors

    PubMed Central

    Furtek, Steffanie L.; Matheson, Christopher J.; Backos, Donald S.; Reigan, Philip

    2016-01-01

    In many forms of cancer the signal transducer and activator of transcription 3 (STAT3) transcription factor remains constitutively active, driving cancer survival and progression. The critical role of STAT3 in tumorigenesis has prompted a campaign of drug discovery programs to identify small molecules that disrupt the function of STAT3, with more recent efforts focusing on direct STAT3 inhibition. There are two target binding sites for direct STAT3 inhibitors: the SH2 dimerization domain and the DNA-binding domain. An in vitro fluorescence polarization assay, using recombinant STAT3 protein, has successfully identified compounds that target the SH2 domain; however, no assay has been reported to identify inhibitors that bind the DNA-binding domain. The lack of such a quantitative assay has limited the identification and development of STAT3 DNA-binding domain inhibitors. Here, we report a modified DNA-binding ELISA to incorporate recombinant STAT3 protein to evaluate small molecules that prevent STAT3-DNA binding. The concomitant use of the ELISA and fluorescence polarization assay enables the classification of direct STAT3 inhibitors by their site of action. Our data provide further support that niclosamide inhibits STAT3 through interaction with the DNA-binding domain. Furthermore, the ELISA can support medicinal chemistry efforts by identifying DNA-binding domain inhibitors and allowing the determination of an IC50 value, supporting the ranking of inhibitors and development of structure-activity relationships. Therefore, we propose a tandem evaluation approach to identify small molecules that target the SH2 domain or the DNA-binding domain of STAT3, which allows for quantitative evaluation of candidate STAT3 inhibitors. PMID:27793003

  10. Evaluation of quantitative assays for the identification of direct signal transducer and activator of transcription 3 (STAT3) inhibitors.

    PubMed

    Furtek, Steffanie L; Matheson, Christopher J; Backos, Donald S; Reigan, Philip

    2016-11-22

    In many forms of cancer the signal transducer and activator of transcription 3 (STAT3) transcription factor remains constitutively active, driving cancer survival and progression. The critical role of STAT3 in tumorigenesis has prompted a campaign of drug discovery programs to identify small molecules that disrupt the function of STAT3, with more recent efforts focusing on direct STAT3 inhibition. There are two target binding sites for direct STAT3 inhibitors: the SH2 dimerization domain and the DNA-binding domain. An in vitro fluorescence polarization assay, using recombinant STAT3 protein, has successfully identified compounds that target the SH2 domain; however, no assay has been reported to identify inhibitors that bind the DNA-binding domain. The lack of such a quantitative assay has limited the identification and development of STAT3 DNA-binding domain inhibitors. Here, we report a modified DNA-binding ELISA to incorporate recombinant STAT3 protein to evaluate small molecules that prevent STAT3-DNA binding. The concomitant use of the ELISA and fluorescence polarization assay enables the classification of direct STAT3 inhibitors by their site of action. Our data provide further support that niclosamide inhibits STAT3 through interaction with the DNA-binding domain. Furthermore, the ELISA can support medicinal chemistry efforts by identifying DNA-binding domain inhibitors and allowing the determination of an IC50 value, supporting the ranking of inhibitors and development of structure-activity relationships. Therefore, we propose a tandem evaluation approach to identify small molecules that target the SH2 domain or the DNA-binding domain of STAT3, which allows for quantitative evaluation of candidate STAT3 inhibitors.

  11. Integrative analysis of RNA polymerase II and transcriptional dynamics upon MYC activation.

    PubMed

    de Pretis, Stefano; Kress, Theresia R; Morelli, Marco J; Sabò, Arianna; Locarno, Chiara; Verrecchia, Alessandro; Doni, Mirko; Campaner, Stefano; Amati, Bruno; Pelizzola, Mattia

    2017-10-01

    Overexpression of the MYC transcription factor causes its widespread interaction with regulatory elements in the genome but leads to the up- and down-regulation of discrete sets of genes. The molecular determinants of these selective transcriptional responses remain elusive. Here, we present an integrated time-course analysis of transcription and mRNA dynamics following MYC activation in proliferating mouse fibroblasts, based on chromatin immunoprecipitation, metabolic labeling of newly synthesized RNA, extensive sequencing, and mathematical modeling. Transcriptional activation correlated with the highest increases in MYC binding at promoters. Repression followed a reciprocal scenario, with the lowest gains in MYC binding. Altogether, the relative abundance (henceforth, "share") of MYC at promoters was the strongest predictor of transcriptional responses in diverse cell types, predominating over MYC's association with the corepressor ZBTB17 (also known as MIZ1). MYC activation elicited immediate loading of RNA polymerase II (RNAPII) at activated promoters, followed by increases in pause-release, while repressed promoters showed opposite effects. Gains and losses in RNAPII loading were proportional to the changes in the MYC share, suggesting that repression by MYC may be partly indirect, owing to competition for limiting amounts of RNAPII. Secondary to the changes in RNAPII loading, the dynamics of elongation and pre-mRNA processing were also rapidly altered at MYC regulated genes, leading to the transient accumulation of partially or aberrantly processed mRNAs. Altogether, our results shed light on how overexpressed MYC alters the various phases of the RNAPII cycle and the resulting transcriptional response. © 2017 de Pretis et al.; Published by Cold Spring Harbor Laboratory Press.

  12. 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

  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. 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

  15. A quantitative validated model reveals two phases of transcriptional regulation for the gap gene giant in Drosophila.

    PubMed

    Hoermann, Astrid; Cicin-Sain, Damjan; Jaeger, Johannes

    2016-03-15

    Understanding eukaryotic transcriptional regulation and its role in development and pattern formation is one of the big challenges in biology today. Most attempts at tackling this problem either focus on the molecular details of transcription factor binding, or aim at genome-wide prediction of expression patterns from sequence through bioinformatics and mathematical modelling. Here we bridge the gap between these two complementary approaches by providing an integrative model of cis-regulatory elements governing the expression of the gap gene giant (gt) in the blastoderm embryo of Drosophila melanogaster. We use a reverse-engineering method, where mathematical models are fit to quantitative spatio-temporal reporter gene expression data to infer the regulatory mechanisms underlying gt expression in its anterior and posterior domains. These models are validated through prediction of gene expression in mutant backgrounds. A detailed analysis of our data and models reveals that gt is regulated by domain-specific CREs at early stages, while a late element drives expression in both the anterior and the posterior domains. Initial gt expression depends exclusively on inputs from maternal factors. Later, gap gene cross-repression and gt auto-activation become increasingly important. We show that auto-regulation creates a positive feedback, which mediates the transition from early to late stages of regulation. We confirm the existence and role of gt auto-activation through targeted mutagenesis of Gt transcription factor binding sites. In summary, our analysis provides a comprehensive picture of spatio-temporal gene regulation by different interacting enhancer elements for an important developmental regulator.

  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. Transcription factor family-specific DNA shape readout revealed by quantitative specificity models.

    PubMed

    Yang, Lin; Orenstein, Yaron; Jolma, Arttu; Yin, Yimeng; Taipale, Jussi; Shamir, Ron; Rohs, Remo

    2017-02-06

    Transcription factors (TFs) achieve DNA-binding specificity through contacts with functional groups of bases (base readout) and readout of structural properties of the double helix (shape readout). Currently, it remains unclear whether DNA shape readout is utilized by only a few selected TF families, or whether this mechanism is used extensively by most TF families. We resequenced data from previously published HT-SELEX experiments, the most extensive mammalian TF-DNA binding data available to date. Using these data, we demonstrated the contributions of DNA shape readout across diverse TF families and its importance in core motif-flanking regions. Statistical machine-learning models combined with feature-selection techniques helped to reveal the nucleotide position-dependent DNA shape readout in TF-binding sites and the TF family-specific position dependence. Based on these results, we proposed novel DNA shape logos to visualize the DNA shape preferences of TFs. Overall, this work suggests a way of obtaining mechanistic insights into TF-DNA binding without relying on experimentally solved all-atom structures. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

  18. Quantitative respirator fit testing: dynamic pressure versus aerosol measurement.

    PubMed

    Carpenter, D R; Willeke, K

    1988-10-01

    A noninvasive, fast, inexpensive new fit testing method has been invented which relates the slope of the pressure decay inside a respirator during breath-holding to the fit of the respirator on the wearer's face. The dynamic pressure test has been compared with the conventional aerosol test at different leakage levels. The results of this comparison show that the sensitivity of the dynamic pressure test is similar to that of the aerosol test. The pressure test, however, is independent of leak site and probe location and can be performed on respirators before and after their use.

  19. Dynamic sorting of nuclear components into distinct nucleolar caps during transcriptional inhibition.

    PubMed

    Shav-Tal, Yaron; Blechman, Janna; Darzacq, Xavier; Montagna, Cristina; Dye, Billy T; Patton, James G; Singer, Robert H; Zipori, Dov

    2005-05-01

    Nucleolar segregation is observed under some physiological conditions of transcriptional arrest. This process can be mimicked by transcriptional arrest after actinomycin D treatment leading to the segregation of nucleolar components and the formation of unique structures termed nucleolar caps surrounding a central body. These nucleolar caps have been proposed to arise from the segregation of nucleolar components. We show that contrary to prevailing notion, a group of nucleoplasmic proteins, mostly RNA binding proteins, relocalized from the nucleoplasm to a specific nucleolar cap during transcriptional inhibition. For instance, an exclusively nucleoplasmic protein, the splicing factor PSF, localized to nucleolar caps under these conditions. This structure also contained pre-rRNA transcripts, but other caps contained either nucleolar proteins, PML, or Cajal body proteins and in addition nucleolar or Cajal body RNAs. In contrast to the capping of the nucleoplasmic components, nucleolar granular component proteins dispersed into the nucleoplasm, although at least two (p14/ARF and MRP RNA) were retained in the central body. The nucleolar caps are dynamic structures as determined using photobleaching and require energy for their formation. These findings demonstrate that the process of nucleolar segregation and capping involves energy-dependent repositioning of nuclear proteins and RNAs and emphasize the dynamic characteristics of nuclear domain formation in response to cellular stress.

  20. An rtt109-independent role for vps75 in transcription-associated nucleosome dynamics.

    PubMed

    Selth, Luke A; Lorch, Yahli; Ocampo-Hafalla, Maria T; Mitter, Richard; Shales, Michael; Krogan, Nevan J; Kornberg, Roger D; Svejstrup, Jesper Q

    2009-08-01

    The histone chaperone Vps75 forms a complex with, and stimulates the activity of, the histone acetyltransferase Rtt109. However, Vps75 can also be isolated on its own and might therefore possess Rtt109-independent functions. Analysis of epistatic miniarray profiles showed that VPS75 genetically interacts with factors involved in transcription regulation whereas RTT109 clusters with genes linked to DNA replication/repair. Additional genetic and biochemical experiments revealed a close relationship between Vps75 and RNA polymerase II. Furthermore, Vps75 is recruited to activated genes in an Rtt109-independent manner, and its genome-wide association with genes correlates with transcription rate. Expression microarray analysis identified a number of genes whose normal expression depends on VPS75. Interestingly, histone H2B dynamics at some of these genes are consistent with a role for Vps75 in histone H2A/H2B eviction/deposition during transcription. Indeed, reconstitution of nucleosome disassembly using the ATP-dependent chromatin remodeler Rsc and Vps75 revealed that these proteins can cooperate to remove H2A/H2B dimers from nucleosomes. These results indicate a role for Vps75 in nucleosome dynamics during transcription, and importantly, this function appears to be largely independent of Rtt109.

  1. Role of transcriptional regulation in the evolution of plant phenotype: A dynamic systems approach.

    PubMed

    Rodríguez-Mega, Emiliano; Piñeyro-Nelson, Alma; Gutierrez, Crisanto; García-Ponce, Berenice; Sánchez, María De La Paz; Zluhan-Martínez, Estephania; Álvarez-Buylla, Elena R; Garay-Arroyo, Adriana

    2015-03-02

    A growing body of evidence suggests that alterations in transcriptional regulation of genes involved in modulating development are an important part of phenotypic evolution, and this can be documented among species and within populations. While the effects of differential transcriptional regulation in organismal development have been preferentially studied in animal systems, this phenomenon has also been addressed in plants. In this review, we summarize evidence for cis-regulatory mutations, trans-regulatory changes and epigenetic modifications as molecular events underlying important phenotypic alterations, and thus shaping the evolution of plant development. We postulate that a mechanistic understanding of why such molecular alterations have a key role in development, morphology and evolution will have to rely on dynamic models of complex regulatory networks that consider the concerted action of genetic and nongenetic components, and that also incorporate the restrictions underlying the genotype to phenotype mapping process. Developmental Dynamics, 2015. © 2015 Wiley Periodicals, Inc.

  2. Dynamic holographic interferometry by photorefractive crystals for quantitative deformation measurements.

    PubMed

    Pouet, B; Krishnaswamy, S

    1996-02-10

    A holographic interferometer that uses two-wave mixing in a photorefractive (Bi12SiO20) crystal under an applied ac field is described. The interferometer uses a repetitive sequence of separate record and readout times to obtain quasi real-time holographic interferograms of vibrating objects. It is shown that a good signal-to-noise ratio of the interferometer is obtained by turning off the object illumination and the applied ac field during readout of the hologram. The good signal-to-noise ratio of the resulting holographic interferograms enables phase measurement, which allows for quantitative deformation analysis.

  3. Transcriptional Dynamics During Human Adipogenesis and Its Link to Adipose Morphology and Distribution.

    PubMed

    Ehrlund, Anna; Mejhert, Niklas; Björk, Christel; Andersson, Robin; Kulyté, Agné; Åström, Gaby; Itoh, Masayoshi; Kawaji, Hideya; Lassmann, Timo; Daub, Carsten O; Carninci, Piero; Forrest, Alistair R R; Hayashizaki, Yoshihide; Sandelin, Albin; Ingelsson, Erik; Rydén, Mikael; Laurencikiene, Jurga; Arner, Peter; Arner, Erik

    2017-01-01

    White adipose tissue (WAT) can develop into several phenotypes with different pathophysiological impact on type 2 diabetes. To better understand the adipogenic process, the transcriptional events that occur during in vitro differentiation of human adipocytes were investigated and the findings linked to WAT phenotypes. Single-molecule transcriptional profiling provided a detailed map of the expressional changes of genes, enhancers, and long noncoding RNAs, where different types of transcripts share common dynamics during differentiation. Common signatures include early downregulated, transient, and late induced transcripts, all of which are linked to distinct developmental processes during adipogenesis. Enhancers expressed during adipogenesis overlap significantly with genetic variants associated with WAT distribution. Transiently expressed and late induced genes are associated with hypertrophic WAT (few but large fat cells), a phenotype closely linked to insulin resistance and type 2 diabetes. Transcription factors that are expressed early or transiently affect differentiation and adipocyte function and are controlled by several well-known upstream regulators such as glucocorticosteroids, insulin, cAMP, and thyroid hormones. Taken together, our results suggest a complex but highly coordinated regulation of adipogenesis. © 2017 by the American Diabetes Association.

  4. Research resource: the dynamic transcriptional profile of sertoli cells during the progression of spermatogenesis.

    PubMed

    Zimmermann, Céline; Stévant, Isabelle; Borel, Christelle; Conne, Béatrice; Pitetti, Jean-Luc; Calvel, Pierre; Kaessmann, Henrik; Jégou, Bernard; Chalmel, Frédéric; Nef, Serge

    2015-04-01

    Sertoli cells (SCs), the only somatic cells within seminiferous tubules, associate intimately with developing germ cells. They not only provide physical and nutritional support but also secrete factors essential to the complex developmental processes of germ cell proliferation and differentiation. The SC transcriptome must therefore adapt rapidly during the different stages of spermatogenesis. We report comprehensive genome-wide expression profiles of pure populations of SCs isolated at 5 distinct stages of the first wave of mouse spermatogenesis, using RNA sequencing technology. We were able to reconstruct about 13 901 high-confidence, nonredundant coding and noncoding transcripts, characterized by complex alternative splicing patterns with more than 45% comprising novel isoforms of known genes. Interestingly, roughly one-fifth (2939) of these genes exhibited a dynamic expression profile reflecting the evolving role of SCs during the progression of spermatogenesis, with stage-specific expression of genes involved in biological processes such as cell cycle regulation, metabolism and energy production, retinoic acid synthesis, and blood-testis barrier biogenesis. Finally, regulatory network analysis identified the transcription factors endothelial PAS domain-containing protein 1 (EPAS1/Hif2α), aryl hydrocarbon receptor nuclear translocator (ARNT/Hif1β), and signal transducer and activator of transcription 1 (STAT1) as potential master regulators driving the SC transcriptional program. Our results highlight the plastic transcriptional landscape of SCs during the progression of spermatogenesis and provide valuable resources to better understand SC function and spermatogenesis and its related disorders, such as male infertility.

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

    PubMed Central

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

    2015-01-01

    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 (DRG). In the present study we investigated 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, X, as well as other brainstem nuclei during different stages of development into adult stage. We find that genetically identified Brn3bKO 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. PMID:26356988

  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. © 2015 Wiley Periodicals, Inc.

  7. Improvements in Diagnostic Accuracy with Quantitative Dynamic Contrast Enhanced MRI

    DTIC Science & Technology

    2012-12-01

    0.1, 0.2, 0.3, 0.4, 0.5 mM Omniscan (Gd- DTPA GE Healthcare). We discovered large variations in proton density values found by fitting variable...Transplanted Rodent Prostate Tumors. MRM 2004; 51:487-494 [2] Tofts P, Modeling Tracer Kinetics in Dynamic Gd- DTPA Imaging. JMRI 2005; 7:91-101 [3

  8. Quantitative Evaluation of the Reticuloendothelial System Function with Dynamic MRI

    PubMed Central

    Liu, Ting; Choi, Hoon; Zhou, Rong; Chen, I-Wei

    2014-01-01

    Purpose To evaluate the reticuloendothelial system (RES) function by real-time imaging blood clearance as well as hepatic uptake of superparamagnetic iron oxide nanoparticle (SPIO) using dynamic magnetic resonance imaging (MRI) with two-compartment pharmacokinetic modeling. Materials and Methods Kinetics of blood clearance and hepatic accumulation were recorded in young adult male 01b74 athymic nude mice by dynamic T2* weighted MRI after the injection of different doses of SPIO nanoparticles (0.5, 3 or 10 mg Fe/kg). Association parameter, Kin, dissociation parameter, Kout, and elimination constant, Ke, derived from dynamic data with two-compartment model, were used to describe active binding to Kupffer cells and extrahepatic clearance. The clodrosome and liposome were utilized to deplete macrophages and block the RES function to evaluate the capability of the kinetic parameters for investigation of macrophage function and density. Results The two-compartment model provided a good description for all data and showed a low sum squared residual for all mice (0.27±0.03). A lower Kin, a lower Kout and a lower Ke were found after clodrosome treatment, whereas a lower Kin, a higher Kout and a lower Ke were observed after liposome treatment in comparison to saline treatment (P<0.005). Conclusion Dynamic SPIO-enhanced MR imaging with two-compartment modeling can provide information on RES function on both a cell number and receptor function level. PMID:25090653

  9. 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. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. Reliable gene expression analysis by reverse transcription-quantitative PCR: reporting and minimizing the uncertainty in data accuracy.

    PubMed

    Remans, Tony; Keunen, Els; Bex, Geert Jan; Smeets, Karen; Vangronsveld, Jaco; Cuypers, Ann

    2014-10-01

    Reverse transcription-quantitative PCR (RT-qPCR) has been widely adopted to measure differences in mRNA levels; however, biological and technical variation strongly affects the accuracy of the reported differences. RT-qPCR specialists have warned that, unless researchers minimize this variability, they may report inaccurate differences and draw incorrect biological conclusions. The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines describe procedures for conducting and reporting RT-qPCR experiments. The MIQE guidelines enable others to judge the reliability of reported results; however, a recent literature survey found low adherence to these guidelines. Additionally, even experiments that use appropriate procedures remain subject to individual variation that statistical methods cannot correct. For example, since ideal reference genes do not exist, the widely used method of normalizing RT-qPCR data to reference genes generates background noise that affects the accuracy of measured changes in mRNA levels. However, current RT-qPCR data reporting styles ignore this source of variation. In this commentary, we direct researchers to appropriate procedures, outline a method to present the remaining uncertainty in data accuracy, and propose an intuitive way to select reference genes to minimize uncertainty. Reporting the uncertainty in data accuracy also serves for quality assessment, enabling researchers and peer reviewers to confidently evaluate the reliability of gene expression data. © 2014 American Society of Plant Biologists. All rights reserved.

  11. Quantitative Analysis of Peripheral Tissue Perfusion Using Spatiotemporal Molecular Dynamics

    PubMed Central

    Lee, Jungsul; Koh, Gou Young; Kwon, Kihwan; Choi, Chulhee

    2009-01-01

    Background Accurate measurement of peripheral tissue perfusion is challenging but necessary to diagnose peripheral vascular insufficiency. Because near infrared (NIR) radiation can penetrate relatively deep into tissue, significant attention has been given to intravital NIR fluorescence imaging. Methodology/Principal Findings We developed a new optical imaging-based strategy for quantitative measurement of peripheral tissue perfusion by time-series analysis of local pharmacokinetics of the NIR fluorophore, indocyanine green (ICG). Time-series NIR fluorescence images were obtained after injecting ICG intravenously in a murine hindlimb ischemia model. Mathematical modeling and computational simulations were used for translating time-series ICG images into quantitative pixel perfusion rates and a perfusion map. We could successfully predict the prognosis of ischemic hindlimbs based on the perfusion profiles obtained immediately after surgery, which were dependent on the preexisting collaterals. This method also reflected increases in perfusion and improvements in prognosis of ischemic hindlimbs induced by treatment with vascular endothelial growth factor and COMP-angiopoietin-1. Conclusions/Significance We propose that this novel NIR-imaging-based strategy is a powerful tool for biomedical studies related to the evaluation of therapeutic interventions directed at stimulating angiogenesis. PMID:19169354

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

    PubMed

    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

    2013-11-01

    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.

  13. 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

  14. Dynamic transcriptional signature and cell fate analysis reveals plasticity of individual neural plate border cells.

    PubMed

    Roellig, Daniela; Tan-Cabugao, Johanna; Esaian, Sevan; Bronner, Marianne E

    2017-03-29

    The 'neural plate border' of vertebrate embryos contains precursors of neural crest and placode cells, both defining vertebrate characteristics. How these lineages segregate from neural and epidermal fates has been a matter of debate. We address this by performing a fine-scale quantitative temporal analysis of transcription factor expression in the neural plate border of chick embryos. The results reveal significant overlap of transcription factors characteristic of multiple lineages in individual border cells from gastrula through neurula stages. Cell fate analysis using a Sox2 (neural) enhancer reveals that cells that are initially Sox2+ cells can contribute not only to neural tube but also to neural crest and epidermis. Moreover, modulating levels of Sox2 or Pax7 alters the apportionment of neural tube versus neural crest fates. Our results resolve a long-standing question and suggest that many individual border cells maintain ability to contribute to multiple ectodermal lineages until or beyond neural tube closure.

