Experimental transmission of equine hepacivirus in horses as a model for hepatitis C virus

USDA-ARS?s Scientific Manuscript database

Equine hepacivirus (EHCV; non-primate hepacivirus) is a hepatotropic member of the Flaviviridae family that infects horses. Although EHCV is the closest known relative to hepatitis C virus (HCV), its complete replication kinetics in vivo have not been described, and direct evidence that it causes he...

Recent Insight into the Kinetic Mechanisms and Conformational Dynamics of Y-Family DNA Polymerases

PubMed Central

2015-01-01

The kinetic mechanisms by which DNA polymerases catalyze DNA replication and repair have long been areas of active research. Recently discovered Y-family DNA polymerases catalyze the bypass of damaged DNA bases that would otherwise block replicative DNA polymerases and stall replication forks. Unlike DNA polymerases from the five other families, the Y-family DNA polymerases have flexible, solvent-accessible active sites that are able to tolerate various types of damaged template bases and allow for efficient lesion bypass. Their promiscuous active sites, however, also lead to fidelities that are much lower than those observed for other DNA polymerases and give rise to interesting mechanistic properties. Additionally, the Y-family DNA polymerases have several other unique structural features and undergo a set of conformational changes during substrate binding and catalysis different from those observed for replicative DNA polymerases. In recent years, pre-steady-state kinetic methods have been extensively employed to reveal a wealth of information about the catalytic properties of these fascinating noncanonical DNA polymerases. Here, we review many of the recent findings on the kinetic mechanisms of DNA polymerization with undamaged and damaged DNA substrates by the Y-family DNA polymerases, and the conformational dynamics employed by these error-prone enzymes during catalysis. PMID:24716482

Human stem cell–derived astrocytes replicate human prions in a PRNP genotype–dependent manner

PubMed Central

Krejciova, Zuzana; Alibhai, James; Zhao, Chen; Rzechorzek, Nina M.; Ullian, Erik M.; Manson, Jean

2017-01-01

Prions are infectious agents that cause neurodegenerative diseases such as Creutzfeldt–Jakob disease (CJD). The absence of a human cell culture model that replicates human prions has hampered prion disease research for decades. In this paper, we show that astrocytes derived from human induced pluripotent stem cells (iPSCs) support the replication of prions from brain samples of CJD patients. For experimental exposure of astrocytes to variant CJD (vCJD), the kinetics of prion replication occur in a prion protein codon 129 genotype–dependent manner, reflecting the genotype-dependent susceptibility to clinical vCJD found in patients. Furthermore, iPSC-derived astrocytes can replicate prions associated with the major sporadic CJD strains found in human patients. Lastly, we demonstrate the subpassage of prions from infected to naive astrocyte cultures, indicating the generation of prion infectivity in vitro. Our study addresses a long-standing gap in the repertoire of human prion disease research, providing a new in vitro system for accelerated mechanistic studies and drug discovery. PMID:29141869

Replication and shedding kinetics of infectious hematopoietic necrosis virus in juvenile rainbow trout

USGS Publications Warehouse

Wargo, Andrew R.; Scott, Robert J.; Kerr, Benjamin; Kurath, Gael

2017-01-01

Viral replication and shedding are key components of transmission and fitness, the kinetics of which are heavily dependent on virus, host, and environmental factors. To date, no studies have quantified the shedding kinetics of infectious hematopoietic necrosis virus (IHNV) in rainbow trout (Oncorhynchus mykiss), or how they are associated with replication, making it difficult to ascertain the transmission dynamics of this pathogen of high agricultural and conservation importance. Here, the replication and shedding kinetics of two M genogroup IHNV genotypes were examined in their naturally co-evolved rainbow trout host. Within host virus replication began rapidly, approaching maximum values by day 3 post-infection, after which viral load was maintained or gradually dropped through day 7. Host innate immune response measured as stimulation of Mx-1 gene expression generally followed within host viral loads. Shedding also began very quickly and peaked within 2 days, defining a generally uniform early peak period of shedding from 1 to 4 days after exposure to virus. This was followed by a post-peak period where shedding declined, such that the majority of fish were no longer shedding by day 12 post-infection. Despite similar kinetics, the average shedding rate over the course of infection was significantly lower in mixed compared to single genotype infections, suggesting a competition effect, however, this did not significantly impact the total amount of virus shed. The data also indicated that the duration of shedding, rather than peak amount of virus shed, was correlated with fish mortality. Generally, the majority of virus produced during infection appeared to be shed into the environment rather than maintained in the host, although there was more retention of within host virus during the post-peak period. Viral virulence was correlated with shedding, such that the more virulent of the two genotypes shed more total virus. This fundamental understanding of IHNV shedding kinetics and variation at the individual fish level could assist with management decisions about how to respond to disease outbreaks when they occur.

Replication and shedding kinetics of infectious hematopoietic necrosis virus in juvenile rainbow trout.

PubMed

Wargo, Andrew R; Scott, Robert J; Kerr, Benjamin; Kurath, Gael

2017-01-02

Viral replication and shedding are key components of transmission and fitness, the kinetics of which are heavily dependent on virus, host, and environmental factors. To date, no studies have quantified the shedding kinetics of infectious hematopoietic necrosis virus (IHNV) in rainbow trout (Oncorhynchus mykiss), or how they are associated with replication, making it difficult to ascertain the transmission dynamics of this pathogen of high agricultural and conservation importance. Here, the replication and shedding kinetics of two M genogroup IHNV genotypes were examined in their naturally co-evolved rainbow trout host. Within host virus replication began rapidly, approaching maximum values by day 3 post-infection, after which viral load was maintained or gradually dropped through day 7. Host innate immune response measured as stimulation of Mx-1 gene expression generally followed within host viral loads. Shedding also began very quickly and peaked within 2days, defining a generally uniform early peak period of shedding from 1 to 4days after exposure to virus. This was followed by a post-peak period where shedding declined, such that the majority of fish were no longer shedding by day 12 post-infection. Despite similar kinetics, the average shedding rate over the course of infection was significantly lower in mixed compared to single genotype infections, suggesting a competition effect, however, this did not significantly impact the total amount of virus shed. The data also indicated that the duration of shedding, rather than peak amount of virus shed, was correlated with fish mortality. Generally, the majority of virus produced during infection appeared to be shed into the environment rather than maintained in the host, although there was more retention of within host virus during the post-peak period. Viral virulence was correlated with shedding, such that the more virulent of the two genotypes shed more total virus. This fundamental understanding of IHNV shedding kinetics and variation at the individual fish level could assist with management decisions about how to respond to disease outbreaks when they occur. Copyright © 2016 Elsevier B.V. All rights reserved.

Replication and shedding kinetics of infectious hematopoietic necrosis virus in juvenile rainbow trout

PubMed Central

Wargo, Andrew R.; Scott, Robert J.; Kerr, Benjamin; Kurath, Gael

2016-01-01

Viral replication and shedding are key components of transmission and fitness, the kinetics of which are heavily dependent on virus, host, and environmental factors. To date, no studies have quantified the shedding kinetics of infectious hematopoietic necrosis virus (IHNV) in rainbow trout (Oncorhynchus mykiss), or how they are associated with replication, making it difficult to ascertain the transmission dynamics of this pathogen of high agricultural and conservation importance. Here, the replication and shedding kinetics of two M genogroup IHNV genotypes were examined in their naturally co-evolved rainbow trout host. Within host virus replication began rapidly, approaching maximum values by day 3 post-infection, after which viral load was maintained or gradually dropped through day 7. Host innate immune response measured as stimulation of Mx-1 gene expression generally followed within host viral loads. Shedding also began very quickly and peaked within 2 days, defining a generally uniform early peak period of shedding from 1 to 4 days after exposure to virus. This was followed by a post-peak period where shedding declined, such that the majority offish were no longer shedding by day 12 post-infection. Despite similar kinetics, the average shedding rate over the course of infection was significantly lower in mixed compared to single genotype infections, suggesting a competition effect, however, this did not significantly impact the total amount of virus shed. The data also indicated that the duration of shedding, rather than peak amount of virus shed, was correlated with fish mortality. Generally, the majority of virus produced during infection appeared to be shed into the environment rather than maintained in the host, although there was more retention of within host virus during the post-peak period. Viral virulence was correlated with shedding, such that the more virulent of the two genotypes shed more total virus. This fundamental understanding of IHNV shedding kinetics and variation at the individual fish level could assist with management decisions about how to respond to disease outbreaks when they occur. PMID:27771253

Kinetics and thermodynamics of living copolymerization processes

PubMed Central

2016-01-01

Theoretical advances are reported on the kinetics and thermodynamics of free and template-directed living copolymerizations. Until recently, the kinetic theory of these processes had only been established in the fully irreversible regime, in which the attachment rates are only considered. However, the entropy production is infinite in this regime and the approach to thermodynamic equilibrium cannot be investigated. For this purpose, the detachment rates should also be included. Inspite of this complication, the kinetics can be exactly solved in the regimes of steady growth and depolymerization. In this way, analytical expressions are obtained for the mean growth velocity, the statistical properties of the copolymer sequences, as well as the thermodynamic entropy production. The results apply to DNA replication, transcription and translation, allowing us to understand important aspects of molecular evolution. This article is part of the themed issue ‘Multiscale modelling at the physics–chemistry–biology interface’. PMID:27698043

Kinetics and thermodynamics of living copolymerization processes.

PubMed

Gaspard, Pierre

2016-11-13

Theoretical advances are reported on the kinetics and thermodynamics of free and template-directed living copolymerizations. Until recently, the kinetic theory of these processes had only been established in the fully irreversible regime, in which the attachment rates are only considered. However, the entropy production is infinite in this regime and the approach to thermodynamic equilibrium cannot be investigated. For this purpose, the detachment rates should also be included. Inspite of this complication, the kinetics can be exactly solved in the regimes of steady growth and depolymerization. In this way, analytical expressions are obtained for the mean growth velocity, the statistical properties of the copolymer sequences, as well as the thermodynamic entropy production. The results apply to DNA replication, transcription and translation, allowing us to understand important aspects of molecular evolution.This article is part of the themed issue 'Multiscale modelling at the physics-chemistry-biology interface'. © 2016 The Author(s).

Kinetics and thermodynamics of living copolymerization processes

NASA Astrophysics Data System (ADS)

Gaspard, Pierre

2016-11-01

Theoretical advances are reported on the kinetics and thermodynamics of free and template-directed living copolymerizations. Until recently, the kinetic theory of these processes had only been established in the fully irreversible regime, in which the attachment rates are only considered. However, the entropy production is infinite in this regime and the approach to thermodynamic equilibrium cannot be investigated. For this purpose, the detachment rates should also be included. Inspite of this complication, the kinetics can be exactly solved in the regimes of steady growth and depolymerization. In this way, analytical expressions are obtained for the mean growth velocity, the statistical properties of the copolymer sequences, as well as the thermodynamic entropy production. The results apply to DNA replication, transcription and translation, allowing us to understand important aspects of molecular evolution. This article is part of the themed issue 'Multiscale modelling at the physics-chemistry-biology interface'.

Ribosome biogenesis in replicating cells: Integration of experiment and theory.

PubMed

Earnest, Tyler M; Cole, John A; Peterson, Joseph R; Hallock, Michael J; Kuhlman, Thomas E; Luthey-Schulten, Zaida

2016-10-01

Ribosomes-the primary macromolecular machines responsible for translating the genetic code into proteins-are complexes of precisely folded RNA and proteins. The ways in which their production and assembly are managed by the living cell is of deep biological importance. Here we extend a recent spatially resolved whole-cell model of ribosome biogenesis in a fixed volume [Earnest et al., Biophys J 2015, 109, 1117-1135] to include the effects of growth, DNA replication, and cell division. All biological processes are described in terms of reaction-diffusion master equations and solved stochastically using the Lattice Microbes simulation software. In order to determine the replication parameters, we construct and analyze a series of Escherichia coli strains with fluorescently labeled genes distributed evenly throughout their chromosomes. By measuring these cells' lengths and number of gene copies at the single-cell level, we could fit a statistical model of the initiation and duration of chromosome replication. We found that for our slow-growing (120 min doubling time) E. coli cells, replication was initiated 42 min into the cell cycle and completed after an additional 42 min. While simulations of the biogenesis model produce the correct ribosome and mRNA counts over the cell cycle, the kinetic parameters for transcription and degradation are lower than anticipated from a recent analytical time dependent model of in vivo mRNA production. Describing expression in terms of a simple chemical master equation, we show that the discrepancies are due to the lack of nonribosomal genes in the extended biogenesis model which effects the competition of mRNA for ribosome binding, and suggest corrections to parameters to be used in the whole-cell model when modeling expression of the entire transcriptome. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 735-751, 2016. © 2016 Wiley Periodicals, Inc.

Use of a highly sensitive strand-specific quantitative PCR to identify abortive replication in the mouse model of respiratory syncytial virus disease

PubMed Central

2010-01-01

Background The BALB/c mouse is commonly used to study RSV infection and disease. However, despite the many advantages of this well-characterised model, the inoculum is large, viral replication is restricted and only a very small amount of virus can be recovered from infected animals. A key question in this model is the fate of the administered virus. Is replication really being measured or is the model measuring the survival of the virus over time? To answer these questions we developed a highly sensitive strand-specific quantitative PCR (QPCR) able to accurately quantify the amount of RSV replication in the BALB/c mouse lung, allowing characterisation of RSV negative and positive strand RNA dynamics. Results In the mouse lung, no increase in RSV genome was seen above the background of the original inoculum whilst only a limited transient increase (< 1 log) in positive strand, replicative intermediate (RI) RNA occurred. This RNA did however persist at detectable levels for 59 days post infection. As expected, ribavirin therapy reduced levels of infectious virus and RI RNA in the mouse lung. However, whilst Palivizumab therapy was also able to reduce levels of infectious virus, it failed to prevent production of intracellular RI RNA. A comparison of RSV RNA kinetics in human (A549) and mouse (KLN205) cell lines demonstrated that RSV replication was also severely delayed and impaired in vitro in the mouse cells. Conclusions This is the first time that such a sensitive strand-specific QPCR technique has been to the RSV mouse system. We have accurately quantified the restricted and abortive nature of RSV replication in the mouse. Further in vitro studies in human and mouse cells suggest this restricted replication is due at least in part to species-specific host cell-viral interactions. PMID:20860795

Cluster-cluster aggregation with particle replication and chemotaxy: a simple model for the growth of animal cells in culture

NASA Astrophysics Data System (ADS)

Alves, S. G.; Martins, M. L.

2010-09-01

Aggregation of animal cells in culture comprises a series of motility, collision and adhesion processes of basic relevance for tissue engineering, bioseparations, oncology research and in vitro drug testing. In the present paper, a cluster-cluster aggregation model with stochastic particle replication and chemotactically driven motility is investigated as a model for the growth of animal cells in culture. The focus is on the scaling laws governing the aggregation kinetics. Our simulations reveal that in the absence of chemotaxy the mean cluster size and the total number of clusters scale in time as stretched exponentials dependent on the particle replication rate. Also, the dynamical cluster size distribution functions are represented by a scaling relation in which the scaling function involves a stretched exponential of the time. The introduction of chemoattraction among the particles leads to distribution functions decaying as power laws with exponents that decrease in time. The fractal dimensions and size distributions of the simulated clusters are qualitatively discussed in terms of those determined experimentally for several normal and tumoral cell lines growing in culture. It is shown that particle replication and chemotaxy account for the simplest cluster size distributions of cellular aggregates observed in culture.

Mechanical-Kinetic Modeling of a Molecular Walker from a Modular Design Principle

NASA Astrophysics Data System (ADS)

Hou, Ruizheng; Loh, Iong Ying; Li, Hongrong; Wang, Zhisong

2017-02-01

Artificial molecular walkers beyond burnt-bridge designs are complex nanomachines that potentially replicate biological walkers in mechanisms and functionalities. Improving the man-made walkers up to performance for widespread applications remains difficult, largely because their biomimetic design principles involve entangled kinetic and mechanical effects to complicate the link between a walker's construction and ultimate performance. Here, a synergic mechanical-kinetic model is developed for a recently reported DNA bipedal walker, which is based on a modular design principle, potentially enabling many directional walkers driven by a length-switching engine. The model reproduces the experimental data of the walker, and identifies its performance-limiting factors. The model also captures features common to the underlying design principle, including counterintuitive performance-construction relations that are explained by detailed balance, entropy production, and bias cancellation. While indicating a low directional fidelity for the present walker, the model suggests the possibility of improving the fidelity above 90% by a more powerful engine, which may be an improved version of the present engine or an entirely new engine motif, thanks to the flexible design principle. The model is readily adaptable to aid these experimental developments towards high-performance molecular walkers.

Human stem cell-derived astrocytes replicate human prions in a PRNP genotype-dependent manner.

PubMed

Krejciova, Zuzana; Alibhai, James; Zhao, Chen; Krencik, Robert; Rzechorzek, Nina M; Ullian, Erik M; Manson, Jean; Ironside, James W; Head, Mark W; Chandran, Siddharthan

2017-12-04

Prions are infectious agents that cause neurodegenerative diseases such as Creutzfeldt-Jakob disease (CJD). The absence of a human cell culture model that replicates human prions has hampered prion disease research for decades. In this paper, we show that astrocytes derived from human induced pluripotent stem cells (iPSCs) support the replication of prions from brain samples of CJD patients. For experimental exposure of astrocytes to variant CJD (vCJD), the kinetics of prion replication occur in a prion protein codon 129 genotype-dependent manner, reflecting the genotype-dependent susceptibility to clinical vCJD found in patients. Furthermore, iPSC-derived astrocytes can replicate prions associated with the major sporadic CJD strains found in human patients. Lastly, we demonstrate the subpassage of prions from infected to naive astrocyte cultures, indicating the generation of prion infectivity in vitro. Our study addresses a long-standing gap in the repertoire of human prion disease research, providing a new in vitro system for accelerated mechanistic studies and drug discovery. © 2017 Krejciova et al.

Mutational analysis of varicella-zoster virus (VZV) immediate early protein (IE62) subdomains and their importance in viral replication

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

Khalil, Mohamed I., E-mail: mkhalil2@stanford.edu; Department of Molecular Biology, National Research Centre, El-Buhouth St., Cairo; Che, Xibing

VZV IE62 is an essential, immediate-early, tegument protein and consists of five domains. We generated recombinant viruses carrying mutations in the first three IE62 domains and tested their influence on VZV replication kinetics. The mutations in domain I did not affect replication kinetics while domain II mutations, disrupting the DNA binding and dimerization domain (DBD), were lethal for VZV replication. Mutations in domain III of the nuclear localization signal (NLS) and the two phosphorylation sites S686A/S722A resulted in slower growth in early and late infection respectively and were associated with IE62 accumulation in the cytoplasm and nucleus respectively. This studymore » mapped the functional domains of IE62 in context of viral infection, indicating that DNA binding and dimerization domain is essential for VZV replication. In addition, the correct localization of IE62, whether nuclear or cytoplasmic, at different points in the viral life cycle, is important for normal progression of VZV replication. - Highlights: • Mutation of IE62 domain I did not affect VZV replication in melanoma cells. • IE62 domain II and III are important for VZV replication in melanoma cells. • Mutations of IE62 domain II (DBD) were lethal for virus replication. • Mutations of IE62 NLS and phosphorylation sites inhibited VZV replication. • NLS and S686A/S722A mutations altered localization of IE62 during early and late infection.« less

Single-molecule FRET studies of the cooperative and non-cooperative binding kinetics of the bacteriophage T4 single-stranded DNA binding protein (gp32) to ssDNA lattices at replication fork junctions

PubMed Central

Lee, Wonbae; Gillies, John P.; Jose, Davis; Israels, Brett A.; von Hippel, Peter H.; Marcus, Andrew H.

2016-01-01

Gene 32 protein (gp32) is the single-stranded (ss) DNA binding protein of the bacteriophage T4. It binds transiently and cooperatively to ssDNA sequences exposed during the DNA replication process and regulates the interactions of the other sub-assemblies of the replication complex during the replication cycle. We here use single-molecule FRET techniques to build on previous thermodynamic studies of gp32 binding to initiate studies of the dynamics of the isolated and cooperative binding of gp32 molecules within the replication complex. DNA primer/template (p/t) constructs are used as models to determine the effects of ssDNA lattice length, gp32 concentration, salt concentration, binding cooperativity and binding polarity at p/t junctions. Hidden Markov models (HMMs) and transition density plots (TDPs) are used to characterize the dynamics of the multi-step assembly pathway of gp32 at p/t junctions of differing polarity, and show that isolated gp32 molecules bind to their ssDNA targets weakly and dissociate quickly, while cooperatively bound dimeric or trimeric clusters of gp32 bind much more tightly, can ‘slide’ on ssDNA sequences, and exhibit binding dynamics that depend on p/t junction polarities. The potential relationships of these binding dynamics to interactions with other components of the T4 DNA replication complex are discussed. PMID:27694621

An attenuated strain of Bacillus anthracis (CDC 684) has a large chromosomal inversion and altered growth kinetics.

PubMed

Okinaka, Richard T; Price, Erin P; Wolken, Spenser R; Gruendike, Jeffrey M; Chung, Wai Kwan; Pearson, Talima; Xie, Gary; Munk, Chris; Hill, Karen K; Challacombe, Jean; Ivins, Bruce E; Schupp, James M; Beckstrom-Sternberg, Stephen M; Friedlander, Arthur; Keim, Paul

2011-09-30

An isolate originally labeled Bacillus megaterium CDC 684 was found to contain both pXO1 and pXO2, was non-hemolytic, sensitive to gamma-phage, and produced both the protective antigen and the poly-D-glutamic acid capsule. These phenotypes prompted Ezzell et al., (J. Clin. Microbiol. 28:223) to reclassify this isolate to Bacillus anthracis in 1990. We demonstrate that despite these B. anthracis features, the isolate is severely attenuated in a guinea pig model. This prompted whole genome sequencing and closure. The comparative analysis of CDC 684 to other sequenced B. anthracis isolates and further analysis reveals: a) CDC 684 is a close relative of a virulent strain, Vollum A0488; b) CDC 684 defines a new B. anthracis lineage (at least 51 SNPs) that includes 15 other isolates; c) the genome of CDC 684 contains a large chromosomal inversion that spans 3.3 Mbp; d) this inversion has caused a displacement of the usual spatial orientation of the origin of replication (ori) to the termination of replication (ter) from 180° in wild-type B. anthracis to 120° in CDC 684 and e) this isolate also has altered growth kinetics in liquid media. We propose two alternative hypotheses explaining the attenuated phenotype of this isolate. Hypothesis 1 suggests that the skewed ori/ter relationship in CDC 684 has altered its DNA replication and/or transcriptome processes resulting in altered growth kinetics and virulence capacity. Hypothesis 2 suggests that one or more of the single nucleotide polymorphisms in CDC 684 has altered the expression of a regulatory element or other genes necessary for virulence.

An attenuated strain of Bacillus anthracis (CDC 684) has a large chromosomal inversion and altered growth kinetics

PubMed Central

2011-01-01

Background An isolate originally labeled Bacillus megaterium CDC 684 was found to contain both pXO1 and pXO2, was non-hemolytic, sensitive to gamma-phage, and produced both the protective antigen and the poly-D-glutamic acid capsule. These phenotypes prompted Ezzell et al., (J. Clin. Microbiol. 28:223) to reclassify this isolate to Bacillus anthracis in 1990. Results We demonstrate that despite these B. anthracis features, the isolate is severely attenuated in a guinea pig model. This prompted whole genome sequencing and closure. The comparative analysis of CDC 684 to other sequenced B. anthracis isolates and further analysis reveals: a) CDC 684 is a close relative of a virulent strain, Vollum A0488; b) CDC 684 defines a new B. anthracis lineage (at least 51 SNPs) that includes 15 other isolates; c) the genome of CDC 684 contains a large chromosomal inversion that spans 3.3 Mbp; d) this inversion has caused a displacement of the usual spatial orientation of the origin of replication (ori) to the termination of replication (ter) from 180° in wild-type B. anthracis to 120° in CDC 684 and e) this isolate also has altered growth kinetics in liquid media. Conclusions We propose two alternative hypotheses explaining the attenuated phenotype of this isolate. Hypothesis 1 suggests that the skewed ori/ter relationship in CDC 684 has altered its DNA replication and/or transcriptome processes resulting in altered growth kinetics and virulence capacity. Hypothesis 2 suggests that one or more of the single nucleotide polymorphisms in CDC 684 has altered the expression of a regulatory element or other genes necessary for virulence. PMID:21962024

Thermodynamic Basis for the Emergence of Genomes during Prebiotic Evolution

PubMed Central

Woo, Hyung-June; Vijaya Satya, Ravi; Reifman, Jaques

2012-01-01

The RNA world hypothesis views modern organisms as descendants of RNA molecules. The earliest RNA molecules must have been random sequences, from which the first genomes that coded for polymerase ribozymes emerged. The quasispecies theory by Eigen predicts the existence of an error threshold limiting genomic stability during such transitions, but does not address the spontaneity of changes. Following a recent theoretical approach, we applied the quasispecies theory combined with kinetic/thermodynamic descriptions of RNA replication to analyze the collective behavior of RNA replicators based on known experimental kinetics data. We find that, with increasing fidelity (relative rate of base-extension for Watson-Crick versus mismatched base pairs), replications without enzymes, with ribozymes, and with protein-based polymerases are above, near, and below a critical point, respectively. The prebiotic evolution therefore must have crossed this critical region. Over large regions of the phase diagram, fitness increases with increasing fidelity, biasing random drifts in sequence space toward ‘crystallization.’ This region encloses the experimental nonenzymatic fidelity value, favoring evolutions toward polymerase sequences with ever higher fidelity, despite error rates above the error catastrophe threshold. Our work shows that experimentally characterized kinetics and thermodynamics of RNA replication allow us to determine the physicochemical conditions required for the spontaneous crystallization of biological information. Our findings also suggest that among many potential oligomers capable of templated replication, RNAs may have evolved to form prebiotic genomes due to the value of their nonenzymatic fidelity. PMID:22693440

Replication Origins and Timing of Temporal Replication in Budding Yeast: How to Solve the Conundrum?

PubMed Central

Barberis, Matteo; Spiesser, Thomas W.; Klipp, Edda

2010-01-01

Similarly to metazoans, the budding yeast Saccharomyces cereviasiae replicates its genome with a defined timing. In this organism, well-defined, site-specific origins, are efficient and fire in almost every round of DNA replication. However, this strategy is neither conserved in the fission yeast Saccharomyces pombe, nor in Xenopus or Drosophila embryos, nor in higher eukaryotes, in which DNA replication initiates asynchronously throughout S phase at random sites. Temporal and spatial controls can contribute to the timing of replication such as Cdk activity, origin localization, epigenetic status or gene expression. However, a debate is going on to answer the question how individual origins are selected to fire in budding yeast. Two opposing theories were proposed: the “replicon paradigm” or “temporal program” vs. the “stochastic firing”. Recent data support the temporal regulation of origin activation, clustering origins into temporal blocks of early and late replication. Contrarily, strong evidences suggest that stochastic processes acting on origins can generate the observed kinetics of replication without requiring a temporal order. In mammalian cells, a spatiotemporal model that accounts for a partially deterministic and partially stochastic order of DNA replication has been proposed. Is this strategy the solution to reconcile the conundrum of having both organized replication timing and stochastic origin firing also for budding yeast? In this review we discuss this possibility in the light of our recent study on the origin activation, suggesting that there might be a stochastic component in the temporal activation of the replication origins, especially under perturbed conditions. PMID:21037857

Exploring the Roles of Nucleobase Desolvation and Shape Complementarity during the Misreplication of O6-Methylguanine

PubMed Central

Chavarria, Delia; Ramos-Serrano, Andrea; Hirao, Ichiro; Berdis, Anthony J.

2011-01-01

O6-methylguanine is a miscoding DNA lesion arising from the alkylation of guanine. This report uses the bacteriophage T4 DNA polymerase as a model to probe the roles hydrogen-bonding interactions, shape/size, and nucleobase desolvation during the replication of this miscoding lesion. This was accomplished by using transient kinetic techniques to monitor the kinetic parameters for incorporating and extending natural and non-natural nucleotides. In general, the efficiency of nucleotide incorporation does not depend on the hydrogen-bonding potential of the incoming nucleotide. Instead, nucleobase hydrophobicity and shape complementarity appear to be the preeminent factors controlling nucleotide incorporation. In addition, shape complementarity plays a large role in controlling the extension of various mispairs containing O6-methylguanine. This is evident as the rate constants for extension correlate with proper interglycosyl distances and symmetry between the base angles of the formed mispair. Base pairs not conforming to an acceptable geometry within the polymerase’s active site are refractory to elongation and are processed via exonuclease proofreading. The collective data set encompassing nucleotide incorporation, extension, and excision is used to generate a model accounting for the mutagenic potential of O6-methylguanine observed in vivo. In addition, kinetic studies monitoring the incorporation and extension of non-natural nucleotides identified an analog that displays high selectivity for incorporation opposite O6-methylguanine compared to unmodified purines. The unusual selectivity of this analog for replicating damaged DNA provides a novel biochemical tool to study translesion DNA synthesis. PMID:21819995

A rabbit ventricular action potential model replicating cardiac dynamics at rapid heart rates.

PubMed

Mahajan, Aman; Shiferaw, Yohannes; Sato, Daisuke; Baher, Ali; Olcese, Riccardo; Xie, Lai-Hua; Yang, Ming-Jim; Chen, Peng-Sheng; Restrepo, Juan G; Karma, Alain; Garfinkel, Alan; Qu, Zhilin; Weiss, James N

2008-01-15

Mathematical modeling of the cardiac action potential has proven to be a powerful tool for illuminating various aspects of cardiac function, including cardiac arrhythmias. However, no currently available detailed action potential model accurately reproduces the dynamics of the cardiac action potential and intracellular calcium (Ca(i)) cycling at rapid heart rates relevant to ventricular tachycardia and fibrillation. The aim of this study was to develop such a model. Using an existing rabbit ventricular action potential model, we modified the L-type calcium (Ca) current (I(Ca,L)) and Ca(i) cycling formulations based on new experimental patch-clamp data obtained in isolated rabbit ventricular myocytes, using the perforated patch configuration at 35-37 degrees C. Incorporating a minimal seven-state Markovian model of I(Ca,L) that reproduced Ca- and voltage-dependent kinetics in combination with our previously published dynamic Ca(i) cycling model, the new model replicates experimentally observed action potential duration and Ca(i) transient alternans at rapid heart rates, and accurately reproduces experimental action potential duration restitution curves obtained by either dynamic or S1S2 pacing.