  15. Dynamical features of deoxyribonucleic acid and configuration transition in the transcription process

    NASA Astrophysics Data System (ADS)

    Pang, Xiao-feng; Feng, Yuan Ping; Zhang, Huai-wu; Assad, S. M.

    2006-10-01

    Biological functions and genetic features of DNA, such as duplication, transcription and gene expression, are mainly determined by its structure, but depend also on the temperature and features of solution, such as salt concentration. We study the influence of temperature and salt concentration on the conformation changes and transcription of DNA by using a new dynamical model. This new model admits three degrees of freedom per base-pair: two displacement variables related to the vibrations of hydrogen atom in the hydrogen bonds and base (nucleotide), respectively, and an angular variable related to the rotation of base. The important role of motion of hydrogen atom in the hydrogen bonds is specially stressed in this model. This is helpful to reveal the mechanism of transcription of DNA. According to their properties of motion, we first give the Hamiltonian of the system, corresponding equations of motion and their soliton-solutions. The solitons are the excitation states formed by the displacements of hydrogen atoms and bases and the rotations of bases, arising from the energy absorbed by DNA, in the systems, respectively. By applying the transfer integral method we obtain the thermodynamic properties (e.g. free energy and entropy) of the thermal excitation state of DNA at the biological temperature in this model. According to the properties of these thermodynamic functions obtained we study the mechanism and processes of melting and transcription of DNA with the aid of the transforms of energy carried by the soliton in such a case. We further give the properties of the transcription of DNA with the help of the average value of the mean square of displacement of hydrogen atom, and the values of subcritical temperature and force of the phase transition are also found. Finally, we conclude that the transcription of DNA not only depends directly on the properties of its structure and of energy absorbed by it, but also is influenced by the temperature and salt

  16. Dynamic and still microcirculatory image analysis for quantitative microcirculation research

    NASA Astrophysics Data System (ADS)

    Ying, Xiaoyou; Xiu, Rui-juan

    1994-05-01

    Based on analyses of various types of digital microcirculatory image (DMCI), we summed up the image features of DMCI, the digitizing demands for digital microcirculatory imaging, and the basic characteristics of the DMCI processing. A dynamic and still imaging separation processing (DSISP) mode was designed for developing a DMCI workstation and the DMCI processing. Original images in this study were clinical microcirculatory images from human finger nail-bed and conjunctiva microvasculature, and intravital microvascular network images from animal tissue or organs. A series of dynamic and still microcirculatory image analysis functions were developed in this study. The experimental results indicate most of the established analog video image analysis methods for microcirculatory measurement could be realized in a more flexible way based on the DMCI. More information can be rapidly extracted from the quality improved DMCI by employing intelligence digital image analysis methods. The DSISP mode is very suitable for building a DMCI workstation.

  17. 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

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

    PubMed

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

    2011-04-18

    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.

  19. 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. PsycINFO Database Record (c) 2013 APA, all rights reserved

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

    PubMed

    Patel, Pranav; Landt, Olfert; Kaiser, Marco; Faye, Oumar; Koppe, Tanja; Lass, Ulrich; Sall, Amadou A; Niedrig, Matthias

    2013-02-14

    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. 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. 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. 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.

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

    PubMed

    Li, Zitong; Sillanpää, Mikko J

    2015-12-01

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

  2. Dynamically monitoring the gene expression of dual fluorophore in the cell cycle with quantitative spectrum analysis

    NASA Astrophysics Data System (ADS)

    Lee, Ja-Yun; Wu, Tzong-Yuan; Hsu, I.-Jen

    2008-04-01

    The cloning and transcription techniques on gene cloned fluorescent proteins have been widely used in many applications. They have been used as reporters of some conditions in a series of reactions. However, it is usually difficult to monitor the specific target with the exactly number of proteins during the process in turbid media, especially at micrometer scales. We successfully revealed an alternative way to monitor the cell cycle behavior and quantitatively analyzed the target cells with green and red fluorescent proteins (GFP and RFP) during different phases of the cell cycle by quantitatively analyzing its behavior and also monitoring its spatial distribution.

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

    PubMed Central

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

    2016-01-01

    Motivation: Rapid advances in live-cell imaging analysis and mathematical modeling have produced a large amount of quantitative data on spatiotemporal dynamics of biological objects ranging from molecules to organisms. There is now a crucial need to bring these large amounts of quantitative biological dynamics data together centrally in a coherent and systematic manner. This will facilitate the reuse of this data for further analysis. Results: We have developed the Systems Science of Biological Dynamics database (SSBD) to store and share quantitative biological dynamics data. SSBD currently provides 311 sets of quantitative data for single molecules, nuclei and whole organisms in a wide variety of model organisms from Escherichia coli to Mus musculus. The data are provided in Biological Dynamics Markup Language format and also through a REST API. In addition, SSBD provides 188 sets of time-lapse microscopy images from which the quantitative data were obtained and software tools for data visualization and analysis. Availability and Implementation: SSBD is accessible at http://ssbd.qbic.riken.jp. Contact: sonami@riken.jp PMID:27412095

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

    PubMed

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

    2016-11-15

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

  5. Spatial-temporal transcriptional dynamics of long non-coding RNAs in human brain.

    PubMed

    Zhang, Xiao-Qin; Wang, Ze-Lin; Poon, Ming-Wai; Yang, Jian-Hua

    2017-08-15

    The functional architecture of the human brain is greatly determined by the temporal and spatial regulation of the transcription process. However, the spatial and temporal transcriptional landscape of long non-coding RNAs (lncRNAs) during human brain development remains poorly understood. Here, we report the genome-wide lncRNA transcriptional analysis in an extensive series of 1340 post-mortem human brain specimens collected from 16 regions spanning the period from early embryo development to late adulthood. We discovered that lncRNA transcriptome dramatically changed during fetal development, while transited to a surprisingly relatively stable state after birth till the late adulthood. We also discovered that the transcription map of lncRNAs was spatially different, and that this spatial difference was developmentally regulated. Of the 16 brain regions explored (cerebellar cortex, thalamus, striatum, amygdala, hippocampus and 11 neocortex areas), cerebellar cortex showed the most distinct lncRNA expression features from all remaining brain regions throughout the whole developmental period, reflecting its unique developmental and functional features. Furthermore, by characterizing the functional modules and cellular processes of the spatial-temporal dynamic lncRNAs, we found that they were significantly associated with the RNA processing, neuron differentiation and synaptic signal transportation processes. Furthermore, we found that many lncRNAs associated with the neurodegenerative Alzheimer and Parkinson diseases were co-expressed in the fetal development of the human brain, and affected the convergent biological processes. In summary, our study provides a comprehensive map for lncRNA transcription dynamics in human brain development, which might shed light on the understanding of the molecular underpinnings of human brain function and disease. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  6. Quantitative Assessment of Dynamic Control of Fingertip Forces After Pollicization

    PubMed Central

    Lightdale-Miric, Nina; Mueske, Nicole M.; Dayanidhi, Sudarshan; Loiselle, Jennifer; Berggren, Jamie; Lawrence, Emily L.; Stevanovic, Milan; Valero-Cuevas, Francisco J.; Wren, Tishya A. L.

    2014-01-01

    Dexterity after finger pollicization (reconstruction to thumb) is critical to functional outcomes. While most tests of hand function evaluate a combination of strength, coordination, and motor control, the Strength-Dexterity (S-D) paradigm focuses on the dynamic control of fingertip forces. We evaluated 10 pollicized and 5 non-pollicized hands from 8 participants ages 4–17 years (2 female, 6 male; 10.6 ± 4.5 years). Participants partially compressed and held an instrumented spring prone to buckling between the thumb and first finger to quantify dynamic control over the direction and magnitude of fingertip forces. They also completed traditional functional tests including grip, lateral pinch, and tripod pinch strength, Box and Blocks, and 9-hole peg test. Six of 10 pollicized hands and all non-pollicized hands had S-D scores comparable to typically developing children. However, dynamical analysis showed that pollicized hands exhibit greater variability in compression force, indicating poorer corrective action. Almost all pollicized hands scored below the normal range for the traditional functional tests. The S-D test Z-scores correlated moderately with Z-scores from the other functional tests (r=0.54–0.61; p= 0.02–0.04) but more weakly than amongst the other functional measures (r=0.58–0.83; p=0.0002–0.02), suggesting that the S-D test captures a different domain of function. A higher incidence of radial absence in the hands with poor S-D scores (3/4 vs. 0/6 in hands with normal S-D scores, p=0.03) was the only clinical characteristic associated with S-D outcome. Overall, these results suggest that while most pollicized hands can control fingertip forces, the nature of that control is altered. PMID:25262333

  7. Interplay of microRNAs, transcription factors and target genes: linking dynamic expression changes to function.

    PubMed

    Nazarov, Petr V; Reinsbach, Susanne E; Muller, Arnaud; Nicot, Nathalie; Philippidou, Demetra; Vallar, Laurent; Kreis, Stephanie

    2013-03-01

    MicroRNAs (miRNAs) are ubiquitously expressed small non-coding RNAs that, in most cases, negatively regulate gene expression at the post-transcriptional level. miRNAs are involved in fine-tuning fundamental cellular processes such as proliferation, cell death and cell cycle control and are believed to confer robustness to biological responses. Here, we investigated simultaneously the transcriptional changes of miRNA and mRNA expression levels over time after activation of the Janus kinase/Signal transducer and activator of transcription (Jak/STAT) pathway by interferon-γ stimulation of melanoma cells. To examine global miRNA and mRNA expression patterns, time-series microarray data were analysed. We observed delayed responses of miRNAs (after 24-48 h) with respect to mRNAs (12-24 h) and identified biological functions involved at each step of the cellular response. Inference of the upstream regulators allowed for identification of transcriptional regulators involved in cellular reactions to interferon-γ stimulation. Linking expression profiles of transcriptional regulators and miRNAs with their annotated functions, we demonstrate the dynamic interplay of miRNAs and upstream regulators with biological functions. Finally, our data revealed network motifs in the form of feed-forward loops involving transcriptional regulators, mRNAs and miRNAs. Additional information obtained from integrating time-series mRNA and miRNA data may represent an important step towards understanding the regulatory principles of gene expression.

  8. Interplay of microRNAs, transcription factors and target genes: linking dynamic expression changes to function

    PubMed Central

    Nazarov, Petr V.; Reinsbach, Susanne E.; Muller, Arnaud; Nicot, Nathalie; Philippidou, Demetra; Vallar, Laurent; Kreis, Stephanie

    2013-01-01

    MicroRNAs (miRNAs) are ubiquitously expressed small non-coding RNAs that, in most cases, negatively regulate gene expression at the post-transcriptional level. miRNAs are involved in fine-tuning fundamental cellular processes such as proliferation, cell death and cell cycle control and are believed to confer robustness to biological responses. Here, we investigated simultaneously the transcriptional changes of miRNA and mRNA expression levels over time after activation of the Janus kinase/Signal transducer and activator of transcription (Jak/STAT) pathway by interferon-γ stimulation of melanoma cells. To examine global miRNA and mRNA expression patterns, time-series microarray data were analysed. We observed delayed responses of miRNAs (after 24–48 h) with respect to mRNAs (12–24 h) and identified biological functions involved at each step of the cellular response. Inference of the upstream regulators allowed for identification of transcriptional regulators involved in cellular reactions to interferon-γ stimulation. Linking expression profiles of transcriptional regulators and miRNAs with their annotated functions, we demonstrate the dynamic interplay of miRNAs and upstream regulators with biological functions. Finally, our data revealed network motifs in the form of feed-forward loops involving transcriptional regulators, mRNAs and miRNAs. Additional information obtained from integrating time-series mRNA and miRNA data may represent an important step towards understanding the regulatory principles of gene expression. PMID:23335783

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

    PubMed

    Kast, Erin J; Nguyen, Minh-Duyen T; Lawrence, Rosalie E; Rabeler, Christina; Kaplinsky, Nicholas J

    2013-10-12

    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. 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. The RootScope system is a simple and powerful platform for investigating the heat shock response in plants.

  10. 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

  11. 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

  12. 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. Copyright © 2014 Elsevier Inc. All rights reserved.

  13. Quantitative analysis of dynamic association in live biological fluorescent samples.

    PubMed

    Ruusuvuori, Pekka; Paavolainen, Lassi; Rutanen, Kalle; Mäki, Anita; Huttunen, Heikki; Marjomäki, Varpu

    2014-01-01

    Determining vesicle localization and association in live microscopy may be challenging due to non-simultaneous imaging of rapidly moving objects with two excitation channels. Besides errors due to movement of objects, imaging may also introduce shifting between the image channels, and traditional colocalization methods cannot handle such situations. Our approach to quantifying the association between tagged proteins is to use an object-based method where the exact match of object locations is not assumed. Point-pattern matching provides a measure of correspondence between two point-sets under various changes between the sets. Thus, it can be used for robust quantitative analysis of vesicle association between image channels. Results for a large set of synthetic images shows that the novel association method based on point-pattern matching demonstrates robust capability to detect association of closely located vesicles in live cell-microscopy where traditional colocalization methods fail to produce results. In addition, the method outperforms compared Iterated Closest Points registration method. Results for fixed and live experimental data shows the association method to perform comparably to traditional methods in colocalization studies for fixed cells and to perform favorably in association studies for live cells.

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

    PubMed Central

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

    2016-01-01

    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. PMID:26986359

  15. Tracking of chromosome dynamics in live Streptococcus pneumoniae reveals that transcription promotes chromosome segregation.

    PubMed

    Kjos, Morten; Veening, Jan-Willem

    2014-03-01

    Chromosome segregation is an essential part of the bacterial cell cycle but is poorly characterized in oval-shaped streptococci. Using time-lapse fluorescence microscopy and total internal reflection fluorescence microscopy, we have tracked the dynamics of chromosome segregation in live cells of the human pathogen Streptococcus pneumoniae. Our observations show that the chromosome segregation process last for two-thirds of the total cell cycle; the origin region segregates rapidly in the early stages of the cell cycle while nucleoid segregation finishes just before cell division. Previously we have demonstrated that the DNA-binding protein ParB and the condensin SMC promote efficient chromosome segregation, likely by an active mechanism. We now show that in the absence of SMC, cell division can occur over the unsegregated chromosomes. However, neither smc nor parB are essential in S. pneumoniae, suggesting the importance of additional mechanisms. Here we have identified the process of transcription as one of these mechanisms important for chromosome segregation in S. pneumoniae. Transcription inhibitors rifampicin and streptolydigin as well as mutants affected in transcription elongation cause chromosome segregation defects. Together, our results highlight the importance of passive (or indirect) processes such as transcription for chromosome segregation in oval-shaped bacteria.

  16. 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.

  17. 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.

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

    PubMed

    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.

  19. Achieving large dynamic range control of gene expression with a compact RNA transcription-translation regulator.

    PubMed

    Westbrook, Alexandra M; Lucks, Julius B

    2017-04-06

    RNA transcriptional regulators are emerging as versatile components for genetic network construction. However, these regulators suffer from incomplete repression in their OFF state, making their dynamic range less than that of their protein counterparts. This incomplete repression causes expression leak, which impedes the construction of larger synthetic regulatory networks as leak propagation can interfere with desired network function. To address this, we demonstrate how naturally derived antisense RNA-mediated transcriptional regulators can be configured to regulate both transcription and translation in a single compact RNA mechanism that functions in Escherichia coli. Using in vivo gene expression assays, we show that a combination of transcriptional termination and ribosome binding site sequestration increases repression from 85% to 98%, or activation from 10-fold to over 900-fold, in response to cognate antisense RNAs. We also show that orthogonal repressive versions of this mechanism can be created through engineering minimal antisense RNAs. Finally, to demonstrate the utility of this mechanism, we use it to reduce network leak in an RNA-only cascade. We anticipate these regulators will find broad use as synthetic biology moves beyond parts engineering to the design and construction of more sophisticated regulatory networks.

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

    PubMed

    Geier, Florian; Lohmann, Jan U; Gerstung, Moritz; Maier, Annette T; Timmer, Jens; Fleck, Christian

    2008-01-01

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

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

    PubMed

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

    2015-07-06

    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.

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

    PubMed

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

    2016-03-24

    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.

  3. 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

  4. 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

  5. Progressive tarsal patterning in the Drosophila by temporally dynamic regulation of transcription factor genes.

    PubMed

    Natori, Kohei; Tajiri, Reiko; Furukawa, Shiori; Kojima, Tetsuya

    2012-01-15

    The morphology of insect appendages, such as the number and proportion of leg tarsal segments, is immensely diverse. In Drosophila melanogaster, adult legs have five tarsal segments. Accumulating evidence indicates that tarsal segments are formed progressively through dynamic changes in the expression of transcription factor genes, such as Bar genes, during development. In this study, to examine further the basis of progressive tarsal patterning, the precise expression pattern and function of several transcription factor genes were investigated in relation to the temporal regulation of Bar expression. The results indicate that nubbin is expressed over a broad region at early stages but gradually disappears from the middle of the tarsal region. This causes the progressive expansion of rotund expression, which in turn progressively represses Bar expression, leading to the formation of the tarsal segment 3. The region corresponding to the tarsal segment 4 is formed when apterous expression is initiated, which renders Bar expression refractory to rotund. In addition, the tarsal segment 2 appears to be derived from the region that expresses Bar at a very early stage. Cessation of Bar expression in this region requires the function of spineless, which also regulates rotund expression. These findings indicate that the temporally dynamic regulatory interaction of these transcription factor genes is the fundamental basis of the progressive patterning of the tarsal region.

  6. 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.

  7. The molecular mechanism and post-transcriptional regulation characteristic of Tetragenococcus halophilus acclimation to osmotic stress revealed by quantitative proteomics.

    PubMed

    Lin, Jieting; Liang, Hebin; Yan, Junwei; Luo, Lixin

    2017-09-25

    Tetragenococcus halophilus is a moderate halophilic bacterium which was widely used in fermentation processes, growing in a broad range of salinity conditions, and can survive a saturated 26.47% w/w NaCl concentration. However, the mechanism of this outstanding ability to acclimate to extracellular osmotic stress still remains unknown. The current study firstly conducted a quantitative proteomic analysis to identify alterations of the cellular proteome under both hypo-osmotic and hyper-osmotic stress conditions. A total of 1405 proteins were identified and differentially accumulated proteins were analyzed, further functional annotations were performed using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes. The results revealed that both hypo- and hyper-osmotic stresses have prominent impacts on the synthesis of proteins involving in multiple cellular functions. Further analyses of the differentially accumulated proteins suggested that the adaptation strategies T. halophilus applies to deal with hypo- and hyper-osmotic stress conditions may be distinct. Comparison of the differentially accumulated proteins in both transcriptomic and proteomic study indicated the existence of post-transcriptional modification during salinity adaptation of T. halophilus. The current study generated a proteomic atlas of differentially accumulated proteins under both hypo- and hyper-osmotic stress conditions, provided an overview of the molecular mechanism of osmotic acclimation of T. halophilus. The current study aimed to reveal how the moderately halophilic Tetragenococcus halophilus adapt to extracellular salinity stress, which is the first proteomic study analyzing the differences in proteome of Tetragenococcus halophilus between hypo- and hyper-osmotic stress to our knowledge. By analyzing the differences in the accumulating levels of the proteome via isobaric labeling-based quantitative proteomic study, we identified proteins with significantly different accumulation

  8. Quantitative and Qualitative Stem Rust Resistance Factors in Barley Are Associated with Transcriptional Suppression of Defense Regulons

    PubMed Central

    Moscou, Matthew J.; Lauter, Nick; Steffenson, Brian; Wise, Roger P.

    2011-01-01

    Stem rust (Puccinia graminis f. sp. tritici; Pgt) is a devastating fungal disease of wheat and barley. Pgt race TTKSK (isolate Ug99) is a serious threat to these Triticeae grain crops because resistance is rare. In barley, the complex Rpg-TTKSK locus on chromosome 5H is presently the only known source of qualitative resistance to this aggressive Pgt race. Segregation for resistance observed on seedlings of the Q21861 × SM89010 (QSM) doubled-haploid (DH) population was found to be predominantly qualitative, with little of the remaining variance explained by loci other than Rpg-TTKSK. In contrast, analysis of adult QSM DH plants infected by field inoculum of Pgt race TTKSK in Njoro, Kenya, revealed several additional quantitative trait loci that contribute to resistance. To molecularly characterize these loci, Barley1 GeneChips were used to measure the expression of 22,792 genes in the QSM population after inoculation with Pgt race TTKSK or mock-inoculation. Comparison of expression Quantitative Trait Loci (eQTL) between treatments revealed an inoculation-dependent expression polymorphism implicating Actin depolymerizing factor3 (within the Rpg-TTKSK locus) as a candidate susceptibility gene. In parallel, we identified a chromosome 2H trans-eQTL hotspot that co-segregates with an enhancer of Rpg-TTKSK-mediated, adult plant resistance discovered through the Njoro field trials. Our genome-wide eQTL studies demonstrate that transcript accumulation of 25% of barley genes is altered following challenge by Pgt race TTKSK, but that few of these genes are regulated by the qualitative Rpg-TTKSK on chromosome 5H. It is instead the chromosome 2H trans-eQTL hotspot that orchestrates the largest inoculation-specific responses, where enhanced resistance is associated with transcriptional suppression of hundreds of genes scattered throughout the genome. Hence, the present study associates the early suppression of genes expressed in this host–pathogen interaction with enhancement

  9. Quantitative and qualitative stem rust resistance factors in barley are associated with transcriptional suppression of defense regulons.

    PubMed

    Moscou, Matthew J; Lauter, Nick; Steffenson, Brian; Wise, Roger P

    2011-07-01

    Stem rust (Puccinia graminis f. sp. tritici; Pgt) is a devastating fungal disease of wheat and barley. Pgt race TTKSK (isolate Ug99) is a serious threat to these Triticeae grain crops because resistance is rare. In barley, the complex Rpg-TTKSK locus on chromosome 5H is presently the only known source of qualitative resistance to this aggressive Pgt race. Segregation for resistance observed on seedlings of the Q21861 × SM89010 (QSM) doubled-haploid (DH) population was found to be predominantly qualitative, with little of the remaining variance explained by loci other than Rpg-TTKSK. In contrast, analysis of adult QSM DH plants infected by field inoculum of Pgt race TTKSK in Njoro, Kenya, revealed several additional quantitative trait loci that contribute to resistance. To molecularly characterize these loci, Barley1 GeneChips were used to measure the expression of 22,792 genes in the QSM population after inoculation with Pgt race TTKSK or mock-inoculation. Comparison of expression Quantitative Trait Loci (eQTL) between treatments revealed an inoculation-dependent expression polymorphism implicating Actin depolymerizing factor3 (within the Rpg-TTKSK locus) as a candidate susceptibility gene. In parallel, we identified a chromosome 2H trans-eQTL hotspot that co-segregates with an enhancer of Rpg-TTKSK-mediated, adult plant resistance discovered through the Njoro field trials. Our genome-wide eQTL studies demonstrate that transcript accumulation of 25% of barley genes is altered following challenge by Pgt race TTKSK, but that few of these genes are regulated by the qualitative Rpg-TTKSK on chromosome 5H. It is instead the chromosome 2H trans-eQTL hotspot that orchestrates the largest inoculation-specific responses, where enhanced resistance is associated with transcriptional suppression of hundreds of genes scattered throughout the genome. Hence, the present study associates the early suppression of genes expressed in this host-pathogen interaction with enhancement

  10. Development of a neutralization assay for influenza virus using an endpoint assessment based on quantitative reverse-transcription PCR.