Proofreading of DNA polymerase: a new kinetic model with higher-order terminal effects

NASA Astrophysics Data System (ADS)

Song, Yong-Shun; Shu, Yao-Gen; Zhou, Xin; Ou-Yang, Zhong-Can; Li, Ming

2017-01-01

The fidelity of DNA replication by DNA polymerase (DNAP) has long been an important issue in biology. While numerous experiments have revealed details of the molecular structure and working mechanism of DNAP which consists of both a polymerase site and an exonuclease (proofreading) site, there were quite a few theoretical studies on the fidelity issue. The first model which explicitly considered both sites was proposed in the 1970s and the basic idea was widely accepted by later models. However, all these models did not systematically investigate the dominant factor on DNAP fidelity, i.e. the higher-order terminal effects through which the polymerization pathway and the proofreading pathway coordinate to achieve high fidelity. In this paper, we propose a new and comprehensive kinetic model of DNAP based on some recent experimental observations, which includes previous models as special cases. We present a rigorous and unified treatment of the corresponding steady-state kinetic equations of any-order terminal effects, and derive analytical expressions for fidelity in terms of kinetic parameters under bio-relevant conditions. These expressions offer new insights on how the higher-order terminal effects contribute substantially to the fidelity in an order-by-order way, and also show that the polymerization-and-proofreading mechanism is dominated only by very few key parameters. We then apply these results to calculate the fidelity of some real DNAPs, which are in good agreements with previous intuitive estimates given by experimentalists.

Pre-steady-state Kinetic Analysis of a Family D DNA Polymerase from Thermococcus sp. 9°N Reveals Mechanisms for Archaeal Genomic Replication and Maintenance*

PubMed Central

Schermerhorn, Kelly M.; Gardner, Andrew F.

2015-01-01

Family D DNA polymerases (polDs) have been implicated as the major replicative polymerase in archaea, excluding the Crenarchaeota branch, and bear little sequence homology to other DNA polymerase families. Here we report a detailed kinetic analysis of nucleotide incorporation and exonuclease activity for a Family D DNA polymerase from Thermococcus sp. 9°N. Pre-steady-state single-turnover nucleotide incorporation assays were performed to obtain the kinetic parameters, kpol and Kd, for correct nucleotide incorporation, incorrect nucleotide incorporation, and ribonucleotide incorporation by exonuclease-deficient polD. Correct nucleotide incorporation kinetics revealed a relatively slow maximal rate of polymerization (kpol ∼2.5 s−1) and especially tight nucleotide binding (Kd(dNTP) ∼1.7 μm), compared with DNA polymerases from Families A, B, C, X, and Y. Furthermore, pre-steady-state nucleotide incorporation assays revealed that polD prevents the incorporation of incorrect nucleotides and ribonucleotides primarily through reduced nucleotide binding affinity. Pre-steady-state single-turnover assays on wild-type 9°N polD were used to examine 3′-5′ exonuclease hydrolysis activity in the presence of Mg2+ and Mn2+. Interestingly, substituting Mn2+ for Mg2+ accelerated hydrolysis rates >40-fold (kexo ≥110 s−1 versus ≥2.5 s−1). Preference for Mn2+ over Mg2+ in exonuclease hydrolysis activity is a property unique to the polD family. The kinetic assays performed in this work provide critical insight into the mechanisms that polD employs to accurately and efficiently replicate the archaeal genome. Furthermore, despite the unique properties of polD, this work suggests that a conserved polymerase kinetic pathway is present in all known DNA polymerase families. PMID:26160179

Seeking the chemical roots of darwinism: bridging between chemistry and biology.

PubMed

Pross, Addy

2009-08-24

Chemistry and biology are intimately connected sciences yet the chemistry-biology interface remains problematic and central issues regarding the very essence of living systems remain unresolved. In this essay we build on a kinetic theory of replicating systems that encompasses the idea that there are two distinct kinds of stability in nature-thermodynamic stability, associated with "regular" chemical systems, and dynamic kinetic stability, associated with replicating systems. That fundamental distinction is utilized to bridge between chemistry and biology by demonstrating that within the parallel world of replicating systems there is a second law analogue to the second law of thermodynamics, and that Darwinian theory may, through scientific reductionism, be related to that second law analogue. Possible implications of these ideas to the origin of life problem and the relationship between chemical emergence and biological evolution are discussed.

RuvAB and RecG are not essential for the recovery of DNA synthesis following UV-induced DNA damage in Escherichia coli.

PubMed Central

Donaldson, Janet R; Courcelle, Charmain T; Courcelle, Justin

2004-01-01

Ultraviolet light induces DNA lesions that block the progression of the replication machinery. Several models speculate that the resumption of replication following disruption by UV-induced DNA damage requires regression of the nascent DNA or migration of the replication machinery away from the blocking lesion to allow repair or bypass of the lesion to occur. Both RuvAB and RecG catalyze branch migration of three- and four-stranded DNA junctions in vitro and are proposed to catalyze fork regression in vivo. To examine this possibility, we characterized the recovery of DNA synthesis in ruvAB and recG mutants. We found that in the absence of either RecG or RuvAB, arrested replication forks are maintained and DNA synthesis is resumed with kinetics that are similar to those in wild-type cells. The data presented here indicate that RecG- or RuvAB-catalyzed fork regression is not essential for DNA synthesis to resume following arrest by UV-induced DNA damage in vivo. PMID:15126385

Amyloid fibrils: formation, replication, and physics behind them

NASA Astrophysics Data System (ADS)

Saric, Andela

The assembly of normally soluble proteins into long fibrils, known as amyloids, is associated with a range of pathologies, including Alzheimer's and Parkinson's diseases. A large number of structurally unrelated proteins form this type of fibrils, and we are in a pursuit of physical principles that underlie the amyloid formation and propagation. We show that small disorders oligomers, which are increasingly believed to be the prime cause for cellular toxicity, serve as nucleation centers for the fibril formation. We then relate experimentally measurable kinetic descriptors of amyloid aggregation to the microscopic mechanisms of the process. Once formed, amyloid fibrils can catalyse the formation of new oligomers and fibrils in a process that resembles self-replication. By combining simulations with biosensing and kinetic measurements of the aggregation of Alzheimer's A β peptide, we propose a mechanistic explanation for the self-replication of protein fibrils, and discuss its thermodynamic signature. Finally, we consider the design of possible inhibitors of the fibril self-replication process. Mechanistic understandings provided here not only have implications for future efforts to control pathological protein aggregation, but are also of interest for the rational assembly of bionanomaterials, where achieving and controlling self-replication is one of the unfulfilled goals.

Developing Inhibitors of Translesion DNA Synthesis as Therapeutic Agents Against Lung Cancer

DTIC Science & Technology

2014-10-01

pol eta when replicating damaged DNA. 1S. SUBJECT TERMS: Mutagenesis, DNA polymerases, nucleoside analogs, chemotherapeutic agents 16. SECURITY ...such as polymerase eta, iota , and kappa that are involved in replicating damaged DNA. Our kinetic data obtained under Task 1B indicates that pol eta

Volterra representation enables modeling of complex synaptic nonlinear dynamics in large-scale simulations.

PubMed

Hu, Eric Y; Bouteiller, Jean-Marie C; Song, Dong; Baudry, Michel; Berger, Theodore W

2015-01-01

Chemical synapses are comprised of a wide collection of intricate signaling pathways involving complex dynamics. These mechanisms are often reduced to simple spikes or exponential representations in order to enable computer simulations at higher spatial levels of complexity. However, these representations cannot capture important nonlinear dynamics found in synaptic transmission. Here, we propose an input-output (IO) synapse model capable of generating complex nonlinear dynamics while maintaining low computational complexity. This IO synapse model is an extension of a detailed mechanistic glutamatergic synapse model capable of capturing the input-output relationships of the mechanistic model using the Volterra functional power series. We demonstrate that the IO synapse model is able to successfully track the nonlinear dynamics of the synapse up to the third order with high accuracy. We also evaluate the accuracy of the IO synapse model at different input frequencies and compared its performance with that of kinetic models in compartmental neuron models. Our results demonstrate that the IO synapse model is capable of efficiently replicating complex nonlinear dynamics that were represented in the original mechanistic model and provide a method to replicate complex and diverse synaptic transmission within neuron network simulations.

Volterra representation enables modeling of complex synaptic nonlinear dynamics in large-scale simulations

PubMed Central

Hu, Eric Y.; Bouteiller, Jean-Marie C.; Song, Dong; Baudry, Michel; Berger, Theodore W.

2015-01-01

Chemical synapses are comprised of a wide collection of intricate signaling pathways involving complex dynamics. These mechanisms are often reduced to simple spikes or exponential representations in order to enable computer simulations at higher spatial levels of complexity. However, these representations cannot capture important nonlinear dynamics found in synaptic transmission. Here, we propose an input-output (IO) synapse model capable of generating complex nonlinear dynamics while maintaining low computational complexity. This IO synapse model is an extension of a detailed mechanistic glutamatergic synapse model capable of capturing the input-output relationships of the mechanistic model using the Volterra functional power series. We demonstrate that the IO synapse model is able to successfully track the nonlinear dynamics of the synapse up to the third order with high accuracy. We also evaluate the accuracy of the IO synapse model at different input frequencies and compared its performance with that of kinetic models in compartmental neuron models. Our results demonstrate that the IO synapse model is capable of efficiently replicating complex nonlinear dynamics that were represented in the original mechanistic model and provide a method to replicate complex and diverse synaptic transmission within neuron network simulations. PMID:26441622

Quasispecies in population of compositional assemblies.

PubMed

Gross, Renan; Fouxon, Itzhak; Lancet, Doron; Markovitch, Omer

2014-12-30

The quasispecies model refers to information carriers that undergo self-replication with errors. A quasispecies is a steady-state population of biopolymer sequence variants generated by mutations from a master sequence. A quasispecies error threshold is a minimal replication accuracy below which the population structure breaks down. Theory and experimentation of this model often refer to biopolymers, e.g. RNA molecules or viral genomes, while its prebiotic context is often associated with an RNA world scenario. Here, we study the possibility that compositional entities which code for compositional information, intrinsically different from biopolymers coding for sequential information, could show quasispecies dynamics. We employed a chemistry-based model, graded autocatalysis replication domain (GARD), which simulates the network dynamics within compositional molecular assemblies. In GARD, a compotype represents a population of similar assemblies that constitute a quasi-stationary state in compositional space. A compotype's center-of-mass is found to be analogous to a master sequence for a sequential quasispecies. Using single-cycle GARD dynamics, we measured the quasispecies transition matrix (Q) for the probabilities of transition from one center-of-mass Euclidean distance to another. Similarly, the quasispecies' growth rate vector (A) was obtained. This allowed computing a steady state distribution of distances to the center of mass, as derived from the quasispecies equation. In parallel, a steady state distribution was obtained via the GARD equation kinetics. Rewardingly, a significant correlation was observed between the distributions obtained by these two methods. This was only seen for distances to the compotype center-of-mass, and not to randomly selected compositions. A similar correspondence was found when comparing the quasispecies time dependent dynamics towards steady state. Further, changing the error rate by modifying basal assembly joining rate of GARD kinetics was found to display an error catastrophe, similar to the standard quasispecies model. Additional augmentation of compositional mutations leads to the complete disappearance of the master-like composition. Our results show that compositional assemblies, as simulated by the GARD formalism, portray significant attributes of quasispecies dynamics. This expands the applicability of the quasispecies model beyond sequence-based entities, and potentially enhances validity of GARD as a model for prebiotic evolution.

Treatment with integrase inhibitor suggests a new interpretation of HIV RNA decay curves that reveals a subset of cells with slow integration

DOE PAGES

Cardozo, Erwing Fabian; Andrade, Adriana; Mellors, John W.; ...

2017-07-05

The kinetics of HIV-1 decay under treatment depends on the class of antiretrovirals used. Mathematical models are useful to interpret the different profiles, providing quantitative information about the kinetics of virus replication and the cell populations contributing to viral decay. We modeled proviral integration in short- and long-lived infected cells to compare viral kinetics under treatment with and without the integrase inhibitor raltegravir (RAL). We fitted the model to data obtained from participants treated with RAL-containing regimes or with a four-drug regimen of protease and reverse transcriptase inhibitors. Our model explains the existence and quantifies the three phases of HIV-1more » RNA decay in RAL-based regimens vs. the two phases observed in therapies without RAL. Our findings indicate that HIV-1 infection is mostly sustained by short-lived infected cells with fast integration and a short viral production period, and by long-lived infected cells with slow integration but an equally short viral production period. We propose that these cells represent activated and resting infected CD4+ T-cells, respectively, and estimate that infection of resting cells represent ~4% of productive reverse transcription events in chronic infection. RAL reveals the kinetics of proviral integration, showing that in short-lived cells the pre-integration population has a half-life of ~7 hours, whereas in long-lived cells this half-life is ~6 weeks. We also show that the efficacy of RAL can be estimated by the difference in viral load at the start of the second phase in protocols with and without RAL. Altogether, we provide a mechanistic model of viral infection that parsimoniously explains the kinetics of viral load decline under multiple classes of antiretrovirals.« less

Treatment with integrase inhibitor suggests a new interpretation of HIV RNA decay curves that reveals a subset of cells with slow integration

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

Cardozo, Erwing Fabian; Andrade, Adriana; Mellors, John W.

The kinetics of HIV-1 decay under treatment depends on the class of antiretrovirals used. Mathematical models are useful to interpret the different profiles, providing quantitative information about the kinetics of virus replication and the cell populations contributing to viral decay. We modeled proviral integration in short- and long-lived infected cells to compare viral kinetics under treatment with and without the integrase inhibitor raltegravir (RAL). We fitted the model to data obtained from participants treated with RAL-containing regimes or with a four-drug regimen of protease and reverse transcriptase inhibitors. Our model explains the existence and quantifies the three phases of HIV-1more » RNA decay in RAL-based regimens vs. the two phases observed in therapies without RAL. Our findings indicate that HIV-1 infection is mostly sustained by short-lived infected cells with fast integration and a short viral production period, and by long-lived infected cells with slow integration but an equally short viral production period. We propose that these cells represent activated and resting infected CD4+ T-cells, respectively, and estimate that infection of resting cells represent ~4% of productive reverse transcription events in chronic infection. RAL reveals the kinetics of proviral integration, showing that in short-lived cells the pre-integration population has a half-life of ~7 hours, whereas in long-lived cells this half-life is ~6 weeks. We also show that the efficacy of RAL can be estimated by the difference in viral load at the start of the second phase in protocols with and without RAL. Altogether, we provide a mechanistic model of viral infection that parsimoniously explains the kinetics of viral load decline under multiple classes of antiretrovirals.« less

Detection of kinetic change points in piece-wise linear single molecule motion

NASA Astrophysics Data System (ADS)

Hill, Flynn R.; van Oijen, Antoine M.; Duderstadt, Karl E.

2018-03-01

Single-molecule approaches present a powerful way to obtain detailed kinetic information at the molecular level. However, the identification of small rate changes is often hindered by the considerable noise present in such single-molecule kinetic data. We present a general method to detect such kinetic change points in trajectories of motion of processive single molecules having Gaussian noise, with a minimum number of parameters and without the need of an assumed kinetic model beyond piece-wise linearity of motion. Kinetic change points are detected using a likelihood ratio test in which the probability of no change is compared to the probability of a change occurring, given the experimental noise. A predetermined confidence interval minimizes the occurrence of false detections. Applying the method recursively to all sub-regions of a single molecule trajectory ensures that all kinetic change points are located. The algorithm presented allows rigorous and quantitative determination of kinetic change points in noisy single molecule observations without the need for filtering or binning, which reduce temporal resolution and obscure dynamics. The statistical framework for the approach and implementation details are discussed. The detection power of the algorithm is assessed using simulations with both single kinetic changes and multiple kinetic changes that typically arise in observations of single-molecule DNA-replication reactions. Implementations of the algorithm are provided in ImageJ plugin format written in Java and in the Julia language for numeric computing, with accompanying Jupyter Notebooks to allow reproduction of the analysis presented here.

Thermodynamic and kinetic modelling of fuel oxidation behaviour in operating defective fuel

NASA Astrophysics Data System (ADS)

Lewis, operating defective fuel B. J.; Thompson, W. T.; Akbari, F.; Thompson, D. M.; Thurgood, C.; Higgs, J.

2004-07-01

A theoretical treatment has been developed to predict the fuel oxidation behaviour in operating defective nuclear fuel elements. The equilibrium stoichiometry deviation in the hyper-stoichiometric fuel has been derived from thermodynamic considerations using a self-consistent set of thermodynamic properties for the U-O system, which emphasizes replication of solubilities and three-phase invariant conditions displayed in the U-O binary phase diagram. The kinetics model accounts for multi-phase transport including interstitial oxygen diffusion in the solid and gas-phase transport of hydrogen and steam in the fuel cracks. The fuel oxidation model is further coupled to a heat conduction model to account for the feedback effect of a reduced thermal conductivity in the hyper-stoichiometric fuel. A numerical solution has been developed using a finite-element technique with the FEMLAB software package. The model has been compared to available data from several in-reactor X-2 loop experiments with defective fuel conducted at the Chalk River Laboratories. The model has also been benchmarked against an O/U profile measurement for a spent defective fuel element discharged from a commercial reactor.

Mathematical Modeling of Protein Misfolding Mechanisms in Neurological Diseases: A Historical Overview.

PubMed

Carbonell, Felix; Iturria-Medina, Yasser; Evans, Alan C

2018-01-01

Protein misfolding refers to a process where proteins become structurally abnormal and lose their specific 3-dimensional spatial configuration. The histopathological presence of misfolded protein (MP) aggregates has been associated as the primary evidence of multiple neurological diseases, including Prion diseases, Alzheimer's disease, Parkinson's disease, and Creutzfeldt-Jacob disease. However, the exact mechanisms of MP aggregation and propagation, as well as their impact in the long-term patient's clinical condition are still not well understood. With this aim, a variety of mathematical models has been proposed for a better insight into the kinetic rate laws that govern the microscopic processes of protein aggregation. Complementary, another class of large-scale models rely on modern molecular imaging techniques for describing the phenomenological effects of MP propagation over the whole brain. Unfortunately, those neuroimaging-based studies do not take full advantage of the tremendous capabilities offered by the chemical kinetics modeling approach. Actually, it has been barely acknowledged that the vast majority of large-scale models have foundations on previous mathematical approaches that describe the chemical kinetics of protein replication and propagation. The purpose of the current manuscript is to present a historical review about the development of mathematical models for describing both microscopic processes that occur during the MP aggregation and large-scale events that characterize the progression of neurodegenerative MP-mediated diseases.

An Integrated Qualitative and Quantitative Biochemical Model Learning Framework Using Evolutionary Strategy and Simulated Annealing.

PubMed

Wu, Zujian; Pang, Wei; Coghill, George M

2015-01-01

Both qualitative and quantitative model learning frameworks for biochemical systems have been studied in computational systems biology. In this research, after introducing two forms of pre-defined component patterns to represent biochemical models, we propose an integrative qualitative and quantitative modelling framework for inferring biochemical systems. In the proposed framework, interactions between reactants in the candidate models for a target biochemical system are evolved and eventually identified by the application of a qualitative model learning approach with an evolution strategy. Kinetic rates of the models generated from qualitative model learning are then further optimised by employing a quantitative approach with simulated annealing. Experimental results indicate that our proposed integrative framework is feasible to learn the relationships between biochemical reactants qualitatively and to make the model replicate the behaviours of the target system by optimising the kinetic rates quantitatively. Moreover, potential reactants of a target biochemical system can be discovered by hypothesising complex reactants in the synthetic models. Based on the biochemical models learned from the proposed framework, biologists can further perform experimental study in wet laboratory. In this way, natural biochemical systems can be better understood.

A teacher-developed inquiry model to teach the molecular basis of hyperbolic kinetics in biological membrane transport.

PubMed

Marcus, Leanne; Plumeri, Julia; Baker, Gary M; Miller, Jon S

2013-06-01

A previously published classroom teaching method for helping students visualize and understand Michaelis-Menten kinetics (19) was used as an anticipatory set with high school and middle school science teachers in an Illinois Math and Science Partnership Program. As part of the activity, the teachers were asked to collect data by replicating the method and to analyze and report the data. All concluded that the rate data they had collected were hyperbolic. As part of a guided inquiry plan, teachers were then prompted to reexamine the method and evaluate its efficacy as a teaching strategy for developing specific kinetic concepts. After further data collection and analysis, the teachers discovered that their data trends were not, in fact, hyperbolic, which led to several teacher-developed revisions aimed at obtaining a true hyperbolic outcome. This article outlines the inquiry process that led to these revisions and illustrates their alignment with several key concepts, such as rapid equilibrium kinetics. Instructional decisions were necessary at several key points, and these are discussed.

Mechanistic Investigation of the Bypass of a Bulky Aromatic DNA Adduct Catalyzed by a Y-family DNA Polymerase

PubMed Central

Gadkari, Varun V.; Tokarsky, E. John; Malik, Chanchal K.; Basu, Ashis K.; Suo, Zucai

2014-01-01

3-Nitrobenzanthrone (3-NBA), a nitropolyaromatic hydrocarbon (NitroPAH) pollutant in diesel exhaust, is a potent mutagen and carcinogen. After metabolic activation, the primary metabolites of 3-NBA react with DNA to form dG and dA adducts. One of the three major adducts identified is N-(2’-deoxyguanosin-8-yl)-3-aminobenzanthrone (dGC8-N-ABA). This bulky adduct likely stalls replicative DNA polymerases but can be traversed by lesion bypass polymerases in vivo. Here, we employed running start assays to show that a site-specifically placed dGC8-N-ABA is bypassed in vitro by Sulfolobus solfataricus DNA polymerase IV (Dpo4), a model Y-family DNA polymerase. However, the nucleotide incorporation rate of Dpo4 was significantly reduced opposite both the lesion and the template position immediately downstream from the lesion site, leading to two strong pause sites. To investigate the kinetic effect of dGC8-N-ABA on polymerization, we utilized pre-steady-state kinetic methods to determine the kinetic parameters for individual nucleotide incorporations upstream, opposite, and downstream from the dGC8-N-ABA lesion. Relative to the replication of the corresponding undamaged DNA template, both nucleotide incorporation efficiency and fidelity of Dpo4 were considerably decreased during dGC8-N-ABA lesion bypass and the subsequent extension step. The lower nucleotide incorporation efficiency caused by the lesion is a result of a significantly reduced dNTP incorporation rate constant and modestly weaker dNTP binding affinity. At both pause sites, nucleotide incorporation followed biphasic kinetics with a fast and a slow phase and their rates varied with nucleotide concentration. In contrast, only the fast phase was observed with undamaged DNA. A kinetic mechanism was proposed for the bypass of dGC8-N-ABA bypass catalyzed by Dpo4. PMID:25048879

Mechanistic investigation of the bypass of a bulky aromatic DNA adduct catalyzed by a Y-family DNA polymerase.

PubMed

Gadkari, Varun V; Tokarsky, E John; Malik, Chanchal K; Basu, Ashis K; Suo, Zucai

2014-09-01

3-Nitrobenzanthrone (3-NBA), a nitropolyaromatic hydrocarbon (NitroPAH) pollutant in diesel exhaust, is a potent mutagen and carcinogen. After metabolic activation, the primary metabolites of 3-NBA react with DNA to form dG and dA adducts. One of the three major adducts identified is N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone (dG(C8-N-ABA)). This bulky adduct likely stalls replicative DNA polymerases but can be traversed by lesion bypass polymerases in vivo. Here, we employed running start assays to show that a site-specifically placed dG(C8-N-ABA) is bypassed in vitro by Sulfolobus solfataricus DNA polymerase IV (Dpo4), a model Y-family DNA polymerase. However, the nucleotide incorporation rate of Dpo4 was significantly reduced opposite both the lesion and the template position immediately downstream from the lesion site, leading to two strong pause sites. To investigate the kinetic effect of dG(C8-N-ABA) on polymerization, we utilized pre-steady-state kinetic methods to determine the kinetic parameters for individual nucleotide incorporations upstream, opposite, and downstream from the dG(C8-N-ABA) lesion. Relative to the replication of the corresponding undamaged DNA template, both nucleotide incorporation efficiency and fidelity of Dpo4 were considerably decreased during dG(C8-N-ABA) lesion bypass and the subsequent extension step. The lower nucleotide incorporation efficiency caused by the lesion is a result of a significantly reduced dNTP incorporation rate constant and modestly weaker dNTP binding affinity. At both pause sites, nucleotide incorporation followed biphasic kinetics with a fast and a slow phase and their rates varied with nucleotide concentration. In contrast, only the fast phase was observed with undamaged DNA. A kinetic mechanism was proposed for the bypass of dG(C8-N-ABA) bypass catalyzed by Dpo4. Copyright © 2014 Elsevier B.V. All rights reserved.

Mutations in Replicative Stress Response Pathways Are Associated with S Phase-specific Defects in Nucleotide Excision Repair*

PubMed Central

Bélanger, François; Angers, Jean-Philippe; Fortier, Émile; Hammond-Martel, Ian; Costantino, Santiago; Drobetsky, Elliot; Wurtele, Hugo

2016-01-01

Nucleotide excision repair (NER) is a highly conserved pathway that removes helix-distorting DNA lesions induced by a plethora of mutagens, including UV light. Our laboratory previously demonstrated that human cells deficient in either ATM and Rad3-related (ATR) kinase or translesion DNA polymerase η (i.e. key proteins that promote the completion of DNA replication in response to UV-induced replicative stress) are characterized by profound inhibition of NER exclusively during S phase. Toward elucidating the mechanistic basis of this phenomenon, we developed a novel assay to quantify NER kinetics as a function of cell cycle in the model organism Saccharomyces cerevisiae. Using this assay, we demonstrate that in yeast, deficiency of the ATR homologue Mec1 or of any among several other proteins involved in the cellular response to replicative stress significantly abrogates NER uniquely during S phase. Moreover, initiation of DNA replication is required for manifestation of this defect, and S phase NER proficiency is correlated with the capacity of individual mutants to respond to replicative stress. Importantly, we demonstrate that partial depletion of Rfa1 recapitulates defective S phase-specific NER in wild type yeast; moreover, ectopic RPA1–3 overexpression rescues such deficiency in either ATR- or polymerase η-deficient human cells. Our results strongly suggest that reduction of NER capacity during periods of enhanced replicative stress, ostensibly caused by inordinate sequestration of RPA at stalled DNA replication forks, represents a conserved feature of the multifaceted eukaryotic DNA damage response. PMID:26578521

The replication of Bangladeshi H9N2 avian influenza viruses carrying genes from H7N3 in mammals

PubMed Central

Shanmuganatham, Karthik K; Jones, Jeremy C; Marathe, Bindumadhav M; Feeroz, Mohammed M; Jones-Engel, Lisa; Walker, David; Turner, Jasmine; Rabiul Alam, S M; Kamrul Hasan, M; Akhtar, Sharmin; Seiler, Patrick; McKenzie, Pamela; Krauss, Scott; Webby, Richard J; Webster, Robert G

2016-01-01

H9N2 avian influenza viruses are continuously monitored by the World Health Organization because they are endemic; they continually reassort with H5N1, H7N9 and H10N8 viruses; and they periodically cause human infections. We characterized H9N2 influenza viruses carrying internal genes from highly pathogenic H7N3 viruses, which were isolated from chickens or quail from live-bird markets in Bangladesh between 2010 and 2013. All of the H9N2 viruses used in this study carried mammalian host-specific mutations. We studied their replication kinetics in normal human bronchoepithelial cells and swine tracheal and lung explants, which exhibit many features of the mammalian airway epithelium and serve as a mammalian host model. All H9N2 viruses replicated to moderate-to-high titers in the normal human bronchoepithelial cells and swine lung explants, but replication was limited in the swine tracheal explants. In Balb/c mice, the H9N2 viruses were nonlethal, replicated to moderately high titers and the infection was confined to the lungs. In the ferret model of human influenza infection and transmission, H9N2 viruses possessing the Q226L substitution in hemagglutinin replicated well without clinical signs and spread via direct contact but not by aerosol. None of the H9N2 viruses tested were resistant to the neuraminidase inhibitors. Our study shows that the Bangladeshi H9N2 viruses have the potential to infect humans and highlights the importance of monitoring and characterizing this influenza subtype to better understand the potential risk these viruses pose to humans. PMID:27094903

The replication of Bangladeshi H9N2 avian influenza viruses carrying genes from H7N3 in mammals.

PubMed

Shanmuganatham, Karthik K; Jones, Jeremy C; Marathe, Bindumadhav M; Feeroz, Mohammed M; Jones-Engel, Lisa; Walker, David; Turner, Jasmine; Rabiul Alam, S M; Kamrul Hasan, M; Akhtar, Sharmin; Seiler, Patrick; McKenzie, Pamela; Krauss, Scott; Webby, Richard J; Webster, Robert G

2016-04-20

H9N2 avian influenza viruses are continuously monitored by the World Health Organization because they are endemic; they continually reassort with H5N1, H7N9 and H10N8 viruses; and they periodically cause human infections. We characterized H9N2 influenza viruses carrying internal genes from highly pathogenic H7N3 viruses, which were isolated from chickens or quail from live-bird markets in Bangladesh between 2010 and 2013. All of the H9N2 viruses used in this study carried mammalian host-specific mutations. We studied their replication kinetics in normal human bronchoepithelial cells and swine tracheal and lung explants, which exhibit many features of the mammalian airway epithelium and serve as a mammalian host model. All H9N2 viruses replicated to moderate-to-high titers in the normal human bronchoepithelial cells and swine lung explants, but replication was limited in the swine tracheal explants. In Balb/c mice, the H9N2 viruses were nonlethal, replicated to moderately high titers and the infection was confined to the lungs. In the ferret model of human influenza infection and transmission, H9N2 viruses possessing the Q226L substitution in hemagglutinin replicated well without clinical signs and spread via direct contact but not by aerosol. None of the H9N2 viruses tested were resistant to the neuraminidase inhibitors. Our study shows that the Bangladeshi H9N2 viruses have the potential to infect humans and highlights the importance of monitoring and characterizing this influenza subtype to better understand the potential risk these viruses pose to humans.

Importance of disentanglement and entanglement during DNA replication and segregation. Comment on: "Disentangling DNA molecules" by Alexander Vologodskii

NASA Astrophysics Data System (ADS)

Bates, David; Pettitt, B. Montgomery; Buck, Gregory R.; Zechiedrich, Lynn

2016-09-01

In the Vologodskii review[19], the accompanying comments, and many other publications, there has been considerable effort to analyze the actions of type II topoisomerases, especially with regard to ;topological simplification; [4]. Whereas these efforts could be characterized as a battle of the models, with each research team arguing for their version of how it might work, each specific kinetic concept adds important considerations to the fundamental question of how these enzymes function. The basic tenet, however, of what is called the ;hooked juxtaposition model [1],; is not a modeling aspect, but is simply a geometric mathematical fact.

Accessory genes confer a high replication rate to virulent feline immunodeficiency virus.

PubMed

Troyer, Ryan M; Thompson, Jesse; Elder, John H; VandeWoude, Sue

2013-07-01

Feline immunodeficiency virus (FIV) is a lentivirus that causes AIDS in domestic cats, similar to human immunodeficiency virus (HIV)/AIDS in humans. The FIV accessory protein Vif abrogates the inhibition of infection by cat APOBEC3 restriction factors. FIV also encodes a multifunctional OrfA accessory protein that has characteristics similar to HIV Tat, Vpu, Vpr, and Nef. To examine the role of vif and orfA accessory genes in FIV replication and pathogenicity, we generated chimeras between two FIV molecular clones with divergent disease potentials: a highly pathogenic isolate that replicates rapidly in vitro and is associated with significant immunopathology in vivo, FIV-C36 (referred to here as high-virulence FIV [HV-FIV]), and a less-pathogenic strain, FIV-PPR (referred to here as low-virulence FIV [LV-FIV]). Using PCR-driven overlap extension, we produced viruses in which vif, orfA, or both genes from virulent HV-FIV replaced equivalent genes in LV-FIV. The generation of these chimeras is more straightforward in FIV than in primate lentiviruses, since FIV accessory gene open reading frames have very little overlap with other genes. All three chimeric viruses exhibited increased replication kinetics in vitro compared to the replication kinetics of LV-FIV. Chimeras containing HV-Vif or Vif/OrfA had replication rates equivalent to those of the virulent HV-FIV parental virus. Furthermore, small interfering RNA knockdown of feline APOBEC3 genes resulted in equalization of replication rates between LV-FIV and LV-FIV encoding HV-FIV Vif. These findings demonstrate that Vif-APOBEC interactions play a key role in controlling the replication and pathogenicity of this immunodeficiency-inducing virus in its native host species and that accessory genes act as mediators of lentiviral strain-specific virulence.

Neutrality of the canonical NF-kappaB-dependent pathway for human and murine cytomegalovirus transcription and replication in vitro.

PubMed

Benedict, Chris A; Angulo, Ana; Patterson, Ginelle; Ha, Sukwon; Huang, Huang; Messerle, Martin; Ware, Carl F; Ghazal, Peter

2004-01-01

Cytomegalovirus (CMV) is known to rapidly induce activation of nuclear factor kappaB (NF-kappaB) after infection of fibroblast and macrophage cells. NF-kappaB response elements are present in the enhancer region of the CMV major immediate-early promoter (MIEP), and activity of the MIEP is strongly upregulated by NF-kappaB in transient-transfection assays. Here we investigate whether the NF-kappaB-dependent pathway is required for initiating or potentiating human and murine CMV replication in vitro. We show that expression of a dominant negative mutant of the inhibitor of NF-kappaB-alpha (IkappaBalphaM) does not alter the replication kinetics of human or mouse CMV in cultured cells. In addition, mouse embryo fibroblasts genetically deficient for p65/RelA actually showed elevated levels of MCMV replication. Mutation of all NF-kappaB response elements within the enhancer of the MIEP in a recombinant mouse CMV containing the human MIEP (hMCMV-ES), which we have previously shown to replicate in murine fibroblasts with kinetics equivalent to that of wild-type mouse CMV, did not negatively affect replication in fibroblasts. Taken together, these data show that, for CMV replication in cultured fibroblasts activation of the canonical NF-kappaB pathway and binding of NF-kappaB to the MIEP are dispensable, and in the case of p65 may even interfere, thus uncovering a previously unrecognized level of complexity in the host regulatory network governing MIE gene expression in the context of a viral infection.

Evidence of a conserved role for Chlamydia HtrA in the replication phase of the chlamydial developmental cycle.

PubMed

Patel, Pooja; De Boer, Leonore; Timms, Peter; Huston, Wilhelmina May

2014-08-01

Identification of the HtrA inhibitor JO146 previously enabled us to demonstrate an essential function for HtrA during the mid-replicative phase of the Chlamydia trachomatis developmental cycle. Here we extend our investigations to other members of the Chlamydia genus. C. trachomatis isolates with distinct replicative phase growth kinetics showed significant loss of viable infectious progeny after HtrA was inhibited during the replicative phase. Mid-replicative phase addition of JO146 was also significantly detrimental to Chlamydia pecorum, Chlamydia suis and Chlamydia cavie. These data combined indicate that HtrA has a conserved critical role during the replicative phase of the chlamydial developmental cycle. Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Essential role of cyclophilin A for hepatitis C virus replication and virus production and possible link to polyprotein cleavage kinetics.

PubMed

Kaul, Artur; Stauffer, Sarah; Berger, Carola; Pertel, Thomas; Schmitt, Jennifer; Kallis, Stephanie; Zayas, Margarita; Lopez, Margarita Zayas; Lohmann, Volker; Luban, Jeremy; Bartenschlager, Ralf

2009-08-01

Viruses are obligate intracellular parasites and therefore their replication completely depends on host cell factors. In case of the hepatitis C virus (HCV), a positive-strand RNA virus that in the majority of infections establishes persistence, cyclophilins are considered to play an important role in RNA replication. Subsequent to the observation that cyclosporines, known to sequester cyclophilins by direct binding, profoundly block HCV replication in cultured human hepatoma cells, conflicting results were obtained as to the particular cyclophilin (Cyp) required for viral RNA replication and the underlying possible mode of action. By using a set of cell lines with stable knock-down of CypA or CypB, we demonstrate in the present work that replication of subgenomic HCV replicons of different genotypes is reduced by CypA depletion up to 1,000-fold whereas knock-down of CypB had no effect. Inhibition of replication was rescued by over-expression of wild type CypA, but not by a mutant lacking isomerase activity. Replication of JFH1-derived full length genomes was even more sensitive to CypA depletion as compared to subgenomic replicons and virus production was completely blocked. These results argue that CypA may target an additional viral factor outside of the minimal replicase contributing to RNA amplification and assembly, presumably nonstructural protein 2. By selecting for resistance against the cyclosporine analogue DEBIO-025 that targets CypA in a dose-dependent manner, we identified two mutations (V2440A and V2440L) close to the cleavage site between nonstructural protein 5A and the RNA-dependent RNA polymerase in nonstructural protein 5B that slow down cleavage kinetics at this site and reduce CypA dependence of viral replication. Further amino acid substitutions at the same cleavage site accelerating processing increase CypA dependence. Our results thus identify an unexpected correlation between HCV polyprotein processing and CypA dependence of HCV replication.

Asexual and sexual replication in sporulating organisms

NASA Astrophysics Data System (ADS)

Lee, Bohyun; Tannenbaum, Emmanuel

2007-08-01

Replication via sporulation is the replication strategy for all multicellular life, and may even be observed in unicellular life (such as with budding yeast). We consider diploid populations replicating via one of two possible sporulation mechanisms. (1) Asexual sporulation, whereby adult organisms produce single-celled diploid spores that grow into adults themselves. (2) Sexual sporulation, whereby adult organisms produce single-celled diploid spores that divide into haploid gametes. The haploid gametes enter a haploid “pool,” where they may recombine with other haploids to form a diploid spore that then grows into an adult. We consider a haploid fusion rate given by second-order reaction kinetics. We work with a simplified model where the diploid genome consists of only two chromosomes, each of which may be rendered defective with a single point mutation of the wild-type. We find that the asexual strategy is favored when the rate of spore production is high compared to the characteristic growth rate from a spore to a reproducing adult. Conversely, the sexual strategy is favored when the rate of spore production is low compared to the characteristic growth rate from a spore to a reproducing adult. As the characteristic growth time increases, or as the population density increases, the critical ratio of spore production rate to organism growth rate at which the asexual strategy overtakes the sexual one is pushed to higher values. Therefore, the results of this model suggest that, for complex multicellular organisms, sexual replication is favored at high population densities and low growth and sporulation rates.

Kinetics of Mismatch Formation opposite Lesions by the Replicative DNA Polymerase from Bacteriophage RB69

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

Hogg, Matthew; Rudnicki, Jean; Midkiff, John

2010-04-12

The fidelity of DNA replication is under constant threat from the formation of lesions within the genome. Oxidation of DNA bases leads to the formation of altered DNA bases such as 8-oxo-7,8-dihydroguanine, commonly called 8-oxoG, and 2-hydroxyadenenine, or 2-OHA. In this work we have examined the incorporation kinetics opposite these two oxidatively derived lesions as well as an abasic site analogue by the replicative DNA polymerase from bacteriophage RB69. We compared the kinetic parameters for both wild type and the low fidelity L561A variant. While nucleotide incorporation rates (k{sub pol}) were generally higher for the variant, the presence of amore » lesion in the templating position reduced the ability of both the wild-type and variant DNA polymerases to form ternary enzyme-DNA-dNTP complexes. Thus, the L561A substitution does not significantly affect the ability of the RB69 DNA polymerase to recognize damaged DNA; instead, the mutation increases the probability that nucleotide incorporation will occur. We have also solved the crystal structure of the L561A variant forming an 8-oxoG {center_dot} dATP mispair and show that the propensity for forming this mispair depends on an enlarged polymerase active site.« less

Variability of interferon-λ induction and antiviral activity in Nipah virus infected differentiated human bronchial epithelial cells of two human donors.

PubMed

Sauerhering, Lucie; Müller, Helena; Behner, Laura; Elvert, Mareike; Fehling, Sarah Katharina; Strecker, Thomas; Maisner, Andrea

2017-10-01

Highly pathogenic Nipah virus (NiV) generally causes severe encephalitis in humans. Respiratory symptoms are infrequently observed, likely reflecting variations in infection kinetics in human airways. Supporting this idea, we recently identified individual differences in NiV replication kinetics in cultured airway epithelia from different human donors. As type III interferons (IFN-λ) represent major players in the defence mechanism against viral infection of the respiratory mucosa, we studied IFN-λ induction and antiviral activity in NiV-infected primary differentiated human bronchial epithelial cells (HBEpCs) cultured under air-liquid interface conditions. Our studies revealed that IFN-λ was upregulated in airway epithelia upon NiV infection. We also show that IFN-λ pretreatment efficiently inhibited NiV replication. Interestingly, the antiviral activity of IFN-λ varied in HBEpCs from two different donors. Increased sensitivity to IFN-λ was associated with higher expression levels of IFN-λ receptors, enhanced phosphorylation of STAT1, as well as enhanced induction of interferon-stimulated gene expression. These findings suggest that individual variations in IFN-λ receptor expression affecting IFN responsiveness can play a functional role for NiV replication kinetics in human respiratory epithelial cells of different donors.

A single Markov-type kinetic model accounting for the macroscopic currents of all human voltage-gated sodium channel isoforms.

PubMed

Balbi, Pietro; Massobrio, Paolo; Hellgren Kotaleski, Jeanette

2017-09-01

Modelling ionic channels represents a fundamental step towards developing biologically detailed neuron models. Until recently, the voltage-gated ion channels have been mainly modelled according to the formalism introduced by the seminal works of Hodgkin and Huxley (HH). However, following the continuing achievements in the biophysical and molecular comprehension of these pore-forming transmembrane proteins, the HH formalism turned out to carry limitations and inconsistencies in reproducing the ion-channels electrophysiological behaviour. At the same time, Markov-type kinetic models have been increasingly proven to successfully replicate both the electrophysiological and biophysical features of different ion channels. However, in order to model even the finest non-conducting molecular conformational change, they are often equipped with a considerable number of states and related transitions, which make them computationally heavy and less suitable for implementation in conductance-based neurons and large networks of those. In this purely modelling study we develop a Markov-type kinetic model for all human voltage-gated sodium channels (VGSCs). The model framework is detailed, unifying (i.e., it accounts for all ion-channel isoforms) and computationally efficient (i.e. with a minimal set of states and transitions). The electrophysiological data to be modelled are gathered from previously published studies on whole-cell patch-clamp experiments in mammalian cell lines heterologously expressing the human VGSC subtypes (from NaV1.1 to NaV1.9). By adopting a minimum sequence of states, and using the same state diagram for all the distinct isoforms, the model ensures the lightest computational load when used in neuron models and neural networks of increasing complexity. The transitions between the states are described by original ordinary differential equations, which represent the rate of the state transitions as a function of voltage (i.e., membrane potential). The kinetic model, developed in the NEURON simulation environment, appears to be the simplest and most parsimonious way for a detailed phenomenological description of the human VGSCs electrophysiological behaviour.

Enterovirus 71 infection of human airway organoids reveals VP1-145 as a viral infectivity determinant.

PubMed

van der Sanden, Sabine M G; Sachs, Norman; Koekkoek, Sylvie M; Koen, Gerrit; Pajkrt, Dasja; Clevers, Hans; Wolthers, Katja C

2018-05-09

Human enteroviruses frequently cause severe diseases in children. Human enteroviruses are transmitted via the fecal-oral route and respiratory droplets, and primary replication occurs in the gastro-intestinal and respiratory tracts; however, how enteroviruses infect these sites is largely unknown. Human intestinal organoids have recently proven to be valuable tools for studying enterovirus-host interactions in the intestinal tract. In this study, we demonstrated the susceptibility of a newly developed human airway organoid model for enterovirus 71 (EV71) infection. We showed for the first time in a human physiological model that EV71 replication kinetics are strain-dependent. A glutamine at position 145 of the VP1 capsid protein was identified as a key determinant of infectivity, and residues VP1-98K and VP1-104D were identified as potential infectivity markers. The results from this study provide new insights into EV71 infectivity in the human airway epithelia and demonstrate the value of organoid technology for virus research.

Causation and the origin of life. Metabolism or replication first?

PubMed

Pross, Addy

2004-06-01

The conceptual gulf that separates the 'metabolism first' and 'replication first' mechanisms for the emergence of life continues to cloud the origin of life debate. In the present paper we analyze this aspect of the origin of life problem and offer arguments in favor of the 'replication first' school. Utilizing Wicken's two-tier approach to causation we argue that a causal connection between replication and metabolism can only be demonstrated if replication would have preceded metabolism. In conjunction with existing empirical evidence and theoretical reasoning, our analysis concludes that there is no substantive evidence for a 'metabolism first' mechanism for life's emergence, while a coherent case can be made for the 'replication first' group of mechanisms. The analysis reaffirms our conviction that life is an extreme expression of kinetic control, and that the emergence of metabolic pathways can be understood by considering life as a manifestation of 'replicative chemistry'.

Towards Self-Replicating Chemical Systems Based on Cytidylic and Guanylic Acids

NASA Technical Reports Server (NTRS)

Kanavarioti, Anastassia; Bernasconi, Claude F.

1997-01-01

This project is aimed towards a better understanding of template-directed reactions and, based on this, towards the development of efficient non-enzymatic RNA replicating systems. These systems could serve as models for the prebiotic synthesis of an RNA world. The major objectives of this project were: (a) To elucidate the mechanistic aspects of template-directed (TD) chemistry, (b) to identify the conditions, environmental and other, that favor "organized chemistry" and stereo selective polymerization of nucleotides and (c) to search and, hopefully, find catalysts that will improve the efficiency of these reactions. Enhanced efficiency is expected to facilitate the road towards a self-replicating chemical system based on all four nucleic acid bases. During the first nine months of the granting period from January 1997 to October 1997, we have made substantial progress towards the first two objectives. During this period our activities were directed towards (1) synthesizing activated nucleotides to be used as substrates, (2) using these substrates in order to determine the effect of the leaving group (imidazole (Im), 2-methylimidazole (2-MeIm), and 2,4-dimethylimidazole (2,4-diMeIm)) in the product distribution, (3) developing techniques for analysis of mixtures by LC/MS, (4) creating a protocol in order to obtain kinetic parameters of the dimerization reaction and (5) analyzing kinetic data obtained with the poly(C)/2-MeImpG system. With the exception of item (5), the experimental work for the projects (1) - (4) is still in progress. A list of publications and manuscripts resulted from this research is enclosed.

Computational study of RNA folding kinetics and thermodynamics

NASA Astrophysics Data System (ADS)

Morgan, Steven Robert

RNA in its many forms is involved in the processes of protein manufacture, gene splicing, catalysis and gene regulation. It is also the store of genetic information in some viruses. The function of the RNA is determined by its structure, and it is the purpose of this thesis to investigate kinetic and thermodynamic properties of RNA secondary structures in order to obtain a better understanding of their formation and function. Our main tenet is that kinetic formation of RNA structure is necessary to explain features found in natural RNA structures, as well as aspects of the biological function of RNA. Firstly we show that examination of the energies of fragments of RNA secondary structure provides evidence for kinetic formation of structure. Local regions of RNA of length less than about 100 nucleotides adopt a conformation with energy near or equal to the minimum possible for those regions, whilst the energies of larger domains are much further from the their respective minima. This is consistent with the patterns that would be expected if RNA structure is folded Idneticatic during transcription. A Monte-Carlo algorithm is then used to model the kinetic folding of RNA during transcriptional growth. The algorithm is capable of finding the correct structure of a natural RNA for which the minimum free energy approach is unsuccessful. In the viral phage MS2 Idneticatic formed RNA structure plays an important role in the regulation of gene expression. The folding algorithm can accurately model this by IdneticaUy controlling access to the gene initiation region. The algorithm is also successfully used to model the control of replication in the ColEl plasmid. Taking a different approach, we then use a simplified model of RNA secondary structure to investigate the size of energy barriers between degenerate minimum energy structures. This model has much in common with physical systems such as spin glasses, and in fact shows similar behaviour to these systems in that energy barriers between structures grow quickly with the length of the RNA sequence. These barriers will serve to trap RNA in non-optimal structures. Together these studies demonstrate the necessity of studying RNA secondary structure from a kinetic point of view, and provide clear directions in which further work may be taken. Kinetic models of RNA secondary structure should continue to prove useful in modelling the structure and function of RNA.

Modelling hepatitis C therapy—predicting effects of treatment

PubMed Central

Perelson, Alan S.; Guedj, Jeremie

2015-01-01

Mathematically modelling HCV RNA changes measured in patients who receive antiviral therapy has yielded many insights into the pathogenesis and effects of treatment on the virus. By determining how rapidly HCV is cleared when viral replication is interrupted by a therapy, one can deduce how rapidly the virus is produced in patients before treatment. This knowledge, coupled with estimates of the HCV mutation rate, enables one to estimate the frequency with which drug resistant variants arise. Modelling HCV also permits the deduction of antiviral agent effectiveness at blocking HCV replication from the magnitude of the initial viral decline. One can also estimate the lifespan of an HCV infected cell from the slope of the subsequent viral decline and, determine the duration of therapy needed to cure infection. Our understanding of HCV RNA decline under IFN-based therapies needs to be revised in order to understand the HCV RNA decline kinetics seen when using direct-acting antiviral agents (DAAs). In this Review, we also discuss the unresolved issues involving understanding therapies with combinations of DAAs, such as whether a sustained virological response necessarily involves elimination of all infected cells PMID:26122475

Computational Replication of the Abnormal Secondary Kinetic Isotope Effects in a Hydride Transfer Reaction in Solution with a Motion Assisted H-Tunneling Model

PubMed Central

2015-01-01

We recently reported abnormal secondary deuterium kinetic isotope effects (2° KIEs) for hydride transfer reactions from alcohols to carbocations in acetonitrile (Chem. Comm. 2012, 48, 11337). Experimental 2° KIE values were found to be inflated on the 9-C position in the xanthylium cation but deflated on the β-C position in 2-propanol with respect to the values predicted by the semi-classical transition-state theory. No primary (1°) isotope effect on 2° KIEs was observed. Herein, the KIEs were replicated by the Marcus-like H-tunneling model that requires a longer donor–acceptor distance (DAD) in a lighter isotope transfer process. The 2° KIEs for a range of potential tunneling-ready-states (TRSs) of different DADs were calculated and fitted to the experiments to find the TRS structure. The observed no effect of 1° isotope on 2° KIEs is explained in terms of the less sterically hindered TRS structure so that the change in DAD due to the change in 1° isotope does not significantly affect the reorganization of the 2° isotope and hence the 2° KIE. The effect of 1° isotope on 2° KIEs may be expected to be more pronounced and thus observable in reactions occurring in restrictive environments such as the crowded and relatively rigid active site of enzymes. PMID:24498946

Effects of RNA interference therapy against herpes simplex virus type 1 encephalitis.

PubMed

da Silva, Alexandre S; Raposo, Jéssica V; Pereira, Tiago C; Pinto, Marcelo A; de Paula, Vanessa S

2016-01-01

Herpetic encephalitis (HSE) is caused mainly by herpes simplex virus type 1 (HSV-1) with an annual incidence of 1-4 cases/million inhabitants. Currently, HSE treatment faces difficulties such as the use of antivirals with elevated toxicity, metabolic side effects and HSV-1 resistance. An alternative to antivirals is the use of small interfering RNA (siRNA) as a viral replication inhibitor. In this work, siRNA targeting the UL-39 region was evaluated for HSE treatment in vivo. BALB/c mice were inoculated with HSV-1 and treated with siRNA. The treatment was evaluated through kinetics of HSV-1 replication inhibition, number of siRNA doses administered and treatment with siRNA plus acyclovir. All groups were evaluated for signs of HSE, mortality and HSV-1 replication inhibition. The treated group of the kinetic experiment demonstrated a reduction of HSE signs and an HSV-1 replication inhibition of 43.6-99.9% in the brain and 53-98% in trigeminal ganglia (TG). Animals treated with one or two doses of siRNA had a prolonged survival time, reduced clinical signs of HSE and HSV-1 replication inhibition of 67.7% in brains and 85.7% in TG of animals treated with two doses of siRNA. Also, animals treated with siRNA plus acyclovir demonstrated reduced signs of HSE and mortality, as well as HSV-1 replication inhibition in the brain (83.2%) and TG (74.5%). These findings demonstrated that siRNA was capable of reducing HSE clinical signs, prolonging survival time and inhibiting HSV-1 replication in mice. Thus, siRNA can be a potential alternative to the standard HSE treatment especially to reduce clinical signs and extend survival time in vivo.

A quasispecies approach to the evolution of sexual replication in unicellular organisms.

PubMed

Tannenbaum, Emmanuel; Fontanari, José F

2008-03-01

This study develops a simplified model describing the evolutionary dynamics of a population composed of obligate sexually and asexually reproducing, unicellular organisms. The model assumes that the organisms have diploid genomes consisting of two chromosomes, and that the sexual organisms replicate by first dividing into haploid intermediates, which then combine with other haploids, followed by the normal mitotic division of the resulting diploid into two new daughter cells. We assume that the fitness landscape of the diploids is analogous to the single-fitness-peak approach often used in single-chromosome studies. That is, we assume a master chromosome that becomes defective with just one point mutation. The diploid fitness then depends on whether the genome has zero, one, or two copies of the master chromosome. We also assume that only pairs of haploids with a master chromosome are capable of combining so as to produce sexual diploid cells, and that this process is described by second-order kinetics. We find that, in a range of intermediate values of the replication fidelity, sexually reproducing cells can outcompete asexual ones, provided the initial abundance of sexual cells is above some threshold value. The range of values where sexual reproduction outcompetes asexual reproduction increases with decreasing replication rate and increasing population density. We critically evaluate a common approach, based on a group selection perspective, used to study the competition between populations and show its flaws in addressing the evolution of sex problem.

Human CST has independent functions during telomere duplex replication and C-strand fill-in

PubMed Central

Wang, Feng; Stewart, Jason A.; Kasbek, Christopher; Zhao, Yong; Wright, Woodring E.; Price, Carolyn M.

2012-01-01

Summary Human CST (CTC1-STN1-TEN1) is an RPA-like complex that is needed for efficient replication through the telomere duplex and genome-wide replication restart after fork stalling. Here we show that STN1/CST has a second function in telomere replication during G-overhang maturation. Analysis of overhang structure after STN1 depletion revealed normal kinetics for telomerase-mediated extension in S-phase but a delay in subsequent overhang shortening. This delay resulted from a defect in C-strand fill-in. Short telomeres exhibited the fill-in defect but normal telomere duplex replication, indicating that STN1/CST functions independently in these processes. Our work also indicates that the requirement for STN1/CST in telomere duplex replication correlates with increasing telomere length and replication stress. Our results provide the first direct evidence that STN1/CST participates in C-strand fill-in. They also demonstrate that STN1/CST participates in two mechanistically separate steps during telomere replication and identify CST as a novel replication factor that solves diverse replication-associated problems. PMID:23142664

Potential-based dynamical reweighting for Markov state models of protein dynamics.

PubMed

Weber, Jeffrey K; Pande, Vijay S

2015-06-09

As simulators attempt to replicate the dynamics of large cellular components in silico, problems related to sampling slow, glassy degrees of freedom in molecular systems will be amplified manyfold. It is tempting to augment simulation techniques with external biases to overcome such barriers with ease; biased simulations, however, offer little utility unless equilibrium properties of interest (both kinetic and thermodynamic) can be recovered from the data generated. In this Article, we present a general scheme that harnesses the power of Markov state models (MSMs) to extract equilibrium kinetic properties from molecular dynamics trajectories collected on biased potential energy surfaces. We first validate our reweighting protocol on a simple two-well potential, and we proceed to test our method on potential-biased simulations of the Trp-cage miniprotein. In both cases, we find that equilibrium populations, time scales, and dynamical processes are reliably reproduced as compared to gold standard, unbiased data sets. We go on to discuss the limitations of our dynamical reweighting approach, and we suggest auspicious target systems for further application.

Mesoscale Thermodynamically motivated Statistical Mechanics based Kinetic Model for Sintering monoliths

NASA Astrophysics Data System (ADS)

Mohan, Nisha

Modeling the evolution of microstructure during sintering is a persistent challenge in ceramics science, although needed as the microstructure impacts properties of an engineered material. Bridging the gap between microscopic and continuum models, kinetic Monte Carlo (kMC) methods provide a stochastic approach towards sintering and microstructure evolution. These kMC models work at the mesoscale, with length and time-scales between those of atomistic and continuum approaches. We develop a sintering/compacting model for the two-phase sintering of boron nitride ceramics and allotropes alike. Our formulation includes mechanisms for phase transformation between h-BN and c-BN and takes into account thermodynamics of pressure and temperature on interaction energies and mechanism rates. In addition to replicating the micro-structure evolution observed in experiments, it also captures the phase diagram of Boron Nitride materials. Results have been analyzed in terms of phase diagrams and crystal growth. It also serves with insights to guide the choice of additives and conditions for the sintering process.While detailed time and spatial resolutions are lost in any MC, the progression of stochastic events still captures plausible local energy minima and long-time temporal developments. DARPA.

Kinetic Analysis of a Molecular Model of the Budding Yeast Cell Cycle

PubMed Central

Chen, Katherine C.; Csikasz-Nagy, Attila; Gyorffy, Bela; Val, John; Novak, Bela; Tyson, John J.

2000-01-01

The molecular machinery of cell cycle control is known in more detail for budding yeast, Saccharomyces cerevisiae, than for any other eukaryotic organism. In recent years, many elegant experiments on budding yeast have dissected the roles of cyclin molecules (Cln1–3 and Clb1–6) in coordinating the events of DNA synthesis, bud emergence, spindle formation, nuclear division, and cell separation. These experimental clues suggest a mechanism for the principal molecular interactions controlling cyclin synthesis and degradation. Using standard techniques of biochemical kinetics, we convert the mechanism into a set of differential equations, which describe the time courses of three major classes of cyclin-dependent kinase activities. Model in hand, we examine the molecular events controlling “Start” (the commitment step to a new round of chromosome replication, bud formation, and mitosis) and “Finish” (the transition from metaphase to anaphase, when sister chromatids are pulled apart and the bud separates from the mother cell) in wild-type cells and 50 mutants. The model accounts for many details of the physiology, biochemistry, and genetics of cell cycle control in budding yeast. PMID:10637314

How and why multiple MCMs are loaded at origins of DNA replication.

PubMed

Das, Shankar P; Rhind, Nicholas

2016-07-01

Recent work suggests that DNA replication origins are regulated by the number of multiple mini-chromosome maintenance (MCM) complexes loaded. Origins are defined by the loading of MCM - the replicative helicase which initiates DNA replication and replication kinetics determined by origin's location and firing times. However, activation of MCM is heterogeneous; different origins firing at different times in different cells. Also, more MCMs are loaded in G1 than are used in S phase. These aspects of MCM biology are explained by the observation that multiple MCMs are loaded at origins. Having more MCMs at early origins makes them more likely to fire, effecting differences in origin efficiency that define replication timing. Nonetheless, multiple MCM loading raises new questions, such as how they are loaded, where these MCMs reside at origins, and how their presence affects replication timing. In this review, we address these questions and discuss future avenues of research. © 2016 WILEY Periodicals, Inc.