    PubMed

    Teferedegne, Belete; Lewis, Andrew M; Peden, Keith; Murata, Haruhiko

    2013-01-01

    A microneutralization assay using an ELISA-based endpoint assessment (ELISA-MN) is widely used to measure the serological response to influenza virus infection and vaccination. We have developed an alternative microneutralization assay for influenza virus using a quantitative reverse transcription PCR-based endpoint assessment (qPCR-MN) in order to improve upon technical limitations associated with ELISA-MN. For qPCR-MN, infected MDCK-London cells in 96-well cell-culture plates are processed with minimal steps such that resulting samples are amenable to high-throughput analysis by downstream one-step quantitative reverse transcription PCR (qRT-PCR; SYBR Green chemistry with primers targeting a conserved region of the M1 gene of influenza A viruses). The growth curves of three recent vaccine strains demonstrated that the qRT-PCR signal detected at 6 hours post-infection reflected an amplification of at least 100-fold over input. Using ferret antisera, we have established the feasibility of measuring virus neutralization at 6 hours post-infection, a duration likely confined to a single virus-replication cycle. The neutralization titer for qPCR-MN was defined as the highest reciprocal serum dilution necessary to achieve a 90% inhibition of the qRT-PCR signal; this endpoint was found to be in agreement with ELISA-MN using the same critical reagents in each assay. qPCR-MN was robust with respect to assay duration (6 hours vs. 12 hours). In addition, qPCR-MN appeared to be compliant with the Percentage Law (i.e., virus neutralization results appear to be consistent over an input virus dose ranging from 500 to 12,000 TCID(50)). Compared with ELISA-MN, qPCR-MN might have inherent properties conducive to reducing intra- and inter-laboratory variability while affording suitability for automation and high-throughput uses. Finally, our qRT-PCR-based approach may be broadly applicable to the development of neutralization assays for a wide variety of viruses.

  11. Development of a Neutralization Assay for Influenza Virus Using an Endpoint Assessment Based on Quantitative Reverse-Transcription PCR

    PubMed Central

    Teferedegne, Belete; Lewis, Andrew M.; Peden, Keith; Murata, Haruhiko

    2013-01-01

    A microneutralization assay using an ELISA-based endpoint assessment (ELISA-MN) is widely used to measure the serological response to influenza virus infection and vaccination. We have developed an alternative microneutralization assay for influenza virus using a quantitative reverse transcription PCR-based endpoint assessment (qPCR-MN) in order to improve upon technical limitations associated with ELISA-MN. For qPCR-MN, infected MDCK-London cells in 96-well cell-culture plates are processed with minimal steps such that resulting samples are amenable to high-throughput analysis by downstream one-step quantitative reverse transcription PCR (qRT-PCR; SYBR Green chemistry with primers targeting a conserved region of the M1 gene of influenza A viruses). The growth curves of three recent vaccine strains demonstrated that the qRT-PCR signal detected at 6 hours post-infection reflected an amplification of at least 100-fold over input. Using ferret antisera, we have established the feasibility of measuring virus neutralization at 6 hours post-infection, a duration likely confined to a single virus-replication cycle. The neutralization titer for qPCR-MN was defined as the highest reciprocal serum dilution necessary to achieve a 90% inhibition of the qRT-PCR signal; this endpoint was found to be in agreement with ELISA-MN using the same critical reagents in each assay. qPCR-MN was robust with respect to assay duration (6 hours vs. 12 hours). In addition, qPCR-MN appeared to be compliant with the Percentage Law (i.e., virus neutralization results appear to be consistent over an input virus dose ranging from 500 to 12,000 TCID50). Compared with ELISA-MN, qPCR-MN might have inherent properties conducive to reducing intra- and inter-laboratory variability while affording suitability for automation and high-throughput uses. Finally, our qRT-PCR-based approach may be broadly applicable to the development of neutralization assays for a wide variety of viruses. PMID:23437084

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

    PubMed

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

    2013-08-13

    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.

  13. Quantitative microbial faecal source tracking with sampling guided by hydrological catchment dynamics

    PubMed Central

    Reischer, G H; Haider, J M; Sommer, R; Stadler, H; Keiblinger, K M; Hornek, R; Zerobin, W; Mach, R L; Farnleitner, A H

    2008-01-01

    The impairment of water quality by faecal pollution is a global public health concern. Microbial source tracking methods help to identify faecal sources but the few recent quantitative microbial source tracking applications disregarded catchment hydrology and pollution dynamics. This quantitative microbial source tracking study, conducted in a large karstic spring catchment potentially influenced by humans and ruminant animals, was based on a tiered sampling approach: a 31-month water quality monitoring (Monitoring) covering seasonal hydrological dynamics and an investigation of flood events (Events) as periods of the strongest pollution. The detection of a ruminant-specific and a human-specific faecal Bacteroidetes marker by quantitative real-time PCR was complemented by standard microbiological and on-line hydrological parameters. Both quantitative microbial source tracking markers were detected in spring water during Monitoring and Events, with preponderance of the ruminant-specific marker. Applying multiparametric analysis of all data allowed linking the ruminant-specific marker to general faecal pollution indicators, especially during Events. Up to 80% of the variation of faecal indicator levels during Events could be explained by ruminant-specific marker levels proving the dominance of ruminant faecal sources in the catchment. Furthermore, soil was ruled out as a source of quantitative microbial source tracking markers. This study demonstrates the applicability of quantitative microbial source tracking methods and highlights the prerequisite of considering hydrological catchment dynamics in source tracking study design. PMID:18564182

  14. 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

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

    USDA-ARS?s Scientific Manuscript database

    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 ...

  16. 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)-...

  17. 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)-...

  18. 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.

  19. Quantitative test of the barrier nucleosome model for statistical positioning of nucleosomes up- and downstream of transcription start sites.

    PubMed

    Möbius, Wolfram; Gerland, Ulrich

    2010-08-19

    The positions of nucleosomes in eukaryotic genomes determine which parts of the DNA sequence are readily accessible for regulatory proteins and which are not. Genome-wide maps of nucleosome positions have revealed a salient pattern around transcription start sites, involving a nucleosome-free region (NFR) flanked by a pronounced periodic pattern in the average nucleosome density. While the periodic pattern clearly reflects well-positioned nucleosomes, the positioning mechanism is less clear. A recent experimental study by Mavrich et al. argued that the pattern observed in Saccharomyces cerevisiae is qualitatively consistent with a "barrier nucleosome model," in which the oscillatory pattern is created by the statistical positioning mechanism of Kornberg and Stryer. On the other hand, there is clear evidence for intrinsic sequence preferences of nucleosomes, and it is unclear to what extent these sequence preferences affect the observed pattern. To test the barrier nucleosome model, we quantitatively analyze yeast nucleosome positioning data both up- and downstream from NFRs. Our analysis is based on the Tonks model of statistical physics which quantifies the interplay between the excluded-volume interaction of nucleosomes and their positional entropy. We find that although the typical patterns on the two sides of the NFR are different, they are both quantitatively described by the same physical model with the same parameters, but different boundary conditions. The inferred boundary conditions suggest that the first nucleosome downstream from the NFR (the +1 nucleosome) is typically directly positioned while the first nucleosome upstream is statistically positioned via a nucleosome-repelling DNA region. These boundary conditions, which can be locally encoded into the genome sequence, significantly shape the statistical distribution of nucleosomes over a range of up to approximately 1,000 bp to each side.

  20. Dynamic Quantitative Sensory Testing to Characterize Central Pain Processing

    PubMed Central

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

    2017-01-01

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

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

    PubMed

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

    2015-08-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2015-08-01

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

  3. Application of Quantitative Real-Time Reverse Transcription-PCR in Assessing Drug Efficacy against the Intracellular Pathogen Cryptosporidium parvum In Vitro

    PubMed Central

    Cai, Xiaomin; Woods, Keith M.; Upton, Steve J.; Zhu, Guan

    2005-01-01

    We report here on a quantitative real-time reverse transcription-PCR (qRT-PCR) assay for assessing drug efficacy against the intracellular pathogen Cryptosporidium parvum. The qRT-PCR assay detects 18S rRNA transcripts from both parasites, that is, the cycle threshold for 18S rRNA from parasites (CT[P18S]) and host cells (CT[H18S]), and evaluates the relative expression between parasite and host rRNA levels (i.e., ΔCT = CT[P18S] − CT[H18S]) to minimize experimental and operational errors. The choice of qRT-PCR over quantitative PCR (qPCR) in this study is based on the observations that (i) the relationship between the logarithm of infected parasites (log[P]) and the normalized relative level of rRNA (ΔΔCT) is linear, with a fourfold dynamic range, by qRT-PCR but sigmoidal (nonlinear) by qPCR; and (ii) the level of RNA represents that of live parasites better than that of DNA, because the decay of RNA (99% in ∼3 h) in dead parasites is faster than that of DNA (99% in ∼24 to 48 h) under in vitro conditions. The reliability of the qRT-PCR method was validated by testing the efficacies of nitazoxanide and paromomycin on the development of two strains of C. parvum (IOWA and KSU-1) in HCT-8 cells in vitro. Both compounds displayed dose-dependent inhibitions. The observed MIC50 values for nitazoxanide and paromomycin were 0.30 to 0.45 μg/ml and 89.7 to 119.0 μg/ml, respectively, comparable to the values reported previously. Using the qRT-PCR assay, we have also observed that pyrazole could inhibit C. parvum development in vitro (MIC50 = 15.8 mM), suggesting that the recently discovered Cryptosporidium alcohol dehydrogenases may be explored as new drug targets. PMID:16251280

  4. Dynamic Transcriptional Regulation of Fis in Salmonella During the Exponential Phase.

    PubMed

    Wang, Hui; Wang, Lei; Li, Ping; Hu, Yilang; Zhang, Wei; Tang, Bo

    2015-12-01

    Fis is one of the most important global regulators and has attracted extensive research attention. Many studies have focused on comparing the Fis global regulatory networks for exploring Fis function during different growth stages, such as the exponential and stationary stages. Although the Fis protein in bacteria is mainly expressed in the exponential phase, the dynamic transcriptional regulation of Fis during the exponential phase remains poorly understood. To address this question, we used RNA-seq technology to identify the Fis-regulated genes in the S. enterica serovar Typhimurium during the early exponential phase, and qRT-PCR was performed to validate the transcriptional data. A total of 1495 Fis-regulated genes were successfully identified, including 987 Fis-repressed genes and 508 Fis-activated genes. Comparing the results of this study with those of our previous study, we found that the transcriptional regulation of Fis was diverse during the early- and mid-exponential phases. The results also showed that the strong positive regulation of Fis on Salmonella pathogenicity island genes in the mid-exponential phase transitioned into insignificant effect in the early exponential phase. To validate these results, we performed a cell infection assay and found that Δfis only exhibited a 1.49-fold decreased capacity compared with the LT2 wild-type strain, indicating a large difference from the 6.31-fold decrease observed in the mid-exponential phase. Our results provide strong evidence for a need to thoroughly understand the dynamic transcriptional regulation of Fis in Salmonella during the exponential phase.

  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. Effect of Dynamic Interaction between microRNA and Transcription Factor on Gene Expression

    PubMed Central

    Liu, Hongsheng; Yao, Chenggui

    2016-01-01

    MicroRNAs (miRNAs) are endogenous noncoding RNAs which participate in diverse biological processes in animals and plants. They are known to join together with transcription factors and downstream gene, forming a complex and highly interconnected regulatory network. To recognize a few overrepresented motifs which are expected to perform important elementary regulatory functions, we constructed a computational model of miRNA-mediated feedforward loops (FFLs) in which a transcription factor (TF) regulates miRNA and targets gene. Based on the different dynamic interactions between miRNA and TF on gene expression, four possible structural topologies of FFLs with two gate functions (AND gate and OR gate) are introduced. We studied the dynamic behaviors of these different motifs. Furthermore, the relationship between the response time and maximal activation velocity of miRNA was investigated. We found that the curve of response time shows nonmonotonic behavior in Co1 loop with OR gate. This may help us to infer the mechanism of miRNA binding to the promoter region. At last we investigated the influence of important parameters on the dynamic response of system. We identified that the stationary levels of target gene in all loops were insensitive to the initial value of miRNA. PMID:27957492

  7. Transcription-driven twin supercoiling of a DNA loop: a Brownian dynamics study.

    PubMed

    Mielke, Steven P; Fink, William H; Krishnan, V V; Grønbech-Jensen, Niels; Benham, Craig J

    2004-10-22

    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 paper, we introduce a dynamic computer model for investigating this phenomenon. Duplex DNA is represented as a chain of hydrodynamic beads interacting through potentials of linearly elastic stretching, bending, and twisting, as well as excluded volume. 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 micros response of a 477-base pair 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, as predicted by the "transcription-induced twin-supercoiled-domain" model [L. F. Liu and J. C. Wang, Proc. Natl. Acad. Sci. U.S.A. 84, 7024 (1987)]. 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.

  8. Rapid Sampling of Escherichia coli After Changing Oxygen Conditions Reveals Transcriptional Dynamics

    PubMed Central

    von Wulffen, Joachim; Ulmer, Andreas; Jäger, Günter; Sawodny, Oliver; Feuer, Ronny

    2017-01-01

    Escherichia coli is able to shift between anaerobic and aerobic metabolism by adapting its gene expression, e.g., of metabolic genes, to the new environment. The dynamics of gene expression that result from environmental shifts are limited, amongst others, by the time needed for regulation and transcription elongation. In this study, we examined gene expression dynamics after an anaerobic-to-aerobic shift on a short time scale (0.5, 1, 2, 5, and 10 min) by RNA sequencing with emphasis on delay times and transcriptional elongation rates (TER). Transient expression patterns and timing of differential expression, characterized by delay and elongation, were identified as key features of the dataset. Gene ontology enrichment analysis revealed early upregulation of respiratory and iron-related gene sets. We inferred specific TERs of 89 operons with a mean TER of 42.0 nt/s and mean delay time of 22.4 s. TERs correlate with sequence features, such as codon bias, whereas delay times correlate with the involvement of regulators. The presented data illustrate that at very short times after a shift in oxygenation, extensional changes of the transcriptome, such as temporary responses, can be observed. Besides regulation, TERs contribute to the dynamics of gene expression. PMID:28264512

  9. 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.

  10. Effect of Dynamic Interaction between microRNA and Transcription Factor on Gene Expression.

    PubMed

    Zhao, Qi; Liu, Hongsheng; Yao, Chenggui; Shuai, Jianwei; Sun, Xiaoqiang

    2016-01-01

    MicroRNAs (miRNAs) are endogenous noncoding RNAs which participate in diverse biological processes in animals and plants. They are known to join together with transcription factors and downstream gene, forming a complex and highly interconnected regulatory network. To recognize a few overrepresented motifs which are expected to perform important elementary regulatory functions, we constructed a computational model of miRNA-mediated feedforward loops (FFLs) in which a transcription factor (TF) regulates miRNA and targets gene. Based on the different dynamic interactions between miRNA and TF on gene expression, four possible structural topologies of FFLs with two gate functions (AND gate and OR gate) are introduced. We studied the dynamic behaviors of these different motifs. Furthermore, the relationship between the response time and maximal activation velocity of miRNA was investigated. We found that the curve of response time shows nonmonotonic behavior in Co1 loop with OR gate. This may help us to infer the mechanism of miRNA binding to the promoter region. At last we investigated the influence of important parameters on the dynamic response of system. We identified that the stationary levels of target gene in all loops were insensitive to the initial value of miRNA.

  11. Development and validation of a reverse transcription quantitative PCR for universal detection of viral hemorrhagic septicemia virus.

    PubMed

    Garver, Kyle A; Hawley, Laura M; McClure, Carol A; Schroeder, Tamara; Aldous, Sandra; Doig, Fiona; Snow, Michael; Edes, Sandra; Baynes, Catherine; Richard, Jon

    2011-06-16

    Viral hemorrhagic septicemia virus (VHSV) infects over 70 fish species inhabiting marine, brackish or freshwater environments throughout the Northern Hemisphere. Over its geographic range, 4 VHSV genotypes and multiple subtypes exist. Here, we describe the development and validation of a rapid, sensitive and specific real-time reverse transcription quantitative PCR assay (RT-qPCR) that amplifies sequence from representative isolates of all VHSV genotypes (I, II, III and IV). The pan-specific VHSV RT-qPCR assay reliably detects 100 copies of VHSV nucleoprotein RNA without cross-reacting with infectious hematopoietic necrosis virus, spring viremia of carp virus or aquatic birnavirus. Test performance characteristics evaluated on experimentally infected Atlantic salmon Salmo salar L. revealed a diagnostic sensitivity (DSe) > or = 93% and specificity (DSp) = 100%. The repeatability and reproducibility of the procedure was exceptionally high, with 93% agreement among test results within and between 2 laboratories. Furthermore, proficiency testing demonstrated the VHSV RT-qPCR assay to be easily transferred to and performed by a total of 9 technicians representing 4 laboratories in 2 countries. The assay performed equivalent to the traditional detection method of virus isolation via cell culture with the advantage of faster turnaround times and high throughput capacity, further suggesting the suitability of the use of this VHSV RT-qPCR in a diagnostic setting.

  12. Quantitative Real-Time Reverse Transcription-PCR Analysis of Deformed Wing Virus Infection in the Honeybee (Apis mellifera L.)

    PubMed Central

    Chen, Y. P.; Higgins, J. A.; Feldlaufer, M. F.

    2005-01-01

    Deformed wing virus (DWV) can cause wing deformity and premature death in adult honeybees, although like many other bee viruses, DWV generally persists as a latent infection with no apparent symptoms. Using reverse transcription (RT)-PCR and Southern hybridization, we detected DWV in all life stages of honeybees, including adults with and without deformed wings. We also found DWV in the parasitic mite Varroa destructor, suggesting that this mite may be involved in the transmission of DWV. However, the detection of the virus in life stages not normally associated with mite parasitism (i.e., eggs and larvae) suggests that there are other modes of transmission. The levels of DWV in different life stages of bees were investigated by using TaqMan real-time quantitative RT-PCR. The amounts of virus varied significantly in these different stages, and the highest levels occurred in pupae and in adult worker bees with deformed wings. The variability in virus titer may reflect the different abilities of bees to resist DWV infection and replication. The epidemiology of DWV is discussed, and factors such as mite infestation, malnutrition, and climate are also considered. PMID:15640219

  13. Identification of suitable reference genes for investigating gene expression in human gallbladder carcinoma using reverse transcription quantitative polymerase chain reaction.

    PubMed

    Yu, Shan; Yang, Qiwei; Yang, Jing Hui; Du, Zhenwu; Zhang, Guizhen

    2015-04-01

    Reverse transcription quantitative polymerase chain reaction (RT‑qPCR) has become a frequently used strategy in gene expression studies. The relative quantification method is an important and commonly used method for the evaluation of RT‑qPCR data. The key aim of this method is to identify an applicable internal reference gene, however, there are currently no suitable reference genes for gene analysis in gallbladder carcinoma. In the present study, screening was performed using 12 common reference genes, which were selected in order to provide an experimental basis for the investigation of gene expression in gallbladder carcinoma. A total of 16 tissue samples of gallbladder carcinoma and their matched normal gallbladder tissues were used. The gene expression stability and applicability of the 12 reference gene candidates were determined using the geNorm, NormFinder and BestKeeper software programs. Following comparison of the results of the three software programs, HPRT1 was identified as the most stably expressed reference gene. In the normal gallbladder group, the relative stably expressed reference gene was PPIA and in the entire sample group, the relatively stably expressed reference gene was PPIA. The present study also demonstrated that the combination of the three reference genes was the most appropriate. The recommended combinations were PPIA + PUM1 + ACTB for the total sample group, GAPDH + PBGD + ALAS1 for the gallbladder carcinoma group and PPIA + PUM1 + TBP for the paired normal gallbladder group.

  14. Real-Time Quantitative PCR (QPCR) and Reverse Transcription-QPCR for Detection and Enumeration of Total Yeasts in Wine▿

    PubMed Central

    Hierro, Núria; Esteve-Zarzoso, Braulio; González, Ángel; Mas, Albert; Guillamón, Jose M.

    2006-01-01

    Real-time PCR, or quantitative PCR (QPCR), has been developed to rapidly detect and quantify the total number of yeasts in wine without culturing. Universal yeast primers were designed from the variable D1/D2 domains of the 26S rRNA gene. These primers showed good specificity with all the wine yeasts tested, and they did not amplify the most representative wine species of acetic acid bacteria and lactic acid bacteria. Numerous standard curves were constructed with different strains and species grown in yeast extract-peptone-dextrose medium or incubated in wine. The small standard errors with these replicas proved that the assay is reproducible and highly robust. This technique was validated with artificially contaminated and natural wine samples. We also performed a reverse transcription-QPCR (RT-QPCR) assay from rRNA for total viable yeast quantification. This technique had a low detection limit and was more accurate than QPCR because the dead cells were not quantified. As far as we know, this is the first time that RT-QPCR has been performed to quantify viable yeasts from rRNA. RT-QPCR is a rapid and accurate technique for enumerating yeasts during industrial wine fermentation and controlling the risk of wine spoilage. PMID:17088381

  15. Real-time quantitative PCR (QPCR) and reverse transcription-QPCR for detection and enumeration of total yeasts in wine.

    PubMed

    Hierro, Núria; Esteve-Zarzoso, Braulio; González, Angel; Mas, Albert; Guillamón, Jose M

    2006-11-01

    Real-time PCR, or quantitative PCR (QPCR), has been developed to rapidly detect and quantify the total number of yeasts in wine without culturing. Universal yeast primers were designed from the variable D1/D2 domains of the 26S rRNA gene. These primers showed good specificity with all the wine yeasts tested, and they did not amplify the most representative wine species of acetic acid bacteria and lactic acid bacteria. Numerous standard curves were constructed with different strains and species grown in yeast extract-peptone-dextrose medium or incubated in wine. The small standard errors with these replicas proved that the assay is reproducible and highly robust. This technique was validated with artificially contaminated and natural wine samples. We also performed a reverse transcription-QPCR (RT-QPCR) assay from rRNA for total viable yeast quantification. This technique had a low detection limit and was more accurate than QPCR because the dead cells were not quantified. As far as we know, this is the first time that RT-QPCR has been performed to quantify viable yeasts from rRNA. RT-QPCR is a rapid and accurate technique for enumerating yeasts during industrial wine fermentation and controlling the risk of wine spoilage.

  16. Analysis of plasma viral RNA levels during acute dengue virus infection using quantitative competitor reverse transcription-polymerase chain reaction.