Molecular Characterization of Feline Infectious Peritonitis Virus Strain DF-2 and Studies of the Role of ORF3abc in Viral Cell Tropism

PubMed Central

Farsang, Attila; Zádori, Zoltán; Hornyák, Ákos; Dencső, László; Almazán, Fernando; Enjuanes, Luis; Belák, Sándor

2012-01-01

The full-length genome of the highly lethal feline infectious peritonitis virus (FIPV) strain DF-2 was sequenced and cloned into a bacterial artificial chromosome (BAC) to study the role of ORF3abc in the FIPV-feline enteric coronavirus (FECV) transition. The reverse genetic system allowed the replacement of the truncated ORF3abc of the original FIPV DF-2 genome with the intact ORF3abc of the canine coronavirus (CCoV) reference strain Elmo/02. The in vitro replication kinetics of these two viruses was studied in CrFK and FCWF-4 cell lines, as well as in feline peripheral blood monocytes. Both viruses showed similar replication kinetics in established cell lines. However, the strain with a full-length ORF3 showed markedly lower replication of more than 2 log10 titers in feline peripheral blood monocytes. Our results suggest that the truncated ORF3abc plays an important role in the efficient macrophage/monocyte tropism of type II FIPV. PMID:22438554

Monitoring Replication Protein A (RPA) dynamics in homologous recombination through site-specific incorporation of non-canonical amino acids

PubMed Central

Pokhrel, Nilisha; Origanti, Sofia; Davenport, Eric Parker; Gandhi, Disha; Kaniecki, Kyle; Mehl, Ryan A.; Greene, Eric C.; Dockendorff, Chris

2017-01-01

Abstract An essential coordinator of all DNA metabolic processes is Replication Protein A (RPA). RPA orchestrates these processes by binding to single-stranded DNA (ssDNA) and interacting with several other DNA binding proteins. Determining the real-time kinetics of single players such as RPA in the presence of multiple DNA processors to better understand the associated mechanistic events is technically challenging. To overcome this hurdle, we utilized non-canonical amino acids and bio-orthogonal chemistry to site-specifically incorporate a chemical fluorophore onto a single subunit of heterotrimeric RPA. Upon binding to ssDNA, this fluorescent RPA (RPAf) generates a quantifiable change in fluorescence, thus serving as a reporter of its dynamics on DNA in the presence of multiple other DNA binding proteins. Using RPAf, we describe the kinetics of facilitated self-exchange and exchange by Rad51 and mediator proteins during various stages in homologous recombination. RPAf is widely applicable to investigate its mechanism of action in processes such as DNA replication, repair and telomere maintenance. PMID:28934470

Mammalian DNA enriched for replication origins is enriched for snap-back sequences.

PubMed

Zannis-Hadjopoulos, M; Kaufmann, G; Martin, R G

1984-11-15

Using the instability of replication loops as a method for the isolation of double-stranded nascent DNA, extruded DNA enriched for replication origins was obtained and denatured. Snap-back DNA, single-stranded DNA with inverted repeats (palindromic sequences), reassociates rapidly into stem-loop structures with zero-order kinetics when conditions are changed from denaturing to renaturing, and can be assayed by chromatography on hydroxyapatite. Origin-enriched nascent DNA strands from mouse, rat and monkey cells growing either synchronously or asynchronously were purified and assayed for the presence of snap-back sequences. The results show that origin-enriched DNA is also enriched for snap-back sequences, implying that some origins for mammalian DNA replication contain or lie near palindromic sequences.

Single-cell analysis of transcription kinetics across the cell cycle

PubMed Central

Skinner, Samuel O; Xu, Heng; Nagarkar-Jaiswal, Sonal; Freire, Pablo R; Zwaka, Thomas P; Golding, Ido

2016-01-01

Transcription is a highly stochastic process. To infer transcription kinetics for a gene-of-interest, researchers commonly compare the distribution of mRNA copy-number to the prediction of a theoretical model. However, the reliability of this procedure is limited because the measured mRNA numbers represent integration over the mRNA lifetime, contribution from multiple gene copies, and mixing of cells from different cell-cycle phases. We address these limitations by simultaneously quantifying nascent and mature mRNA in individual cells, and incorporating cell-cycle effects in the analysis of mRNA statistics. We demonstrate our approach on Oct4 and Nanog in mouse embryonic stem cells. Both genes follow similar two-state kinetics. However, Nanog exhibits slower ON/OFF switching, resulting in increased cell-to-cell variability in mRNA levels. Early in the cell cycle, the two copies of each gene exhibit independent activity. After gene replication, the probability of each gene copy to be active diminishes, resulting in dosage compensation. DOI: http://dx.doi.org/10.7554/eLife.12175.001 PMID:26824388

Prereplicative events involving simian virus 40 DNA in permissive cells

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

Rinaldy, A.; Feunteun, J.; Rosenberg, B.H.

1982-01-01

Simian virus 40 DNA molecules were found to be unable to replicate for 9 h after infection, even in cells that were already replicating the DNA of preinfecting simian virus 40; after 9 h, the ability of the DNA to replicate began to rise sharply. The kinetics of activation indicated that each DNA molecule undergoes a series of slow consecutive reactions, not involving T-antigen, before it can replicate. These pre-replicative molecular transformations probably involve configurational changes; their nature and their relation to the initiation of viral DNA synthesis is discussed. Observation of the replicative behavior of one viral DNA inmore » the presence of another was made possible by the use of two different mutants with distinguishable DNAs: a viable deletion mutant containing DNA insensitive to TaqI restriction enzyme was used to provide viral functions required for replication, and is a tsA mutant with TaqI-sensitive DNA was introduced at various times as a probe to determine the ability of the DNA to replicate under different conditions.« less

Impairment of DNA Methylation Maintenance Is the Main Cause of Global Demethylation in Naive Embryonic Stem Cells.

PubMed

von Meyenn, Ferdinand; Iurlaro, Mario; Habibi, Ehsan; Liu, Ning Qing; Salehzadeh-Yazdi, Ali; Santos, Fátima; Petrini, Edoardo; Milagre, Inês; Yu, Miao; Xie, Zhenqing; Kroeze, Leonie I; Nesterova, Tatyana B; Jansen, Joop H; Xie, Hehuang; He, Chuan; Reik, Wolf; Stunnenberg, Hendrik G

2016-06-16

Global demethylation is part of a conserved program of epigenetic reprogramming to naive pluripotency. The transition from primed hypermethylated embryonic stem cells (ESCs) to naive hypomethylated ones (serum-to-2i) is a valuable model system for epigenetic reprogramming. We present a mathematical model, which accurately predicts global DNA demethylation kinetics. Experimentally, we show that the main drivers of global demethylation are neither active mechanisms (Aicda, Tdg, and Tet1-3) nor the reduction of de novo methylation. UHRF1 protein, the essential targeting factor for DNMT1, is reduced upon transition to 2i, and so is recruitment of the maintenance methylation machinery to replication foci. Concurrently, there is global loss of H3K9me2, which is needed for chromatin binding of UHRF1. These mechanisms synergistically enforce global DNA hypomethylation in a replication-coupled fashion. Our observations establish the molecular mechanism for global demethylation in naive ESCs, which has key parallels with those operating in primordial germ cells and early embryos. Crown Copyright © 2016. Published by Elsevier Inc. All rights reserved.

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

Perelson, Alan S.; Guedj, Jeremie

Mathematically modelling changes in HCV RNA levels measured in patients who receive antiviral therapy has yielded many insights into the pathogenesis and effects of treatment on the virus. By determining how rapidly HCV is cleared when viral replication is interrupted by a therapy, one can deduce how rapidly the virus is produced in patients before treatment. This knowledge, coupled with estimates of the HCV mutation rate, enables one to estimate the frequency with which drug resistant variants arise. Modelling HCV also permits the deduction of the effectiveness of an antiviral agent at blocking HCV replication from the magnitude of themore » initial viral decline. One can also estimate the lifespan of an HCV-infected cell from the slope of the subsequent viral decline and determine the duration of therapy needed to cure infection. The original understanding of HCV RNA decline under interferon-based therapies obtained by modelling needed to be revised in order to interpret the HCV RNA decline kinetics seen when using direct-acting antiviral agents (DAAs). In addition, there also exist unresolved issues involving understanding therapies with combinations of DAAs, such as the presence of detectable HCV RNA at the end of therapy in patients who nonetheless have a sustained virologic response.« less

Behavioral, metabolic, and immune consequences of chronic alcohol or cannabinoids on HIV/AIDs: Studies in the Non-Human Primate SIV model

PubMed Central

Molina, Patricia E.; Amedee, Angela M.; Winsauer, Peter; Nelson, Steve; Bagby, Gregory; Simon, Liz

2015-01-01

HIV-associated mortality has been significantly reduced with antiretroviral therapy (ART), and HIV infection has become a chronic disease that frequently coexists with many disorders, including substance abuse (Azar et al. 2010; Phillips et al. 2001). Alcohol and drugs of abuse may modify host-pathogen interactions at various levels including behavioral, metabolic, and immune consequences of HIV infection, as well as the ability of the virus to integrate into the genome and replicate in host cells. Identifying mechanisms responsible for these interactions is complicated by many factors, such as the tissue specific responses to viral infection, multiple cellular mechanisms involved in inflammatory responses, neuroendocrine and localized responses to infection, and kinetics of viral replication. An integrated physiological analysis of the biomedical consequences of chronic alcohol and drug use or abuse on disease progression is possible using rhesus macaques infected with simian immunodeficiency virus (SIV), a relevant model of HIV infection. This review will provide an overview of the data gathered using this model to show that chronic administration of two of the most commonly abused substances, alcohol and cannabinoids (Δ9-Tetrahydrocannabinol; THC), affect host-pathogen interactions. PMID:25795088

A kinetic approach to evaluate salinity effects on carbon mineralization in a plant residue-amended soil*

PubMed Central

Nourbakhsh, Farshid; Sheikh-Hosseini, Ahmad R.

2006-01-01

The interaction of salinity stress and plant residue quality on C mineralization kinetics in soil is not well understood. A laboratory experiment was conducted to study the effects of salinity stress on C mineralization kinetics in a soil amended with alfalfa, wheat and corn residues. A factorial combination of two salinity levels (0.97 and 18.2 dS/m) and four levels of plant residues (control, alfalfa, wheat and corn) with three replications was performed. A first order kinetic model was used to describe the C mineralization and to calculate the potentially mineralizable C. The CO2-C evolved under non-saline condition, ranged from 814.6 to 4842.4 mg CO2-C/kg in control and alfalfa residue-amended soils, respectively. Salinization reduced the rates of CO2 evolution by 18.7%, 6.2% and 5.2% in alfalfa, wheat and corn residue-amended soils, respectively. Potentially mineralizable C (C 0) was reduced significantly in salinized alfalfa residue-treated soils whereas, no significant difference was observed for control treatments as well as wheat and corn residue-treated soils. We concluded that the response pattern of C mineralization to salinity stress depended on the plant residue quality and duration of incubation. PMID:16972320

Pan-Genotype Hepatitis E Virus Replication in Stem Cell-Derived Hepatocellular Systems.

PubMed

Wu, Xianfang; Dao Thi, Viet Loan; Liu, Peng; Takacs, Constantin N; Xiang, Kuanhui; Andrus, Linda; Gouttenoire, Jérôme; Moradpour, Darius; Rice, Charles M

2018-02-01

The 4 genotypes of hepatitis E virus (HEV) that infect humans (genotypes 1-4) vary in geographical distribution, transmission, and pathogenesis. Little is known about the properties of HEV or its hosts that contribute to these variations. Primary isolates grow poorly in cell culture; most studies have relied on variants adapted to cancer cell lines, which likely alter virus biology. We investigated the infection and replication of primary isolates of HEV in hepatocyte-like cells (HLCs) derived from human embryonic and induced pluripotent stem cells. Using a cell culture-adapted genotype 3 strain and primary isolates of genotypes 1 to 4, we compared viral replication kinetics, sensitivity to drugs, and ability of HEV to activate the innate immune response. We studied HLCs using quantitative reverse-transcriptase polymerase chain reaction and immunofluorescence assay and enzyme-linked immunosorbent assays. We used an embryonic stem cell line that can be induced to express the CRISPR-Cas9 machinery to disrupt the peptidylprolyl isomerase A gene, encoding cyclophilin A (CYPA), a protein reported to inhibit replication of cell culture-adapted HEV. We further modified this line to rescue expression of CYPA before terminal differentiation to HLCs and performed HEV infection studies. HLCs were permissive for infection by nonadapted, primary isolates of HEV genotypes 1 to 4. HEV infection of HLCs induced a replication-dependent type III interferon response. Replication of primary HEV isolates, unlike the cell culture-adapted strain, was not affected by disruption of the peptidylprolyl isomerase A gene or exposure to the CYPA inhibitor cyclosporine A. Cell culture adaptations alter the replicative capacities of HEV. HLCs offer an improved, physiologically relevant, and genetically tractable system for studying the replication of primary HEV isolates. HLCs could provide a model to aid development of HEV drugs and a system to guide personalized regimens, especially for patients with chronic hepatitis E who have developed resistance to ribavirin. Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.

Dynamic remodeling of lipids coincides with dengue virus replication in the midgut of Aedes aegypti mosquitoes.

PubMed

Chotiwan, Nunya; Andre, Barbara G; Sanchez-Vargas, Irma; Islam, M Nurul; Grabowski, Jeffrey M; Hopf-Jannasch, Amber; Gough, Erik; Nakayasu, Ernesto; Blair, Carol D; Belisle, John T; Hill, Catherine A; Kuhn, Richard J; Perera, Rushika

2018-02-01

We describe the first comprehensive analysis of the midgut metabolome of Aedes aegypti, the primary mosquito vector for arboviruses such as dengue, Zika, chikungunya and yellow fever viruses. Transmission of these viruses depends on their ability to infect, replicate and disseminate from several tissues in the mosquito vector. The metabolic environments within these tissues play crucial roles in these processes. Since these viruses are enveloped, viral replication, assembly and release occur on cellular membranes primed through the manipulation of host metabolism. Interference with this virus infection-induced metabolic environment is detrimental to viral replication in human and mosquito cell culture models. Here we present the first insight into the metabolic environment induced during arbovirus replication in Aedes aegypti. Using high-resolution mass spectrometry, we have analyzed the temporal metabolic perturbations that occur following dengue virus infection of the midgut tissue. This is the primary site of infection and replication, preceding systemic viral dissemination and transmission. We identified metabolites that exhibited a dynamic-profile across early-, mid- and late-infection time points. We observed a marked increase in the lipid content. An increase in glycerophospholipids, sphingolipids and fatty acyls was coincident with the kinetics of viral replication. Elevation of glycerolipid levels suggested a diversion of resources during infection from energy storage to synthetic pathways. Elevated levels of acyl-carnitines were observed, signaling disruptions in mitochondrial function and possible diversion of energy production. A central hub in the sphingolipid pathway that influenced dihydroceramide to ceramide ratios was identified as critical for the virus life cycle. This study also resulted in the first reconstruction of the sphingolipid pathway in Aedes aegypti. Given conservation in the replication mechanisms of several flaviviruses transmitted by this vector, our results highlight biochemical choke points that could be targeted to disrupt transmission of multiple pathogens by these mosquitoes.

Dynamic remodeling of lipids coincides with dengue virus replication in the midgut of Aedes aegypti mosquitoes

PubMed Central

Chotiwan, Nunya; Andre, Barbara G.; Sanchez-Vargas, Irma; Islam, M. Nurul; Grabowski, Jeffrey M.; Hopf-Jannasch, Amber; Gough, Erik; Nakayasu, Ernesto; Blair, Carol D.; Hill, Catherine A.; Kuhn, Richard J.

2018-01-01

We describe the first comprehensive analysis of the midgut metabolome of Aedes aegypti, the primary mosquito vector for arboviruses such as dengue, Zika, chikungunya and yellow fever viruses. Transmission of these viruses depends on their ability to infect, replicate and disseminate from several tissues in the mosquito vector. The metabolic environments within these tissues play crucial roles in these processes. Since these viruses are enveloped, viral replication, assembly and release occur on cellular membranes primed through the manipulation of host metabolism. Interference with this virus infection-induced metabolic environment is detrimental to viral replication in human and mosquito cell culture models. Here we present the first insight into the metabolic environment induced during arbovirus replication in Aedes aegypti. Using high-resolution mass spectrometry, we have analyzed the temporal metabolic perturbations that occur following dengue virus infection of the midgut tissue. This is the primary site of infection and replication, preceding systemic viral dissemination and transmission. We identified metabolites that exhibited a dynamic-profile across early-, mid- and late-infection time points. We observed a marked increase in the lipid content. An increase in glycerophospholipids, sphingolipids and fatty acyls was coincident with the kinetics of viral replication. Elevation of glycerolipid levels suggested a diversion of resources during infection from energy storage to synthetic pathways. Elevated levels of acyl-carnitines were observed, signaling disruptions in mitochondrial function and possible diversion of energy production. A central hub in the sphingolipid pathway that influenced dihydroceramide to ceramide ratios was identified as critical for the virus life cycle. This study also resulted in the first reconstruction of the sphingolipid pathway in Aedes aegypti. Given conservation in the replication mechanisms of several flaviviruses transmitted by this vector, our results highlight biochemical choke points that could be targeted to disrupt transmission of multiple pathogens by these mosquitoes. PMID:29447265

The eukaryotic bell-shaped temporal rate of DNA replication origin firing emanates from a balance between origin activation and passivation.

PubMed

Arbona, Jean-Michel; Goldar, Arach; Hyrien, Olivier; Arneodo, Alain; Audit, Benjamin

2018-06-01

The time-dependent rate I(t) of origin firing per length of unreplicated DNA presents a universal bell shape in eukaryotes that has been interpreted as the result of a complex time-evolving interaction between origins and limiting firing factors. Here we show that a normal diffusion of replication fork components towards localized potential replication origins (p-oris) can more simply account for the I(t) universal bell shape, as a consequence of a competition between the origin firing time and the time needed to replicate DNA separating two neighboring p-oris . We predict the I(t) maximal value to be the product of the replication fork speed with the squared p-ori density. We show that this relation is robustly observed in simulations and in experimental data for several eukaryotes. Our work underlines that fork-component recycling and potential origins localization are sufficient spatial ingredients to explain the universality of DNA replication kinetics. © 2018, Arbona et al.

RFWD3-Dependent Ubiquitination of RPA Regulates Repair at Stalled Replication Forks.

PubMed

Elia, Andrew E H; Wang, David C; Willis, Nicholas A; Boardman, Alexander P; Hajdu, Ildiko; Adeyemi, Richard O; Lowry, Elizabeth; Gygi, Steven P; Scully, Ralph; Elledge, Stephen J

2015-10-15

We have used quantitative proteomics to profile ubiquitination in the DNA damage response (DDR). We demonstrate that RPA, which functions as a protein scaffold in the replication stress response, is multiply ubiquitinated upon replication fork stalling. Ubiquitination of RPA occurs on chromatin, involves sites outside its DNA binding channel, does not cause proteasomal degradation, and increases under conditions of fork collapse, suggesting a role in repair at stalled forks. We demonstrate that the E3 ligase RFWD3 mediates RPA ubiquitination. RFWD3 is necessary for replication fork restart, normal repair kinetics during replication stress, and homologous recombination (HR) at stalled replication forks. Mutational analysis suggests that multisite ubiquitination of the entire RPA complex is responsible for repair at stalled forks. Multisite protein group sumoylation is known to promote HR in yeast. Our findings reveal a similar requirement for multisite protein group ubiquitination during HR at stalled forks in mammalian cells. Copyright © 2015 Elsevier Inc. All rights reserved.

Crimean-Congo haemorrhagic fever virus replication in adult Hyalomma truncatum and Amblyomma variegatum ticks.

PubMed

Gonzalez, J P; Cornet, J P; Wilson, M L; Camicas, J L

1991-01-01

The kinetics of the replication of the Crimean-Congo haemorrhagic fever virus (CCHFV) was studied in intra-anally inoculated adult Hyalomma truncatum and Amblyomma variegatum ticks. The virus was re-isolated by suckling mouse inoculation and revealed by antigen capture with ground ticks and indirect immunofluorescence of haemolymph. The virus was detected in ticks in the first hours post-inoculation (p.i.) and its replication was observed from 36 h p.i. onwards. Virus titre reached a maximum within 3-5 days then decreased slowly to a level of at 2 log LD50/ml for several months until the end of observations. Several specific, non-identified factors seem to favour CCHFV replication in H. truncatum. Long-term virus persistence seems to occur in CCHFV-infected adult ticks.

Modelling hepatitis C therapy—predicting effects of treatment

DOE PAGES

Perelson, Alan S.; Guedj, Jeremie

2015-06-30

Mathematically modelling changes in HCV RNA levels measured in patients who receive antiviral therapy has yielded many insights into the pathogenesis and effects of treatment on the virus. By determining how rapidly HCV is cleared when viral replication is interrupted by a therapy, one can deduce how rapidly the virus is produced in patients before treatment. This knowledge, coupled with estimates of the HCV mutation rate, enables one to estimate the frequency with which drug resistant variants arise. Modelling HCV also permits the deduction of the effectiveness of an antiviral agent at blocking HCV replication from the magnitude of themore » initial viral decline. One can also estimate the lifespan of an HCV-infected cell from the slope of the subsequent viral decline and determine the duration of therapy needed to cure infection. The original understanding of HCV RNA decline under interferon-based therapies obtained by modelling needed to be revised in order to interpret the HCV RNA decline kinetics seen when using direct-acting antiviral agents (DAAs). In addition, there also exist unresolved issues involving understanding therapies with combinations of DAAs, such as the presence of detectable HCV RNA at the end of therapy in patients who nonetheless have a sustained virologic response.« less

CYP2E1 hydroxylation of aniline involves negative cooperativity.

PubMed

Hartman, Jessica H; Knott, Katie; Miller, Grover P

2014-02-01

CYP2E1 plays a role in the metabolic activation and elimination of aniline, yet there are conflicting reports on its mechanism of action, and hence relevance, in aniline metabolism. Based on our work with similar compounds, we hypothesized that aniline binds two CYP2E1 sites during metabolism resulting in cooperative reaction kinetics and tested this hypothesis through rigorous in vitro studies. The kinetic profile for recombinant CYP2E1 demonstrated significant negative cooperativity based on a fit of data to the Hill equation (n=0.56). Mechanistically, the data were best explained through a two-binding site cooperative model in which aniline binds with high affinity (K(s)=30 μM) followed by a second weaker binding event (K(ss)=1100 uM) resulting in a threefold increase in the oxidation rate. Binding sites for aniline were confirmed by inhibition studies with 4-methylpyrazole. Inhibitor phenotyping experiments with human liver microsomes validated the central role for CYP2E1 in aniline hydroxylation and indicated minor roles for CYP2A6 and CYP2C9. Importantly, inhibition of minor metabolic pathways resulted in a kinetic profile for microsomal CYP2E1 that replicated the preferred mechanism and parameters observed with the recombinant enzyme. Scaled modeling of in vitro CYP2E1 metabolism of aniline to in vivo clearance, especially at low aniline levels, led to significant deviations from the traditional model based on non-cooperative, Michaelis-Menten kinetics. These findings provide a critical mechanistic perspective on the potential importance of CYP2E1 in the metabolic activation and elimination of aniline as well as the first experimental evidence of a negatively cooperative metabolic reaction catalyzed by CYP2E1. Copyright © 2013 Elsevier Inc. All rights reserved.

The role of the C-domain of bacteriophage T4 gene 32 protein in ssDNA binding and dsDNA helix-destabilization: Kinetic, single-molecule, and cross-linking studies

PubMed Central

Pant, Kiran; Anderson, Brian; Perdana, Hendrik; Malinowski, Matthew A.; Win, Aye T.; Williams, Mark C.

2018-01-01

The model single-stranded DNA binding protein of bacteriophage T4, gene 32 protein (gp32) has well-established roles in DNA replication, recombination, and repair. gp32 is a single-chain polypeptide consisting of three domains. Based on thermodynamics and kinetics measurements, we have proposed that gp32 can undergo a conformational change where the acidic C-terminal domain binds internally to or near the single-stranded (ss) DNA binding surface in the core (central) domain, blocking ssDNA interaction. To test this model, we have employed a variety of experimental approaches and gp32 variants to characterize this conformational change. Utilizing stopped-flow methods, the association kinetics of wild type and truncated forms of gp32 with ssDNA were measured. When the C-domain is present, the log-log plot of k vs. [NaCl] shows a positive slope, whereas when it is absent (*I protein), there is little rate change with salt concentration, as expected for this model.A gp32 variant lacking residues 292–296 within the C-domain, ΔPR201, displays kinetic properties intermediate between gp32 and *I. The single molecule force-induced DNA helix-destabilizing activitiesas well as the single- and double-stranded DNA affinities of ΔPR201 and gp32 truncated at residue 295 also fall between full-length protein and *I. Finally, chemical cross-linking of recombinant C-domain and gp32 lacking both N- and C-terminal domains is inhibited by increasing concentrations of a short single-stranded oligonucleotide, and the salt dependence of cross-linking mirrors that expected for the model. Taken together, these results provide the first evidence in support of this model that have been obtained through structural probes. PMID:29634784

Substrate inhibition kinetic model for West Nile virus NS2B-NS3 protease.

PubMed

Tomlinson, Suzanne M; Watowich, Stanley J

2008-11-11

West Nile virus (WNV) has recently emerged in North America as a significant disease threat to humans and animals. Unfortunately, no approved antiviral drugs exist to combat WNV or other members of the genus Flavivirus in humans. The WNV NS2B-NS3 protease has been one of the primary targets for anti-WNV drug discovery and design since it is required for virus replication. As part of our efforts to develop effective WNV inhibitors, we reexamined the reaction kinetics of the NS2B-NS3 protease and the inhibition mechanisms of newly discovered inhibitors. The WNV protease showed substrate inhibition in assays utilizing fluorophore-linked peptide substrates GRR, GKR, and DFASGKR. Moreover, a substrate inhibition reaction step was required to accurately model kinetic data generated from protease assays with a peptide inhibitor. The substrate inhibition model suggested that peptide substrates could bind to two binding sites on the protease. Reaction product analogues also showed inhibition of the protease, demonstrating product inhibition in addition to and distinct from substrate inhibition. We propose that small peptide substrates and inhibitors may interact with protease residues that form either the P3-P1 binding surface (i.e., the S3-S1 sites) or the P1'-P3' interaction surface (i.e., the S1'-S3' sites). Optimization of substrate analogue inhibitors that target these two independent sites may lead to novel anti-WNV drugs.

Monitoring Replication Protein A (RPA) dynamics in homologous recombination through site-specific incorporation of non-canonical amino acids.

PubMed

Pokhrel, Nilisha; Origanti, Sofia; Davenport, Eric Parker; Gandhi, Disha; Kaniecki, Kyle; Mehl, Ryan A; Greene, Eric C; Dockendorff, Chris; Antony, Edwin

2017-09-19

An essential coordinator of all DNA metabolic processes is Replication Protein A (RPA). RPA orchestrates these processes by binding to single-stranded DNA (ssDNA) and interacting with several other DNA binding proteins. Determining the real-time kinetics of single players such as RPA in the presence of multiple DNA processors to better understand the associated mechanistic events is technically challenging. To overcome this hurdle, we utilized non-canonical amino acids and bio-orthogonal chemistry to site-specifically incorporate a chemical fluorophore onto a single subunit of heterotrimeric RPA. Upon binding to ssDNA, this fluorescent RPA (RPAf) generates a quantifiable change in fluorescence, thus serving as a reporter of its dynamics on DNA in the presence of multiple other DNA binding proteins. Using RPAf, we describe the kinetics of facilitated self-exchange and exchange by Rad51 and mediator proteins during various stages in homologous recombination. RPAf is widely applicable to investigate its mechanism of action in processes such as DNA replication, repair and telomere maintenance. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

The sequential tissue distribution of duck Tembusu virus in adult ducks.

PubMed

Wu, Li; Liu, Jinxiong; Chen, Pucheng; Jiang, Yongping; Ding, Leilei; Lin, Yuan; Li, Qimeng; He, Xijun; Chen, Qiusheng; Chen, Hualan

2014-01-01

In 2010, a novel Tembusu virus (TMUV) that caused a severe decrease in the egg production of ducks was isolated in southeast China. Given the novelty of this duck pathogen, little information is available regarding its pathogenesis. Here, we systematically investigated the replication kinetics of TMUV PTD2010 in adult male and female ducks. We found that PTD2010 was detectable in most of the parenchymatous organs as well as the oviduct and intestinal tract from days 1 to 7 after inoculation. Viral titers were maintained at high levels for at least 9 days in the spleen, kidney, bursa of Fabricius, brain, and ovary. No virus was detected in any of these organs or tissues at 18 days after inoculation. PTD2010, thus, causes systemic infections in male and female ducks; its replication kinetics show similar patterns in most organs, with the exception of the ovaries and testes.

Use of quantitative real-time RT-PCR to investigate the correlation between viremia and viral shedding of canine distemper virus, and infection outcomes in experimentally infected dogs.

PubMed

Sehata, Go; Sato, Hiroaki; Ito, Toshihiro; Imaizumi, Yoshitaka; Noro, Taichi; Oishi, Eiji

2015-07-01

We used real-time RT-PCR and virus titration to examine canine distemper virus (CDV) kinetics in peripheral blood and rectal and nasal secretions from 12 experimentally infected dogs. Real-time RT-PCR proved extremely sensitive, and the correlation between the two methods for rectal and nasal (r=0.78, 0.80) samples on the peak day of viral RNA was good. Although the dogs showed diverse symptoms, viral RNA kinetics were similar; the peak of viral RNA in the symptomatic dogs was consistent with the onset of symptoms. These results indicate that real-time RT-PCR is sufficiently sensitive to monitor CDV replication in experimentally infected dogs regardless of the degree of clinical manifestation and suggest that the peak of viral RNA reflects active CDV replication.

Use of quantitative real-time RT-PCR to investigate the correlation between viremia and viral shedding of canine distemper virus, and infection outcomes in experimentally infected dogs

PubMed Central

SEHATA, Go; SATO, Hiroaki; ITO, Toshihiro; IMAIZUMI, Yoshitaka; NORO, Taichi; OISHI, Eiji

2015-01-01

We used real-time RT-PCR and virus titration to examine canine distemper virus (CDV) kinetics in peripheral blood and rectal and nasal secretions from 12 experimentally infected dogs. Real-time RT-PCR proved extremely sensitive, and the correlation between the two methods for rectal and nasal (r=0.78, 0.80) samples on the peak day of viral RNA was good. Although the dogs showed diverse symptoms, viral RNA kinetics were similar; the peak of viral RNA in the symptomatic dogs was consistent with the onset of symptoms. These results indicate that real-time RT-PCR is sufficiently sensitive to monitor CDV replication in experimentally infected dogs regardless of the degree of clinical manifestation and suggest that the peak of viral RNA reflects active CDV replication. PMID:25728411

Multiplicity of genome equivalents in the radiation-resistant bacterium Micrococcus radiodurans.