    PubMed

    Sudiro, T M; Zivny, J; Ishiko, H; Green, S; Vaughn, D W; Kalayanarooj, S; Nisalak, A; Norman, J E; Ennis, F A; Rothman, A L

    2001-01-01

    There is increasing recognition of the potential importance of viral burden in the pathogenesis of dengue hemorrhagic fever (DHF). There is little data available, however, describing the kinetics of viral replication in humans with natural dengue virus (DV) infection. Standard procedures for measuring titers of infectious virus in clinical specimens are either laborious or insensitive. We developed a method for measurement of DV RNA in plasma samples based on reverse transcription-polymerase chain reaction (RT-PCR) using a mutant RNA target as a competitor. This technique was reproducible and accurate for samples containing any of the four DV serotypes, and could be applied to samples containing as few as 250 copies of RNA per reaction. We examined plasma viral RNA levels in 80 children with acute DV infection; sequential plasma samples were tested in 34 of these children. Plasma viral RNA levels ranged as high as 10(9) RNA copies/ml, and correlated with titers of infectious virus measured in mosquitoes (r= 0.69). Plasma viral RNA levels fell rapidly during the last several days of the febrile period. We did not find a significant difference in maximal plasma viral RNA levels between children with DHF and children with dengue fever, but peak viral RNA levels were identified in only 16 subjects. We conclude that this quantitative RT-PCR method will be valuable for further studies of natural DV infections.

  17. Identification of appropriate reference genes for normalizing transcript expression by quantitative real-time PCR in Litsea cubeba.

    PubMed

    Lin, Liyuan; Han, Xiaojiao; Chen, Yicun; Wu, Qingke; Wang, Yangdong

    2013-12-01

    Quantitative real-time PCR has emerged as a highly sensitive and widely used method for detection of gene expression profiles, via which accurate detection depends on reliable normalization. Since no single control is appropriate for all experimental treatments, it is generally advocated to select suitable internal controls prior to use for normalization. This study reported the evaluation of the expression stability of twelve potential reference genes in different tissue/organs and six fruit developmental stages of Litsea cubeba in order to screen the superior internal reference genes for data normalization. Two softwares-geNorm, and NormFinder-were used to identify stability of these candidate genes. The cycle threshold difference and coefficient of variance were also calculated to evaluate the expression stability of candidate genes. F-BOX, EF1α, UBC, and TUA were selected as the most stable reference genes across 11 sample pools. F-BOX, EF1α, and EIF4α exhibited the highest expression stability in different tissue/organs and different fruit developmental stages. Besides, a combination of two stable reference genes would be sufficient for gene expression normalization in different fruit developmental stages. In addition, the relative expression profiles of DXS and DXR were evaluated by EF1α, UBC, and SAMDC. The results further validated the reliability of stable reference genes and also highlighted the importance of selecting suitable internal controls for L. cubeba. These reference genes will be of great importance for transcript normalization in future gene expression studies on L. cubeba.

  18. 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.

  19. Development of a quantitative competitive reverse transcription polymerase chain reaction (QC-RT-PCR) for detection and quantitation of Chikungunya virus.

    PubMed

    Sharma, Shashi; Dash, Paban Kumar; Santhosh, S R; Shukla, Jyoti; Parida, Manmohan; Rao, P V Lakshmana

    2010-05-01

    Chikungunya is one of the most important emerging arboviral infections of public health significance. Due to lack of a licensed vaccine, rapid diagnosis plays an important role in early management of patients. In this study, a QC-RT-PCR assay was developed to quantify Chikungunya virus (CHIKV) RNA by targeting the conserved region of E1 gene. A competitor molecule containing an internal insertion was generated, which provided a stringent control of the quantification process. The introduction of 10-fold serially diluted competitor in each reaction was further used to determine sensitivity. The applicability of this assay for quantification of CHIKV RNA was evaluated with human clinical samples, and the results were compared with real-time quantitative RT-PCR. The sensitivity of this assay was estimated to be 100 RNA copies per reaction with a dynamic detection range of 10(2) to 10(10) copies. Specificity was confirmed using closely related alpha and flaviviruses. The comparison of QC-RT-PCR result with real-time RT-PCR revealed 100% concordance for the detection of CHIKV in clinical samples. These findings demonstrated that the reported assay is convenient, sensitive and accurate method and has the potential usefulness for clinical diagnosis due to simultaneous detection and quantification of CHIKV in acute-phase serum samples.

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

    PubMed

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

    2017-08-01

    Objective: To explore the differential between the value of dynamic contrast-enhanced MRI quantitative pharmacokinetic parameters and relative pharmacokinetic quantitative parameters in breast lesions. Methods: Retrospective analysis of 255 patients(262 breast lesions) who was obtained by clinical palpation , ultrasound or full-field digital mammography , and then all lessions were pathologically confirmed in Zhongda Hospital, Southeast University from May 2012 to May 2016. A 3.0 T MRI scanner was used to obtain the quantitative MR pharmacokinetic parameters: volume transfer constant (K(trans)), exchange rate constant (k(ep))and extravascular extracellular volume fraction (V(e)). And measured the quantitative pharmacokinetic parameters of normal glands tissues which on the same side of the same level of the lesions; and then calculated the value of relative pharmacokinetic parameters: rK(rans)、rk(ep) and rV(e).To explore the diagnostic value of two pharmacokinetic parameters in differential diagnosis of benign and malignant breast lesions using receiver operating curves and model of logistic regression. Results: (1)There were significant differences between benign lesions and malignant lesions in K(trans) and k(ep) (t=15.489, 15.022, respectively, P<0.05), there were no significant differences between benign lesions and malignant lesions in V(e)(t=-2.346, P>0.05). The areas under the ROC curve(AUC)of K(trans), k(ep) and V(e) between malignant and benign lesions were 0.933, 0.948 and 0.387, the sensitivity of K(trans), k(ep) and V(e) were 77.1%, 85.0%, 51.0% , and the specificity of K(trans), k(ep) and V(e) were 96.3%, 93.6%, 60.8% for the differential diagnosis of breast lesions if taken the maximum Youden's index as cut-off. (2)There were significant differences between benign lesions and malignant lesions in rK(trans), rk(ep) and rV(e) (t=14.177, 11.726, 2.477, respectively, P<0.05). The AUC of rK(trans), rk(ep) and rV(e) between malignant and benign lesions

  1. 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.

  2. 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

  3. 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.

  4. 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.

  5. Dynamic quantitative phase imaging for biological objects using a pixelated phase mask

    PubMed Central

    Creath, Katherine; Goldstein, Goldie

    2012-01-01

    This paper describes research in developing a dynamic quantitative phase imaging microscope providing instantaneous measurements of dynamic motions within and among live cells without labels or contrast agents. It utilizes a pixelated phase mask enabling simultaneous measurement of multiple interference patterns derived using the polarization properties of light to track dynamic motions and morphological changes. Optical path difference (OPD) and optical thickness (OT) data are obtained from phase images. Two different processing routines are presented to remove background surface shape to enable quantification of changes in cell position and volume over time. Data from a number of different moving biological organisms and cell cultures are presented. PMID:23162725

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

    NASA Astrophysics Data System (ADS)

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

    2016-11-01

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

  7. Optical tweezers studies of transcription by eukaryotic RNA polymerases.

    PubMed

    Lisica, Ana; Grill, Stephan W

    2017-02-21

    Transcription is the first step in the expression of genetic information and it is carried out by large macromolecular enzymes called RNA polymerases. Transcription has been studied for many years and with a myriad of experimental techniques, ranging from bulk studies to high-resolution transcript sequencing. In this review, we emphasise the advantages of using single-molecule techniques, particularly optical tweezers, to study transcription dynamics. We give an overview of the latest results in the single-molecule transcription field, focusing on transcription by eukaryotic RNA polymerases. Finally, we evaluate recent quantitative models that describe the biophysics of RNA polymerase translocation and backtracking dynamics.

  8. 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

  9. 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.

  10. 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

  11. Quantifying β-catenin subcellular dynamics and cyclin D1 mRNA transcription during Wnt signaling in single living cells

    PubMed Central

    Kafri, Pinhas; Hasenson, Sarah E; Kanter, Itamar; Sheinberger, Jonathan; Kinor, Noa; Yunger, Sharon; Shav-Tal, Yaron

    2016-01-01

    Signal propagation from the cell membrane to a promoter can induce gene expression. To examine signal transmission through sub-cellular compartments and its effect on transcription levels in individual cells within a population, we used the Wnt/β-catenin signaling pathway as a model system. Wnt signaling orchestrates a response through nuclear accumulation of β-catenin in the cell population. However, quantitative live-cell measurements in individual cells showed variability in nuclear β-catenin accumulation, which could occur in two waves, followed by slow clearance. Nuclear accumulation dynamics were initially rapid, cell cycle independent and differed substantially from LiCl stimulation, presumed to mimic Wnt signaling. β-catenin levels increased simultaneously at adherens junctions and the centrosome, and a membrane-centrosome transport system was revealed. Correlating β-catenin nuclear dynamics to cyclin D1 transcriptional activation showed that the nuclear accumulation rate of change of the signaling factor, and not actual protein levels, correlated with the transcriptional output of the pathway. DOI: http://dx.doi.org/10.7554/eLife.16748.001 PMID:27879202

  12. Diversity of Intestinal Clostridium coccoides Group in the Japanese Population, as Demonstrated by Reverse Transcription-Quantitative PCR.

    PubMed

    Kurakawa, Takashi; Ogata, Kiyohito; Matsuda, Kazunori; Tsuji, Hirokazu; Kubota, Hiroyuki; Takada, Toshihiko; Kado, Yukiko; Asahara, Takashi; Takahashi, Takuya; Nomoto, Koji

    2015-01-01

    We used sensitive rRNA-targeted reverse transcription-quantitative PCR (RT-qPCR) to quantify the Clostridium coccoides group, which is a major anaerobic population in the human intestine. For this purpose, the C. coccoides group was classified into 3 subgroups and 19 species for expediency in accordance with the existing database, and specific primers were newly developed to evaluate them. Population levels of the C. coccoides group in human feces determined by RT-qPCR were equivalent to those determined by fluorescence in situ hybridization. RT-qPCR analysis of fecal samples from 96 volunteers (32 young children, 32 adults and 32 elderly) by using the 22 new primer sets together with the C. coccoides group-specific primer setm revealed that (i) total counts obtained as the sum of the 3 subgroups and 19 species were equivalent to the results obtained by using the C. coccoides group-specific primer set; (ii) total C. coccoides-group counts in the elderly were significantly lower than those in young children and adults; (iii) genus Blautia was the most common subgroup in the human intestinal C. coccoides-group populations at all age populations tested; (iv) the prevalences of Fusicatenibacter saccharivorans and genus Dorea were significantly higher in adults than in young children and the elderly; and (v) the prevalences of C. scindens and C. hylemonae, both of which produce secondary bile acid in the human intestine, were significantly higher in the elderly than in young children and adults. Hierarchical clustering and principal component analysis showed clear separation of the bacterial components between adult and elderly populations. Taken together, these data suggest that aging plays an important role in the diversity of C. coccoides-group populations in human intestinal microbiota; changes in this diversity likely influence the health of the host.

  13. 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.

  14. Selection of endogenous reference microRNA genes for quantitative reverse transcription polymerase chain reaction studies of boar spermatozoa cryopreservation.

    PubMed

    Zhang, Yan; Zeng, Chang-Jun; He, Lian; Ding, Li; Tang, Ke-Yi; Peng, Wen-Pei

    2015-03-01

    It is important to select high-quality reference genes for the accurate interpretation of quantitative reverse transcription polymerase chain reaction data, in particular for certain miRNAs that may demonstrate unstable expression. Although several studies have attempted to validate reference miRNA genes in the porcine testis, spermatozoa, and other tissues, no validation studies have been carried out on cryopreserved boar spermatozoa. In this study, 15 commonly used reference miRNA genes (5S, let-7c-5p, ssc-miR-16-5p, ssc-miR-17-5p, ssc-miR-20a, ssc-miR-23a, ssc-miR-24-3p, ssc-miR-26a, ssc-miR-27a-3p, ssc-miR-92a, ssc-miR-103-3p, ssc-miR-106a, ssc-miR-107-3p, ssc-miR-186, and ssc-miR-221-3p) were selected to evaluate the expression stability of target miRNAs in boar spermatozoa under different experimental conditions and concentrations. The stability of the expression of these reference miRNAs across each sample was evaluated using geNorm, NormFinder, and BestKeeper software. The results showed that ssc-miR-186 (mean rank value = 5.00), ssc-miR-23a (5.33), and ssc-miR-27a (5.33) were the most suitable reference genes using three different statistical algorithms and comprehensive ranking. The identification of these reference miRNAs will allow for more accurate quantification of the changes in miRNA expression during cryopreservation of boar spermatozoa.

  15. Dynamic transcriptional signature and cell fate analysis reveals plasticity of individual neural plate border cells

    PubMed Central

    Roellig, Daniela; Tan-Cabugao, Johanna; Esaian, Sevan; Bronner, Marianne E

    2017-01-01

    The ‘neural plate border’ of vertebrate embryos contains precursors of neural crest and placode cells, both defining vertebrate characteristics. How these lineages segregate from neural and epidermal fates has been a matter of debate. We address this by performing a fine-scale quantitative temporal analysis of transcription factor expression in the neural plate border of chick embryos. The results reveal significant overlap of transcription factors characteristic of multiple lineages in individual border cells from gastrula through neurula stages. Cell fate analysis using a Sox2 (neural) enhancer reveals that cells that are initially Sox2+ cells can contribute not only to neural tube but also to neural crest and epidermis. Moreover, modulating levels of Sox2 or Pax7 alters the apportionment of neural tube versus neural crest fates. Our results resolve a long-standing question and suggest that many individual border cells maintain ability to contribute to multiple ectodermal lineages until or beyond neural tube closure. DOI: http://dx.doi.org/10.7554/eLife.21620.001 PMID:28355135

  16. Ribosome and transcript copy numbers, polysome occupancy and enzyme dynamics in Arabidopsis

    PubMed Central

    Piques, Maria; Schulze, Waltraud X; Höhne, Melanie; Usadel, Björn; Gibon, Yves; Rohwer, Johann; Stitt, Mark

    2009-01-01

    Plants are exposed to continual changes in the environment. The daily alternation between light and darkness results in massive recurring changes in the carbon budget, and leads to widespread changes in transcript levels. These diurnal changes are superimposed on slower changes in the environment. Quantitative molecular information about the numbers of ribosomes, of transcripts for 35 enzymes in central metabolism and their loading into polysomes is used to estimate translation rates in Arabidopsis rosettes, and explore the consequences for important sub-processes in plant growth. Translation rates for individual enzyme are compared with their abundance in the rosette to predict which enzymes are subject to rapid turnover every day, and which are synthesized at rates that would allow only slow adjustments to sustained changes of the environment, or resemble those needed to support the observed rate of growth. Global translation rates are used to estimate the energy costs of protein synthesis and relate them to the plant carbon budget, in particular the rates of starch degradation and respiration at night. PMID:19888209

  17. 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

  18. Robust dynamic balance of AP-1 transcription factors in a neuronal gene regulatory network

    PubMed Central

    2010-01-01

    Background The octapeptide Angiotensin II is a key hormone that acts via its receptor AT1R in the brainstem to modulate the blood pressure control circuits and thus plays a central role in the cardiac and respiratory homeostasis. This modulation occurs via activation of a complex network of signaling proteins and transcription factors, leading to changes in levels of key genes and proteins. AT1R initiated activity in the nucleus tractus solitarius (NTS), which regulates blood pressure, has been the subject of extensive molecular analysis. But the adaptive network interactions in the NTS response to AT1R, plausibly related to the development of hypertension, are not understood. Results We developed and analyzed a mathematical model of AT1R-activated signaling kinases and a downstream gene regulatory network, with structural basis in our transcriptomic data analysis and literature. To our knowledge, our report presents the first computational model of this key regulatory network. Our simulations and analysis reveal a dynamic balance among distinct dimers of the AP-1 family of transcription factors. We investigated the robustness of this behavior to simultaneous perturbations in the network parameters using a novel multivariate approach that integrates global sensitivity analysis with decision-tree methods. Our analysis implicates a subset of Fos and Jun dependent mechanisms, with dynamic sensitivities shifting from Fos-regulating kinase (FRK)-mediated processes to those downstream of c-Jun N-terminal kinase (JNK). Decision-tree analysis indicated that while there may be a large combinatorial functional space feasible for neuronal states and parameters, the network behavior is constrained to a small set of AP-1 response profiles. Many of the paths through the combinatorial parameter space lead to a dynamic balance of AP-1 dimer forms, yielding a robust AP-1 response counteracting the biological variability. Conclusions Based on the simulation and analysis results, we

  19. Dynamic analysis of pathogen-infected host cells using quantitative phase microscopy

    NASA Astrophysics Data System (ADS)

    Lee, Seungrag; Kim, Young Ran; Lee, Ji Yong; Rhee, Joon Haeng; Park, Chang-Soo; Kim, Dug Young

    2011-03-01

    We present the real-time quantitative analysis of Vibrio vulnificus-infected host cells using quantitative phase microscopy (QPM) based on interferometric techniques. This provides the ability to retrieve the phase or optical path-length distribution over the cell with nanometer path-length sensitivity from a single interferogram image. We have used QPM to study dynamic cell morphologic changes and to noninvasively quantify the cell volumes of rat basophilic leukemia RBL-2H3 cells infected with V. vulnificus strains: wild type (MO6-24/O) and RtxA1 toxin mutant (CMM770). During the process of V. vulnificus infection in RBL-2H3 cells, the dynamic changes of quantitative phase images, cell volumes, and areas were observed in real time using QPM. In contrast, dramatic changes were not detected in RBL-2H3 cells infected with the noncytotoxic RtxA1 toxin mutant. The results showed good correlation between QPM analysis and biochemical assays, such as lactate dehydrogenase assay or β-hexosaminidase release assay. We suggest that QPM is a powerful quantitative method to study the dynamic process of host cells infected with pathogens in a noninvasive manner.

  20. 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.

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

    PubMed

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

    2017-04-01

    Variability across devices, patients, and time still hinders widespread recognition of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) as quantitative biomarker. The purpose of this work was to introduce and characterize a dedicated microchannel phantom as a model for quantitative DCE-MRI measurements. A perfusable, MR-compatible microchannel network was constructed on the basis of sacrificial melt-spun sugar fibers embedded in a block of epoxy resin. Structural analysis was performed on the basis of light microscopy images before DCE-MRI experiments. During dynamic acquisition the capillary network was perfused with a standard contrast agent injection system. Flow-dependency, as well as inter- and intrascanner reproducibility of the computed DCE parameters were evaluated using a 3.0 T whole-body MRI. Semi-quantitative and quantitative flow-related parameters exhibited the expected proportionality to the set flow rate (mean Pearson correlation coefficient: 0.991, P < 2.5e-5). The volume fraction was approximately independent from changes of the applied flow rate through the phantom. Repeatability and reproducibility experiments yielded maximum intrascanner coefficients of variation (CV) of 4.6% for quantitative parameters. All evaluated parameters were well in the range of known in vivo results for the applied flow rates. The constructed phantom enables reproducible, flow-dependent, contrast-enhanced MR measurements with the potential to facilitate standardization and comparability of DCE-MRI examinations. © 2017 American Association of Physicists in Medicine.

  2. 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

  3. 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

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

    PubMed

    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.

  5. 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

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

    PubMed

    Tanaka, Rie; Sanada, Shigeru; Sakuta, Keita; Kawashima, Hiroki

    2015-04-01

    An image-processing technique for separating bones from soft tissue in static chest radiographs has been developed. The present study was performed to evaluate the usefulness of dynamic bone images in quantitative analysis of rib movement. Dynamic chest radiographs of 16 patients were obtained using a dynamic flat-panel detector and processed to create bone images by using commercial software (Clear Read BS, Riverain Technologies). Velocity vectors were measured in local areas on the dynamic 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 a reduced rib velocity field, resulting in an asymmetrical distribution of rib movement. Vector maps in all normal cases exhibited left/right symmetric distributions of the velocity field, whereas those in abnormal cases showed asymmetric distributions because of locally limited rib movements. Dynamic bone images were useful for accurate quantitative analysis of rib movements. The present method has a potential for an additional functional examination in chest radiography.

  7. Transcription-driven twin supercoiling of a DNA loop: A Brownian dynamics study

    NASA Astrophysics Data System (ADS)

    Mielke, Steven P.; Fink, William H.; Krishnan, V. V.; Grønbech-Jensen, Niels; Benham, Craig J.

    2004-10-01

    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 paper, we introduce a dynamic computer model for investigating this phenomenon. Duplex DNA is represented as a chain of hydrodynamic beads interacting through potentials of linearly elastic stretching, bending, and twisting, as well as excluded volume. 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 μs response of a 477-base pair 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, as predicted by the "transcription-induced twin-supercoiled-domain" model [L. F. Liu and J. C. Wang, Proc. Natl. Acad. Sci. U.S.A. 84, 7024 (1987)]. 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.

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

    PubMed

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

    2006-02-01

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

  9. 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.

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

    DTIC Science & Technology

    2015-07-24

    NOS3 ,  DOCK11,  NT5E,  DHCR24,  RBM45,  RAB4B,  CBR4,   ADK,  TOP3A...SLC22A4,  EEF2K,  TUBB5,  TUBB6,  GUCY1A3,   NOS3 ,  SLC22A5,  SIK1,  DOCK11,  DHCR24,  RAP2B,   SRPK2,  RAP2A,  AIFM2,  LYN...ACVRL1,  NUAK2,  ATP10A,   CAMKK1,  CAMKK2,  CKB,  ACTR3,  ADCK1,  SLC22A4,  EEF2K,   NOS3 ,  SLC22A5,  SIK1,  DHCR24,

  11. Reference genes for high-throughput quantitative reverse transcription-PCR analysis of gene expression in organs and tissues of Eucalyptus grown in various environmental conditions.

    PubMed

    Cassan-Wang, Hua; Soler, Marçal; Yu, Hong; Camargo, Eduardo Leal O; Carocha, Victor; Ladouce, Nathalie; Savelli, Bruno; Paiva, Jorge A P; Leplé, Jean-Charles; Grima-Pettenati, Jacqueline

    2012-12-01

    Interest in the genomics of Eucalyptus has skyrocketed thanks to the recent sequencing of the genome of Eucalyptus grandis and to a growing number of large-scale transcriptomic studies. Quantitative reverse transcription-PCR (RT-PCR) is the method of choice for gene expression analysis and can now also be used as a high-throughput method. The selection of appropriate internal controls is becoming of utmost importance to ensure accurate expression results in Eucalyptus. To this end, we selected 21 candidate reference genes and used high-throughput microfluidic dynamic arrays to assess their expression among a large panel of developmental and environmental conditions with a special focus on wood-forming tissues. We analyzed the expression stability of these genes by using three distinct statistical algorithms (geNorm, NormFinder and ΔCt), and used principal component analysis to compare methods and rankings. We showed that the most stable genes identified depended not only on the panel of biological samples considered but also on the statistical method used. We then developed a comprehensive integration of the rankings generated by the three methods and identified the optimal reference genes for 17 distinct experimental sets covering 13 organs and tissues, as well as various developmental and environmental conditions. The expression patterns of Eucalyptus master genes EgMYB1 and EgMYB2 experimentally validated our selection. Our findings provide an important resource for the selection of appropriate reference genes for accurate and reliable normalization of gene expression data in the organs and tissues of Eucalyptus trees grown in a range of conditions including abiotic stresses.

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. Dynamic microRNA gene transcription and processing during T cell development

    PubMed Central

    Kirigin, Francis F.; Lindstedt, Kenneth; Sellars, Maclean; Ciofani, Maria; Low, Siao Li; Jones, Lachlan; Bell, Fiona; Pauli, Florencia; Bonneau, Richard; Myers, Richard M.; Littman, Dan R.; Chong, Mark M.W.

    2014-01-01

    By disrupting microRNA (miRNA) biogenesis, we previously showed that this pathway is critical for the differentiation and function of T cells. While various cloning studies have shown that many miRNAs are expressed during T cell development, and in a dynamic manner, it was unclear how comprehensive these earlier analyses were. We therefore decided to profile miRNA expression by means of Next Generation Sequencing. Furthermore, we profiled miRNA expression starting from the hematopoietic stem cell. This analysis revealed that miRNA expression during T cell development is extremely dynamic, with 645 miRNAs sequenced, and the expression of some varying by as much as 3 orders of magnitude. Furthermore, changes in precursor processing led to altered mature miRNA sequences. We also analyzed the structures of the primary miRNA transcripts expressed in T cells, and found that many were extremely long. The longest was pri-mir-29b-1/29a at ~168kb. All the long pri-miRNAs also displayed extensive splicing. Our findings indicate that miRNA expression during T cell development is both a highly dynamic and a highly regulated process. PMID:22379031

  18. Dynamic Transcript Profiling of Candida albicans Infection in Zebrafish: A Pathogen-Host Interaction Study

    PubMed Central

    Liu, Fu-Chen; Hsu, Po-Chen; Chen, Hsueh-Fen; Peng, Shih-Chi; Chuang, Yung-Jen; Lan, Chung-Yu; Hsieh, Wen-Ping; Wong, David Shan Hill

    2013-01-01

    Candida albicans is responsible for a number of life-threatening infections and causes considerable morbidity and mortality in immunocompromised patients. Previous studies of C. albicans pathogenesis have suggested several steps must occur before virulent infection, including early adhesion, invasion, and late tissue damage. However, the mechanism that triggers C. albicans transformation from yeast to hyphae form during infection has yet to be fully elucidated. This study used a systems biology approach to investigate C. albicans infection in zebrafish. The surviving fish were sampled at different post-infection time points to obtain time-lapsed, genome-wide transcriptomic data from both organisms, which were accompanied with in sync histological analyses. Principal component analysis (PCA) was used to analyze the dynamic gene expression profiles of significant variations in both C. albicans and zebrafish. The results categorized C. albicans infection into three progressing phases: adhesion, invasion, and damage. Such findings were highly supported by the corresponding histological analysis. Furthermore, the dynamic interspecies transcript profiling revealed that C. albicans activated its filamentous formation during invasion and the iron scavenging functions during the damage phases, whereas zebrafish ceased its iron homeostasis function following massive hemorrhage during the later stages of infection. Most of the immune related genes were expressed as the infection progressed from invasion to the damage phase. Such global, inter-species evidence of virulence-immune and iron competition dynamics during C. albicans infection could be crucial in understanding control fungal pathogenesis. PMID:24019870

  19. Quantitative study of polymer conformation and dynamics by single-particle tracking.

    PubMed Central

    Qian, H; Elson, E L

    1999-01-01

    We present a new method for analyzing the dynamics of conformational fluctuations of individual flexible polymer molecules. In single-particle tracking (SPT), one end of the polymer molecule is tethered to an immobile substratum. A microsphere attached to the other end serves as an optical marker. The conformational fluctuations of the polymer molecule can be measured by optical microscopy via the motion of the microsphere. The bead-and-spring theory for polymer dynamics is further developed to account for the microsphere, and together the measurement and the theory yield quantitative information about molecular conformations and dynamics under nonperturbing conditions. Applying the method to measurements carried out on DNA molecules provides information complementary to recent studies of single DNA molecules under extensional force. Combining high precision measurements with the theoretical analysis presented here creates a powerful tool for studying conformational dynamics of biological and synthetic macromolecules at the single-molecule level. PMID:10049340

  20. Chromatin code, local non-equilibrium dynamics, and the emergence of transcription regulatory programs

    NASA Astrophysics Data System (ADS)

    Benecke, A.

    2006-03-01

    Chromatin is a, if not the, hallmark of eukaryotic life. Any molecular process entailing genomic DNA or the nucleus by default provokes or depends on chromatin structural dynamics on various space and time scales. Chromatin dynamics are result of changes in the physico-chemical properties of the chromatin constituents themselves or the nuclear environment. Chromatin has been found in the former case to undergo many different covalent enzyme-mediated chemical modifications. Their identification sheds light on the molecular mechanisms and the physico-chemical properties underlying chromatin dynamics, and allows the development of quantitative models for the chromatin fiber. The abundance of the different modifications, their dynamics, and short- as well as long-range correlation phenomena between different modifications also point to a second layer of genomic coding implemented at the level of chromatin. Especially, gene regulatory coding seems to depend on such a second-level code. The information-theoretical properties of chromatin in the context of gene regulatory coding are discussed. A model for the emergence of cellular differentiation from the intricate interplay between genomic and chromatin code is presented and discussed in light of recent experimental insights.

  1. Quantitative Image Analysis of Single-Molecule mRNA Dynamics in Living Cells.

    PubMed

    Rino, José; de Jesus, Ana C; Carmo-Fonseca, Maria

    2017-01-01

    Single mRNA molecules can be imaged in living cells by a method that consists in genetically inserting binding sites for a bacteriophage protein in the gene of interest. The resulting reporter transgene is then integrated in the genome of cells that express the phage protein fused to a fluorescent tag. Upon transcription, binding of the fluorescent protein to its target sequence makes the RNA visible. With this approach it is possible to track, in real time, the life cycle of a precursor mRNA at the site of transcription in the nucleus and transport of mature mRNA to the cytoplasm. In order to measure the fluorescence associated with individual RNA molecules over time, we developed a semi-automated quantitative image analysis tool termed STaQTool. We describe in detail the implementation and application of the STaQTool software package, which is a generic tool able to process large 4D datasets allowing quantitative studies of different steps in gene expression.

  2. QIN: Quantitative Assessment of Dynamic PET Imaging Data in Cancer Imaging

    PubMed Central

    Muzi, Mark; O’Sullivan, Finbarr; Mankoff, David; Doot, Robert; Pierce, Larry; Kurland, Brenda; Linden, Hannah; Kinahan, Paul

    2012-01-01

    Purpose Clinical imaging in PET is often performed using single time point estimates of tracer uptake or static imaging that provides a spatial map of regional tracer concentration. However, dynamic tracer imaging can provide considerably more information about in vivo biology by delineating both the temporal and spatial pattern of tracer uptake. In addition several potential sources of error that occur in static imaging can be mitigated. This review focuses of the application of dynamic PET imaging to measuring regional cancer biologic features and especially in using dynamic PET imaging for quantitative therapeutic response monitoring in cancer clinical trials. Dynamic PET imaging output parameters, particularly transport (flow) and overall metabolic rate, have provided imaging endpoints for clinical trials at single center institutions for years. However dynamic imaging poses many challenges for multi-center clinical trial implementations from cross-center calibration to the inadequacy of a common informatics infrastructure. Underlying principles and methodology of PET dynamic imaging are first reviewed, followed by an examination of current approaches to dynamic PET image analysis with a specific case example of dynamic FLT imaging to illustrate the approach. PMID:22819579

  3. Transcription factor assisted loading and enhancer dynamics dictate the hepatic fasting response

    PubMed Central

    Goldstein, Ido; Baek, Songjoon; Presman, Diego M.; Paakinaho, Ville; Swinstead, Erin E.; Hager, Gordon L.

    2017-01-01

    Fasting elicits transcriptional programs in hepatocytes leading to glucose and ketone production. This transcriptional program is regulated by many transcription factors (TFs). To understand how this complex network regulates the metabolic response to fasting, we aimed at isolating the enhancers and TFs dictating it. Measuring chromatin accessibility revealed that fasting massively reorganizes liver chromatin, exposing numerous fasting-induced enhancers. By utilizing computational methods in combination with dissecting enhancer features and TF cistromes, we implicated four key TFs regulating the fasting response: glucocorticoid receptor (GR), cAMP responsive element binding protein 1 (CREB1), peroxisome proliferator activated receptor alpha (PPARA), and CCAAT/enhancer binding protein beta (CEBPB). These TFs regulate fuel production by two distinctly operating modules, each controlling a separate metabolic pathway. The gluconeogenic module operates through assisted loading, whereby GR doubles the number of sites occupied by CREB1 as well as enhances CREB1 binding intensity and increases accessibility of CREB1 binding sites. Importantly, this GR-assisted CREB1 binding was enhancer-selective and did not affect all CREB1-bound enhancers. Single-molecule tracking revealed that GR increases the number and DNA residence time of a portion of chromatin-bound CREB1 molecules. These events collectively result in rapid synergistic gene expression and higher hepatic glucose production. Conversely, the ketogenic module operates via a GR-induced TF cascade, whereby PPARA levels are increased following GR activation, facilitating gradual enhancer maturation next to PPARA target genes and delayed ketogenic gene expression. Our findings reveal a complex network of enhancers and TFs that dynamically cooperate to restore homeostasis upon fasting. PMID:28031249

  4. Noise and interlocking signaling pathways promote distinct transcription factor dynamics in response to different stresses

    PubMed Central

    Petrenko, Natalia; Chereji, Raˇzvan V.; McClean, Megan N.; Morozov, Alexandre V.; Broach, James R.

    2013-01-01

    All cells perceive and respond to environmental stresses through elaborate stress-sensing networks. Yeast cells sense stress through diverse signaling pathways that converge on the transcription factors Msn2 and Msn4, which respond by initiating rapid, idiosyncratic cycles into and out of the nucleus. To understand the role of Msn2/4 nuclear localization dynamics, we combined time-lapse studies of Msn2-GFP localization in living cells with computational modeling of stress-sensing signaling networks. We find that several signaling pathways, including Ras/protein kinase A, AMP-activated kinase, the high-osmolarity response mitogen-activated protein kinase pathway, and protein phosphatase 1, regulate activation of Msn2 in distinct ways in response to different stresses. Moreover, we find that bursts of nuclear localization elicit a more robust transcriptional response than does sustained nuclear localization. Using stochastic modeling, we reproduce in silico the responses of Msn2 to different stresses, and demonstrate that bursts of localization arise from noise in the signaling pathways amplified by the small number of Msn2 molecules in the cell. This noise imparts diverse behaviors to genetically identical cells, allowing cell populations to “hedge their bets” in responding to an uncertain future, and to balance growth and survival in an unpredictable environment. PMID:23615444

  5. Integrating single-molecule experiments and discrete stochastic models to understand heterogeneous gene transcription dynamics.

    PubMed

    Munsky, Brian; Fox, Zachary; Neuert, Gregor

    2015-09-01

    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 as 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). Copyright © 2015. Published by Elsevier Inc.

  6. Noise and interlocking signaling pathways promote distinct transcription factor dynamics in response to different stresses.

    PubMed

    Petrenko, Natalia; Chereji, Razvan V; McClean, Megan N; Morozov, Alexandre V; Broach, James R

    2013-06-01

    All cells perceive and respond to environmental stresses through elaborate stress-sensing networks. Yeast cells sense stress through diverse signaling pathways that converge on the transcription factors Msn2 and Msn4, which respond by initiating rapid, idiosyncratic cycles into and out of the nucleus. To understand the role of Msn2/4 nuclear localization dynamics, we combined time-lapse studies of Msn2-GFP localization in living cells with computational modeling of stress-sensing signaling networks. We find that several signaling pathways, including Ras/protein kinase A, AMP-activated kinase, the high-osmolarity response mitogen-activated protein kinase pathway, and protein phosphatase 1, regulate activation of Msn2 in distinct ways in response to different stresses. Moreover, we find that bursts of nuclear localization elicit a more robust transcriptional response than does sustained nuclear localization. Using stochastic modeling, we reproduce in silico the responses of Msn2 to different stresses, and demonstrate that bursts of localization arise from noise in the signaling pathways amplified by the small number of Msn2 molecules in the cell. This noise imparts diverse behaviors to genetically identical cells, allowing cell populations to "hedge their bets" in responding to an uncertain future, and to balance growth and survival in an unpredictable environment.

  7. 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

  8. Application of long-range and binding reverse transcription-quantitative PCR to indicate the viral integrities of noroviruses.

    PubMed

    Li, Dan; De Keuckelaere, Ann; Uyttendaele, Mieke

    2014-10-01

    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/cm(2), 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. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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

    PubMed Central

    Dror, Iris; Rohs, Remo

    2016-01-01

    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

  10. Extensive Rewiring and Complex Evolutionary Dynamics in a C. elegans Multiparameter Transcription Factor Network

    PubMed Central

    Reece-Hoyes, John S.; Pons, Carles; Diallo, Alos; Mori, Akihiro; Shrestha, Shaleen; Kadreppa, Sreenath; Nelson, Justin; DiPrima, Stephanie; Dricot, Amelie; Lajoie, Bryan R.; Ribeiro, Philippe Souza Moraes; Weirauch, Matthew T.; Hill, David E.; Hughes, Timothy R.; Myers, Chad L.; Walhout, Albertha J.M.

    2013-01-01

    SUMMARY Gene duplication results in two identical paralogs that diverge through mutation, leading to loss or gain of interactions with other biomolecules. Here, we comprehensively characterize such network rewiring for C. elegans transcription factors (TFs) within and across four newly delineated molecular networks. Remarkably, we find that even highly similar TFs often have different interaction degree and partners. In addition, we find that most TF families have a member that is highly connected in multiple networks. Further, different TF families have opposing correlations between network connectivity and phylogenetic age, suggesting that they are subject to different evolutionary pressures. Finally, TFs that have similar partners in one network generally do not in another, indicating a lack of pressure to retain cross-network similarity. Our multiparameter analyses provide an unprecedented glimpse into the evolutionary dynamics that shaped TF networks. PMID:23791784

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

    PubMed

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

    2016-05-26

    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.

  12. Complex genomic interactions in the dynamic regulation of transcription by the glucocorticoid receptor.

    PubMed

    Miranda, Tina B; Morris, Stephanie A; Hager, Gordon L

    2013-11-05

    The glucocorticoid receptor regulates transcriptional output through complex interactions with the genome. These events require continuous remodeling of chromatin, interactions of the glucocorticoid receptor with chaperones and other accessory factors, and recycling of the receptor by the proteasome. Therefore, the cohort of factors expressed in a particular cell type can determine the physiological outcome upon treatment with glucocorticoid hormones. In addition, circadian and ultradian cycling of hormones can also affect GR response. Here we will discuss revision of the classical static model of GR binding to response elements to incorporate recent findings from single cell and genome-wide analyses of GR regulation. We will highlight how these studies have changed our views on the dynamics of GR recruitment and its modulation of gene expression.

  13. A quantitative ratiometric sensor for time-resolved analysis of auxin dynamics

    PubMed Central

    Wend, Sabrina; Bosco, Cristina Dal; Kämpf, Michael M.; Ren, Fugang; Palme, Klaus; Weber, Wilfried; Dovzhenko, Alexander; Zurbriggen, Matias D.

    2013-01-01

    Time-resolved quantitative analysis of auxin-mediated processes in plant cells is as of yet limited. By applying a synergistic mammalian and plant synthetic biology approach, we have developed a novel ratiometric luminescent biosensor with wide applicability in the study of auxin metabolism, transport, and signalling. The sensitivity and kinetic properties of our genetically encoded biosensor open new perspectives for the analysis of highly complex auxin dynamics in plant growth and development. PMID:23787479

  14. Systems analysis of dynamic transcription factor activity identifies targets for treatment in Olaparib resistant cancer cells.

    PubMed

    Decker, Joseph T; Hobson, Eric C; Zhang, Yining; Shin, Seungjin; Thomas, Alexandra L; Jeruss, Jacqueline S; Arnold, Kelly B; Shea, Lonnie D

    2017-09-01

    The development of resistance to targeted therapeutics is a challenging issue for the treatment of cancer. Cancers that have mutations in BRCA, a DNA repair protein, have been treated with poly(ADP-ribose) polymerase (PARP) inhibitors, which target a second DNA repair mechanism with the aim of inducing synthetic lethality. While these inhibitors have shown promise clinically, the development of resistance can limit their effectiveness as a therapy. This study investigated mechanisms of resistance in BRCA-mutated cancer cells (HCC1937) to Olaparib (AZD2281) using TRACER, a technique for measuring dynamics of transcription factor (TF) activity in living cells. TF activity was monitored in the parental HCC1937 cell line and two distinct resistant cell lines, one with restored wild-type BRCA1 and one with acquired resistance independent of BRCA1 for 48 h during treatment with Olaparib. Partial least squares discriminant analysis (PLSDA) was used to categorize the three cell types based on TF activity, and network analysis was used to investigate the mechanism of early response to Olaparib in the study cells. NOTCH signaling was identified as a common pathway linked to resistance in both Olaparib-resistant cell types. Western blotting confirmed upregulation of NOTCH protein, and sensitivity to Olaparib was restored through co-treatment with a gamma secretase inhibitor. The identification of NOTCH signaling as a common pathway contributing to PARP inhibitor resistance by TRACER indicates the efficacy of transcription factor dynamics in identifying targets for intervention in treatment-resistant cancer and provides a new method for determining effective strategies for directed chemotherapy. Biotechnol. Bioeng. 2017;114: 2085-2095. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

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

    PubMed

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

    2013-10-01

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

  16. Extensions and evaluations of a general quantitative theory of forest structure and dynamics

    PubMed Central

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

    2009-01-01

    Here, we present the second part of a quantitative theory for the structure and dynamics of forests under demographic and resource steady state. The theory is based on individual-level allometric scaling relations for how trees use resources, fill space, and grow. These scale up to determine emergent properties of diverse forests, including size–frequency distributions, spacing relations, canopy configurations, mortality rates, population dynamics, successional dynamics, and resource flux rates. The theory uniquely makes quantitative predictions for both stand-level scaling exponents and normalizations. We evaluate these predictions by compiling and analyzing macroecological datasets from several tropical forests. The close match between theoretical predictions and data suggests that forests are organized by a set of very general scaling rules. Our mechanistic theory is based on allometric scaling relations, is complementary to “demographic theory,” but is fundamentally different in approach. It provides a quantitative baseline for understanding deviations from predictions due to other factors, including disturbance, variation in branching architecture, asymmetric competition, resource limitation, and other sources of mortality, which are not included in the deliberately simplified theory. The theory should apply to a wide range of forests despite large differences in abiotic environment, species diversity, and taxonomic and functional composition. PMID:19363161

  17. 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

    2016-07-05

    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. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  18. 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

  19. 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.

  20. [Application of Real-time Quantitative PCR in Detecting Atypical BCR/ABL mRNA Transcripts in Chronic Myelocytic Leukemia].

    PubMed

    Zou, Yuan; DU, Cui; Chen, Hong-Mei; Guo, Fu-Xiao; Cheng, Jian-Bing; Tang, Yuan-Yan; Wu, Wei; Xia, Cheng-Qing

    2017-08-01

    To detect atypical BCR/ABL mRNA transcript by real-time quantitative PCR in CML patients without e13a2/e14a2,e19a2 or e1a2 transcripts, and investigate its value of clinical application. Twelve cases of CML with positive for t(9;22) translocation, but negative for common major and minor breakpoint cluster regions comfirmed by chromosome karyotyping or FISH analysis, were collected from July 2012 to December 2015. These 12 cases were then detected for b2a3(e13a3), b3a3(e14a3), e6a2, e8a2 and e1a3 fusion variants by real-time quantitative PCR. Among 12 cases 4 variant transcripts were detected, including e1a3 in 1 case (8.33%), e8a2 in 2 cases (16.67%), b2a3 in 5 cases (41.67%) and b3a3 in 4 cases (33.33%), with total positivity of 100%, moreover b2a3 and b3a3 were predominant. The detecting atypical BCR/ABL mRNA transcripts by real-time quantitative PCR is suitable for the diagnosis of CML negative for P210, P190 and P230 by standard real-time PCR test, and this detection is still the standard and economic method for monitoring minimal residual disease in CML patients with variants of BCR/ABL fusion gene.

  1. Evaluation of transcription levels of inlA, inlB, hly, bsh and prfA genes in Listeria monocytogenes strains using quantitative reverse-transcription PCR and ability of invasion into human CaCo-2 cells.

    PubMed

    Tamburro, Manuela; Sammarco, Michela Lucia; Ammendolia, Maria Grazia; Fanelli, Incoronata; Minelli, Fabio; Ripabelli, Giancarlo

    2015-03-01

    Listeria monocytogenes virulence depends on the activity of well-characterized virulence factors. In this study, transcription levels of inlA, inlB, hly, bsh and prfA genes in L. monocytogenes strains, and the ability of invasion into CaCo-2 cells were investigated. Serotyping, multiplex-PCR for serovar identification and restriction fragment analysis of inlA were performed. Transcription levels and invasiveness were evaluated by quantitative reverse-transcription PCR and by in vitro assays, respectively. The isolates were of serovars 1/2a, 4b, 1/2c, 1/2b and 3a. Full-length inlA profiles were found for nine of ten clinical isolates, while five of seven cultures from foods showed truncated profile. The analysis of transcription levels of virulence factors encoding genes demonstrated a substantial inter-strain heterogeneity, with clinical strains showing higher levels for almost all genes than isolates from food. A correlation between transcription levels of inlA and inlB, as well as between bsh and prfA, was observed. Significant differences between clinical strains and food isolates in the invasion of CaCo-2 cells were found. Analysis of gene transcription and invasiveness of human cells suggests different virulence phenotypes among L. monocytogenes populations, and this characterization could be a useful tool for risk assessment purposes and for the development of public health strategies. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  2. 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.

  3. 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.