PubMed Central

Hansen, M T

1978-01-01

The complexity of the genome of Micrococcus radiodurans was determined to be (2.0 +/- 0.3) X 10(9) daltons by DNA renaturation kinetics. The number of genome equivalents of DNA per cell was calculated from the complexity and the content of DNA. A lower limit of four genome equivalents per cell was approached with decreasing growth rate. Thus, no haploid stage appeared to be realized in this organism. The replication time was estimated from the kinetics and amount of residual DNA synthesis after inhibiting initiation of new rounds of replication. From this, the redundancy of terminal genetic markers was calculated to vary with growth rate from four to approximately eight copies per cell. All genetic material, including the least abundant, is thus multiply represented in each cell. The potential significance of the maintenance in each cell of multiple gene copies is discussed in relation to the extreme radiation resistance of M. radiodurans. PMID:649572

Modeling Non-Steady Isotopologue and Isotopomer Speciation and Fractionation during Denitrification in Soils

NASA Astrophysics Data System (ADS)

Maggi, F.; Riley, W. J.

2009-12-01

The composition and location of 15N atoms on N2O isotopomers and isotopologues during isotope speciation has been used to characterize soil biological N cycling and N2O surface emissions. Although there exist few experimental observations, no attempt has been made to model N2O isotopomer speciation. The mathematical treatment of biological kinetic reactions in isotopic applications normally makes use of first-order and quasi steady-state complexation assumptions without taking into account changes in enzyme concentration, reaction stoichiometry, and isotopologue and isotopomer speciation. When multiatomic isotopically-labeled reactants are used in a multi-molecurar reaction, these assumptions may fail since they always lead to a constant fractionation factor and cannot describe speciation of isotopologues and isotopomers. We have developed a mathematical framework that is capable of describing isotopologue and isotopmer speciation and fractionation under the assumption of non-steady complexation during biological kinetic reactions that overcome the limitations mentioned above. This framework was applied to a case study of non-steady (variable and inverse) isotopic effects observed during N2O production and consumption in soils. Our mathematical treatment has led to generalized kinetic equations which replicate experimental observations with high accuracy and help interpret non-steady isotopic effects and isotopologue and isotopomer speciation. The kinetic equations introduced and applied here have general validity in describing isotopic effects in any biochemical reactions by considering: changing enzyme concentrations, mass and isotope conservation, and reaction stoichiometry. The equations also describe speciation of any isotopologue and isotopomer product from any isotopologue and isotopmer reactant.

Kinetics and thermodynamics of exonuclease-deficient DNA polymerases

NASA Astrophysics Data System (ADS)

Gaspard, Pierre

2016-04-01

A kinetic theory is developed for exonuclease-deficient DNA polymerases, based on the experimental observation that the rates depend not only on the newly incorporated nucleotide, but also on the previous one, leading to the growth of Markovian DNA sequences from a Bernoullian template. The dependencies on nucleotide concentrations and template sequence are explicitly taken into account. In this framework, the kinetic and thermodynamic properties of DNA replication, in particular, the mean growth velocity, the error probability, and the entropy production are calculated analytically in terms of the rate constants and the concentrations. Theory is compared with numerical simulations for the DNA polymerases of T7 viruses and human mitochondria.

Analysis of mutagenic DNA repair in a thermoconditional mutant of Saccharomyces cerevisiae. IV. Influence of DNA replication and excision repair on REV2 dependent UV-mutagenesis and repair.

PubMed

Siede, W; Eckardt, F

1986-01-01

A double mutant being thermoconditionally defective in mutation induction as well as in repair of pre-lethal UV-induced DNA damage (rev2ts) and deficient in excision repair (rad3-2) was studied in temperature-shift experiments. The influence of inhibitors of DNA replication (hydroxyurea, aphidicolin) was determined. Additionally, an analysis of the dose-response pattern of mutation induction ("mutation kinetics") at several ochre alleles was carried out. It was concluded that the UV-inducible REV2 dependent mutagenic repair process is not induced in excision-deficient cells. In excision-deficient cells, REV2 dependent mutation fixation is slow and mostly post-replicative though not dependent on DNA replication. The REV2 mediated mutagenic process could be separated from the repair function.

Universal Sequence Replication, Reversible Polymerization and Early Functional Biopolymers: A Model for the Initiation of Prebiotic Sequence Evolution

PubMed Central

Walker, Sara Imari; Grover, Martha A.; Hud, Nicholas V.

2012-01-01

Many models for the origin of life have focused on understanding how evolution can drive the refinement of a preexisting enzyme, such as the evolution of efficient replicase activity. Here we present a model for what was, arguably, an even earlier stage of chemical evolution, when polymer sequence diversity was generated and sustained before, and during, the onset of functional selection. The model includes regular environmental cycles (e.g. hydration-dehydration cycles) that drive polymers between times of replication and functional activity, which coincide with times of different monomer and polymer diffusivity. Template-directed replication of informational polymers, which takes place during the dehydration stage of each cycle, is considered to be sequence-independent. New sequences are generated by spontaneous polymer formation, and all sequences compete for a finite monomer resource that is recycled via reversible polymerization. Kinetic Monte Carlo simulations demonstrate that this proposed prebiotic scenario provides a robust mechanism for the exploration of sequence space. Introduction of a polymer sequence with monomer synthetase activity illustrates that functional sequences can become established in a preexisting pool of otherwise non-functional sequences. Functional selection does not dominate system dynamics and sequence diversity remains high, permitting the emergence and spread of more than one functional sequence. It is also observed that polymers spontaneously form clusters in simulations where polymers diffuse more slowly than monomers, a feature that is reminiscent of a previous proposal that the earliest stages of life could have been defined by the collective evolution of a system-wide cooperation of polymer aggregates. Overall, the results presented demonstrate the merits of considering plausible prebiotic polymer chemistries and environments that would have allowed for the rapid turnover of monomer resources and for regularly varying monomer/polymer diffusivities. PMID:22493682

T135I substitution in the nonstructural protein 2C enhances foot-and-mouth disease virus replication.

PubMed

Yuan, Tiangang; Wang, Haiwei; Li, Chen; Yang, Decheng; Zhou, Guohui; Yu, Li

2017-12-01

The foot-and-mouth disease virus (FMDV) nonstructural protein 3A plays an important role in viral replication, virulence, and host range. It has been shown that deletions of 10 or 19-20 amino acids in the C-terminal half of 3A attenuate serotype O and C FMDVs, which replicate poorly in bovine cells but normally in porcine-derived cells, and the C-terminal half of 3A is not essential for serotype Asia1 FMDV replication in BHK-21 cells. In this study, we constructed a 3A deletion FMDV mutant based on a serotype O FMDV, the wild-type virus O/YS/CHA/05, with a 60-amino acid deletion in the 3A protein sequence, between residues 84 and 143. The rescued virus O/YS/CHA/05-Δ3A exhibited slower growth kinetics and formed smaller plaques compared to O/YS/CHA/05 in both BHK-21 and IBRS-2 cells, indicating that the 60-amino acid deletion in the 3A protein impaired FMDV replication. After 14 passages in BHK-21 cells, the replication capacity of the passaged virus O/YS/CHA/05-Δ3A-P14 returned to a level similar to the wild-type virus, suggesting that amino acid substitutions responsible for the enhanced replication capacity occurred in the genome of O/YS/CHA/05-Δ3A-P14. By sequence analysis, two amino acid substitutions, P153L in VP1 and T135I in 2C, were found in the O/YS/CHA/05-Δ3A-P14 genome compared to the O/YS/CHA/05-Δ3A genome. Subsequently, the amino acid substitutions VP1 P153L and 2C T135I were separately introduced into O/YS/CHA/05-Δ3A to rescue mutant viruses for examining their growth kinetics. Results showed that the 2C T135I instead of the VP1 P153L enhanced the virus replication capacity. The 2C T135I substitution also improved the replication of the wild-type virus, indicating that the effect of 2C T135I substitution on FMDV replication is not associated with the 3A deletion. Furthermore, our results showed that the T135I substitution in the nonstructural protein 2C enhanced O/YS/CHA/05 replication through promoting viral RNA synthesis.

Prototype Biology-Based Radiation Risk Module Project

NASA Technical Reports Server (NTRS)

Terrier, Douglas; Clayton, Ronald G.; Patel, Zarana; Hu, Shaowen; Huff, Janice

2015-01-01

Biological effects of space radiation and risk mitigation are strategic knowledge gaps for the Evolvable Mars Campaign. The current epidemiology-based NASA Space Cancer Risk (NSCR) model contains large uncertainties (HAT #6.5a) due to lack of information on the radiobiology of galactic cosmic rays (GCR) and lack of human data. The use of experimental models that most accurately replicate the response of human tissues is critical for precision in risk projections. Our proposed study will compare DNA damage, histological, and cell kinetic parameters after irradiation in normal 2D human cells versus 3D tissue models, and it will use a multi-scale computational model (CHASTE) to investigate various biological processes that may contribute to carcinogenesis, including radiation-induced cellular signaling pathways. This cross-disciplinary work, with biological validation of an evolvable mathematical computational model, will help reduce uncertainties within NSCR and aid risk mitigation for radiation-induced carcinogenesis.

Structure of the replication fork in ultraviolet light-irradiated human cells.

PubMed Central

Cordeiro-Stone, M; Schumacher, R I; Meneghini, R

1979-01-01

The DNA extracted from xeroderma pigmentosum human fibroblasts previously irradiated with 12.5 J/m2 of UV light and pulse-labeled for 45 min with radioactive and (or) heavy precursors, was used to determine the structural characteristics of the replication fork. Density equilibrium centrifugation experiments showed that a fork moved 6 micrometer in 45 min and bypassed 3 pyrimidine dimers in both strands. The same length was covered in 15-20 min in control cells. The delay in irradiated cells was apparently due to pyrimidine dimers acting as temporary blocks to the fork movement. Evidence for this interpretation comes from kinetics of incorporation of [3H]thymidine into DNA, which show that the time necessary to attain a new stable level of DNA synthesis in irradiated cells is equivalent to that required for the replication fork to cover the interdimer distance in one strand. On the other hand, the action of S1 nuclease on DNA synthesized soon after irradiation gives rise to a bimodal distribution in neutral sucrose gradients, one peak corresponding to 43 X 10(6) daltons and the other to 3 X 10(6) daltons. These two DNA species are generated by the attack of the S1 nuclease on single-stranded regions associated with the replication fork. A possible explanation for these results is given by a model according to which there is a delayed bypass of the dimer in the leading strand and the appearance of gaps opposite pyrimidine dimers in the lagging strand, as a direct consequence of the discontinuous mode of DNA replication. In terms of the model, the DNA of 43 X 10(6) daltons corresponds to the leading strand, linked to the unreplicated branch of the forks, whereas the piece of 3 X 10(6) daltons is the intergap DNA coming from the lagging strand. Pulse and chase experiments reveal that the low molecular weight DNA grows in a pattern that suggests that more than one gap may be formed per replication fork. PMID:233582

Structure of the replication fork in ultraviolet light-irradiated human cells.

PubMed

Cordeiro-Stone, M; Schumacher, R I; Meneghini, R

1979-08-01

The DNA extracted from xeroderma pigmentosum human fibroblasts previously irradiated with 12.5 J/m2 of UV light and pulse-labeled for 45 min with radioactive and (or) heavy precursors, was used to determine the structural characteristics of the replication fork. Density equilibrium centrifugation experiments showed that a fork moved 6 micrometer in 45 min and bypassed 3 pyrimidine dimers in both strands. The same length was covered in 15-20 min in control cells. The delay in irradiated cells was apparently due to pyrimidine dimers acting as temporary blocks to the fork movement. Evidence for this interpretation comes from kinetics of incorporation of [3H]thymidine into DNA, which show that the time necessary to attain a new stable level of DNA synthesis in irradiated cells is equivalent to that required for the replication fork to cover the interdimer distance in one strand. On the other hand, the action of S1 nuclease on DNA synthesized soon after irradiation gives rise to a bimodal distribution in neutral sucrose gradients, one peak corresponding to 43 X 10(6) daltons and the other to 3 X 10(6) daltons. These two DNA species are generated by the attack of the S1 nuclease on single-stranded regions associated with the replication fork. A possible explanation for these results is given by a model according to which there is a delayed bypass of the dimer in the leading strand and the appearance of gaps opposite pyrimidine dimers in the lagging strand, as a direct consequence of the discontinuous mode of DNA replication. In terms of the model, the DNA of 43 X 10(6) daltons corresponds to the leading strand, linked to the unreplicated branch of the forks, whereas the piece of 3 X 10(6) daltons is the intergap DNA coming from the lagging strand. Pulse and chase experiments reveal that the low molecular weight DNA grows in a pattern that suggests that more than one gap may be formed per replication fork.

Kinetics and thermodynamics of DNA polymerases with exonuclease proofreading

NASA Astrophysics Data System (ADS)

Gaspard, Pierre

2016-04-01

Kinetic theory and thermodynamics are applied to DNA polymerases with exonuclease activity, taking into account the dependence of the rates on the previously incorporated nucleotide. The replication fidelity is shown to increase significantly thanks to this dependence at the basis of the mechanism of exonuclease proofreading. In particular, this dependence can provide up to a 100-fold lowering of the error probability under physiological conditions. Theory is compared with numerical simulations for the DNA polymerases of T7 viruses and human mitochondria.

Inside the lifestyle of the virophage.

PubMed

Desnues, C; Raoult, D

2010-01-01

We sought to better characterize Sputnik, the first isolated virophage, and to analyze its parasitic lifestyle during co-infection with Marseillevirus (a new giant virus) in Acanthamoeba castellanii. A combination of electron microscopy, immunofluorescence microscopy, and real-time PCR was used to characterize the kinetics of the viral replication cycle. RT-PCR was performed to detect RNAs inside the Sputnik virions. Sputnik is a new viral entity carrying an almost complete ready-to-use set of viral RNAs (20 out of 21). Sputnik does not replicate with Marseillevirus but delays its replication cycle. While Marseillevirus is successfully internalized by A. castellanii following co-infections with Mamavirus and Sputnik, it does not initiate a replication cycle. In contrast, both Marseillevirus and Mamavirus can replicate in the amoeba in case of co-infection, but the development of one is exclusive from the other inside a single amoeba cell. This work provides new insight into the Sputnik replication cycle with another giant virus and confirms that Sputnik is a virophage. It shows new dimensions of the interactions existing among giant viruses. Copyright 2010 S. Karger AG, Basel.

Viral evolution in response to the broad-based retroviral protease inhibitor TL-3.

PubMed

Bühler, B; Lin, Y C; Morris, G; Olson, A J; Wong, C H; Richman, D D; Elder, J H; Torbett, B E

2001-10-01

TL-3 is a protease inhibitor developed using the feline immunodeficiency virus protease as a model. It has been shown to efficiently inhibit replication of human, simian, and feline immunodeficiency viruses and therefore has broad-based activity. We now demonstrate that TL-3 efficiently inhibits the replication of 6 of 12 isolates with confirmed resistance mutations to known protease inhibitors. To dissect the spectrum of molecular changes in protease and viral properties associated with resistance to TL-3, a panel of chronological in vitro escape variants was generated. We have virologically and biochemically characterized mutants with one (V82A), three (M46I/F53L/V82A), or six (L24I/M46I/F53L/L63P/V77I/V82A) changes in the protease and structurally modeled the protease mutant containing six changes. Virus containing six changes was found to be 17-fold more resistant to TL-3 in cell culture than was wild-type virus but maintained similar in vitro replication kinetics compared to the wild-type virus. Analyses of enzyme activity of protease variants with one, three, and six changes indicated that these enzymes, compared to wild-type protease, retained 40, 47, and 61% activity, respectively. These results suggest that deficient protease enzymatic activity is sufficient for function, and the observed protease restoration might imply a selective advantage, at least in vitro, for increased protease activity.

Viral Evolution in Response to the Broad-Based Retroviral Protease Inhibitor TL-3†

PubMed Central

Bühler, Bernd; Lin, Ying-Chuan; Morris, Garrett; Olson, Arthur J.; Wong, Chi-Huey; Richman, Douglas D.; Elder, John H.; Torbett, Bruce E.

2001-01-01

TL-3 is a protease inhibitor developed using the feline immunodeficiency virus protease as a model. It has been shown to efficiently inhibit replication of human, simian, and feline immunodeficiency viruses and therefore has broad-based activity. We now demonstrate that TL-3 efficiently inhibits the replication of 6 of 12 isolates with confirmed resistance mutations to known protease inhibitors. To dissect the spectrum of molecular changes in protease and viral properties associated with resistance to TL-3, a panel of chronological in vitro escape variants was generated. We have virologically and biochemically characterized mutants with one (V82A), three (M46I/F53L/V82A), or six (L24I/M46I/F53L/L63P/V77I/V82A) changes in the protease and structurally modeled the protease mutant containing six changes. Virus containing six changes was found to be 17-fold more resistant to TL-3 in cell culture than was wild-type virus but maintained similar in vitro replication kinetics compared to the wild-type virus. Analyses of enzyme activity of protease variants with one, three, and six changes indicated that these enzymes, compared to wild-type protease, retained 40, 47, and 61% activity, respectively. These results suggest that deficient protease enzymatic activity is sufficient for function, and the observed protease restoration might imply a selective advantage, at least in vitro, for increased protease activity. PMID:11533212

Assessment of Molecular, Antigenic, and Pathological Features of Canine Influenza A(H3N2) Viruses That Emerged in the United States

PubMed Central

Pulit-Penaloza, Joanna A.; Simpson, Natosha; Yang, Hua; Creager, Hannah M.; Jones, Joyce; Carney, Paul; Belser, Jessica A.; Yang, Genyan; Chang, Jessie; Zeng, Hui; Thor, Sharmi; Jang, Yunho; Killian, Mary Lea; Jenkins-Moore, Melinda; Janas-Martindale, Alicia; Dubovi, Edward; Wentworth, David E.; Stevens, James; Tumpey, Terrence M.; Davis, C. Todd; Maines, Taronna R.

2017-01-01

Background A single subtype of canine influenza virus (CIV), A(H3N8), was circulating in the United States until a new subtype, A(H3N2), was detected in Illinois in spring 2015. Since then, this CIV has caused thousands of infections in dogs in multiple states. Methods In this study, genetic and antigenic properties of the new CIV were evaluated. In addition, structural and glycan array binding features of the recombinant hemagglutinin were determined. Replication kinetics in human airway cells and pathogenesis and transmissibility in animal models were also assessed. Results A(H3N2) CIVs maintained molecular and antigenic features related to low pathogenicity avian influenza A(H3N2) viruses and were distinct from A(H3N8) CIVs. The structural and glycan array binding profile confirmed these findings and revealed avian-like receptor-binding specificity. While replication kinetics in human airway epithelial cells was on par with that of seasonal influenza viruses, mild-to-moderate disease was observed in infected mice and ferrets, and the virus was inefficiently transmitted among cohoused ferrets. Conclusions Further adaptation is needed for A(H3N2) CIVs to present a likely threat to humans. However, the potential for coinfection of dogs and possible reassortment of human and other animal influenza A viruses presents an ongoing risk to public health. PMID:28934454

Reverse genetics in high throughput: rapid generation of complete negative strand RNA virus cDNA clones and recombinant viruses thereof.

PubMed

Nolden, T; Pfaff, F; Nemitz, S; Freuling, C M; Höper, D; Müller, T; Finke, Stefan

2016-04-05

Reverse genetics approaches are indispensable tools for proof of concepts in virus replication and pathogenesis. For negative strand RNA viruses (NSVs) the limited number of infectious cDNA clones represents a bottleneck as clones are often generated from cell culture adapted or attenuated viruses, with limited potential for pathogenesis research. We developed a system in which cDNA copies of complete NSV genomes were directly cloned into reverse genetics vectors by linear-to-linear RedE/T recombination. Rapid cloning of multiple rabies virus (RABV) full length genomes and identification of clones identical to field virus consensus sequence confirmed the approache's reliability. Recombinant viruses were recovered from field virus cDNA clones. Similar growth kinetics of parental and recombinant viruses, preservation of field virus characters in cell type specific replication and virulence in the mouse model were confirmed. Reduced titers after reporter gene insertion indicated that the low level of field virus replication is affected by gene insertions. The flexibility of the strategy was demonstrated by cloning multiple copies of an orthobunyavirus L genome segment. This important step in reverse genetics technology development opens novel avenues for the analysis of virus variability combined with phenotypical characterization of recombinant viruses at a clonal level.

Multiphysics Model of Palladium Hydride Isotope Exchange Accounting for Higher Dimensionality

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

Gharagozloo, Patricia E.; Eliassi, Mehdi; Bon, Bradley Luis

2015-03-01

This report summarizes computational model developm ent and simulations results for a series of isotope exchange dynamics experiments i ncluding long and thin isothermal beds similar to the Foltz and Melius beds and a lar ger non-isothermal experiment on the NENG7 test bed. The multiphysics 2D axi-symmetr ic model simulates the temperature and pressure dependent exchange reactio n kinetics, pressure and isotope dependent stoichiometry, heat generation from the r eaction, reacting gas flow through porous media, and non-uniformities in the bed perme ability. The new model is now able to replicate the curved reaction front and asy mmetry of themore » exit gas mass fractions over time. The improved understanding of the exchange process and its dependence on the non-uniform bed properties and te mperatures in these larger systems is critical to the future design of such sy stems.« less

Development of a Model of Geophysical and Geochemical Controls on Abiotic Carbon Cycling on Earth-Like Planets

NASA Astrophysics Data System (ADS)

Neveu, M.; Felton, R.; Domagal-Goldman, S. D.; Desch, S. J.; Arney, G. N.

2017-12-01

About 20 Earth-sized planets (0.6-1.6 Earth masses and radii) have now been discovered beyond our solar system [1]. Although such planets are prime targets in the upcoming search for atmospheric biosignatures, their composition, geology, and climate are essentially unconstrained. Yet, developing an understanding of how these factors influence planetary evolution through time and space is essential to establishing abiotic backgrounds against which any deviations can provide evidence for biological activity. To this end, we are building coupled geophysical-geochemical models of abiotic carbon cycling on such planets. Our models are controlled by atmospheric factors such as temperature and composition, and compute interior inputs to atmospheric species. They account for crustal weathering, ocean-atmosphere equilibria, and exchange with the deep interior as a function of planet composition and size (and, eventually, age).Planets in other solar systems differ from the Earth not only in their bulk physical properties, but also likely in their bulk chemical composition [2], which influences key parameters such as the vigor of mantle convection and the near-surface redox state. Therefore, simulating how variations in such parameters affect carbon cycling requires us to simulate the above processes from first principles, rather than by using arbitrary parameterizations derived from observations as is often done with models of carbon cycling on Earth [3] or extrapolations thereof [4]. As a first step, we have developed a kinetic model of crustal weathering using the PHREEQC code [5] and kinetic data from [6]. We will present the ability of such a model to replicate Earth's carbon cycle using, for the time being, parameterizations for surface-interior-atmosphere exchange processes such as volcanism (e.g., [7]).[1] exoplanet.eu, 7/28/2017.[2] Young et al. (2014) Astrobiology 14, 603-626.[3] Lerman & Wu (2008) Kinetics of Global Geochemical Cycles. In Kinetics of Water-Rock Interaction (Brantley et al., eds.), Springer, New York.[4] Edson et al. (2012) Astrobiology 12, 562-571.[5] Parkhurst & Appelo (2013) USGS Techniques and Methods 6-A43.[6] Palandri & Kharaka (2008) USGS Report 2004-1068.[7] Kite et al. (2009) ApJ 700, 1732-1749.

Kinetic measurement of 2-aminopurine X cytosine and 2-aminopurine X thymine base pairs as a test of DNA polymerase fidelity mechanisms.

PubMed Central

Watanabe, S M; Goodman, M F

1982-01-01

Enzyme kinetic measurements are presented showing that Km rather than maximum velocity (Vmax) discrimination governs the frequency of forming 2-aminopurine X cytosine base mispairs by DNA polymerase alpha. An in vitro system is used in which incorporation of dTMP or dCMP occurs opposite a template 2-aminopurine, and values for Km and Vmax are obtained. Results from a previous study in which dTTP and dCTP were competing simultaneously for insertion opposite 2-aminopurine indicated that dTMP is inserted 22 times more frequently than dCMP. We now report that the ratio of Km values KCm/KTm = 25 +/- 6, which agrees quantitatively with the dTMP/dCMP incorporation ratio obtained previously. We also report that VCmax is indistinguishable from VTmax. These Km and Vmax data are consistent with predictions from a model, the Km discrimination model, in which replication fidelity is determined by free energy differences between matched and mismatched base pairs. Central to this model is the prediction that the ratio of Km values for insertion of correct and incorrect nucleotides specifies the insertion fidelity, and the maximum velocities of insertion are the same for both nucleotides. PMID:6959128

A spatial focusing model for G protein signals. Regulator of G protein signaling (RGS) protien-mediated kinetic scaffolding.

PubMed

Zhong, Huailing; Wade, Susan M; Woolf, Peter J; Linderman, Jennifer J; Traynor, John R; Neubig, Richard R

2003-02-28

Regulators of G protein signaling (RGS) are GTPase-accelerating proteins (GAPs), which can inhibit heterotrimeric G protein pathways. In this study, we provide experimental and theoretical evidence that high concentrations of receptors (as at a synapse) can lead to saturation of GDP-GTP exchange making GTP hydrolysis rate-limiting. This results in local depletion of inactive heterotrimeric G-GDP, which is reversed by RGS GAP activity. Thus, RGS enhances receptor-mediated G protein activation even as it deactivates the G protein. Evidence supporting this model includes a GTP-dependent enhancement of guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding to G(i) by RGS. The RGS domain of RGS4 is sufficient for this, not requiring the NH(2)- or COOH-terminal extensions. Furthermore, a kinetic model including only the GAP activity of RGS replicates the GTP-dependent enhancement of GTPgammaS binding observed experimentally. Finally in a Monte Carlo model, this mechanism results in a dramatic "spatial focusing" of active G protein. Near the receptor, G protein activity is maintained even with RGS due to the ability of RGS to reduce depletion of local Galpha-GDP levels permitting rapid recoupling to receptor and maintained G protein activation near the receptor. In contrast, distant signals are suppressed by the RGS, since Galpha-GDP is not depleted there. Thus, a novel RGS-mediated "kinetic scaffolding" mechanism is proposed which narrows the spatial range of active G protein around a cluster of receptors limiting the spill-over of G protein signals to more distant effector molecules, thus enhancing the specificity of G(i) protein signals.

Functional centromeres determine the activation time of pericentric origins of DNA replication in Saccharomyces cerevisiae.

PubMed

Pohl, Thomas J; Brewer, Bonita J; Raghuraman, M K

2012-01-01

The centromeric regions of all Saccharomyces cerevisiae chromosomes are found in early replicating domains, a property conserved among centromeres in fungi and some higher eukaryotes. Surprisingly, little is known about the biological significance or the mechanism of early centromere replication; however, the extensive conservation suggests that it is important for chromosome maintenance. Do centromeres ensure their early replication by promoting early activation of nearby origins, or have they migrated over evolutionary time to reside in early replicating regions? In Candida albicans, a neocentromere contains an early firing origin, supporting the first hypothesis but not addressing whether the new origin is intrinsically early firing or whether the centromere influences replication time. Because the activation time of individual origins is not an intrinsic property of S. cerevisiae origins, but is influenced by surrounding sequences, we sought to test the hypothesis that centromeres influence replication time by moving a centromere to a late replication domain. We used a modified Meselson-Stahl density transfer assay to measure the kinetics of replication for regions of chromosome XIV in which either the functional centromere or a point-mutated version had been moved near origins that reside in a late replication region. We show that a functional centromere acts in cis over a distance as great as 19 kb to advance the initiation time of origins. Our results constitute a direct link between establishment of the kinetochore and the replication initiation machinery, and suggest that the proposed higher-order structure of the pericentric chromatin influences replication initiation.

Functional Centromeres Determine the Activation Time of Pericentric Origins of DNA Replication in Saccharomyces cerevisiae

PubMed Central

Pohl, Thomas J.; Brewer, Bonita J.; Raghuraman, M. K.

2012-01-01

The centromeric regions of all Saccharomyces cerevisiae chromosomes are found in early replicating domains, a property conserved among centromeres in fungi and some higher eukaryotes. Surprisingly, little is known about the biological significance or the mechanism of early centromere replication; however, the extensive conservation suggests that it is important for chromosome maintenance. Do centromeres ensure their early replication by promoting early activation of nearby origins, or have they migrated over evolutionary time to reside in early replicating regions? In Candida albicans, a neocentromere contains an early firing origin, supporting the first hypothesis but not addressing whether the new origin is intrinsically early firing or whether the centromere influences replication time. Because the activation time of individual origins is not an intrinsic property of S. cerevisiae origins, but is influenced by surrounding sequences, we sought to test the hypothesis that centromeres influence replication time by moving a centromere to a late replication domain. We used a modified Meselson-Stahl density transfer assay to measure the kinetics of replication for regions of chromosome XIV in which either the functional centromere or a point-mutated version had been moved near origins that reside in a late replication region. We show that a functional centromere acts in cis over a distance as great as 19 kb to advance the initiation time of origins. Our results constitute a direct link between establishment of the kinetochore and the replication initiation machinery, and suggest that the proposed higher-order structure of the pericentric chromatin influences replication initiation. PMID:22589733

Activation of human herpesvirus replication by apoptosis.