  4. 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

  5. Quantitative cw Overhauser effect dynamic nuclear polarization for the analysis of local water dynamics.

    PubMed

    Franck, John M; Pavlova, Anna; Scott, John A; Han, Songi

    2013-10-01

    Liquid state Overhauser effect Dynamic Nuclear Polarization (ODNP) has experienced a recent resurgence of interest. The ODNP technique described here relies on the double resonance of electron spin resonance (ESR) at the most common, i.e. X-band (∼10GHz), frequency and ¹H nuclear magnetic resonance (NMR) at ∼15 MHz. It requires only a standard continuous wave (cw) ESR spectrometer with an NMR probe inserted or built into an X-band cavity. We focus on reviewing a new and powerful manifestation of ODNP as a high frequency NMR relaxometry tool that probes dipolar cross relaxation between the electron spins and the ¹H nuclear spins at X-band frequencies. This technique selectively measures the translational mobility of water within a volume extending 0.5-1.5 nm outward from a nitroxide radical spin probe that is attached to a targeted site of a macromolecule. It allows one to study the dynamics of water that hydrates or permeates the surface or interior of proteins, polymers, and lipid membrane vesicles. We begin by reviewing the recent advances that have helped develop ODNP into a tool for mapping the dynamic landscape of hydration water with sub-nanometer locality. In order to bind this work coherently together and to place it in the context of the extensive body of research in the field of NMR relaxometry, we then rephrase the analytical model and extend the description of the ODNP-derived NMR signal enhancements. This extended model highlights several aspects of ODNP data analysis, including the importance of considering all possible effects of microwave sample heating, the need to consider the error associated with various relaxation rates, and the unique ability of ODNP to probe the electron-¹H cross-relaxation process, which is uniquely sensitive to fast (tens of ps) dynamical processes. By implementing the relevant corrections in a stepwise fashion, this paper draws a consensus result from previous ODNP procedures and then shows how such data can be

  6. Quantitative cw Overhauser Dynamic Nuclear Polarization for the Analysis of Local Water Dynamics

    PubMed Central

    Franck, John M.; Pavlova, Anna; Scott, John A.; Han, Songi

    2013-01-01

    Liquid state Overhauser Effect Dynamic Nuclear Polarization (ODNP) has experienced a recent resurgence of interest. The ODNP technique described here relies on the double resonance of electron spin resonance (ESR) at the most common, i.e. X-band (~ 10 GHz), frequency and 1H nuclear magnetic resonance (NMR) at ~ 15 MHz. It requires only a standard continuous wave (cw) ESR spectrometer with an NMR probe inserted or built into an X-band cavity. Our focus lies on reviewing a new and powerful manifestation of ODNP as a high frequency NMR relaxometry tool that probes dipolar cross relaxation between the electron spins and the 1H nuclear spins at X-band frequencies. This technique selectively measures the translational mobility of water within a volume extending 0.5–1.5 nm outward from a nitroxide radical spin probe that is attached to a targeted site of a macromolecule. This method has been applied to study the dynamics of water that hydrates or permeates the surface or interior of proteins, polymers, and lipid membrane vesicles. We begin by reviewing the recent advances that have helped develop ODNP into a tool for mapping the dynamic landscape of hydration water with sub-nanometer locality. In order to bind this work coherently together, and to place it in the context of the extensive body of research in the field of NMR relaxometry, we then rephrase the analytical model and extend the description of the ODNP-derived NMR signal enhancements. This extended model highlights several aspects of ODNP data analysis, including the importance of considering all possible effects of microwave sample heating, the need to consider the error associated with various relaxation rates, and the unique ability of ODNP to probe the electron–1H cross-relaxation process, which is uniquely sensitive to fast (tens of ps) dynamical processes. By implementing the relevant corrections in a stepwise fashion, this paper draws a consensus result from previous ODNP procedures, and then shows

  7. 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

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

    PubMed Central

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

    2016-01-01

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

  9. 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.

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

    NASA Astrophysics Data System (ADS)

    Lee, SangYun; Kim, Kyoohyun; Park, YongKeun

    2017-02-01

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

  11. Stability of reference genes for normalization of reverse transcription quantitative real-time PCR (RT-qPCR) data in bovine blastocysts produced by IVF, ICSI and SCNT.

    PubMed

    Luchsinger, Charlotte; Arias, María Elena; Vargas, Tamara; Paredes, Marcos; Sánchez, Raúl; Felmer, Ricardo

    2014-11-01

    Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a sensitive and accurate tool for quantitative estimation of gene transcription levels in preimplantation embryos. To control for possible experimental variations, gene expression data must be normalized using internal control genes commonly known as reference genes. However, the stability of reference genes can vary depending on the state of development and/or experimental conditions; hence the assessment of their stability is essential before initiating a gene expression analysis. In the present study, we used RT-qPCR to measure the transcript levels of 10 commonly used reference genes and analyzed their expression stability in bovine blastocysts produced by in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI) and somatic cell nuclear transfer (SCNT). Using the geNorm program, we found the best combination of genes to normalize gene expression data in bovine embryos at the blastocyst stage produced by IVF (HMBS, SF3A1, and HPRT1), ICSI (H2A, HMBS, and GAPDH), SCNT (ACTB, SF3A1, and SDHA) and/or between blastocysts produced by these methods (GAPDH, HMBS and EEF1A2). We also demonstrated that not only the culture conditions may affect the expression patterns in bovine blastocysts but also the choice of embryo production method may have an important effect.

  12. 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

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

    PubMed

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

    2016-04-20

    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.

  14. Rapid Temporal Dynamics of Transcription, Protein Synthesis, and Secretion during Macrophage Activation*

    PubMed Central

    Eichelbaum, Katrin; Krijgsveld, Jeroen

    2014-01-01

    Macrophages provide the first line of host defense with their capacity to react to an array of cytokines and bacterial components requiring tight regulation of protein expression and secretion to invoke a properly tuned innate immune response. To capture the dynamics of this system, we introduce a novel method combining pulsed stable isotope labeling with amino acids in cell culture (SILAC) with pulse labeling using the methionine analog azidohomoalanine that allows the enrichment of newly synthesized proteins via click-chemistry followed by their identification and quantification by mass spectrometry. We show that this permits the analysis of proteome changes on a rapid time scale, as evidenced by the detection of 4852 newly synthesized proteins after only a 20-min SILAC pulse. We have applied this methodology to study proteome response during macrophage activation in a time-course manner. We have combined this with full proteome, transcriptome, and secretome analyses, producing an integrative analysis of the first 3 h of lipopolysaccharide-induced macrophage activation. We observed the rapid induction of multiple processes well known to TLR4 signaling, as well as anti-inflammatory proteins and proteins not previously associated with immune response. By correlating transcriptional, translational, and secretory events, we derived novel mechanistic principles of processes specifically induced by lipopolysaccharides, including ectodomain shedding and proteolytic processing of transmembrane and extracellular proteins and protein secretion independent of transcription. In conclusion, we demonstrate that the combination of pulsed azidohomoalanine and pulsed SILAC permits the detailed characterization of proteomic events on a rapid time scale. We anticipate that this approach will be very useful in probing the immediate effects of cellular stimuli and will provide mechanistic insight into cellular perturbation in multiple biological systems. The data have been deposited

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

    PubMed

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

    2011-01-01

    Analytical platforms for the fast detection, identification and quantification of circulating drugs with a narrow therapeutic range are vital in clinical pharmacology. As a result of low drug concentrations, analytical tools need to provide high sensitivity and specificity. Dynamic nuclear polarization-NMR (DNP-NMR) in the form of the hyperpolarization-dissolution method should afford the sensitivity and spectral resolution for the direct detection and quantification of numerous isotopically labeled circulating drugs and their metabolites in single liquid-state NMR transients. This study explores the capability of quantitative in vitro DNP-NMR to assay drug metabolites in blood plasma. The lower limit of detection for the anti-epileptic drug (13)C-carbamazepine and its pharmacologically active metabolite (13)C-carbamazepine-10,11-epoxide is 0.08 µg/mL in rabbit blood plasma analyzed by single-scan (13)C DNP-NMR. An internal standard is used for the accurate quantification of drug and metabolite. Comparison of quantitative DNP-NMR data with an established analytical method (liquid chromatography-mass spectrometry) yields a Pearson correlation coefficient r of 0.99. Notably, all DNP-NMR determinations were performed without analyte derivatization or sample purification other than plasma protein precipitation. Quantitative DNP-NMR is an emerging methodology which requires little sample preparation and yields quantitative data with high sensitivity for therapeutic drug monitoring. Copyright © 2010 John Wiley & Sons, Ltd.

  16. Dynamic and quantitative method of analyzing service consistency evolution based on extended hierarchical finite state automata.

    PubMed

    Fan, Linjun; Tang, Jun; Ling, Yunxiang; Li, Benxian

    2014-01-01

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

  17. Surfactant uptake dynamics in mammalian cells elucidated with quantitative coherent anti-stokes Raman scattering microspectroscopy.

    PubMed

    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.

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

    PubMed Central

    Fan, Linjun; Tang, Jun; Ling, Yunxiang; Li, Benxian

    2014-01-01

    This paper is concerned with the dynamic evolution analysis and quantitative measurement of primary factors that cause service inconsistency in service-oriented distributed simulation applications (SODSA). Traditional methods are mostly qualitative and empirical, and they do not consider the dynamic disturbances among factors in service's evolution behaviors such as producing, publishing, calling, and maintenance. Moreover, SODSA are rapidly evolving in terms of large-scale, reusable, compositional, pervasive, and flexible features, which presents difficulties in the usage of traditional analysis methods. To resolve these problems, a novel dynamic evolution model extended hierarchical service-finite state automata (EHS-FSA) is constructed based on finite state automata (FSA), which formally depict overall changing processes of service consistency states. And also the service consistency evolution algorithms (SCEAs) based on EHS-FSA are developed to quantitatively assess these impact factors. Experimental results show that the bad reusability (17.93% on average) is the biggest influential factor, the noncomposition of atomic services (13.12%) is the second biggest one, and the service version's confusion (1.2%) is the smallest one. Compared with previous qualitative analysis, SCEAs present good effectiveness and feasibility. This research can guide the engineers of service consistency technologies toward obtaining a higher level of consistency in SODSA. PMID:24772033

  19. A new 3-dimensional dynamic quantitative analysis system of facial motion: an establishment and reliability test.

    PubMed

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

    2014-01-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

    Schierz, Philipp; Zierenberg, Johannes; Janke, Wolfhard

    2015-10-07

    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.

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

    PubMed

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

    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.

  3. 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.

  4. 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

  5. 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.

  6. Quantitative protein expression profiling reveals extensive post-transcriptional regulation and post-translational modifications in schizont-stage malaria parasites

    PubMed Central

    Foth, Bernardo J; Zhang, Neng; Mok, Sachel; Preiser, Peter R; Bozdech, Zbynek

    2008-01-01

    Background Malaria is a one of the most important infectious diseases and is caused by parasitic protozoa of the genus Plasmodium. Previously, quantitative characterization of the P. falciparum transcriptome demonstrated that the strictly controlled progression of these parasites through their intra-erythrocytic developmental cycle is accompanied by a continuous cascade of gene expression. Although such analyses have proven immensely useful, the correlations between abundance of transcripts and their cognate proteins remain poorly characterized. Results Here, we present a quantitative time-course analysis of relative protein abundance for schizont-stage parasites (34 to 46 hours after invasion) based on two-dimensional differential gel electrophoresis of protein samples labeled with fluorescent dyes. For this purpose we analyzed parasite samples taken at 4-hour intervals from a tightly synchronized culture and established more than 500 individual protein abundance profiles with high temporal resolution and quantitative reproducibility. Approximately half of all profiles exhibit a significant change in abundance and 12% display an expression peak during the observed 12-hour time interval. Intriguingly, identification of 54 protein spots by mass spectrometry revealed that 58% of the corresponding proteins - including actin-I, enolase, eukaryotic initiation factor (eIF)4A, eIF5A, and several heat shock proteins - are represented by more than one isoform, presumably caused by post-translational modifications, with the various isoforms of a given protein frequently showing different expression patterns. Furthermore, comparisons with transcriptome data generated from the same parasite samples reveal evidence of significant post-transcriptional gene expression regulation. Conclusions Together, our data indicate that both post-transcriptional and post-translational events are widespread and of presumably great biological significance during the intra

  7. CytoModeler: a tool for bridging large-scale network analysis and dynamic quantitative modeling

    PubMed Central

    Xia, Tian; Van Hemert, John; Dickerson, Julie A.

    2011-01-01

    Summary: CytoModeler is an open-source Java application based on the Cytoscape platform. It integrates large-scale network analysis and quantitative modeling by combining omics analysis on the Cytoscape platform, access to deterministic and stochastic simulators, and static and dynamic network context visualizations of simulation results. Availability: Implemented in Java, CytoModeler runs with Cytoscape 2.6 and 2.7. Binaries, documentation and video walkthroughs are freely available at http://vrac.iastate.edu/~jlv/cytomodeler/. Contact: julied@iastate.edu; netscape@iastate.edu Supplementary Information: Supplementary data are available at Bioinformatics online. PMID:21511714

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

    PubMed

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

    2013-01-01

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

  9. Quantitative comparison of dynamic flux distribution of magnetic couplers for roadway electric vehicle wireless charging system

    NASA Astrophysics Data System (ADS)

    Qiu, Chun; Chau, K. T.; Liu, Chunhua; Li, Wenlong; Lin, Fei

    2014-05-01

    This paper gives a quantitative comparison of magnetic couplers for electric vehicle (EV) wireless charging applications. Circular pad with ferrite spokes and coreless rectangular coils are specially selected for analysis. The dynamic flux density between couplers under high misalignment is studied by calculating the uncompensated power of the pick-up coupler. By using finite element analysis, the performance of each type of coupler is evaluated, and its adaptation to on-road EV charging are compared according to the flux distribution and effective charging area.

  10. Nonhost Resistance of Barley to Different Fungal Pathogens Is Associated with Largely Distinct, Quantitative Transcriptional Responses1[W][OA

    PubMed Central

    Zellerhoff, Nina; Himmelbach, Axel; Dong, Wubei; Bieri, Stephane; Schaffrath, Ulrich; Schweizer, Patrick

    2010-01-01

    Nonhost resistance protects plants against attack by the vast majority of potential pathogens, including phytopathogenic fungi. Despite its high biological importance, the molecular architecture of nonhost resistance has remained largely unexplored. Here, we describe the transcriptional responses of one particular genotype of barley (Hordeum vulgare subsp. vulgare ‘Ingrid’) to three different pairs of adapted (host) and nonadapted (nonhost) isolates of fungal pathogens, which belong to the genera Blumeria (powdery mildew), Puccinia (rust), and Magnaporthe (blast). Nonhost resistance against each of these pathogens was associated with changes in transcript abundance of distinct sets of nonhost-specific genes, although general (not nonhost-associated) transcriptional responses to the different pathogens overlapped considerably. The powdery mildew- and blast-induced differences in transcript abundance between host and nonhost interactions were significantly correlated with differences between a near-isogenic pair of barley lines that carry either the Mlo wild-type allele or the mutated mlo5 allele, which mediates basal resistance to powdery mildew. Moreover, during the interactions of barley with the different host or nonhost pathogens, similar patterns of overrepresented and underrepresented functional categories of genes were found. The results suggest that nonhost resistance and basal host defense of barley are functionally related and that nonhost resistance to different fungal pathogens is associated with more robust regulation of complex but largely nonoverlapping sets of pathogen-responsive genes involved in similar metabolic or signaling pathways. PMID:20172964

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

    PubMed

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

    2012-04-01

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

  12. 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

  13. 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

    2016-03-07

    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.

  14. Quantitative levels of Deficiens and Globosa during late petal development show a complex transcriptional network topology of B function.

    PubMed

    Manchado-Rojo, María; Delgado-Benarroch, Luciana; Roca, María J; Weiss, Julia; Egea-Cortines, Marcos

    2012-10-01

    The transcriptional network topology of B function in Antirrhinum, required for petal and stamen development, is thought to rely on initial activation of transcription of DEFICIENS (DEF) and GLOBOSA (GLO), followed by a positive autoregulatory loop maintaining gene expression levels. Here, we show that the mutant compacta (co), whose vegetative growth and petal size are affected, plays a role in B function. Late events in petal morphogenesis such as development of conical cell area and scent emissions were reduced in co and def (nicotianoides) (def (nic) ), and absent in co def (nic) double mutants, suggesting a role for CO in petal identity. Expression of DEF was down-regulated in co but surprisingly GLO was not affected. We investigated the levels of DEF and GLO at late stages of petal development in the co, def (nic) and glo-1 mutants, and established a reliable transformation protocol that yielded RNAi-DEF lines. We show that the threshold levels of DEF or GLO required to obtain petal tissue are approximately 11% of wild-type. The relationship between DEF and GLO transcripts is not equal or constant and changes during development. Furthermore, down-regulation of DEF or GLO does not cause parallel down-regulation of the partner. Our results demonstrate that, at late stages of petal development, the B function transcriptional network topology is not based on positive autoregulation, and has additional components of transcriptional maintenance. Our results suggest changes in network topology that may allow changes in protein complexes that would explain the fact that not all petal traits appear early in development.

  15. An alternative method for the synthesis of competitor RNA transcripts useful for specific detection and quantitation of dengue virus serotype 2 genome and replicative intermediate RNA.

    PubMed

    Vaughan, Gilberto; Gonzalez-Hernandez, Yolanda; Gudino, Jose-Carmen; Olivera, Hiram; Landa-Piedra, Abraham; Escobar-Gutierrez, Alejandro

    2008-09-01

    The development of a quantitative-competitive reverse transcription-PCR (RT-PCR) assay to quantify dengue virus (DEN) genome (vRNA) and its replicative intermediate RNA (vRI) is described. A highly conserved region located on the DEN capsid-premembrane genes was used to produce a competitor RNA molecule which contains an internal deletion of 70 nucleotides. The competitor provides a suitable internal control useful to quantify viral RNA from all four dengue virus (DEN 1-4) serotypes. The detection limit of the assay was found to be 100 copies per reaction. This is a rapid, simple, sensitive, inexpensive and easy method for quantitation of DEN RNA species.

  16. 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.

  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. 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

  19. 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.

  20. 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.

  1. Characterization of expression dynamics of WOX homeodomain transcription factors during somatic embryogenesis in Vitis vinifera.

    PubMed

    Gambino, Giorgio; Minuto, Martina; Boccacci, Paolo; Perrone, Irene; Vallania, Rosalina; Gribaudo, Ivana

    2011-01-01

    Different cultivars of Vitis vinifera vary in their potential to form embryogenic tissues. The WUSCHEL (WUS)-related homeobox (WOX) genes have been shown to play an important role in coordinating the gene transcription involved in the early phases of embryogenesis. The expression dynamics of 12 VvWOX genes present in the V. vinifera genome in embryogenic and other tissues of 'Chardonnay' were analysed. In order to understand the influence of WOX genes on the somatic embryogenic process, their expression profiles were compared in two cultivars of V. vinifera ('Chardonnay' and 'Cabernet Sauvignon') that show different aptitudes for embryogenesis. The expression of all VvWOX genes was influenced by culture conditions. VvWOX2 and VvWOX9 were the principal WOX genes expressed during the somatic embryogenesis process, and the low aptitude for embryogenesis of 'Cabernet Sauvignon' was generally correlated with the low expression levels of these VvWOX genes. VvWOX3 and VvWOX11 were strongly activated in correspondence to torpedo and cotyledonary stages of somatic embryos, with low expression in the earlier developmental stages (pre-embryogenic masses and globular embryos) and during embryo germination. VvWOX genes appeared to be key regulators of somatic embryogenesis in grapevine, and the regulation of these genes during early phases of somatic embryogenesis differed between the two cultivars of the same species.

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

    PubMed Central

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

    2016-01-01

    Abstract. 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 0.1  ml/min/g) compared to linearly interpreted static assessment (0.45 to 0.48  ml/min/g), 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 (p<0.05) in stratifying patients, especially for larger patients and lower contrast doses [area under the curve (AUC)=0.95 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. PMID:27175377

  3. 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

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

    PubMed

    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.

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

    DOE PAGES

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

    2017-04-07

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

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

    PubMed Central

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

    2017-01-01

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

  7. 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

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

    PubMed

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

    2017-04-07

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

  9. 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

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

    PubMed

    Rapp, Paul E; Cellucci, Christopher J; Gilpin, Adele M K; Jiménez-Montaño, Miguel A; Korslund, Kathryn E

    2011-07-27

    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. 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. 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). 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 value in the examination of therapies

  11. 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

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. Quantitative measurement of dynamic nanostrain based on a phase-sensitive optical time domain reflectometer.

    PubMed

    Dong, Yongkang; Chen, Xi; Liu, Erhu; Fu, Cheng; Zhang, Hongying; Lu, Zhiwei

    2016-10-01

    A sensing system is proposed for quantitative measurement of large-range dynamic nanostrain based on a phase-sensitive optical time domain reflectometer, where the coherent detection and I/Q demodulation methods are employed to demodulate both the phase and the amplitude of the Rayleigh scattering light in real time. A nanopositioning translation stage is utilized to apply precise nanostrain to fiber. By measuring phase differences between two adjacent sections, the quantitative nanostrain with a large measurement range is demonstrated; this is also a method to measure the strain parameter of refractive index. For the Panda polarization-maintaining fiber under test in the experiment, the strain parameter of phase difference is measured to be 8.714  mrad/(nε·m), while the strain parameter of refractive index is measured to be -0.3751ε-1. As a proof of the concept, the dynamic strain sensing with a range of 10-1000 nε is experimentally demonstrated, and the strain resolution is 1 or 2 nε, corresponding to 5 or 2.5 m spatial resolution, respectively. The experimental measurement also shows a triangular wave with a 12-Hz vibrating frequency and a 100-nε strain amplitude as well as a 188-Hz resonant signal of the tensile section.

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

    PubMed Central

    Xu, Xiangqun; Zhu, Jiang; Chen, Zhongping

    2016-01-01

    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. PMID:27090437

  18. 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.

  19. Fast and Quantitative T1ρ-weighted Dynamic Glucose Enhanced MRI

    PubMed Central

    Schuenke, Patrick; Paech, Daniel; Koehler, Christina; Windschuh, Johannes; Bachert, Peter; Ladd, Mark E.; Schlemmer, Heinz-Peter; Radbruch, Alexander; Zaiss, Moritz

    2017-01-01

    Common medical imaging techniques usually employ contrast agents that are chemically labeled, e.g. with radioisotopes in the case of PET, iodine in the case of CT or paramagnetic metals in the case of MRI to visualize the heterogeneity of the tumor microenvironment. Recently, it was shown that natural unlabeled D-glucose can be used as a nontoxic biodegradable contrast agent in Chemical Exchange sensitive Spin-Lock (CESL) magnetic resonance imaging (MRI) to detect the glucose uptake and potentially the metabolism of tumors. As an important step to fulfill the clinical needs for practicability, reproducibility and imaging speed we present here a robust and quantitative T1ρ-weighted technique for dynamic glucose enhanced MRI (DGE-MRI) with a temporal resolution of less than 7 seconds. Applied to a brain tumor patient, the new technique provided a distinct DGE contrast between tumor and healthy brain tissue and showed the detailed dynamics of the glucose enhancement after intravenous injection. Development of this fast and quantitative DGE-MRI technique allows for a more detailed analysis of DGE correlations in the future and potentially enables non-invasive diagnosis, staging and monitoring of tumor response to therapy. PMID:28169369

  20. Identification of novel transcriptional regulators of PKA subunits in Saccharomyces cerevisiae by quantitative promoter-reporter screening.

    PubMed

    Pautasso, Constanza; Reca, Sol; Chatfield-Reed, Kate; Chua, Gordon; Galello, Fiorella; Portela, Paula; Zaremberg, Vanina; Rossi, Silvia

    2016-08-01

    The cAMP-dependent protein kinase (PKA) signaling is a broad pathway that plays important roles in the transduction of environmental signals triggering precise physiological responses. However, how PKA achieves the cAMP-signal transduction specificity is still in study. The regulation of expression of subunits of PKA should contribute to the signal specificity. Saccharomyces cerevisiae PKA holoenzyme contains two catalytic subunits encoded by TPK1, TPK2 and TPK3 genes, and two regulatory subunits encoded by BCY1 gene. We studied the activity of these gene promoters using a fluorescent reporter synthetic genetic array screen, with the goal of systematically identifying novel regulators of expression of PKA subunits. Gene ontology analysis of the identified modulators showed enrichment not only in the category of transcriptional regulators, but also in less expected categories such as lipid and phosphate metabolism. Inositol, choline and phosphate were identified as novel upstream signals that regulate transcription of PKA subunit genes. The results support the role of transcription regulation of PKA subunits in cAMP specificity signaling. Interestingly, known targets of PKA phosphorylation are associated with the identified pathways opening the possibility of a reciprocal regulation. PKA would be coordinating different metabolic pathways and these processes would in turn regulate expression of the kinase subunits. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  1. Quantitative analysis of proteome and lipidome dynamics reveals functional regulation of global lipid metabolism.