PubMed

Prasad, Alka; Remick, Jill; Zeichner, Steven L

2013-10-01

A central feature of herpesvirus biology is the ability of herpesviruses to remain latent within host cells. Classically, exposure to inducing agents, like activating cytokines or phorbol esters that stimulate host cell signal transduction events, and epigenetic agents (e.g., butyrate) was thought to end latency. We recently showed that Kaposi's sarcoma-associated herpesvirus (KSHV, or human herpesvirus-8 [HHV-8]) has another, alternative emergency escape replication pathway that is triggered when KSHV's host cell undergoes apoptosis, characterized by the lack of a requirement for the replication and transcription activator (RTA) protein, accelerated late gene kinetics, and production of virus with decreased infectivity. Caspase-3 is necessary and sufficient to initiate the alternative replication program. HSV-1 was also recently shown to initiate replication in response to host cell apoptosis. These observations suggested that an alternative apoptosis-triggered replication program might be a general feature of herpesvirus biology and that apoptosis-initiated herpesvirus replication may have clinical implications, particularly for herpesviruses that almost universally infect humans. To explore whether an alternative apoptosis-initiated replication program is a common feature of herpesvirus biology, we studied cell lines latently infected with Epstein-Barr virus/HHV-4, HHV-6A, HHV-6B, HHV-7, and KSHV. We found that apoptosis triggers replication for each HHV studied, with caspase-3 being necessary and sufficient for HHV replication. An alternative apoptosis-initiated replication program appears to be a common feature of HHV biology. We also found that commonly used cytotoxic chemotherapeutic agents activate HHV replication, which suggests that treatments that promote apoptosis may lead to activation of latent herpesviruses, with potential clinical significance.

Activation of Human Herpesvirus Replication by Apoptosis

PubMed Central

Prasad, Alka; Remick, Jill

2013-01-01

A central feature of herpesvirus biology is the ability of herpesviruses to remain latent within host cells. Classically, exposure to inducing agents, like activating cytokines or phorbol esters that stimulate host cell signal transduction events, and epigenetic agents (e.g., butyrate) was thought to end latency. We recently showed that Kaposi's sarcoma-associated herpesvirus (KSHV, or human herpesvirus-8 [HHV-8]) has another, alternative emergency escape replication pathway that is triggered when KSHV's host cell undergoes apoptosis, characterized by the lack of a requirement for the replication and transcription activator (RTA) protein, accelerated late gene kinetics, and production of virus with decreased infectivity. Caspase-3 is necessary and sufficient to initiate the alternative replication program. HSV-1 was also recently shown to initiate replication in response to host cell apoptosis. These observations suggested that an alternative apoptosis-triggered replication program might be a general feature of herpesvirus biology and that apoptosis-initiated herpesvirus replication may have clinical implications, particularly for herpesviruses that almost universally infect humans. To explore whether an alternative apoptosis-initiated replication program is a common feature of herpesvirus biology, we studied cell lines latently infected with Epstein-Barr virus/HHV-4, HHV-6A, HHV-6B, HHV-7, and KSHV. We found that apoptosis triggers replication for each HHV studied, with caspase-3 being necessary and sufficient for HHV replication. An alternative apoptosis-initiated replication program appears to be a common feature of HHV biology. We also found that commonly used cytotoxic chemotherapeutic agents activate HHV replication, which suggests that treatments that promote apoptosis may lead to activation of latent herpesviruses, with potential clinical significance. PMID:23885073

Zika virus preferentially replicates in the female reproductive tract after vaginal inoculation of rhesus macaques.

PubMed

Carroll, Timothy; Lo, Ming; Lanteri, Marion; Dutra, Joseph; Zarbock, Katie; Silveira, Paola; Rourke, Tracy; Ma, Zhong-Min; Fritts, Linda; O'Connor, Shelby; Busch, Michael; Miller, Christopher J

2017-07-01

Zika virus (ZIKV) is a mosquito-transmitted virus that can cause severe defects in an infected fetus. ZIKV is also transmitted by sexual contact, although the relative importance of sexual transmission is unclear. To better understand the role of sexual transmission in ZIKV pathogenesis, a nonhuman primate (NHP) model of vaginal transmission was developed. ZIKV was readily transmitted to mature cycling female rhesus macaque (RM) by vaginal inoculation with 104-106 plaque-forming units (PFU). However, there was variability in susceptibility between the individual RM with 1->8 vaginal inoculations required to establish infection. After treatment with Depoprovera, a widely used contraceptive progestin, two RM that initially resisted 8 vaginal ZIKV inoculations became infected after one ZIKV inoculation. Thus, Depoprovera seemed to enhance susceptibility to vaginal ZIKV transmission. Unexpectedly, the kinetics of virus replication and dissemination after intravaginal ZIKV inoculation were markedly different from RM infected with ZIKV by subcutaneous (SQ) virus inoculation. Several groups have reported that after SQ ZIKV inoculation vRNA is rapidly detected in blood plasma with vRNA less common in urine and saliva and only rarely detected in female reproductive tract (FRT) secretions. In contrast, in vaginally inoculated RM, plasma vRNA is delayed for several days and ZIKV replication in, and vRNA shedding from, the FRT was found in all 6 animals. Further, after intravaginal transmission ZIKV RNA shedding from FRT secretions was detected before or simultaneously with plasma vRNA, and persisted for at least as long. Thus, ZIKV replication in the FRT was independent of, and often preceded virus replication in the tissues contributing to plasma vRNA. These results support the conclusion that ZIKV preferentially replicates in the FRT after vaginal transmission, but not after SQ transmission, and raise the possibility that there is enhanced fetal infection and pathology after vaginal ZIKV transmission compared to a mosquito transmitted ZIKV.

In Vitro Synthesized RNA Generated from cDNA Clones of Both Genomic Components of Cucurbit yellow stunting disorder virus Replicates in Cucumber Protoplasts

PubMed Central

Owen, Carolyn A.; Moukarzel, Romy; Huang, Xiao; Kassem, Mona A.; Eliasco, Eleonora; Aranda, Miguel A.; Coutts, Robert H. A.; Livieratos, Ioannis C.

2016-01-01

Cucurbit yellow stunting disorder virus (CYSDV), a bipartite whitefly-transmitted virus, constitutes a major threat to commercial cucurbit production worldwide. Here, construction of full-length CYSDV RNA1 and RNA2 cDNA clones allowed the in vitro synthesis of RNA transcripts able to replicate in cucumber protoplasts. CYSDV RNA1 proved competent for replication; transcription of both polarities of the genomic RNA was detectable 24 h post inoculation. Hybridization of total RNA extracted from transfected protoplasts or from naturally CYSDV-infected cucurbits revealed high-level transcription of the p22 subgenomic RNA species. Replication of CYSDV RNA2 following co-transfection with RNA1 was also observed, with similar transcription kinetics. A CYSDV RNA2 cDNA clone (T3CM8Δ) comprising the 5′- and 3′-UTRs plus the 3′-terminal gene, generated a 2.8 kb RNA able to replicate to high levels in protoplasts in the presence of CYSDV RNA1. The clone T3CM8Δ will facilitate reverse genetics studies of CYSDV gene function and RNA replication determinants. PMID:27314380

Single Cell Analysis of Human RAD18-Dependent DNA Post-Replication Repair by Alkaline Bromodeoxyuridine Comet Assay

PubMed Central

Mórocz, Mónika; Gali, Himabindu; Raskó, István; Downes, C. Stephen; Haracska, Lajos

2013-01-01

Damage to DNA can block replication progression resulting in gaps in the newly synthesized DNA. Cells utilize a number of post-replication repair (PRR) mechanisms such as the RAD18 controlled translesion synthesis or template switching to overcome the discontinuities formed opposite the DNA lesions and to complete DNA replication. Gaining more insights into the role of PRR genes promotes better understanding of DNA damage tolerance and of how their malfunction can lead to increased genome instability and cancer. However, a simple and efficient method to characterise gene specific PRR deficiencies at a single cell level has not been developed. Here we describe the so named BrdU comet PRR assay to test the contribution of human RAD18 to PRR at a single cell level, by which we kinetically characterized the consequences of the deletion of human RAD18 on the replication of UV-damaged DNA. Moreover, we demonstrate the capability of our method to evaluate PRR at a single cell level in unsynchronized cell population. PMID:23936422

Species-specific and individual differences in Nipah virus replication in porcine and human airway epithelial cells.

PubMed

Sauerhering, Lucie; Zickler, Martin; Elvert, Mareike; Behner, Laura; Matrosovich, Tatyana; Erbar, Stephanie; Matrosovich, Mikhail; Maisner, Andrea

2016-07-01

Highly pathogenic Nipah virus (NiV) causes symptomatic infections in pigs and humans. The severity of respiratory symptoms is much more pronounced in pigs than in humans, suggesting species-specific differences of NiV replication in porcine and human airways. Here, we present a comparative study on productive NiV replication in primary airway epithelial cell cultures of the two species. We reveal that NiV growth substantially differs in primary cells between pigs and humans, with a more rapid spread of infection in human airway epithelia. Increased replication, correlated with higher endogenous expression levels of the main NiV entry receptor ephrin-B2, not only significantly differed between airway cells of the two species but also varied between cells from different human donors. To our knowledge, our study provides the first experimental evidence of species-specific and individual differences in NiV receptor expression and replication kinetics in primary airway epithelial cells. It remains to be determined whether and how these differences contribute to the viral host range and pathogenicity.

A biokinetic model to describe consequences of inhibition/stimulation in DNA-proofreading and repair-1. Development of the model.

PubMed

Haschke, H

2001-10-21

A biokinetic model is described which deals with the mathematical consequences of the inhibition or stimulation of DNA proofreading. It demonstrates the development of the number of DNA mismatch-dependent cells (e.g. cells with a malignant phenotype), where such mismatches arise by the in situ interaction of various substances with nucleotides of the DNA. The model can test for consequences by a logic gating on an "if-then" type of analysis in relation to the separate and consecutive processes of proofreading and repair. In particular, the consequences are considered in cases where either (i) the efficacy of proofreading and repair are reduced/prevented (inhibited) or (ii) are increased by some form of stimulation. On the chosen kinetic parameters, the model is accessible to manipulation as new data arising from further investigations become available and are introduced. The model is based on recently published data which show that an increased "mutant fraction" (see note on terms) arises in DNA replication when intracellular nucleotide pools show "asymmetries" (see note on terms). Extraordinarily high mutant fractions can be predicted/have been recorded in the presence of proofreading inhibitors. The model expresses data in mathematical terms of the competition between the development of mismatch-dependent cells and those with authentic genetic information. (Feedback and metastasis-effects and those of wild-type replicates are included.) A computerized (numerical) integration of the corresponding set of differential equations is offered. (A diskette with the program CANCER.xls is available upon request.) Copyright 2001 Academic Press

Pathogenesis, Transmissibility, and Tropism of a Highly Pathogenic Avian Influenza A(H7N7) Virus Associated With Human Conjunctivitis in Italy, 2013.

PubMed

Belser, Jessica A; Creager, Hannah M; Zeng, Hui; Maines, Taronna R; Tumpey, Terrence M

2017-09-15

H7 subtype influenza viruses represent a persistent public health threat because of their continued detection in poultry and ability to cause human infection. An outbreak of highly pathogenic avian influenza H7N7 virus in Italy during 2013 resulted in 3 cases of human conjunctivitis. We determined the pathogenicity and transmissibility of influenza A/Italy/3/2013 virus in mouse and ferret models and examined the replication kinetics of this virus in several human epithelial cell types. The moderate virulence observed in mammalian models and the capacity for transmission in a direct contact model underscore the need for continued study of H7 subtype viruses. Published by Oxford University Press for the Infectious Diseases Society of America 2017. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Requirements for rapid plasmid ColE1 copy number adjustments: a mathematical model of inhibition modes and RNA turnover rates.

PubMed

Paulsson, J; Nordström, K; Ehrenberg, M

1998-01-01

The random distribution of ColE1 plasmids between the daughter cells at cell division introduces large copy number variations. Statistic variation associated with limited copy number in single cells also causes fluctuations to emerge spontaneously during the cell cycle. Efficient replication control out of steady state is therefore important to tame such stochastic effects of small numbers. In the present model, the dynamic features of copy number control are divided into two parts: first, how sharply the replication frequency per plasmid responds to changes in the concentration of the plasmid-coded inhibitor, RNA I, and second, how tightly RNA I and plasmid concentrations are coupled. Single (hyperbolic)- and multiple (exponential)-step inhibition mechanisms are compared out of steady state and it is shown how the response in replication frequency depends on the mode of inhibition. For both mechanisms, sensitivity of inhibition is "bought" at the expense of a rapid turnover of a replication preprimer, RNA II. Conventional, single-step, inhibition kinetics gives a sloppy replication control even at high RNA II turnover rates, whereas multiple-step inhibition has the potential of working with unlimited precision. When plasmid concentration changes rapidly, RNA I must be degraded rapidly to be "up to date" with the change. Adjustment to steady state is drastically impaired when the turnover rate constants of RNA I decrease below certain thresholds, but is basically unaffected for a corresponding increase. Several features of copy number control that are shown to be crucial for the understanding of ColE1-type plasmids still remain to be experimentally characterized. It is shown how steady-state properties reflect dynamics at the heart of regulation and therefore can be used to discriminate between fundamentally different copy number control mechanisms. The experimental tests of the predictions made require carefully planned assays, and some suggestions for suitable experiments arise naturally from the present work. It is also discussed how the presence of the Rom protein may affect dynamic qualities of copy number control. Copyright 1998 Academic Press.

An Analysis of Enzyme Kinetics Data for Mitochondrial DNA Strand Termination by Nucleoside Reverse Transcription Inhibitors

PubMed Central

Wendelsdorf, Katherine V.; Song, Zhuo; Cao, Yang; Samuels, David C.

2009-01-01

Nucleoside analogs used in antiretroviral treatment have been associated with mitochondrial toxicity. The polymerase-γ hypothesis states that this toxicity stems from the analogs' inhibition of the mitochondrial DNA polymerase (polymerase-γ) leading to mitochondrial DNA (mtDNA) depletion. We have constructed a computational model of the interaction of polymerase-γ with activated nucleoside and nucleotide analog drugs, based on experimentally measured reaction rates and base excision rates, together with the mtDNA genome size, the human mtDNA sequence, and mitochondrial dNTP concentrations. The model predicts an approximately 1000-fold difference in the activated drug concentration required for a 50% probability of mtDNA strand termination between the activated di-deoxy analogs d4T, ddC, and ddI (activated to ddA) and the activated forms of the analogs 3TC, TDF, AZT, FTC, and ABC. These predictions are supported by experimental and clinical data showing significantly greater mtDNA depletion in cell culture and patient samples caused by the di-deoxy analog drugs. For zidovudine (AZT) we calculated a very low mtDNA replication termination probability, in contrast to its reported mitochondrial toxicity in vitro and clinically. Therefore AZT mitochondrial toxicity is likely due to a mechanism that does not involve strand termination of mtDNA replication. PMID:19132079

Differential virulence mechanisms of infectious hematopoietic necrosis virus in rainbow trout (Oncorhynchus mykiss) include host entry and virus replication kinetics

USGS Publications Warehouse

Penaranda, M.M.D.; Purcell, M.K.; Kurath, G.

2009-01-01

Host specificity is a phenomenon exhibited by all viruses. For the fish rhabdovirus infectious hematopoietic necrosis virus (IHNV), differential specificity of virus strains from the U and M genogroups has been established both in the field and in experimental challenges. In rainbow trout (Oncorhynchus mykiss), M IHNV strains are consistently more prevalent and more virulent than U IHNV. The basis of the differential ability of these two IHNV genogroups to cause disease in rainbow trout was investigated in live infection challenges with representative U and M IHNV strains. When IHNV was delivered by intraperitoneal injection, the mortality caused by U IHNV increased, indicating that the low virulence of U IHNV is partly due to inefficiency in entering the trout host. Analyses of in vivo replication showed that U IHNV consistently had lower prevalence and lower viral load than M IHNV during the course of infection. In analyses of the host immune response, M IHNV-infected fish consistently had higher and longer expression of innate immune-related genes such as Mx-1. This suggests that the higher virulence of M IHNV is not due to suppression of the immune response in rainbow trout. Taken together, the results support a kinetics hypothesis wherein faster replication enables M IHNV to rapidly achieve a threshold level of virus necessary to override the strong host innate immune response. ?? 2009 SGM.

A ribonucleotide Origin for Life - Fluctuation and Near-ideal Reactions

NASA Astrophysics Data System (ADS)

Yarus, Michael

2013-02-01

Oligoribonucleotides are potentially capable of Darwinian evolution - they may replicate and can express an independent chemical phenotype, as embodied in modern enzymatic cofactors. Using quantitative chemical kinetics on a sporadically fed ribonucleotide pool, unreliable supplies of unstable activated ribonucleotides A and B at low concentrations recurrently yield a replicating AB polymer with a potential chemical phenotype. Self-complementary replication in the pool occurs during a minority (here ≈ 35 %) of synthetic episodes that exploit coincidental overlaps between 4, 5 or 6 spikes of arbitrarily arriving substrates. Such uniquely productive synthetic episodes, in which near-ideal reaction sequences recur at random, account for most AB oligonucleotide synthesis, and therefore underlie the emergence of net replication under realistic primordial conditions. Because overlapping substrate spikes are unexpectedly frequent, and in addition, complex spike sequences appear disproportionately, a sporadically fed pool can host unexpectedly complex syntheses. Thus, primordial substrate fluctuations are not necessarily a barrier to Darwinism, but instead can facilitate early evolution.

A ribonucleotide Origin for Life--fluctuation and near-ideal reactions.

PubMed

Yarus, Michael

2013-02-01

Oligoribonucleotides are potentially capable of Darwinian evolution - they may replicate and can express an independent chemical phenotype, as embodied in modern enzymatic cofactors. Using quantitative chemical kinetics on a sporadically fed ribonucleotide pool, unreliable supplies of unstable activated ribonucleotides A and B at low concentrations recurrently yield a replicating AB polymer with a potential chemical phenotype. Self-complementary replication in the pool occurs during a minority (here ≈ 35 %) of synthetic episodes that exploit coincidental overlaps between 4, 5 or 6 spikes of arbitrarily arriving substrates. Such uniquely productive synthetic episodes, in which near-ideal reaction sequences recur at random, account for most AB oligonucleotide synthesis, and therefore underlie the emergence of net replication under realistic primordial conditions. Because overlapping substrate spikes are unexpectedly frequent, and in addition, complex spike sequences appear disproportionately, a sporadically fed pool can host unexpectedly complex syntheses. Thus, primordial substrate fluctuations are not necessarily a barrier to Darwinism, but instead can facilitate early evolution.

Development of bioluminescence imaging of respiratory syncytial virus (RSV) in virus-infected live mice and its use for evaluation of therapeutics and vaccines.

PubMed

Fuentes, Sandra; Arenas, Diego; Moore, Martin M; Golding, Hana; Khurana, Surender

2017-01-23

Respiratory Syncytial virus (RSV) is one of the leading causes of pneumonia among infants with no human vaccine or efficient curative treatments. Efforts are underway to develop new RSV vaccines and therapeutics. There is a dire need for animal models for preclinical evaluation and selection of products against RSV. Herein, we developed a whole body bioluminescence imaging to follow replication of RSV A2 virus strain expressing firefly luciferase (RSVA2-line19-FFL) in live BALB/c mice that can be used as an extremely sensitive readout for studying effects of antiviral and vaccines in living mice. Strong bioluminescence signal was detected in the nasal cavity and in the lungs following intranasal infection of mice with RSVA2-line19-FFL. The kinetics of viral replication in lungs quantified by daily live imaging strongly correlated with viral titers measured by ex-vivo plaque assay and by assessing viral RNA by qRT-PCR. Vaccination of mice with a pre-fusion F protein elicited high neutralizing antibody titers conferring strong protective immunity against virus replication in the nasal cavity and lungs. In contrast, post-challenge treatment of mice with the monoclonal antibody Palivizumab two days after infection reduced viral replication in the nasal cavity at day 4, but only modestly reduced virus loads in the lungs by day 5. In contrast to RSV bioluminescence, plaque assay did not detect viral titers in lungs on day 5 in Palivizumab-treated animals. This difference between viral loads measured by the two assays was found to be due to coating of virions with the Palivizumab that blocked infection of target cells in vitro and shows importance of live imaging in evaluation of RSV therapeutics. This recombinant RSV based live imaging animal model is convenient and valuable tool that can be used to study host dissemination of RSV and evaluation of antiviral compounds and vaccines against RSV. Published by Elsevier Ltd.

Comparative analysis of seven viral nuclear export signals (NESs) reveals the crucial role of nuclear export mediated by the third NES consensus sequence of nucleoprotein (NP) in influenza A virus replication.

PubMed

Chutiwitoonchai, Nopporn; Kakisaka, Michinori; Yamada, Kazunori; Aida, Yoko

2014-01-01

The assembly of influenza virus progeny virions requires machinery that exports viral genomic ribonucleoproteins from the cell nucleus. Currently, seven nuclear export signal (NES) consensus sequences have been identified in different viral proteins, including NS1, NS2, M1, and NP. The present study examined the roles of viral NES consensus sequences and their significance in terms of viral replication and nuclear export. Mutation of the NP-NES3 consensus sequence resulted in a failure to rescue viruses using a reverse genetics approach, whereas mutation of the NS2-NES1 and NS2-NES2 sequences led to a strong reduction in viral replication kinetics compared with the wild-type sequence. While the viral replication kinetics for other NES mutant viruses were also lower than those of the wild-type, the difference was not so marked. Immunofluorescence analysis after transient expression of NP-NES3, NS2-NES1, or NS2-NES2 proteins in host cells showed that they accumulated in the cell nucleus. These results suggest that the NP-NES3 consensus sequence is mostly required for viral replication. Therefore, each of the hydrophobic (Φ) residues within this NES consensus sequence (Φ1, Φ2, Φ3, or Φ4) was mutated, and its viral replication and nuclear export function were analyzed. No viruses harboring NP-NES3 Φ2 or Φ3 mutants could be rescued. Consistent with this, the NP-NES3 Φ2 and Φ3 mutants showed reduced binding affinity with CRM1 in a pull-down assay, and both accumulated in the cell nucleus. Indeed, a nuclear export assay revealed that these mutant proteins showed lower nuclear export activity than the wild-type protein. Moreover, the Φ2 and Φ3 residues (along with other Φ residues) within the NP-NES3 consensus were highly conserved among different influenza A viruses, including human, avian, and swine. Taken together, these results suggest that the Φ2 and Φ3 residues within the NP-NES3 protein are important for its nuclear export function during viral replication.

Primordial evolvability: Impasses and challenges.

PubMed

Vasas, Vera; Fernando, Chrisantha; Szilágyi, András; Zachár, István; Santos, Mauro; Szathmáry, Eörs

2015-09-21

While it is generally agreed that some kind of replicating non-living compounds were the precursors of life, there is much debate over their possible chemical nature. Metabolism-first approaches propose that mutually catalytic sets of simple organic molecules could be capable of self-replication and rudimentary chemical evolution. In particular, the graded autocatalysis replication domain (GARD) model, depicting assemblies of amphiphilic molecules, has received considerable interest. The system propagates compositional information across generations and is suggested to be a target of natural selection. However, evolutionary simulations indicate that the system lacks selectability (i.e. selection has negligible effect on the equilibrium concentrations). We elaborate on the lessons learnt from the example of the GARD model and, more widely, on the issue of evolvability, and discuss the implications for similar metabolism-first scenarios. We found that simple incorporation-type chemistry based on non-covalent bonds, as assumed in GARD, is unlikely to result in alternative autocatalytic cycles when catalytic interactions are randomly distributed. An even more serious problem stems from the lognormal distribution of catalytic factors, causing inherent kinetic instability of such loops, due to the dominance of efficiently catalyzed components that fail to return catalytic aid. Accordingly, the dynamics of the GARD model is dominated by strongly catalytic, but not auto-catalytic, molecules. Without effective autocatalysis, stable hereditary propagation is not possible. Many repetitions and different scaling of the model come to no rescue. Despite all attempts to show the contrary, the GARD model is not evolvable, in contrast to reflexively autocatalytic networks, complemented by rare uncatalyzed reactions and compartmentation. The latter networks, resting on the creation and breakage of chemical bonds, can generate novel ('mutant') autocatalytic loops from a given set of environmentally available compounds. Real chemical reactions that make or break covalent bonds, rather than mere incorporation of components, are necessary for open-ended evolvability. The issue of whether or not several concrete chemical systems (rather than singular curiosities) could realize reflexively autocatalytic macromolecular networks will ultimately determine the relevance of metabolism-first approaches to the origin of life, as stepping stones towards true open-endedness that requires the combination of rich combinatorial chemistry controlled by information stored in template replicators. Copyright © 2015 Elsevier Ltd. All rights reserved.

In Vitro Lesion Bypass Studies of O(4)-Alkylthymidines with Human DNA Polymerase η.

PubMed

Williams, Nicole L; Wang, Pengcheng; Wu, Jiabin; Wang, Yinsheng

2016-04-18

Environmental exposure and endogenous metabolism can give rise to DNA alkylation. Among alkylated nucleosides, O(4)-alkylthymidine (O(4)-alkyldT) lesions are poorly repaired in mammalian systems and may compromise the efficiency and fidelity of cellular DNA replication. To cope with replication-stalling DNA lesions, cells are equipped with translesion synthesis DNA polymerases that are capable of bypassing various DNA lesions. In this study, we assessed human DNA polymerase η (Pol η)-mediated bypass of various O(4)-alkyldT lesions, with the alkyl group being Me, Et, nPr, iPr, nBu, iBu, (R)-sBu, or (S)-sBu, in template DNA by conducting primer extension and steady-state kinetic assays. Our primer extension assay results revealed that human Pol η, but not human polymerases κ and ι or yeast polymerase ζ, was capable of bypassing all O(4)-alkyldT lesions and extending the primer to generate full-length replication products. Data from steady-state kinetic measurements showed that Pol η preferentially misincorporated dGMP opposite O(4)-alkyldT lesions with a straight-chain alkyl group. The nucleotide misincorporation opposite most lesions with a branched-chain alkyl group was, however, not selective, where dCMP, dGMP, and dTMP were inserted at similar efficiencies opposite O(4)-iPrdT, O(4)-iBudT, and O(4)-(R)-sBudT. These results provide important knowledge about the effects of the length and structure of the alkyl group in O(4)-alkyldT lesions on the fidelity and efficiency of DNA replication mediated by human Pol η.

Adeno-associated virus rep protein synthesis during productive infection

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

Redemann, B.E.; Mendelson, E.; Carter, B.J.

1989-02-01

Adeno-associated virus (AAV) Rep proteins mediate viral DNA replication and can regulate expression from AAV genes. The authors studied the kinetics of synthesis of the four Rep proteins, Rep78, Rep68, Rep52, and Rep40, during infection of human 293 or KB cells with AAV and helper adenovirus by in vivo labeling with (/sup 35/S)methionine, immunoprecipitation, and immunoblotting analyses. Rep78 and Rep52 were readily detected concomitantly with detection of viral monomer duplex DNA replicating about 10 to 12 h after infection, and Rep68 and Rep40 were detected 2 h later. Rep78 and Rep52 were more abundant than Rep68 and Rep40 owing tomore » a higher synthesis rate throughout the infectious cycle. In some experiments, very low levels of Rep78 could be detected as early as 4 h after infection. The synthesis rates of Rep proteins were maximal between 14 and 24 h and then decreased later after infection. Isotopic pulse-chase experiments showed that each of the Rep proteins was synthesized independently and was stable for at least 15 h. A slower-migrating, modified form of Rep78 was identified late after infection. AAV capsid protein synthesis was detected at 10 to 12 h after infection and also exhibited synthesis kinetics similar to those of the Rep proteins. AAV DNA replication showed at least two clearly defined stages. Bulk duplex replicating DNA accumulation began around 10 to 12 h and reached a maximum level at about 20 h when Rep and capsid protein synthesis was maximal. Progeny single-stranded DNA accumulation began about 12 to 13 h, but most of this DNA accumulated after 24 h when Rep and capsid protein synthesis had decreased.« less

Transition-state destabilization reveals how human DNA polymerase β proceeds across the chemically unstable lesion N7-methylguanine

PubMed Central

Ouzon-Shubeita, Hala; Lee, Seongmin

2014-01-01

N7-Methyl-2′-deoxyguanosine (m7dG) is the predominant lesion formed by methylating agents. A systematic investigation on the effect of m7dG on DNA replication has been difficult due to the chemical instability of m7dG. To gain insights into the m7dG effect, we employed a 2′-fluorine-mediated transition-state destabilzation strategy. Specifically, we determined kinetic parameters for dCTP insertion opposite a chemically stable m7dG analogue, 2′-fluoro-m7dG (Fm7dG), by human DNA polymerase β (polβ) and solved three X-ray structures of polβ in complex with the templating Fm7dG paired with incoming dCTP or dTTP analogues. The kinetic studies reveal that the templating Fm7dG slows polβ catalysis ∼300-fold, suggesting that m7dG in genomic DNA may impede replication by some DNA polymerases. The structural analysis reveals that Fm7dG forms a canonical Watson–Crick base pair with dCTP, but metal ion coordination is suboptimal for catalysis in the polβ-Fm7dG:dCTP complex, which partially explains the slow insertion of dCTP opposite Fm7dG by polβ. In addition, the polβ-Fm7dG:dTTP structure shows open protein conformations and staggered base pair conformations, indicating that N7-methylation of dG does not promote a promutagenic replication. Overall, the first systematic studies on the effect of m7dG on DNA replication reveal that polβ catalysis across m7dG is slow, yet highly accurate. PMID:24966350

Zika Virus Antagonizes Type I Interferon Responses during Infection of Human Dendritic Cells

PubMed Central

Maddur, Mohan S.; O’Neal, Justin T.; Fedorova, Nadia B.; Puri, Vinita; Pulendran, Bali; Suthar, Mehul S.

2017-01-01

Zika virus (ZIKV) is an emerging mosquito-borne flavivirus that is causally linked to severe neonatal birth defects, including microcephaly, and is associated with Guillain-Barre syndrome in adults. Dendritic cells (DCs) are an important cell type during infection by multiple mosquito-borne flaviviruses, including dengue virus, West Nile virus, Japanese encephalitis virus, and yellow fever virus. Despite this, the interplay between ZIKV and DCs remains poorly defined. Here, we found human DCs supported productive infection by a contemporary Puerto Rican isolate with considerable variability in viral replication, but not viral binding, between DCs from different donors. Historic isolates from Africa and Asia also infected DCs with distinct viral replication kinetics between strains. African lineage viruses displayed more rapid replication kinetics and infection magnitude as compared to Asian lineage viruses, and uniquely induced cell death. Infection of DCs with both contemporary and historic ZIKV isolates led to minimal up-regulation of T cell co-stimulatory and MHC molecules, along with limited secretion of inflammatory cytokines. Inhibition of type I interferon (IFN) protein translation was observed during ZIKV infection, despite strong induction at the RNA transcript level and up-regulation of other host antiviral proteins. Treatment of human DCs with RIG-I agonist potently restricted ZIKV replication, while type I IFN had only modest effects. Mechanistically, we found all strains of ZIKV antagonized type I IFN-mediated phosphorylation of STAT1 and STAT2. Combined, our findings show that ZIKV subverts DC immunogenicity during infection, in part through evasion of type I IFN responses, but that the RLR signaling pathway is still capable of inducing an antiviral state, and therefore may serve as an antiviral therapeutic target. PMID:28152048

Replication and Immunogenicity of Swine, Equine, and Avian H3 Subtype Influenza Viruses in Mice and Ferrets

PubMed Central

Baz, Mariana; Paskel, Myeisha; Matsuoka, Yumiko; Zengel, James; Cheng, Xing; Jin, Hong

2013-01-01

Since it is difficult to predict which influenza virus subtype will cause an influenza pandemic, it is important to prepare influenza virus vaccines against different subtypes and evaluate the safety and immunogenicity of candidate vaccines in preclinical and clinical studies prior to a pandemic. In addition to infecting humans, H3 influenza viruses commonly infect pigs, horses, and avian species. We selected 11 swine, equine, and avian H3 influenza viruses and evaluated their kinetics of replication and ability to induce a broadly cross-reactive antibody response in mice and ferrets. The swine and equine viruses replicated well in the upper respiratory tract of mice. With the exception of one avian virus that replicated poorly in the lower respiratory tract, all of the viruses replicated in mouse lungs. In ferrets, all of the viruses replicated well in the upper respiratory tract, but the equine viruses replicated poorly in the lungs. Extrapulmonary spread was not observed in either mice or ferrets. No single virus elicited antibodies that cross-reacted with viruses from all three animal sources. Avian and equine H3 viruses elicited broadly cross-reactive antibodies against heterologous viruses isolated from the same or other species, but the swine viruses did not. We selected an equine and an avian H3 influenza virus for further development as vaccines. PMID:23576512

Replicating Health Economic Models: Firm Foundations or a House of Cards?

PubMed

Bermejo, Inigo; Tappenden, Paul; Youn, Ji-Hee

2017-11-01

Health economic evaluation is a framework for the comparative analysis of the incremental health gains and costs associated with competing decision alternatives. The process of developing health economic models is usually complex, financially expensive and time-consuming. For these reasons, model development is sometimes based on previous model-based analyses; this endeavour is usually referred to as model replication. Such model replication activity may involve the comprehensive reproduction of an existing model or 'borrowing' all or part of a previously developed model structure. Generally speaking, the replication of an existing model may require substantially less effort than developing a new de novo model by bypassing, or undertaking in only a perfunctory manner, certain aspects of model development such as the development of a complete conceptual model and/or comprehensive literature searching for model parameters. A further motivation for model replication may be to draw on the credibility or prestige of previous analyses that have been published and/or used to inform decision making. The acceptability and appropriateness of replicating models depends on the decision-making context: there exists a trade-off between the 'savings' afforded by model replication and the potential 'costs' associated with reduced model credibility due to the omission of certain stages of model development. This paper provides an overview of the different levels of, and motivations for, replicating health economic models, and discusses the advantages, disadvantages and caveats associated with this type of modelling activity. Irrespective of whether replicated models should be considered appropriate or not, complete replicability is generally accepted as a desirable property of health economic models, as reflected in critical appraisal checklists and good practice guidelines. To this end, the feasibility of comprehensive model replication is explored empirically across a small number of recent case studies. Recommendations are put forward for improving reporting standards to enhance comprehensive model replicability.

Expression kinetics of key genes in the early innate immune response to Great Lakes viral hemorrhagic septicemia virus IVb infection in yellow perch (Perca flavescens)

USGS Publications Warehouse

Olson, Wendy; Emmenegger, Eveline; Glenn, Jolene; Simchick, Crystal; Winton, Jim; Goetz, Frederick

2013-01-01

The recently discovered strain of viral hemorrhagic septicemia virus, VHSV-IVb, represents an example of the introduction of an extremely pathogenic rhabdovirus capable of infecting a wide variety of new fish species in a new host-environment. The goal of the present study was to delineate the expression kinetics of key genes in the innate immune response relative to the very early stages of VHSV-IVb infection using the yellow perch (Perca flavescens) as a model. Administration of VHSV-IVb by IP-injection into juvenile yellow perch resulted in 84% cumulative mortality, indicating their high susceptibility to this disease. In fish sampled in the very early stages of infection, a significant up-regulation of Mx gene expression in the liver, as well as IL-1β and SAA activation in the head kidney, spleen, and liver was directly correlated to viral load. The potential down-regulation of Mx in the hematopoietic tissues, head kidney and spleen, may represent a strategy utilized by the virus to increase replication.

Simulation of the M13 life cycle I: Assembly of a genetically-structured deterministic chemical kinetic simulation.

PubMed

Smeal, Steven W; Schmitt, Margaret A; Pereira, Ronnie Rodrigues; Prasad, Ashok; Fisk, John D

2017-01-01

To expand the quantitative, systems level understanding and foster the expansion of the biotechnological applications of the filamentous bacteriophage M13, we have unified the accumulated quantitative information on M13 biology into a genetically-structured, experimentally-based computational simulation of the entire phage life cycle. The deterministic chemical kinetic simulation explicitly includes the molecular details of DNA replication, mRNA transcription, protein translation and particle assembly, as well as the competing protein-protein and protein-nucleic acid interactions that control the timing and extent of phage production. The simulation reproduces the holistic behavior of M13, closely matching experimentally reported values of the intracellular levels of phage species and the timing of events in the M13 life cycle. The computational model provides a quantitative description of phage biology, highlights gaps in the present understanding of M13, and offers a framework for exploring alternative mechanisms of regulation in the context of the complete M13 life cycle. Copyright © 2016 Elsevier Inc. All rights reserved.

3D replicon distributions arise from stochastic initiation and domino-like DNA replication progression.

PubMed

Löb, D; Lengert, N; Chagin, V O; Reinhart, M; Casas-Delucchi, C S; Cardoso, M C; Drossel, B

2016-04-07

DNA replication dynamics in cells from higher eukaryotes follows very complex but highly efficient mechanisms. However, the principles behind initiation of potential replication origins and emergence of typical patterns of nuclear replication sites remain unclear. Here, we propose a comprehensive model of DNA replication in human cells that is based on stochastic, proximity-induced replication initiation. Critical model features are: spontaneous stochastic firing of individual origins in euchromatin and facultative heterochromatin, inhibition of firing at distances below the size of chromatin loops and a domino-like effect by which replication forks induce firing of nearby origins. The model reproduces the empirical temporal and chromatin-related properties of DNA replication in human cells. We advance the one-dimensional DNA replication model to a spatial model by taking into account chromatin folding in the nucleus, and we are able to reproduce the spatial and temporal characteristics of the replication foci distribution throughout S-phase.

Second-order advantage obtained from standard addition first-order instrumental data and multivariate curve resolution-alternating least squares. Calculation of the feasible bands of results.

PubMed

Mohseni, Naimeh; Bahram, Morteza; Olivieri, Alejandro C

2014-03-25

In order to achieve the second-order advantage, second-order data per sample is usually required, e.g., kinetic-spectrophotometric data. In this study, instead of monitoring the time evolution of spectra (and collecting the kinetic-spectrophotometric data) replicate spectra are used to build a virtual second order data. This data matrix (replicate mode×λ) is rank deficient. Augmentation of these data with standard addition data [or standard sample(s)] will break the rank deficiency, making the quantification of the analyte of interest possible. The MCR-ALS algorithm was applied for the resolution and quantitation of the analyte in both simulated and experimental data sets. In order to evaluate the rotational ambiguity in the retrieved solutions, the MCR-BANDS algorithm was employed. It has been shown that the reliability of the quantitative results significantly depends on the amount of spectral overlap in the spectral region of occurrence of the compound of interest and the remaining constituent(s). Copyright © 2013 Elsevier B.V. All rights reserved.

The significance of intergranular diffusion to the mechanisms and kinetics of porphyroblast crystallization

NASA Astrophysics Data System (ADS)

Carlson, William D.

1989-09-01

The spatial disposition, compositional zoning profiles, and size distributions of garnet crystals in 11 specimens of pelitic schist from the Picuris Range of New Mexico (USA) demonstrate that the kinetics of intergranular diffusion controlled the nucleation and growth mechanisms of porphyroblasts in these rocks. An ordered disposition of garnet centers and a significant correlation between crystal radius and near-neighbor distances manifest suppressed nucleation of new crystals in diffusionally depleted zones surrounding pre-existing crystals. Compositional zoning profiles require diffusionally controlled growth, the rate of which increases exponentially as temperature increases with time; an acceleration factor for growth rate can be estimated from a comparison of compositional profiles for crystals of different sizes in each specimen. Crystal size distributions are interpreted as the result of nucleation rates that accelerate exponentially with increasing temperature early in the crystallization process, but decline in the later stages because of suppression effects in the vicinity of earlier-formed nuclei. Simulations of porphyroblast crystallization, based upon thermally accelerated diffusionally influenced nucleation kinetics and diffusionally controlled growth kinetics, quantitatively replicate textural relations in the rocks. The simulations employ only two variable parameters, which are evaluated by fitting of crystal size distributions. Both have physical significance. The first is an acceleration factor for nucleation, with a magnitude reflecting the prograde increase during the nucleation interval of the chemical affinity for the reaction in undepleted regions of the rock. The second is a measure of the relative sizes of the porphyroblast and the diffusionally depleted zone surrounding it. Crystal size distributions for the Picuris Range garnets correspond very closely to those in the literature from a variety of other localities for garnet and other minerals. The same kinetic model accounts quantitatively for crystal size distributions of porphyroblastic garnet, phlogopite, sphene, and pyroxene in rocks from both regional and contact metamorphic occurrences. These commonalities indicate that intergranular diffusion may be the dominant kinetic factor in the crystallization of porphyroblasts in a wide variety of metamorphic environments.

Identification of novel inhibitors of non-replicating Mycobacterium tuberculosis using a carbon starvation model

PubMed Central

Grant, Sarah Schmidt; Kawate, Tomohiko; Nag, Partha P.; Silvis, Melanie R.; Gordon, Katherine; Stanley, Sarah A.; Kazyanskaya, Ed; Nietupski, Ray; Golas, Aaron; Fitzgerald, Michael; Cho, Sanghyun; Franzblau, Scott G.; Hung, Deborah T.

2013-01-01

During Mycobacterium tuberculosis infection, a population of bacteria is thought to exist in a non-replicating state, refractory to antibiotics, which may contribute to the need for prolonged antibiotic therapy. The identification of inhibitors of the non-replicating state provides tools that can be used to probe this hypothesis and the physiology of this state. The development of such inhibitors also has the potential to shorten the duration of antibiotic therapy required. Here we describe the development of a novel non-replicating assay amenable to high-throughput chemical screening coupled with secondary assays that use carbon starvation as the in vitro model. Together these assays identify compounds with activity against replicating and non-replicating M. tuberculosis as well as compounds that inhibit the transition from non-replicating to replicating stages of growth. Using these assays we successfully screened over 300,000 compounds and identified 786 inhibitors of non-replicating M. tuberculosis. In order to understand the relationship among different non-replicating models, we teste 52 of these molecules in a hypoxia model and four different chemical scaffolds in a stochastic persist model and a streptomycin dependent model. We found that compounds display varying levels of activity in different models for the non-replicating state, suggesting important differences in bacterial physiology between models. Therefore, chemical tools identified in this assay may be useful for determining the relevance of different non-replicating in vitro models to in vivo M. tuberculosis infection. Given our current limited understanding, molecules that are active across multiple models may represent more promising candidates for further development. PMID:23898841

3D replicon distributions arise from stochastic initiation and domino-like DNA replication progression

PubMed Central

Löb, D.; Lengert, N.; Chagin, V. O.; Reinhart, M.; Casas-Delucchi, C. S.; Cardoso, M. C.; Drossel, B.

2016-01-01

DNA replication dynamics in cells from higher eukaryotes follows very complex but highly efficient mechanisms. However, the principles behind initiation of potential replication origins and emergence of typical patterns of nuclear replication sites remain unclear. Here, we propose a comprehensive model of DNA replication in human cells that is based on stochastic, proximity-induced replication initiation. Critical model features are: spontaneous stochastic firing of individual origins in euchromatin and facultative heterochromatin, inhibition of firing at distances below the size of chromatin loops and a domino-like effect by which replication forks induce firing of nearby origins. The model reproduces the empirical temporal and chromatin-related properties of DNA replication in human cells. We advance the one-dimensional DNA replication model to a spatial model by taking into account chromatin folding in the nucleus, and we are able to reproduce the spatial and temporal characteristics of the replication foci distribution throughout S-phase. PMID:27052359

Cancer-adipose tissue interaction and fluid flow synergistically modulate cell kinetics, HER2 expression, and trastuzumab efficacy in gastric cancer.

PubMed

Akutagawa, Takashi; Aoki, Shigehisa; Yamamoto-Rikitake, Mihoko; Iwakiri, Ryuichi; Fujimoto, Kazuma; Toda, Shuji

2018-04-25

Early local tumor invasion in gastric cancer results in likely encounters between cancer cells and submucosal and subserosal adipose tissue, but these interactions remain to be clarified. Microenvironmental mechanical forces, such as fluid flow, are known to modulate normal cell kinetics, but the effects of fluid flow on gastric cancer cells are poorly understood. We analyzed the cell kinetics and chemosensitivity in gastric cancer using a simple in vitro model that simultaneously replicated the cancer-adipocyte interaction and physical microenvironment. Gastric cancer cells (MKN7 and MKN74) were seeded on rat adipose tissue fragment-embedded discs or collagen discs alone. To generate fluid flow, samples were placed on a rotatory shaker in a CO 2 incubator. Proliferation, apoptosis, invasion, and motility-related molecules were analyzed by morphometry and immunostaining. Proteins were evaluated by western blot analysis. Chemosensitivity was investigated by trastuzumab treatment. Adipose tissue and fluid flow had a positive synergistic effect on the proliferative potential and invasive capacity of gastric cancer cells, and adipose tissue inhibited apoptosis in these cells. Adipose tissue upregulated ERK1/2 signaling in gastric cancer cells, but downregulated p38 signaling. Notably, adipose tissue and fluid flow promoted membranous and cytoplasmic HER2 expression and modulated chemosensitivity to trastuzumab in gastric cancer cells. We have demonstrated that cancer-adipocyte interaction and physical microenvironment mutually modulate gastric cancer cell kinetics. Further elucidation of the microenvironmental regulation in gastric cancer will be very important for the development of strategies involving molecular targeted therapy.

Pre-clinical evaluation of a replication-competent recombinant adenovirus serotype 4 vaccine expressing influenza H5 hemagglutinin.

PubMed

Alexander, Jeff; Ward, Simone; Mendy, Jason; Manayani, Darly J; Farness, Peggy; Avanzini, Jenny B; Guenther, Ben; Garduno, Fermin; Jow, Lily; Snarsky, Victoria; Ishioka, Glenn; Dong, Xin; Vang, Lo; Newman, Mark J; Mayall, Tim

2012-01-01

Influenza virus remains a significant health and social concern in part because of newly emerging strains, such as avian H5N1 virus. We have developed a prototype H5N1 vaccine using a recombinant, replication-competent Adenovirus serotype 4 (Ad4) vector, derived from the U.S. military Ad4 vaccine strain, to express the hemagglutinin (HA) gene from A/Vietnam/1194/2004 influenza virus (Ad4-H5-Vtn). Our hypothesis is that a mucosally-delivered replicating Ad4-H5-Vtn recombinant vector will be safe and induce protective immunity against H5N1 influenza virus infection and disease pathogenesis. The Ad4-H5-Vtn vaccine was designed with a partial deletion of the E3 region of Ad4 to accommodate the influenza HA gene. Replication and growth kinetics of the vaccine virus in multiple human cell lines indicated that the vaccine virus is attenuated relative to the wild type virus. Expression of the HA transgene in infected cells was documented by flow cytometry, western blot analysis and induction of HA-specific antibody and cellular immune responses in mice. Of particular note, mice immunized intranasally with the Ad4-H5-Vtn vaccine were protected against lethal H5N1 reassortant viral challenge even in the presence of pre-existing immunity to the Ad4 wild type virus. Several non-clinical attributes of this vaccine including safety, induction of HA-specific humoral and cellular immunity, and efficacy were demonstrated using an animal model to support Phase 1 clinical trial evaluation of this new vaccine.

Cell cycle- and chaperone-mediated regulation of H3K56ac incorporation in yeast.

PubMed

Kaplan, Tommy; Liu, Chih Long; Erkmann, Judith A; Holik, John; Grunstein, Michael; Kaufman, Paul D; Friedman, Nir; Rando, Oliver J

2008-11-01

Acetylation of histone H3 lysine 56 is a covalent modification best known as a mark of newly replicated chromatin, but it has also been linked to replication-independent histone replacement. Here, we measured H3K56ac levels at single-nucleosome resolution in asynchronously growing yeast cultures, as well as in yeast proceeding synchronously through the cell cycle. We developed a quantitative model of H3K56ac kinetics, which shows that H3K56ac is largely explained by the genomic replication timing and the turnover rate of each nucleosome, suggesting that cell cycle profiles of H3K56ac should reveal most first-time nucleosome incorporation events. However, since the deacetylases Hst3/4 prevent use of H3K56ac as a marker for histone deposition during M phase, we also directly measured M phase histone replacement rates. We report a global decrease in turnover rates during M phase and a further specific decrease in turnover at several early origins of replication, which switch from rapidly replaced in G1 phase to stably bound during M phase. Finally, by measuring H3 replacement in yeast deleted for the H3K56 acetyltransferase Rtt109 and its two co-chaperones Asf1 and Vps75, we find evidence that Rtt109 and Asf1 preferentially enhance histone replacement at rapidly replaced nucleosomes, whereas Vps75 appears to inhibit histone turnover at those loci. These results provide a broad perspective on histone replacement/incorporation throughout the cell cycle and suggest that H3K56 acetylation provides a positive-feedback loop by which replacement of a nucleosome enhances subsequent replacement at the same location.

Comparative Study of Influenza Virus Replication in MDCK Cells and in Primary Cells Derived from Adenoids and Airway Epithelium

PubMed Central

Ikizler, Mine R.; Kawaoka, Yoshihiro; Rudenko, Larisa G.; Treanor, John J.; Subbarao, Kanta; Wright, Peter F.

2012-01-01

Although clinical trials with human subjects are essential for determination of safety, infectivity, and immunogenicity, it is desirable to know in advance the infectiousness of potential candidate live attenuated influenza vaccine strains for human use. We compared the replication kinetics of wild-type and live attenuated influenza viruses, including H1N1, H3N2, H9N2, and B strains, in Madin-Darby canine kidney (MDCK) cells, primary epithelial cells derived from human adenoids, and human bronchial epithelium (NHBE cells). Our data showed that despite the fact that all tissue culture models lack a functional adaptive immune system, differentiated cultures of human epithelium exhibited the greatest restriction for all H1N1, H3N2, and B vaccine viruses studied among three cell types tested and the best correlation with their levels of attenuation seen in clinical trials with humans. In contrast, the data obtained with MDCK cells were the least predictive of restricted viral replication of live attenuated vaccine viruses in humans. We were able to detect a statistically significant difference between the replication abilities of the U.S. (A/Ann Arbor/6/60) and Russian (A/Leningrad/134/17/57) cold-adapted vaccine donor strains in NHBE cultures. Since live attenuated pandemic influenza vaccines may potentially express a hemagglutinin and neuraminidase from a non-human influenza virus, we assessed which of the three cell cultures could be used to optimally evaluate the infectivity and cellular tropism of viruses derived from different hosts. Among the three cell types tested, NHBE cultures most adequately reflected the infectivity and cellular tropism of influenza virus strains with different receptor specificities. NHBE cultures could be considered for use as a screening step for evaluating the restricted replication of influenza vaccine candidates. PMID:22915797

Replication of surface features from a master model to an amorphous metallic article

DOEpatents

Johnson, William L.; Bakke, Eric; Peker, Atakan

1999-01-01

The surface features of an article are replicated by preparing a master model having a preselected surface feature thereon which is to be replicated, and replicating the preselected surface feature of the master model. The replication is accomplished by providing a piece of a bulk-solidifying amorphous metallic alloy, contacting the piece of the bulk-solidifying amorphous metallic alloy to the surface of the master model at an elevated replication temperature to transfer a negative copy of the preselected surface feature of the master model to the piece, and separating the piece having the negative copy of the preselected surface feature from the master model.

A kinetic clutch governs uncoiling by type IB topoisomerases

NASA Astrophysics Data System (ADS)

Neuman, Keir

2013-03-01

Type IB topoisomerases (Top1B) are essential enzymes that relax excessive DNA supercoiling associated with replication and transcription and are important drug targets for cancer chemotherapy. The natural compound camptothecin (CPT) and the cancer chemotherapeutics derived from it, irinotecan and topotecan, are highly specific inhibitors of human nuclear Type IB topoisomerase (nTop1). We employed a magnetic-tweezers based single-molecule DNA supercoil relaxation assay to measure the torque dependence of human nuclear Top1 relaxation (nTop1) and inhibition by CPT. For comparison, we examined the human mitochondrial (Top1mt) topoisomerase and an N-terminal deletion mutant of nTop1 (Top68). Despite substantial sequence homology in their core domains, nTop1 and Top1mt exhibit dramatic differences in sensitivity to torque and CPT, with Top68 betraying intermediate characteristics. In particular, nTop1 displays nearly torque-independent religation probability, distinguishing it from other Top1B enzymes studied to date. Kinetic modeling reveals a hitherto unobserved torque-independent transition linking the DNA rotation and religation phases of the enzymatic cycle. The parameters of this transition determine the torque sensitivity of religation, and the efficiency of CPT binding. This ``kinetic clutch'' mechanism explains the molecular basis of CPT sensitivity and more generally provides a framework with which to interpret Top1B activity and inhibition.

The formation of double-strand breaks at multiply damaged sites is driven by the kinetics of excision/incision at base damage in eukaryotic cells

PubMed Central

Kozmin, Stanislav G.; Sedletska, Yuliya; Reynaud-Angelin, Anne; Gasparutto, Didier; Sage, Evelyne

2009-01-01

It has been stipulated that repair of clustered DNA lesions may be compromised, possibly leading to the formation of double-strand breaks (DSB) and, thus, to deleterious events. Using a variety of model multiply damaged sites (MDS), we investigated parameters that govern the formation of DSB during the processing of MDS. Duplexes carrying MDS were inserted into replicative or integrative vectors, and used to transform yeast Saccharomyces cerevisiae. Formation of DSB was assessed by a relevant plasmid survival assay. Kinetics of excision/incision and DSB formation at MDS was explored using yeast cell extracts. We show that MDS composed of two uracils or abasic sites, were rapidly incised and readily converted into DSB in yeast cells. In marked contrast, none of the MDS carrying opposed oG and hU separated by 3–8 bp gave rise to DSB, despite the fact that some of them contained preexisting single-strand break (a 1-nt gap). Interestingly, the absence of DSB formation in this case correlated with slow excision/incision rates of lesions. We propose that the kinetics of the initial repair steps at MDS is a major parameter that direct towards the conversion of MDS into DSB. Data provides clues to the biological consequences of MDS in eukaryotic cells. PMID:19174565

Excellent amino acid racemization results from Holocene sand dollars

NASA Astrophysics Data System (ADS)

Kosnik, M.; Kaufman, D. S.; Kowalewski, M.; Whitacre, K.

2015-12-01

Amino acid racemization (AAR) is widely used as a cost-effective method to date molluscs in time-averaging and taphonomic studies, but it has not been attempted for echinoderms despite their paleobiological importance. Here we demonstrate the feasibility of AAR geochronology in Holocene aged Peronella peronii (Echinodermata: Echinoidea) collected from Sydney Harbour (Australia). Using standard HPLC methods we determined the extent of AAR in 74 Peronella tests and performed replicate analyses on 18 tests. We sampled multiple areas of two individuals and identified the outer edge as a good sampling location. Multiple replicate analyses from the outer edge of 18 tests spanning the observed range of D/Ls yielded median coefficients of variation < 4% for Asp, Phe, Ala, and Glu D/L values, which overlaps with the analytical precision. Correlations between D/L values across 155 HPLC injections sampled from 74 individuals are also very high (pearson r2 > 0.95) for these four amino acids. The ages of 11 individuals spanning the observed range of D/L values were determined using 14C analyses, and Bayesian model averaging was used to determine the best AAR age model. The averaged age model was mainly composed of time-dependent reaction kinetics models (TDK, 71%) based on phenylalanine (Phe, 94%). Modelled ages ranged from 14 to 5539 yrs, and the median 95% confidence interval for the 74 analysed individuals is ±28% of the modelled age. In comparison, the median 95% confidence interval for the 11 calibrated 14C ages was ±9% of the median age estimate. Overall Peronella yields exceptionally high-quality AAR D/L values and appears to be an excellent substrate for AAR geochronology. This work opens the way for time-averaging and taphonomic studies of echinoderms similar to those in molluscs.

Cross-Neutralization between Human and African Bat Mumps Viruses.

PubMed

Katoh, Hiroshi; Kubota, Toru; Ihara, Toshiaki; Maeda, Ken; Takeda, Makoto; Kidokoro, Minoru

2016-04-01

Recently, a new paramyxovirus closely related to human mumps virus (MuV) was detected in bats. We generated recombinant MuVs carrying either or both of the fusion and hemagglutinin-neuraminidase bat virus glycoproteins. These viruses showed replication kinetics similar to human MuV in cultured cells and were neutralized efficiently by serum from healthy humans.

Two Asian highly pathogenic strains of Type 2 PRRSV in United States swine

USDA-ARS?s Scientific Manuscript database

Highly pathogenic PRRSV (HP-PRRSV) has been circulating in Asia for 7 years. rJXwn06 and rSRV07 were rescued from infectious clones of two HP-PRRSV for investigation at the National Animal Disease Center. The clinical disease and viral replication kinetics of HP-PRRSV were compared to prototype stra...

Cosegregation of chromosomes containing immortal DNA strands in cells that cycle with asymmetric stem cell kinetics.

PubMed

Merok, Joshua R; Lansita, Janice A; Tunstead, James R; Sherley, James L

2002-12-01

A long-standing intriguing hypothesis in cancer biology is that adult stem cells avoid mutations from DNA replication errors by a unique pattern of chromosome segregation. At each asymmetric cell division, adult stem cells have been postulated to selectively retain a set of chromosomes that contain old template DNA strands (i.e., "immortal DNA strands"). Using cultured cells that cycle with asymmetric cell kinetics, we confirmed both the existence of immortal DNA strands and the cosegregation of chromosomes that bear them. Our findings also lead us to propose a role for immortal DNA strands in tissue aging as well as cancer.

Generation of Recombinant Polioviruses Harboring RNA Affinity Tags in the 5′ and 3′ Noncoding Regions of Genomic RNAs

PubMed Central

Flather, Dylan; Cathcart, Andrea L.; Cruz, Casey; Baggs, Eric; Ngo, Tuan; Gershon, Paul D.; Semler, Bert L.

2016-01-01

Despite being intensely studied for more than 50 years, a complete understanding of the enterovirus replication cycle remains elusive. Specifically, only a handful of cellular proteins have been shown to be involved in the RNA replication cycle of these viruses. In an effort to isolate and identify additional cellular proteins that function in enteroviral RNA replication, we have generated multiple recombinant polioviruses containing RNA affinity tags within the 3′ or 5′ noncoding region of the genome. These recombinant viruses retained RNA affinity sequences within the genome while remaining viable and infectious over multiple passages in cell culture. Further characterization of these viruses demonstrated that viral protein production and growth kinetics were unchanged or only slightly altered relative to wild type poliovirus. However, attempts to isolate these genetically-tagged viral genomes from infected cells have been hindered by high levels of co-purification of nonspecific proteins and the limited matrix-binding efficiency of RNA affinity sequences. Regardless, these recombinant viruses represent a step toward more thorough characterization of enterovirus ribonucleoprotein complexes involved in RNA replication. PMID:26861382

CCR5 Signal Transduction in Macrophages by Human Immunodeficiency Virus and Simian Immunodeficiency Virus Envelopes

PubMed Central

Arthos, James; Rubbert, Andrea; Rabin, Ronald L.; Cicala, Claudia; Machado, Elizabeth; Wildt, Kathryne; Hanbach, Meredith; Steenbeke, Tavis D.; Swofford, Ruth; Farber, Joshua M.; Fauci, Anthony S.

2000-01-01

The capacity of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) envelopes to transduce signals through chemokine coreceptors on macrophages was examined by measuring the ability of recombinant envelope proteins to mobilize intracellular calcium stores. Both HIV and SIV envelopes mobilized calcium via interactions with CCR5. The kinetics of these responses were similar to those observed when macrophages were treated with MIP-1β. Distinct differences in the capacity of envelopes to mediate calcium mobilization were observed. Envelopes derived from viruses capable of replicating in macrophages mobilized relatively high levels of calcium, while envelopes derived from viruses incapable of replicating in macrophages mobilized relatively low levels of calcium. The failure to efficiently mobilize calcium was not restricted to envelopes derived from CXCR4-utilizing isolates but also included envelopes derived from CCR5-utilizing isolates that fail to replicate in macrophages. We characterized one CCR5-utilizing isolate, 92MW959, which entered macrophages but failed to replicate. A recombinant envelope derived from this virus mobilized low levels of calcium. When macrophages were inoculated with 92MW959 in the presence of MIP-1α, viral replication was observed, indicating that a CC chemokine-mediated signal provided the necessary stimulus to allow the virus to complete its replication cycle. Although the role that envelope-CCR5 signal transduction plays in viral replication is not yet understood, it has been suggested that envelope-mediated signals facilitate early postfusion events in viral replication. The data presented here are consistent with this hypothesis and suggest that the differential capacity of viral envelopes to signal through CCR5 may influence their ability to replicate in macrophages. PMID:10864653

CCR5 signal transduction in macrophages by human immunodeficiency virus and simian immunodeficiency virus envelopes.