    PubMed

    Casanovas, Albert; Sprenger, Richard R; Tarasov, Kirill; Ruckerbauer, David E; Hannibal-Bach, Hans Kristian; Zanghellini, Jürgen; Jensen, Ole N; Ejsing, Christer S

    2015-03-19

    Elucidating how and to what extent lipid metabolism is remodeled under changing conditions is essential for understanding cellular physiology. Here, we analyzed proteome and lipidome dynamics to investigate how regulation of lipid metabolism at the global scale supports remodeling of cellular architecture and processes during physiological adaptations in yeast. Our results reveal that activation of cardiolipin synthesis and remodeling supports mitochondrial biogenesis in the transition from fermentative to respiratory metabolism, that down-regulation of de novo sterol synthesis machinery prompts differential turnover of lipid droplet-associated triacylglycerols and sterol esters during respiratory growth, that sphingolipid metabolism is regulated in a previously unrecognized growth stage-specific manner, and that endogenous synthesis of unsaturated fatty acids constitutes an in vivo upstream activator of peroxisomal biogenesis, via the heterodimeric Oaf1/Pip2 transcription factor. Our work demonstrates the pivotal role of lipid metabolism in adaptive processes and provides a resource to investigate its regulation at the cellular level.

  2. Live imaging of nascent RNA dynamics reveals distinct types of transcriptional pulse regulation

    PubMed Central

    Muramoto, Tetsuya; Cannon, Danielle; Gierliński, Marek; Corrigan, Adam; Barton, Geoffrey J.; Chubb, Jonathan R.

    2012-01-01

    Transcription of genes can be discontinuous, occurring in pulses or bursts. It is not clear how properties of transcriptional pulses vary between different genes. We compared the pulsing of five housekeeping and five developmentally induced genes by direct imaging of single gene transcriptional events in individual living Dictyostelium cells. Each gene displayed its own transcriptional signature, differing in probability of firing and pulse duration, frequency, and intensity. In contrast to the prevailing view from both prokaryotes and eukaryotes that transcription displays binary behavior, strongly expressed housekeeping genes altered the magnitude of their transcriptional pulses during development. These nonbinary “tunable” responses may be better suited than stochastic switch behavior for housekeeping functions. Analysis of RNA synthesis kinetics using fluorescence recovery after photobleaching implied modulation of housekeeping-gene pulse strength occurs at the level of transcription initiation rather than elongation. In addition, disparities between single cell and population measures of transcript production suggested differences in RNA stability between gene classes. Analysis of stability using RNAseq revealed no major global differences in stability between developmental and housekeeping transcripts, although strongly induced RNAs showed unusually rapid decay, indicating tight regulation of expression. PMID:22529358

  3. Live imaging of nascent RNA dynamics reveals distinct types of transcriptional pulse regulation.

    PubMed

    Muramoto, Tetsuya; Cannon, Danielle; Gierlinski, Marek; Corrigan, Adam; Barton, Geoffrey J; Chubb, Jonathan R

    2012-05-08

    Transcription of genes can be discontinuous, occurring in pulses or bursts. It is not clear how properties of transcriptional pulses vary between different genes. We compared the pulsing of five housekeeping and five developmentally induced genes by direct imaging of single gene transcriptional events in individual living Dictyostelium cells. Each gene displayed its own transcriptional signature, differing in probability of firing and pulse duration, frequency, and intensity. In contrast to the prevailing view from both prokaryotes and eukaryotes that transcription displays binary behavior, strongly expressed housekeeping genes altered the magnitude of their transcriptional pulses during development. These nonbinary "tunable" responses may be better suited than stochastic switch behavior for housekeeping functions. Analysis of RNA synthesis kinetics using fluorescence recovery after photobleaching implied modulation of housekeeping-gene pulse strength occurs at the level of transcription initiation rather than elongation. In addition, disparities between single cell and population measures of transcript production suggested differences in RNA stability between gene classes. Analysis of stability using RNAseq revealed no major global differences in stability between developmental and housekeeping transcripts, although strongly induced RNAs showed unusually rapid decay, indicating tight regulation of expression.

  4. Confocal reflectance quantitative phase microscope system for cellular membranes dynamics study (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Singh, Vijay Raj; Yaqoob, Zahid; So, Peter T. C.

    2017-02-01

    Quantitative phase microscopy (QPM) techniques developed so far primarily belongs to high speed transmitted light based systems that has enough sensitivity to resolve membrane fluctuations and dynamics, but has no depth resolution. Therefore, most biomechanics studies using QPM today is confined to simple cells, such as RBCs, without internal organelles. An important instrument that will greatly extend the biomedical applications of QPM is to develop next generation microscope with 3D capability and sufficient temporal resolution to study biomechanics of complex eukaryotic cells including the mechanics of their internal compartments. For eukaryotic cells, the depth sectioning capability is critical and should be sufficient to distinguish nucleic membrane fluctuations from plasma membrane fluctuations. Further, this microscope must provide high temporal resolution since typical eukaryotes membranes are substantially stiffer than RBCs. A confocal reflectance quantitative phase microscope is presented based on multi-pinhole scanning, with the capabilities of higher temporal resolution and sensitivity for nucleic and plasma membranes of eukaryotic cells. System hardware is developed based on an array of confocal pinhole generated by using the `ON' state of subset of micro-mirrors of digital micro-mirror device (DMD, from Texas Instruments) and high-speed raster scanning provides 14ms imaging speed in wide-field mode. A common path interferometer is integrated at the imaging arm for detection of specimens' quantitative phase information. Theoretical investigation of quantitative phase reconstructed from system is investigated and application of system is presented for dimensional fluctuations measurements of both cellular plasma and nucleic membranes of embryonic stem cells.

  5. Gait dynamics in trisomic mice: quantitative neurological traits of Down syndrome.

    PubMed

    Hampton, Thomas G; Stasko, Melissa R; Kale, Ajit; Amende, Ivo; Costa, Alberto C S

    2004-09-15

    The segmentally trisomic mouse Ts65Dn is a model of Down syndrome (DS). Gait abnormalities are almost universal in persons with DS. We applied a noninvasive imaging method to quantitatively compare the gait dynamics of Ts65Dn mice (n=10) to their euploid littermates (controls) (n=10). The braking duration of the hind limbs in Ts65Dn mice was prolonged compared to that in control mice (60+/-3 ms vs. 49+/-2 ms, P<.05) at a slow walking speed (18 cm/s). Stride length and stride frequency of forelimbs and hind limbs were comparable between Ts65Dn mice and control mice. Stride dynamics were significantly different in Ts65Dn mice at a faster walking speed (36 cm/s). Stride length was shorter in Ts65Dn mice (5.9+/-0.1 vs. 6.3+/-0.3 cm, P<.05), and stride frequency was higher in Ts65Dn compared to control mice (5.9+/-0.1 vs. 5.3+/-0.1 strides/s, P<.05). Hind limb swing duration was prolonged in Ts65Dn mice compared to control mice (93+/-3 vs. 76+/-3 ms, P<.05). Propulsion of the forelimbs contributed to a significantly larger percentage of stride duration in Ts65Dn mice than in control mice at the faster walking speed. Indices of gait dynamics in Ts65Dn mice correspond to previously reported findings in children with DS. The methods used in the present study provide quantitative markers for genotype and phenotype relationship studies in DS. This technique may provide opportunities for testing the efficacy of therapies for motor dysfunction in persons with DS.

  6. Transcription Factors in Fungi: TFome Dynamics, Three Major Families, and Dual-Specificity TFs.

    PubMed

    Shelest, Ekaterina

    2017-01-01

    Transcription factors (TFs) are essential regulators of gene expression in a cell; the entire repertoire of TFs (TFome) of a species reflects its regulatory potential and the evolutionary history of the regulatory mechanisms. In this work, I give an overview of fungal TFs, analyze TFome dynamics, and discuss TF families and types of particular interest. Whole-genome annotation of TFs in more than 200 fungal species revealed ~80 families of TFs that are typically found in fungi. Almost half of the considered genomes belonged to basidiomycetes and zygomycetes, which have been underrepresented in earlier annotations due to dearth of sequenced genomes. The TFomes were analyzed in terms of expansion strategies genome- and lineage-wise. Generally, TFomes are known to correlate with genome size; but what happens to particular families when a TFome is expanding? By dissecting TFomes into single families and estimating the impact of each of them, I show that in fungi the TFome increment is largely limited to three families (C6 Zn clusters, C2H2-like Zn fingers, and homeodomain-like). To see whether this is a fungal peculiarity or a ubiquitous eukaryotic feature, I also analyzed metazoan TFomes, where I observed a similar trend (limited number of TFome-shaping families) but also some important differences connected mostly with the increased complexity in animals. The expansion strategies of TF families are lineage-specific; I demonstrate how the patterns of the TF families' distributions, designated as "TF signatures," can be used as a taxonomic feature, e.g., for allocation of uncertain phyla. In addition, both fungal and metazoan genomes contain an intriguing type of TFs. While usually TFs have a single DNA-binding domain, these TFs possess two (or more) different DNA-binding specificities. I demonstrate that dual-specific TFs comprising various combinations of all major TF families are a typical feature of fungal and animal genomes and have an interesting evolutionary

  7. Transcription Factors Downstream of IL-4 and TGF-β Signals: Analysis by Quantitative PCR, Western Blot, and Flow Cytometry.

    PubMed

    Sugimoto, Atsushi; Kawakami, Ryoji; Mikami, Norihisa

    2017-01-01

    IL-9-producing Th9 cell is a novel Th cell subset involved in type II allergic inflammations such as asthma. Th9 cells can be induced from naïve Th cells in the presence of IL-4 and TGF-β. It is also well established that downstream signals of IL-4 and TGF-β, including STAT6, IRF4, Smad, and PU.1, directly mediate IL-9 production in Th9 cells. In this chapter we describe the methods of flow cytometry, qPCR and western blot analysis to determine the expression or activation of these transcription factors downstream of IL-4 and TGF-β.

  8. Landscape and Dynamics of Transcription Initiation in the Malaria Parasite Plasmodium falciparum.

    PubMed

    Adjalley, Sophie H; Chabbert, Christophe D; Klaus, Bernd; Pelechano, Vicent; Steinmetz, Lars M

    2016-03-15

    A comprehensive map of transcription start sites (TSSs) across the highly AT-rich genome of P. falciparum would aid progress toward deciphering the molecular mechanisms that underlie the timely regulation of gene expression in this malaria parasite. Using high-throughput sequencing technologies, we generated a comprehensive atlas of transcription initiation events at single-nucleotide resolution during the parasite intra-erythrocytic developmental cycle. This detailed analysis of TSS usage enabled us to define architectural features of plasmodial promoters. We demonstrate that TSS selection and strength are constrained by local nucleotide composition. Furthermore, we provide evidence for coordinate and stage-specific TSS usage from distinct sites within the same transcription unit, thereby producing transcript isoforms, a subset of which are developmentally regulated. This work offers a framework for further investigations into the interactions between genomic sequences and regulatory factors governing the complex transcriptional program of this major human pathogen.

  9. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2013-11-01

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

  11. Quantitative analysis of coronary dynamics by time-dependent ultrasound imaging

    NASA Astrophysics Data System (ADS)

    Marraccini, Paolo; Salvetti, Ovidio; Braccini, Giovanni; Bragagni, Paolo; Levorato, Dianora; L'Abbate, Antonio; Marzilli, Mario

    1995-04-01

    Intravascular ultrasound imaging is a new technique that displays information on lumen and arterial walls, and is capable of providing real-time monitoring of cross-sectional high- resolution images. This technique has potential application for studying the dynamics of the arterial wall with respect to the presence or absence of pathology and the vascular response to physiological or pharmacological stimuli. Although the extraction of information related to coronary dynamics and wall pathologies is possible by manual procedures it is very time consuming and influenced by intra- and interobserver errors. We developed an evaluation system for analyzing 3D spaces defined by digitized cross-sectional ultrasound images of coronaries quantifying the vasomotion in relation to the morphology of the arterial wall. Sequences of echographic images were obtained and recorded as ordered stacks of 2D frames on a VHS videotape. For each image, an automatic lumen edge segmentation was performed, then 3D reconstruction was obtained to evaluate time-dependent lumen and vessel wall changes. These 3D representations serve to demonstrate dynamic phenomena and to perform quantitative analyses (e.g., area/hemidiameter variations, projections, sections, 'carving,' etc.).

  12. Post-transcriptional modifications modulate conformational dynamics in human U2-U6 snRNA complex.

    PubMed

    Karunatilaka, Krishanthi S; Rueda, David

    2014-01-01

    The spliceosome catalyzes precursor-mRNA splicing in all eukaryotes. It consists of over 100 proteins and five small nuclear RNAs (snRNAs), including U2 and U6 snRNAs, which are essential for catalysis. Human and yeast snRNAs share structural similarities despite the fact that human snRNAs contain numerous post-transcriptional modifications. Although functions for these modifications have been proposed, their exact roles are still not well understood. To help elucidate these roles in pre-mRNA splicing, we have used single-molecule fluorescence to characterize the effect of several post-transcriptional modifications in U2 snRNA on the conformation and dynamics of the U2-U6 complex in vitro. Consistent with yeast, the human U2-U6 complex reveals the presence of a magnesium-dependent dynamic equilibrium among three conformations. Interestingly, our data show that modifications in human U2 stem I modulate the dynamic equilibrium of the U2-U6 complex by stabilizing the four-helix structure. However, the small magnitude of this effect suggests that post-transcriptional modifications in human snRNAs may have a primary role in the mediation of specific RNA-protein interactions in vivo.

  13. Post-transcriptional modifications modulate conformational dynamics in human U2–U6 snRNA complex

    PubMed Central

    Karunatilaka, Krishanthi S.; Rueda, David

    2014-01-01

    The spliceosome catalyzes precursor-mRNA splicing in all eukaryotes. It consists of over 100 proteins and five small nuclear RNAs (snRNAs), including U2 and U6 snRNAs, which are essential for catalysis. Human and yeast snRNAs share structural similarities despite the fact that human snRNAs contain numerous post-transcriptional modifications. Although functions for these modifications have been proposed, their exact roles are still not well understood. To help elucidate these roles in pre-mRNA splicing, we have used single-molecule fluorescence to characterize the effect of several post-transcriptional modifications in U2 snRNA on the conformation and dynamics of the U2–U6 complex in vitro. Consistent with yeast, the human U2–U6 complex reveals the presence of a magnesium-dependent dynamic equilibrium among three conformations. Interestingly, our data show that modifications in human U2 stem I modulate the dynamic equilibrium of the U2–U6 complex by stabilizing the four-helix structure. However, the small magnitude of this effect suggests that post-transcriptional modifications in human snRNAs may have a primary role in the mediation of specific RNA–protein interactions in vivo. PMID:24243115

  14. Quantitative detection of Citrus tristeza virus in citrus and aphids by real-time reverse transcription-PCR (TaqMan).

    PubMed

    Saponari, Maria; Manjunath, Keremane; Yokomi, Raymond K

    2008-01-01

    A quantitative and multiplex real-time RT-PCR assay was developed to detect Citrus tristeza virus (CTV) along with plant mRNA, which serves as an internal control to ascertain RNA extraction quality. The real-time technique was validated against 39 CTV strains from around the world as well as with the aphid vector, Aphis gossypii, given a 48 h acquisition access period on a CTV source plant. The assay was effective for quantitation of the viral template in infected plants and in single aphids. CTV detection was compared from different plant tissues and for different RNA isolation methods from aphids. Less than 1 fg was consistently detected when RNA transcripts were diluted in extracts from healthy plants while RNA copies carried by single aphids were estimated to be between 12,000 and 13,000,000. The assay was more sensitive and less time consuming than ELISA or traditional RT-PCR. The real-time RT-PCR assay developed is a valuable new tool for detection and titer quantitation of CTV.

  15. 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.

  16. Promoter decoding of transcription factor dynamics involves a trade-off between noise and control of gene expression.

    PubMed

    Hansen, Anders S; O'Shea, Erin K

    2013-11-05

    Numerous transcription factors (TFs) encode information about upstream signals in the dynamics of their activation, but how downstream genes decode these dynamics remains poorly understood. Using microfluidics to control the nucleocytoplasmic translocation dynamics of the budding yeast TF Msn2, we elucidate the principles that govern how different promoters convert dynamical Msn2 input into gene expression output in single cells. Combining modeling and experiments, we classify promoters according to their signal-processing behavior and reveal that multiple, distinct gene expression programs can be encoded in the dynamics of Msn2. We show that both oscillatory TF dynamics and slow promoter kinetics lead to higher noise in gene expression. Furthermore, we show that the promoter activation timescale is related to nucleosome remodeling. Our findings imply a fundamental trade-off: although the cell can exploit different promoter classes to differentially control gene expression using TF dynamics, gene expression noise fundamentally limits how much information can be encoded in the dynamics of a single TF and reliably decoded by promoters.

  17. 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.

  18. Probing dynamic fluorescence properties of single and clustered quantum dots toward quantitative biomedical imaging of cells.

    PubMed

    Kang, Hyeong-Gon; Tokumasu, Fuyuki; Clarke, Matthew; Zhou, Zhenping; Tang, Jianyong; Nguyen, Tinh; Hwang, Jeeseong

    2010-01-01

    We present results on the dynamic fluorescence properties of bioconjugated nanocrystals or quantum dots (QDs) in different chemical and physical environments. A variety of QD samples was prepared and compared: isolated individual QDs, QD aggregates, and QDs conjugated to other nanoscale materials, such as single-wall carbon nanotubes (SWCNTs) and human erythrocyte plasma membrane proteins. We discuss plausible scenarios to explain the results obtained for the fluorescence characteristics of QDs in these samples, especially for the excitation time-dependent fluorescence emission from clustered QDs. We also qualitatively demonstrate enhanced fluorescence emission signals from clustered QDs and deduce that the band 3 membrane proteins in erythrocytes are clustered. This approach is promising for the development of QD-based quantitative molecular imaging techniques for biomedical studies involving biomolecule clustering.

  19. Developmentally-Dynamic Murine Brain Proteomes and Phosphoproteomes Revealed by Quantitative Proteomics

    PubMed Central

    Doubleday, Peter F.; Ballif, Bryan A.

    2014-01-01

    Developmental processes are governed by a diverse suite of signaling pathways employing reversible phosphorylation. Recent advances in large-scale phosphoproteomic methodologies have made possible the identification and quantification of hundreds to thousands of phosphorylation sites from primary tissues. Towards a global characterization of proteomic changes across brain development, we present the results of a large-scale quantitative mass spectrometry study comparing embryonic, newborn and adult murine brain. Using anti-phosphotyrosine immuno-affinity chromatography and strong cation exchange (SCX) chromatography, coupled to immobilized metal affinity chromatography (IMAC), we identified and quantified over 1,750 phosphorylation sites and over 1,300 proteins between three developmental states. Bioinformatic analyses highlight functions associated with the identified proteins and phosphoproteins and their enrichment at distinct developmental stages. These results serve as a primary reference resource and reveal dynamic developmental profiles of proteins and phosphoproteins from the developing murine brain. PMID:25177544

  20. 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.

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

    PubMed

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

    2016-04-05

    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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. Toward quantitative estimation of material properties with dynamic mode atomic force microscopy: a comparative study

    NASA Astrophysics Data System (ADS)

    Ghosal, Sayan; Gannepalli, Anil; Salapaka, Murti

    2017-08-01

    In this article, we explore methods that enable estimation of material properties with the dynamic mode atomic force microscopy suitable for soft matter investigation. The article presents the viewpoint of casting the system, comprising of a flexure probe interacting with the sample, as an equivalent cantilever system and compares a steady-state analysis based method with a recursive estimation technique for determining the parameters of the equivalent cantilever system in real time. The steady-state analysis of the equivalent cantilever model, which has been implicitly assumed in studies on material property determination, is validated analytically and experimentally. We show that the steady-state based technique yields results that quantitatively agree with the recursive method in the domain of its validity. The steady-state technique is considerably simpler to implement, however, slower compared to the recursive technique. The parameters of the equivalent system are utilized to interpret storage and dissipative properties of the sample. Finally, the article identifies key pitfalls that need to be avoided toward the quantitative estimation of material properties.

  7. Quantitative proteomics and transcriptomics of anaerobic and aerobic yeast cultures reveals post-transcriptional regulation of key cellular processes.

    PubMed

    de Groot, Marco J L; Daran-Lapujade, Pascale; van Breukelen, Bas; Knijnenburg, Theo A; de Hulster, Erik A F; Reinders, Marcel J T; Pronk, Jack T; Heck, Albert J R; Slijper, Monique

    2007-11-01

    Saccharomyces cerevisiae is unique among yeasts in its ability to grow rapidly in the complete absence of oxygen. S. cerevisiae is therefore an ideal eukaryotic model to study physiological adaptation to anaerobiosis. Recent transcriptome analyses have identified hundreds of genes that are transcriptionally regulated by oxygen availability but the relevance of this cellular response has not been systematically investigated at the key control level of the proteome. Therefore, the proteomic response of S. cerevisiae to anaerobiosis was investigated using metabolic stable-isotope labelling in aerobic and anaerobic glucose-limited chemostat cultures, followed by relative quantification of protein expression. Using independent replicate cultures and stringent statistical filtering, a robust dataset of 474 quantified proteins was generated, of which 249 showed differential expression levels. While some of these changes were consistent with previous transcriptome studies, many of the responses of S. cerevisiae to oxygen availability were, to our knowledge, previously unreported. Comparison of transcriptomes and proteomes from identical cultivations yielded strong evidence for post-transcriptional regulation of key cellular processes, including glycolysis, amino-acyl-tRNA synthesis, purine nucleotide synthesis and amino acid biosynthesis. The use of chemostat cultures provided well-controlled and reproducible culture conditions, which are essential for generating robust datasets at different cellular information levels. Integration of transcriptome and proteome data led to new insights into the physiology of anaerobically growing yeast that would not have been apparent from differential analyses at either the mRNA or protein level alone, thus illustrating the power of multi-level studies in yeast systems biology.

  8. Light and an exogenous transcription factor qualitatively and quantitatively affect the biosynthetic pathway of condensed tannins in Lotus corniculatus leaves.

    PubMed

    Paolocci, Francesco; Bovone, Tessa; Tosti, Nicola; Arcioni, Sergio; Damiani, Francesco

    2005-04-01

    The effects of increasing light and of a heterologous bHLH transcription factor on the accumulation of condensed tannins (CT) were investigated in leaves of Lotus corniculatus, a model legume species which accumulates these secondary metabolites in leaves as well as reproductive tissues. Light and expression of the transgene increased the level of CT in a synergistic way. To monitor how the changes in accumulation of condensed tannins were achieved, the level of expression of four key genes in the flavonoid pathway was estimated by real-time RT-PCR analysis. Early genes of the pathway (PAL and CHS) were affected less in their expression and so appeared to be less involved in influencing the final level of CT than later genes in the pathway (DFR and ANS). Steady-state levels of DFR and ANS transcripts showed a strong positive correlation with CT and these genes might be considered the first rate-limiting steps in CT biosynthesis in Lotus leaves. However, additional factors mediated by light are limiting CT accumulation once these genes are up-regulated by the transgene. Therefore, the increment of the steady-state mRNA level for DFR and ANS might not be sufficient to up-regulate condensed tannins in leaves. The real-time RT-PCR approach adopted showed that members within the CHS and DFR gene families are differentially regulated by the exogenous bHLH gene and light. This finding is discussed in relation to the approaches for controlling CT biosynthesis and for studying the expression profile of multi-gene families.

  9. Microprocessor dynamics shows co- and post-transcriptional processing of pri-miRNAs.

    PubMed

    Louloupi, Annita; Ntini, Evgenia; Liz, Julia; Ørom, Ulf Andersson

    2017-03-01

    miRNAs are small regulatory RNAs involved in the regulation of translation of target transcripts. miRNA biogenesis is a multi-step process starting with the cleavage of the primary miRNA transcript in the nucleus by the Microprocessor complex. Endogenous processing of pri-miRNAs is challenging to study and the in vivo kinetics of this process is not known. Here, we present a method for determining the processing kinetics of pri-miRNAs within intact cells over time using a pulse-chase approach to label transcribed RNA during 15 minutes and follow the processing within a 1-hour window after labeling with bromouridine. We show, that pri-miRNAs exhibit different processing kinetics ranging from fast over intermediate to slow processing and provide evidence that pri-miRNA processing can occur both co-transcriptionally and post-transcriptionally.

  10. 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

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

    PubMed

    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).

  12. In situ dynamics of O2, pH and cyanobacterial transcripts associated with CCM, photosynthesis and detoxification of ROS

    PubMed Central

    Jensen, Sheila I; Steunou, Anne-Soisig; Bhaya, Devaki; Kühl, Michael; Grossman, Arthur R

    2011-01-01

    The relative abundance of transcripts encoding proteins involved in inorganic carbon concentrating mechanisms (CCM), detoxification of reactive oxygen species (ROS) and photosynthesis in the thermophilic cyanobacterium Synechococcus OS-B′ was measured in hot spring microbial mats over two diel cycles, and was coupled with in situ determinations of incoming irradiance and microenvironmental dynamics of O2 and pH. Fluctuations in pH and O2 in the mats were largely driven by the diel cycle of solar irradiance, with a pH variation from ∼7.0 to ∼9.5, and O2 levels ranging from anoxia to supersaturation during night and day, respectively. Levels of various transcripts from mat cyanobacteria revealed several patterns that correlated with incident irradiance, O2 and pH within the mat matrix. Transcript abundances for most genes increased during the morning dark–light transition. Some transcripts remained at a near constant level throughout the light period, whereas others showed an additional increase in abundance as the mat underwent transition from low-to-high light (potentially reflecting changes in O2 concentration and pH), followed by either a decreased abundance in the early afternoon, or a gradual decline during the early afternoon and into the evening. One specific transcipt, psbA1, was the lowest during mid-day under high irradiance and increased when the light levels declined. We discuss these complex in situ transcriptional patterns with respect to environmental and endogenous cues that might impact and regulate transcription over the diel cycle. PMID:20740024

  13. Quantitation of cytosine deaminase mRNA by real-time reverse transcription polymerase chain reaction: a sensitive method for assessing 5-fluorocytosine toxicity in vitro.

    PubMed

    Miller, C Ryan; Gustin, Allen N; Buchsbaum, Donald J; Vickers, Selwyn M; Manne, Upender; Grizzle, William E; Cloud, Gretchen A; Diasio, Robert B; Johnson, Martin R

    2002-02-15

    Cytosine deaminase/5-fluorocytosine (CD/5-FC) is a promising strategy for local cancer gene therapy. We hypothesized that CD expression within tumor cells would be directly related to efficacy and that quantitation of markers of CD expression such as mRNA, protein, and enzyme activity would therefore facilitate prediction of 5-FC toxicity. These three markers were thus quantitated by real-time quantitative reverse transcription polymerase chain reaction (Q-RT-PCR), semiquantitative immunocytochemistry (ICC), and 5-[(3)H]FC enzyme assay, respectively. Results with human colon (LS174T) cancer cells infected with a replication-incompetent adenovirus encoding CD (AdCMVCD) demonstrated a significant correlation between CD mRNA and enzyme activity up to 24 h postinfection. A direct correlation was found between CD dose (AdCMVCD PFU/cell) and CD mRNA and protein expression (P < 0.002) in both LS174T and BxPC-3 pancreatic cancer cells, but the relationship with enzyme activity was less strong in LS174T cells (P = 0.09). A remarkable concordance existed among Q-RT-PCR, ICC and enzyme assays with both cell lines. Importantly, CD dose and mRNA and protein expression inversely correlated with 5-FC IC(50) (P < 0.02). Quantitation of CD markers also facilitated identification of factors governing differential susceptibility to CD/5-FC. These results suggest that Q-RT-PCR will be useful for monitoring transgene expression in future studies using improved CD-based expression vectors and may also be useful in predicting the response to CD/5-FC therapy, which is likely to be heterogeneous in the patient population.

  14. 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.

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

    PubMed Central

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

    2015-01-01

    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

  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. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

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

    PubMed

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

    2016-07-01

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

  18. 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.

  19. Quantitative screening of genes regulating tryptophan hydroxylase transcription in Caenorhabditis elegans using microfluidics and an adaptive algorithm.

    PubMed

    Lee, Hyewon; Crane, Matthew M; Zhang, Yun; Lu, Hang

    2013-02-01

    Forward genetic screening via mutagenesis is a powerful method for identifying regulatory factors in target pathways in model organisms such as the soil-dwelling free-living nematode Caenorhabditis elegans (C. elegans). Currently manual microscopy is the standard technique for conducting such screens; however, it is labor-intensive and time-consuming because screening requires imaging thousands of animals. Recently microfluidic chips have been developed to increase the throughput of some of such experiments; nonetheless, most of these chips are multilayer devices and complicated to fabricate and therefore prone to failure during fabrication and operation. In addition, most sorting decisions are made manually and the criteria used for sorting are subjective. To overcome these limitations, we developed a simple single-layer microfluidic device and an adaptive algorithm to make sorting decisions. The one-layer device greatly improves the reliability, while quantitative analysis with the adaptive algorithm allows for the identification of mutations that generate subtle changes in expression, which would have been hard to detect by eye. The screening criterion is set based on the mutagenized population, not separate control populations measured prior to actual screening experiments, to account for stochasticity and day-to-day variations of gene expression in mutagenized worms. Moreover, during each experiment, the threshold is constantly updated to reflect the balance between maximizing sorting rate and minimizing false-positive rate. Using this system, we screened for mutants that have altered expression levels of tryptophan hydroxylase, a key enzyme for serotonin synthesis in a CaMKII gain-of-function background. We found several putative mutants in this screen. Furthermore, this microfluidic system and quantitative analysis can be easily adapted to study other pathways in C. elegans.

  20. Rapid detection and quantification of Ebola Zaire virus by one-step real-time quantitative reverse transcription-polymerase chain reaction.

    PubMed

    Ro, Young-Tae; Ticer, Anysha; Carrion, Ricardo; Patterson, Jean L

    2017-04-01

    Given that Ebola virus causes severe hemorrhagic fever in humans with mortality rates as high as 90%, rapid and accurate detection of this virus is essential both for controlling infection and preventing further transmission. Here, a one-step qRT-PCR assay for rapid and quantitative detection of an Ebola Zaire strain using GP, VP24 or VP40 genes as a target is introduced. Routine assay conditions for hydrolysis probe detection were established from the manufacturer's protocol used in the assays. The analytical specificity and sensitivity of each assay was evaluated using in vitro synthesized viral RNA transcripts. The assays were highly specific for the RNA transcripts, no cross-reactivity being observed among them. The limits of detection of the assays ranged from 10(2) to 10(3) copies per reaction. The assays were also evaluated using viral RNAs extracted from cell culture-propagated viruses (Ebola Zaire, Sudan and Reston strains), confirming that they are gene- and strain-specific. The RT-PCR assays detected viral RNAs in blood samples from virus-infected animal, suggesting that they can be also a useful method for identifying Ebola virus in clinical samples. © 2017 The Societies and John Wiley & Sons Australia, Ltd.

  1. 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.

  2. Transcriptional bursting in Drosophila development: Stochastic dynamics of eve stripe 2 expression.

    PubMed

    Holloway, David M; Spirov, Alexander V

    2017-01-01

    Anterior-posterior (AP) body segmentation of the fruit fly (Drosophila) is first seen in the 7-stripe spatial expression patterns of the pair-rule genes, which regulate downstream genes determining specific segment identities. Regulation of pair-rule expression has been extensively studied for the even-skipped (eve) gene. Recent live imaging, of a reporter for the 2nd eve stripe, has demonstrated the stochastic nature of this process, with 'bursts' in the number of RNA transcripts being made over time. We developed a stochastic model of the spatial and temporal expression of eve stripe 2 (binding by transcriptional activators (Bicoid and Hunchback proteins) and repressors (Giant and Krüppel proteins), transcriptional initiation and termination; with all rate parameters constrained by features of the experimental data) in order to analyze the noisy experimental time series and test hypotheses for how eve transcription is regulated. These include whether eve transcription is simply OFF or ON, with a single ON rate, or whether it proceeds by a more complex mechanism, with multiple ON rates. We find that both mechanisms can produce long (multi-minute) RNA bursts, but that the short-time (minute-to-minute) statistics of the data is indicative of eve being transcribed with at least two distinct ON rates, consistent with data on the joint activation of eve by Bicoid and Hunchback. We also predict distinct statistical signatures for cases in which eve is repressed (e.g. along the edges of the stripe) vs. cases in which activation is reduced (e.g. by mutagenesis of transcription factor binding sites). Fundamental developmental processes such as gene transcription are intrinsically noisy; our approach presents a new way to quantify and analyze time series data during developmental patterning in order to understand regulatory mechanisms and how they propagate noise and impact embryonic robustness.

  3. Dynamic Effects of Topoisomerase I Inhibition on R-Loops and Short Transcripts at Active Promoters.

    PubMed

    Marinello, Jessica; Bertoncini, Stefania; Aloisi, Iris; Cristini, Agnese; Malagoli Tagliazucchi, Guidantonio; Forcato, Mattia; Sordet, Olivier; Capranico, Giovanni

    2016-01-01

    Topoisomerase I-DNA-cleavage complexes (Top1cc) stabilized by camptothecin (CPT) have specific effects at transcriptional levels. We recently reported that Top1cc increase antisense transcript (aRNAs) levels at divergent CpG-island promoters and, transiently, DNA/RNA hybrids (R-loop) in nuclear and mitochondrial genomes of colon cancer HCT116 cells. However, the relationship between R-loops and aRNAs was not established. Here, we show that aRNAs can form R-loops in N-TERA-2 cells under physiological conditions, and that promoter-associated R-loops are somewhat increased and extended in length immediately upon cell exposure to CPT. In contrast, persistent Top1ccs reduce the majority of R-loops suggesting that CPT-accumulated aRNAs are not commonly involved in R-loops. The enhancement of aRNAs by Top1ccs is present both in human colon cancer HCT116 cells and WI38 fibroblasts suggesting a common response of cancer and normal cells. Although Top1ccs lead to DSB and DDR kinases activation, we do not detect a dependence of aRNA accumulation on ATM or DNA-PK activation. However, we showed that the cell response to persistent Top1ccs can involve an impairment of aRNA turnover rather than a higher synthesis rate. Finally, a genome-wide analysis shows that persistent Top1ccs also determine an accumulation of sense transcripts at 5'-end gene regions suggesting an increased occurrence of truncated transcripts. Taken together, the results indicate that Top1 may regulate transcription initiation by modulating RNA polymerase-generated negative supercoils, which can in turn favor R-loop formation at promoters, and that transcript accumulation at TSS is a response to persistent transcriptional stress by Top1 poisoning.

  4. Variables influencing the efficiency and interpretation of reverse transcription quantitative PCR (RT-qPCR): An empirical study using Bacteriophage MS2.

    PubMed

    Miranda, Jaclyn A; Steward, Grieg F

    2017-03-01

    Reverse transcription, quantitative PCR (RT-qPCR) is a sensitive method for quantification of specific RNA targets, but the first step of the assay, reverse transcription, is notoriously variable and sensitive to reaction conditions. In this study, we used purified Bacteriophage MS2 genomic RNA as a model virus target to test two different RT enzymes (SuperScript II and SuperScript III), two RT-priming strategies (gene-specific primers and random hexamers), and varying background RNA concentrations (0-50ngμl(-1)) to determine how these variables influence the efficiency of reverse transcription over a range of target concentrations (10(1)-10(7) copies μl(-1)). The efficiency of the RT reaction was greatly improved by increasing both background RNA and primer concentrations, but the benefit provided by background RNA was source dependent. At a given target concentration, similar RT efficiencies were achieved with gene-specific primers and random hexamers, but the latter required much higher concentrations. With random hexamers, we observed a systematic variation in RT reaction efficiency as a function of target concentration. Using an RNA standard curve that was also subject to RT effectively normalized for this systematic variability, but the assay accuracy depended critically on the length of the standard RNA extending to the 3' end of the qPCR target site. Our results shed some light on previous contradictory conclusions in the literature, and provide insights that may aid in the design of RT-qPCR assays and the design of synthetic RNA standards when full-length material is not available.

  5. Expression of steroidogenic enzymes and their transcription factors in cortisol-producing adrenocortical adenomas: immunohistochemical analysis and quantitative real-time polymerase chain reaction studies.

    PubMed

    Kubota-Nakayama, Fumie; Nakamura, Yasuhiro; Konosu-Fukaya, Sachiko; Azmahani, Abdullah; Ise, Kazue; Yamazaki, Yuto; Kitawaki, Yuko; Felizola, Saulo J A; Ono, Yoshikiyo; Omata, Kei; Morimoto, Ryo; Iwama, Noriyuki; Satoh, Fumitoshi; Sasano, Hironobu

    2016-08-01

    Adrenal Cushing syndrome (CS) is caused by the overproduction of cortisol in adrenocortical tumors including adrenal cortisol-producing adenoma (CPA). In CS, steroidogenic enzymes such as 17α-hydroxylase/17, 20-lase (CYP17A1), 3β-hydroxysteroid dehydrogenase (HSD3B), and 11β-hydroxylase (CYP11B1) are abundantly expressed in tumor cells. In addition, several transcriptional factors have been reported to play pivotal roles in the regulation of these enzymes in CPA, but their correlations with those enzymes above have still remained largely unknown. Therefore, in this study, we examined the status of steroidogenic enzymes and their transcriptional factors in 78 and 15 CPA cases by using immunohistochemistry and quantitative real-time polymerase chain reaction (qPCR), respectively. Immunoreactivity of HSD3B2, CYP11B1, CYP17A1, steroidogenic factor-1 (SF1[NR5A1]), GATA6, and nerve growth factor induced-B (NGFIB[NR4A1]) was detected in tumor cells. Results of qPCR analysis revealed that expression of HSD3B2 mRNA was significantly higher than that of HSD3B1, and CYP11B1 mRNA was significantly higher than CYP11B2. In addition, the expression of CYP11B1 mRNA was positively correlated with those of NR5A1, GATA6, and NR4A1. These results all indicated that HSD3B2 but not HSD3B1 was mainly involved in cortisol overproduction in CPA. In addition, NR5A1, GATA6, and NR4A1 were all considered to play important roles in cortisol overproduction through regulating CYP11B1 gene transcription. Copyright © 2016. Published by Elsevier Inc.

  6. Protein–DNA interfaces: a molecular dynamics analysis of time-dependent recognition processes for three transcription factors

    PubMed Central

    Etheve, Loïc; Martin, Juliette; Lavery, Richard

    2016-01-01

    We have studied the dynamics of three transcription factor–DNA complexes using all-atom, microsecond-scale MD simulations. In each case, the salt bridges and hydrogen bond interactions formed at the protein–DNA interface are found to be dynamic, with lifetimes typically in the range of tens to hundreds of picoseconds, although some interactions, notably those involving specific binding to DNA bases, can be a hundred times longer lived. Depending on the complex studied, this dynamics may or may not lead to the existence of distinct conformational substates. Using a sequence threading technique, it has been possible to determine whether DNA sequence recognition is sensitive or not to such conformational changes, and, in one case, to show that recognition appears to be locally dependent on protein-mediated cation distributions. PMID:27658967

  7. Dynamic transcriptional signatures and network responses for clinical symptoms in influenza-infected human subjects using systems biology approaches

    PubMed Central

    Linel, Patrice; Wu, Shuang; Deng, Nan

    2015-01-01

    Recent studies demonstrate that human blood transcriptional signatures may be used to support diagnosis and clinical decisions for acute respiratory viral infections such as influenza. In this article, we propose to use a newly developed systems biology approach for time course gene expression data to identify significant dynamically response genes and dynamic gene network responses to viral infection. We illustrate the methodological pipeline by reanalyzing the time course gene expression data from a study with healthy human subjects challenged by live influenza virus. We observed clear differences in the number of significant dynamic response genes (DRGs) between the symptomatic and asymptomatic subjects and also identified DRG signatures for symptomatic subjects with influenza infection. The 505 common DRGs shared by the symptomatic subjects have high consistency with the signature genes for predicting viral infection identified in previous works. The temporal response patterns and network response features were carefully analyzed and investigated. PMID:25015847

  8. Dynamic transcriptional signatures and network responses for clinical symptoms in influenza-infected human subjects using systems biology approaches.

    PubMed

    Linel, Patrice; Wu, Shuang; Deng, Nan; Wu, Hulin

    2014-10-01

    Recent studies demonstrate that human blood transcriptional signatures may be used to support diagnosis and clinical decisions for acute respiratory viral infections such as influenza. In this article, we propose to use a newly developed systems biology approach for time course gene expression data to identify significant dynamically response genes and dynamic gene network responses to viral infection. We illustrate the methodological pipeline by reanalyzing the time course gene expression data from a study with healthy human subjects challenged by live influenza virus. We observed clear differences in the number of significant dynamic response genes (DRGs) between the symptomatic and asymptomatic subjects and also identified DRG signatures for symptomatic subjects with influenza infection. The 505 common DRGs shared by the symptomatic subjects have high consistency with the signature genes for predicting viral infection identified in previous works. The temporal response patterns and network response features were carefully analyzed and investigated.

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

    PubMed

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

    2017-03-21

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

  10. 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

  11. Coupled pre-mRNA and mRNA dynamics unveil operational strategies underlying transcriptional responses to stimuli

    PubMed Central

    Zeisel, Amit; Köstler, Wolfgang J; Molotski, Natali; Tsai, Jonathan M; Krauthgamer, Rita; Jacob-Hirsch, Jasmine; Rechavi, Gideon; Soen, Yoav; Jung, Steffen; Yarden, Yosef; Domany, Eytan

    2011-01-01

    Transcriptional responses to extracellular stimuli involve tuning the rates of transcript production and degradation. Here, we show that the time-dependent profiles of these rates can be inferred from simultaneous measurements of precursor mRNA (pre-mRNA) and mature mRNA profiles. Transcriptome-wide measurements demonstrate that genes with similar mRNA profiles often exhibit marked differences in the amplitude and onset of their production rate. The latter is characterized by a large dynamic range, with a group of genes exhibiting an unexpectedly strong transient production overshoot, thereby accelerating their induction and, when combined with time-dependent degradation, shaping transient responses with precise timing and amplitude. PMID:21915116

  12. 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.

  13. 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.

  14. Establishment of a sensitive system for analysis of human vaginal microbiota on the basis of rRNA-targeted reverse transcription-quantitative PCR.

    PubMed

    Kurakawa, Takashi; Ogata, Kiyohito; Tsuji, Hirokazu; Kado, Yukiko; Takahashi, Takuya; Kida, Yumi; Ito, Masahiro; Okada, Nobuhiko; Nomoto, Koji

    2015-04-01

    Ten specific primer sets, for Lactobacillus gasseri, Lactobacillus crispatus, Atopobium vaginae, Gardnerella vaginalis, Mobiluncus curtisii, Chlamydia trachomatis/muridarum, Bifidobacterium longum subsp. longum, Bifidobacterium longum subsp. infantis, Bifidobacterium adolescentis, and Bifidobacterium angulatum, were developed for quantitative analysis of vaginal microbiota. rRNA-targeted reverse transcription-quantitative PCR (RT-qPCR) analysis of the vaginal samples from 12 healthy Japanese volunteers using the new primer sets together with 25 existing primer sets revealed the diversity of their vaginal microbiota: Lactobacilli such as L. crispatus, L. gasseri, Lactobacillus jensenii, Lactobacillus iners, and Lactobacillus vaginalis, as the major populations at 10(7) cells/ml vaginal fluid, were followed by facultative anaerobes such as Streptococcus and strict anaerobes at lower population levels of 10(4) cells/ml or less. Certain bacterial vaginosis (BV)-related bacteria, such as G. vaginalis, A. vaginae, M. curtisii, and Prevotella, were also detected in some subjects. Especially in one subject, both G. vaginalis and A. vaginae were detected at high population levels of 10(8.8) and 10(8.9) cells/ml vaginal fluid, suggesting that she is an asymptomatic BV patient. These results suggest that the RT-qPCR system is effective for accurate analysis of major vaginal commensals and diagnosis of several vaginal infections. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. [Determination of the biological attribute of evidence at the scene using reverse transcription PCR and real-time fluorescent quantitative PCR].

    PubMed

    Xu, Yan; Zhang, Chen; Xu, Qing-Wen; Huang, Jiang-Ping; Liu, Ya-Nan; Zou, Kai-Nan; Ping, Yuan; Zhou, Huai-Gu

    2013-08-01

    To explore the feasibility of biological method to identify the biological attribute of samples at crime scene. Thirty samples of ten blood stains, ten saliva stains and ten semen stains were selected, and all the samples were processed by the routine method and biomolecular method, respectively. Both RNA and DNA were isolated using DNA-RNA co-extraction technology and the mRNA was converted into cDNA using reverse transcription PCR (RT-PCR). Three pairs of specific primers were designed for blood stain, saliva stain and semen stain based on the different target genes in three specific tissues and the primers were amplified using real-time fluorescent quantitative PCR. The differences in these biological samples were evaluated by melting temperature (Tm) values and the size of the amplification fragment. The Tm values of blood stain, saliva stain and semen stain were (84.5 +/- 0.2) degrees C, (76.9 +/- 0.3) degrees C and (88.5 +/- 0.2) degrees C, respectively. The length of PCR fragments of them was 177bp, 134bp and 294bp, respectively. Compared with the routine method, RT-PCR with real-time fluorescent quantitative PCR is highly specific, sensitive and reliable to identify the biological attribute of evidence, and can be potentially applied to determine evidence attribute in forensic practice.

  16. Quantitati