PubMed

Arthos, J; Rubbert, A; Rabin, R L; Cicala, C; Machado, E; Wildt, K; Hanbach, M; Steenbeke, T D; Swofford, R; Farber, J M; Fauci, A S

2000-07-01

The capacity of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) envelopes to transduce signals through chemokine coreceptors on macrophages was examined by measuring the ability of recombinant envelope proteins to mobilize intracellular calcium stores. Both HIV and SIV envelopes mobilized calcium via interactions with CCR5. The kinetics of these responses were similar to those observed when macrophages were treated with MIP-1beta. Distinct differences in the capacity of envelopes to mediate calcium mobilization were observed. Envelopes derived from viruses capable of replicating in macrophages mobilized relatively high levels of calcium, while envelopes derived from viruses incapable of replicating in macrophages mobilized relatively low levels of calcium. The failure to efficiently mobilize calcium was not restricted to envelopes derived from CXCR4-utilizing isolates but also included envelopes derived from CCR5-utilizing isolates that fail to replicate in macrophages. We characterized one CCR5-utilizing isolate, 92MW959, which entered macrophages but failed to replicate. A recombinant envelope derived from this virus mobilized low levels of calcium. When macrophages were inoculated with 92MW959 in the presence of MIP-1alpha, viral replication was observed, indicating that a CC chemokine-mediated signal provided the necessary stimulus to allow the virus to complete its replication cycle. Although the role that envelope-CCR5 signal transduction plays in viral replication is not yet understood, it has been suggested that envelope-mediated signals facilitate early postfusion events in viral replication. The data presented here are consistent with this hypothesis and suggest that the differential capacity of viral envelopes to signal through CCR5 may influence their ability to replicate in macrophages.

Replication-Competent Influenza A and B Viruses Expressing a Fluorescent Dynamic Timer Protein for In Vitro and In Vivo Studies

PubMed Central

Baker, Steven F.; Perez, Daniel R.; Martínez-Sobrido, Luis

2016-01-01

Influenza A and B viruses (IAV and IBV, respectively) cause annual seasonal human respiratory disease epidemics. In addition, IAVs have been implicated in occasional pandemics with inordinate health and economic consequences. Studying influenza viruses in vitro or in vivo requires the use of laborious secondary methodologies to identify infected cells. To circumvent this requirement, replication-competent infectious influenza viruses expressing an easily traceable fluorescent reporter protein can be used. Timer is a fluorescent protein that undergoes a time-dependent color emission conversion from green to red. The rate of spectral change is independent of Timer protein concentration and can be used to chronologically measure the duration of its expression. Here, we describe the generation of replication-competent IAV and IBV where the viral non-structural protein 1 (NS1) was fused to the fluorescent dynamic Timer protein. Timer-expressing IAV and IBV displayed similar plaque phenotypes and growth kinetics to wild-type viruses in tissue culture. Within infected cells, Timer’s spectral shift can be used to measure the rate and cell-to-cell spread of infection using fluorescent microscopy, plate readers, or flow cytometry. The progression of Timer-expressing IAV infection was also evaluated in a mouse model, demonstrating the feasibility to characterize IAV cell-to-cell infections in vivo. By providing the ability to chronologically track viral spread, Timer-expressing influenza viruses are an excellent option to evaluate the in vitro and in vivo dynamics of viral infection. PMID:26809059

Replication-Competent Influenza A and B Viruses Expressing a Fluorescent Dynamic Timer Protein for In Vitro and In Vivo Studies.

PubMed

Breen, Michael; Nogales, Aitor; Baker, Steven F; Perez, Daniel R; Martínez-Sobrido, Luis

2016-01-01

Influenza A and B viruses (IAV and IBV, respectively) cause annual seasonal human respiratory disease epidemics. In addition, IAVs have been implicated in occasional pandemics with inordinate health and economic consequences. Studying influenza viruses in vitro or in vivo requires the use of laborious secondary methodologies to identify infected cells. To circumvent this requirement, replication-competent infectious influenza viruses expressing an easily traceable fluorescent reporter protein can be used. Timer is a fluorescent protein that undergoes a time-dependent color emission conversion from green to red. The rate of spectral change is independent of Timer protein concentration and can be used to chronologically measure the duration of its expression. Here, we describe the generation of replication-competent IAV and IBV where the viral non-structural protein 1 (NS1) was fused to the fluorescent dynamic Timer protein. Timer-expressing IAV and IBV displayed similar plaque phenotypes and growth kinetics to wild-type viruses in tissue culture. Within infected cells, Timer's spectral shift can be used to measure the rate and cell-to-cell spread of infection using fluorescent microscopy, plate readers, or flow cytometry. The progression of Timer-expressing IAV infection was also evaluated in a mouse model, demonstrating the feasibility to characterize IAV cell-to-cell infections in vivo. By providing the ability to chronologically track viral spread, Timer-expressing influenza viruses are an excellent option to evaluate the in vitro and in vivo dynamics of viral infection.

Using Model Replication to Improve the Reliability of Agent-Based Models

NASA Astrophysics Data System (ADS)

Zhong, Wei; Kim, Yushim

The basic presupposition of model replication activities for a computational model such as an agent-based model (ABM) is that, as a robust and reliable tool, it must be replicable in other computing settings. This assumption has recently gained attention in the community of artificial society and simulation due to the challenges of model verification and validation. Illustrating the replication of an ABM representing fraudulent behavior in a public service delivery system originally developed in the Java-based MASON toolkit for NetLogo by a different author, this paper exemplifies how model replication exercises provide unique opportunities for model verification and validation process. At the same time, it helps accumulate best practices and patterns of model replication and contributes to the agenda of developing a standard methodological protocol for agent-based social simulation.

In vivo imaging of cidofovir treatment of cowpox virus infection.

PubMed

Goff, Arthur; Twenhafel, Nancy; Garrison, Aura; Mucker, Eric; Lawler, James; Paragas, Jason

2007-09-01

Variola virus and other members of the genus Orthopoxviruses constitute a prominent bioterrorism and public health threat. Treatment with the anti-viral drug cidofovir inhibits replication of orthopoxviruses in vitro and in vivo. In this study, we visualized the effect of cidofovir on viral kinetics in orthopoxvirus infected mice by using whole-body fluorescence imaging (FI). We engineered a cowpox virus (CPV) expressing the enhanced green fluorescent protein (GFP). Single-step growth curves and calculated 50% lethal doses (LD(50)) of wild-type CPX (Wt-CPV) and GFP-expressing CPX (GFP-CPV) were comparable. Whole-body FI first detected GFP fluorescence in the mesenteric tissue of untreated animals on post-infection day (PID) 1. On PID 3 GFP signal was detected throughout the mesentery, in all abdominal organs by PID 5 and in most major organs, except for the heart and brain by PID 6. Infected animals treated with 25mg/kg of cidofovir also began showing signs of viral replication on PID 1, however, the fluorescent signal was limited only to discrete foci throughout the course of the infection. This work describes the first use of an established Orthopox model of infection to evaluate drug efficacy and track virus progression on a macroscopic level.

BinQuasi: a peak detection method for ChIP-sequencing data with biological replicates.

PubMed

Goren, Emily; Liu, Peng; Wang, Chao; Wang, Chong

2018-04-19

ChIP-seq experiments that are aimed at detecting DNA-protein interactions require biological replication to draw inferential conclusions, however there is no current consensus on how to analyze ChIP-seq data with biological replicates. Very few methodologies exist for the joint analysis of replicated ChIP-seq data, with approaches ranging from combining the results of analyzing replicates individually to joint modeling of all replicates. Combining the results of individual replicates analyzed separately can lead to reduced peak classification performance compared to joint modeling. Currently available methods for joint analysis may fail to control the false discovery rate at the nominal level. We propose BinQuasi, a peak caller for replicated ChIP-seq data, that jointly models biological replicates using a generalized linear model framework and employs a one-sided quasi-likelihood ratio test to detect peaks. When applied to simulated data and real datasets, BinQuasi performs favorably compared to existing methods, including better control of false discovery rate than existing joint modeling approaches. BinQuasi offers a flexible approach to joint modeling of replicated ChIP-seq data which is preferable to combining the results of replicates analyzed individually. Source code is freely available for download at https://cran.r-project.org/package=BinQuasi, implemented in R. pliu@iastate.edu or egoren@iastate.edu. Supplementary material is available at Bioinformatics online.

Experimental transmission of equine hepacivirus in horses as a model for hepatitis C virus.

PubMed

Ramsay, Joshua D; Evanoff, Ryan; Wilkinson, Tom E; Divers, Thomas J; Knowles, Donald P; Mealey, Robert H

2015-05-01

Equine hepacivirus (EHCV; nonprimate hepacivirus) is a hepatotropic member of the Flaviviridae family that infects horses. Although EHCV is the closest known relative to hepatitis C virus (HCV), its complete replication kinetics in vivo have not been described, and direct evidence that it causes hepatitis has been lacking. In this study, we detected EHCV in 2 horses that developed post-transfusion hepatitis. Plasma and serum from these horses were used to experimentally transmit EHCV to 4 young adult Arabian horses, two 1-month-old foals (1 Arabian and 1 Arabian-pony cross), and 2 foals (1 Arabian and 1 Arabian-pony cross) with severe combined immunodeficiency (SCID). Our results demonstrated that EHCV had infection kinetics similar to HCV and that infection was associated with acute and chronic liver disease as measured by elevations of liver-specific enzymes and/or by histopathology. Although most of these animals were coinfected with equine pegivirus (EPgV), also a flavivirus, EPgV viral loads were much lower and often undetectable in both liver and blood. Three additional young adult Arabian-pony crosses and 1 SCID foal were then inoculated with plasma containing only EHCV, and evidence of mild hepatocellular damage was observed. The different levels of liver-specific enzyme elevation, hepatic inflammation, and duration of viremia observed during EHCV infection suggested that the magnitude and course of liver disease was mediated by the virus inoculum and/or by host factors, including breed, age, and adaptive immune status. This work documents the complete infection kinetics and liver pathology associated with acute and chronic EHCV infection in horses and further justifies it as a large animal model for HCV. © 2015 by the American Association for the Study of Liver Diseases.

Porcine circovirus type 2 replication is impaired by inhibition of the extracellular signal-regulated kinase (ERK) signaling pathway

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

Wei Li; Liu Jue

Postweaning multisystemic wasting syndrome, which is primarily caused by porcine circovirus type 2 (PCV2), is an emerging and important swine disease. We have recently shown that PCV2 induces nuclear factor kappa B activation and its activation is required for active replication, but the other cellular factors involved in PCV2 replication are not well defined. The extracellular signal-regulated kinase (ERK) which served as an important component of cellular signal transduction pathways has been shown to regulate many viral infections. In this report, we show that PCV2 activates ERK1/2 in PCV2-infected PK15 cells dependent on viral replication. The PCV2-induced ERK1/2 leads tomore » phosphorylation of the ternary complex factor Elk-1, which kinetically paralleled ERK1/2 activation. Inhibition of ERK activation with U0126, a specific MEK1/2 inhibitor, significantly reduced viral progeny release. Investigations into the mechanism of ERK1/2 regulation revealed that inhibition of ERK activation leads to decreased viral transcription and lower virus protein expression. These data indicate that the ERK signaling pathway is involved in PCV2 infection and beneficial to PCV2 replication in the cultured cells.« less

Dynamic binding of replication protein a is required for DNA repair

PubMed Central

Chen, Ran; Subramanyam, Shyamal; Elcock, Adrian H.; Spies, Maria; Wold, Marc S.

2016-01-01

Replication protein A (RPA), the major eukaryotic single-stranded DNA (ssDNA) binding protein, is essential for replication, repair and recombination. High-affinity ssDNA-binding by RPA depends on two DNA binding domains in the large subunit of RPA. Mutation of the evolutionarily conserved aromatic residues in these two domains results in a separation-of-function phenotype: aromatic residue mutants support DNA replication but are defective in DNA repair. We used biochemical and single-molecule analyses, and Brownian Dynamics simulations to determine the molecular basis of this phenotype. Our studies demonstrated that RPA binds to ssDNA in at least two modes characterized by different dissociation kinetics. We also showed that the aromatic residues contribute to the formation of the longer-lived state, are required for stable binding to short ssDNA regions and are needed for RPA melting of partially duplex DNA structures. We conclude that stable binding and/or the melting of secondary DNA structures by RPA is required for DNA repair, including RAD51 mediated DNA strand exchange, but is dispensable for DNA replication. It is likely that the binding modes are in equilibrium and reflect dynamics in the RPA–DNA complex. This suggests that dynamic binding of RPA to DNA is necessary for different cellular functions. PMID:27131385

The Characteristics of Turbulent Flows on Forested Floodplains

NASA Astrophysics Data System (ADS)

Darby, S. E.; Richardson, K.; Sear, D. A.

2008-12-01

Forested floodplain environments represent the undisturbed land cover of most river systems, but they are under threat from human activities. An understanding of forest floodplain processes therefore has relevance to ecosystem conservation and restoration, and the interpretation of pre-historic river and floodplain evolution. However, relatively little research has been undertaken within forested floodplain environments, a particular limitation being an absence of empirical data regarding the hydraulic characteristics of over bank flows, which inhibits the development of flow, sediment and solute transport models. Forest floodplain flows are strongly modified by floodplain topography and the presence of vegetation and organic debris on the woodland floor. In such instances flow blockage and diversions are common, and there is the possibility of intense turbulence generation, both by wakes and by shear. To address this gap we have undertaken a study based on a floodplain reach located in the Highland Water Research Catchment (southern England), a UK national reference site for lowland floodplain forest streams. Given the difficulties of acquiring spatially-distributed hydraulic data sets during floods, our methodological approach has been to attempt to replicate over bank flow observed at the study site within a laboratory flume. This is necessary to acquire flow velocity data at sufficiently high spatial resolution to evaluate the underlying flow mechanics and has been achieved using (i) a large (21m) flume to achieve 1:1 hydraulic scaling and (ii) a novel method of precisely replicating the floodplain topography within the flume. Specifically, accurate replication of a representative floodplain patch was achieved by creating a 1:1 scale Physical Terrain Model (PTM) from high-density polyurethane using a computer-controlled milling process based on Digital Terrain Model (DTM) data, the latter acquired via terrestrial laser scanning (TLS) survey. The PTM was deployed within the flume immediately downstream of a 8m long hydraulically smooth 'run-in' section with a steady discharge replicating an over bank flow observed in the field, thus achieving 1:1 hydraulic scaling. Above the PTM 3D flow velocity time-series were acquired at each node on a dense (5-10cm horizontal spatial resolution) sampling grid using Acoustic Doppler Velocimeters (ADVs). The data were analysed by visualising the 3D structure of flow velocity and derivative statistics (turbulent intensity, turbulent kinetic energy, Reynolds stresses, etc), combined with quadrant analysis to identify the spatial variation of each quadrant's contribution to the turbulence intensity. These analyses have been used to delineate flow regions dominated by different structures, and construct an empirical model that will be helpful in defining relevant modelling strategies in future research.

Mechanism of error-free DNA synthesis across N1-methyl-deoxyadenosine by human DNA polymerase-ι

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

Jain, Rinku; Choudhury, Jayati Roy; Buku, Angeliki

N1-methyl-deoxyadenosine (1-MeA) is formed by methylation of deoxyadenosine at the N1 atom. 1-MeA presents a block to replicative DNA polymerases due to its inability to participate in Watson-Crick (W-C) base pairing. Here we determine how human DNA polymerase-ι (Polι) promotes error-free replication across 1-MeA. Steady state kinetic analyses indicate that Polι is ~100 fold more efficient in incorporating the correct nucleotide T versus the incorrect nucleotide C opposite 1-MeA. To understand the basis of this selectivity, we determined ternary structures of Polι bound to template 1-MeA and incoming dTTP or dCTP. In both structures, template 1-MeA rotates to the synmore » conformation but pairs differently with dTTP versus dCTP. Thus, whereas dTTP partakes in stable Hoogsteen base pairing with 1-MeA, dCTP fails to gain a “foothold” and is largely disordered. Together, our kinetic and structural studies show how Polι maintains discrimination between correct and incorrect incoming nucleotide opposite 1-MeA in preserving genome integrity.« less

Dynamic basis for dG•dT misincorporation via tautomerization and ionization

NASA Astrophysics Data System (ADS)

Kimsey, Isaac J.; Szymanski, Eric S.; Zahurancik, Walter J.; Shakya, Anisha; Xue, Yi; Chu, Chia-Chieh; Sathyamoorthy, Bharathwaj; Suo, Zucai; Al-Hashimi, Hashim M.

2018-02-01

Tautomeric and anionic Watson-Crick-like mismatches have important roles in replication and translation errors through mechanisms that are not fully understood. Here, using NMR relaxation dispersion, we resolve a sequence-dependent kinetic network connecting G•T/U wobbles with three distinct Watson-Crick mismatches: two rapidly exchanging tautomeric species (Genol•T/UG•Tenol/Uenol population less than 0.4%) and one anionic species (G•T-/U- population around 0.001% at neutral pH). The sequence-dependent tautomerization or ionization step was inserted into a minimal kinetic mechanism for correct incorporation during replication after the initial binding of the nucleotide, leading to accurate predictions of the probability of dG•dT misincorporation across different polymerases and pH conditions and for a chemically modified nucleotide, and providing mechanisms for sequence-dependent misincorporation. Our results indicate that the energetic penalty for tautomerization and/or ionization accounts for an approximately 10-2 to 10-3-fold discrimination against misincorporation, which proceeds primarily via tautomeric dGenol•dT and dG•dTenol, with contributions from anionic dG•dT- dominant at pH 8.4 and above or for some mutagenic nucleotides.

Establishment of an immortal turkey turbinate cell line suitable for avian metapneumovirus propagation.

PubMed

Kong, Byung-Whi; Foster, Linda K; Foster, Douglas N

2007-07-01

Until recently, there has not been a homologous avian cellular substrate which could continuously produce high titer avian metapneumovirus (AMPV); development of such a cell line should provide an excellent model system for studying AMPV infection. We have established a non-tumorigenic immortal turkey turbinate cell line (TT-1) to propagate sufficiently high AMPV titers. Currently, immortal TT-1 cells are growing continuously at 1.2-1.4 population doublings per day and are at passage 160. Kinetic analysis suggests that AMPV can infect and replicate more rapidly in TT-1 compared to Vero cells, although both cell types undergo apoptosis upon infection. The non-tumorigenic, reverse transcriptase negative TT-1 cell line can serve as an excellent homologous cellular substrate for virus propagation.

Assimilation of Biophysical Neuronal Dynamics in Neuromorphic VLSI.

PubMed

Wang, Jun; Breen, Daniel; Akinin, Abraham; Broccard, Frederic; Abarbanel, Henry D I; Cauwenberghs, Gert

2017-12-01

Representing the biophysics of neuronal dynamics and behavior offers a principled analysis-by-synthesis approach toward understanding mechanisms of nervous system functions. We report on a set of procedures assimilating and emulating neurobiological data on a neuromorphic very large scale integrated (VLSI) circuit. The analog VLSI chip, NeuroDyn, features 384 digitally programmable parameters specifying for 4 generalized Hodgkin-Huxley neurons coupled through 12 conductance-based chemical synapses. The parameters also describe reversal potentials, maximal conductances, and spline regressed kinetic functions for ion channel gating variables. In one set of experiments, we assimilated membrane potential recorded from one of the neurons on the chip to the model structure upon which NeuroDyn was designed using the known current input sequence. We arrived at the programmed parameters except for model errors due to analog imperfections in the chip fabrication. In a related set of experiments, we replicated songbird individual neuron dynamics on NeuroDyn by estimating and configuring parameters extracted using data assimilation from intracellular neural recordings. Faithful emulation of detailed biophysical neural dynamics will enable the use of NeuroDyn as a tool to probe electrical and molecular properties of functional neural circuits. Neuroscience applications include studying the relationship between molecular properties of neurons and the emergence of different spike patterns or different brain behaviors. Clinical applications include studying and predicting effects of neuromodulators or neurodegenerative diseases on ion channel kinetics.

Kinetic Study of Yellow Fever 17DD Viral Infection in Gallus gallus domesticus Embryos

PubMed Central

Manso, Pedro Paulo de Abreu; E. P. Dias de Oliveira, Bárbara Cristina; Carvalho de Sequeira, Patrícia; Rodrigues Maia de Souza, Yuli; dos Santos Ferro, Jessica Maria; da Silva, Igor José; Gonçalves Caputo, Luzia Fátima; Tavares Guedes, Priscila; Araujo Cunha dos Santos, Alexandre; da Silva Freire, Marcos; Bonaldo, Myrna Cristina; Pelajo Machado, Marcelo

2016-01-01

Yellow fever continues to be an important epidemiological problem in Africa and South America even though the disease can be controlled by vaccination. The vaccine has been produced since 1937 and is based on YFV 17DD chicken embryo infection. However, little is known about the histopathological background of virus infection and replication in this model. Here we show by morphological and molecular methods (brightfield and confocal microscopies, immunofluorescence, nested-PCR and sequencing) the kinetics of YFV 17DD infection in chicken embryos with 9 days of development, encompassing 24 to 96 hours post infection. Our principal findings indicate that the main cells involved in virus production are myoblasts with a mesenchymal shape, which also are the first cells to express virus proteins in Gallus gallus embryos at 48 hours after infection. At 72 hours post infection, we observed an increase of infected cells in embryos. Many sites are thus affected in the infection sequence, especially the skeletal muscle. We were also able to confirm an increase of nervous system infection at 96 hours post infection. Our data contribute to the comprehension of the pathogenesis of YF 17DD virus infection in Gallus gallus embryos. PMID:27158977

Model of early self-replication based on covalent complementarity for a copolymer of glycerate-3-phosphate and glycerol-3-phosphate

NASA Technical Reports Server (NTRS)

Weber, Arthur L.

1989-01-01

Glyceraldehyde-3-phosphate acts as the substrate in a model of early self-replication of a phosphodiester copolymer of glycerate-3-phosphate and glycerol-3-phosphate. This model of self-replication is based on covalent complementarity in which information transfer is mediated by a single covalent bond, in contrast to multiple weak interactions that establish complementarity in nucleic acid replication. This replication model is connected to contemporary biochemistry through its use of glyceraldehyde-3-phosphate, a central metabolite of glycolysis and photosynthesis.

Cytometry of DNA Replication and RNA Synthesis: Historical Perspective and Recent Advances Based on “Click Chemistry”

PubMed Central

Darzynkiewicz, Zbigniew; Traganos, Frank; Zhao, Hong; Halicka, H. Dorota; Li, Jiangwei

2011-01-01

This review covers progress in the development of cytometric methodologies designed to assess DNA replication and RNA synthesis. The early approaches utilizing autoradiography to detect incorporation of 3H- or 14C-labeled thymidine were able to identify the four fundamental phases of the cell cycle G1, S, G2, and M, and by analysis of the fraction of labeled mitosis (FLM), to precisely define the kinetics of cell progression through these phases. Analysis of 3H-uridine incorporation and RNA content provided the means to distinguish quiescent G0 from cycling G1 cells. Subsequent progress in analysis of DNA replication was based on the use of BrdU as a DNA precursor and its detection by the quenching of the fluorescence intensity of DNA-bound fluorochromes such as Hoechst 33358 or acridine orange as measured by flow cytometry. Several variants of this methodology have been designed and used in studies to detect anticancer drug-induced perturbations of cell cycle kinetics. The next phase of method development, which was particularly useful in studies of the cell cycle in vivo, including clinical applications, relied on immunocytochemical detection of incorporated halogenated DNA or RNA precursors. This approach however was hampered by the need for DNA denaturation, which made it difficult to concurrently detect other cell constituents for multiparametric analysis. The recently introduced “click chemistry” approach has no such limitation and is the method of choice for analysis of DNA replication and RNA synthesis. This method is based on the use of 5-ethynyl-2′deoxyuridine (EdU) as a DNA precursor or 5-ethynyluridine (EU) as an RNA precursor and their detection with fluorochrome-tagged azides utilizing a copper (I) catalyzed [3+2] cycloaddition. Several examples are presented that illustrate incorporation of EdU or EU in cells subjected to DNA damage detected as histone H2AX phosphorylation that have been analyzed by flow or laser scanning cytometry. PMID:21425239

The Controversial Role of Inter-diffusion in Glass Alteration

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

Gin, Stephane; Neill, Lindsay; Fournier, M.

2016-11-15

Current kinetic models for nuclear waste glasses (e.g. GM2001, GRAAL) are based on a set of mechanisms that have been generally agreed upon within the international waste glass community. These mechanisms are: hydration of the glass, ion exchange reactions (the two processes are referred as inter-diffusion), hydrolysis of the silicate network, and condensation/precipitation of partly or completely hydrolyzed species that produces a porous and amorphous layer and crystalline phases on surface of the altered glass. Recently, a new idea with origins in the mineral dissolution community has been proposed that excludes inter-diffusion process as a potential rate-limiting mechanism. To understandmore » how the so-called interfacial dissolution/precipitation model can change the current understanding of glass behavior, a key experiment used to account for this model was replicated to further revisit the interpretation. This experiment was performed at 50°C, with SON68 glass, in static mode, deionized water and S/V ratio of 10 m-1 for 6 months. It turn out that glass alters in an intermediate kinetic regime between the forward and the residual rate. According to previous and new solid characterizations, it is concluded that neither a simple inter-diffusion model nor the interfacial dissolution precipitation model can account for the observed elemental profiles within the alteration layer. More generally, far and close-to-saturation conditions must be distinguished and literature provides evidences that inter-diffusion takes place in slightly acidic conditions and far from saturation. However, closer to saturation, when a sufficiently dense layer is formed, a new approach is proposed requiring a full description of chemical reactions taking place within the alteration layer and involving water molecules as it is thought that water accessibility to the pristine glass is the rate-limiting process.« less

Effect of heating rate and kinetic model selection on activation energy of nonisothermal crystallization of amorphous felodipine.

PubMed

Chattoraj, Sayantan; Bhugra, Chandan; Li, Zheng Jane; Sun, Changquan Calvin

2014-12-01

The nonisothermal crystallization kinetics of amorphous materials is routinely analyzed by statistically fitting the crystallization data to kinetic models. In this work, we systematically evaluate how the model-dependent crystallization kinetics is impacted by variations in the heating rate and the selection of the kinetic model, two key factors that can lead to significant differences in the crystallization activation energy (Ea ) of an amorphous material. Using amorphous felodipine, we show that the Ea decreases with increase in the heating rate, irrespective of the kinetic model evaluated in this work. The model that best describes the crystallization phenomenon cannot be identified readily through the statistical fitting approach because several kinetic models yield comparable R(2) . Here, we propose an alternate paired model-fitting model-free (PMFMF) approach for identifying the most suitable kinetic model, where Ea obtained from model-dependent kinetics is compared with those obtained from model-free kinetics. The most suitable kinetic model is identified as the one that yields Ea values comparable with the model-free kinetics. Through this PMFMF approach, nucleation and growth is identified as the main mechanism that controls the crystallization kinetics of felodipine. Using this PMFMF approach, we further demonstrate that crystallization mechanism from amorphous phase varies with heating rate. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

Spatial aspects of prebiotic replicator coexistence and community stability in a surface-bound RNA world model

PubMed Central

2013-01-01

Background The coexistence of macromolecular replicators and thus the stability of presumed prebiotic replicator communities have been shown to critically depend on spatially constrained catalytic cooperation among RNA-like modular replicators. The necessary spatial constraints might have been supplied by mineral surfaces initially, preceding the more effective compartmentalization in membrane vesicles which must have been a later development of chemical evolution. Results Using our surface-bound RNA world model – the Metabolic Replicator Model (MRM) platform – we show that the mobilities on the mineral substrate surface of both the macromolecular replicators and the small molecules of metabolites they produce catalytically are the key factors determining the stable persistence of an evolvable metabolic replicator community. Conclusion The effects of replicator mobility and metabolite diffusion on different aspects of replicator coexistence in MRM are determined, including the maximum attainable size of the metabolic replicator system and its resistance to the invasion of parasitic replicators. We suggest a chemically plausible hypothetical scenario for the evolution of the first protocell starting from the surface-bound MRM system. PMID:24053177

Molecular analysis of the replication program in unicellular model organisms.

PubMed

Raghuraman, M K; Brewer, Bonita J

2010-01-01

Eukaryotes have long been reported to show temporal programs of replication, different portions of the genome being replicated at different times in S phase, with the added possibility of developmentally regulated changes in this pattern depending on species and cell type. Unicellular model organisms, primarily the budding yeast Saccharomyces cerevisiae, have been central to our current understanding of the mechanisms underlying the regulation of replication origins and the temporal program of replication in particular. But what exactly is a temporal program of replication, and how might it arise? In this article, we explore this question, drawing again on the wealth of experimental information in unicellular model organisms.

Effects of the deletion of early region 4 (E4) open reading frame 1 (orf1), orf1-2, orf1-3 and orf1-4 on virus-host cell interaction, transgene expression, and immunogenicity of replicating adenovirus HIV vaccine vectors.

PubMed

Thomas, Michael A; Song, Rui; Demberg, Thorsten; Vargas-Inchaustegui, Diego A; Venzon, David; Robert-Guroff, Marjorie

2013-01-01

The global health burden engendered by human immunodeficiency virus (HIV)-induced acquired immunodeficiency syndrome (AIDS) is a sobering reminder of the pressing need for a preventative vaccine. In non-human primate models replicating adenovirus (Ad)-HIV/SIV recombinant vaccine vectors have been shown to stimulate potent immune responses culminating in protection against challenge exposures. Nonetheless, an increase in the transgene carrying capacity of these Ad vectors, currently limited to approximately 3000 base pairs, would greatly enhance their utility. Using a replicating, E3-deleted Ad type 5 host range mutant (Ad5 hr) encoding full-length single-chain HIVBaLgp120 linked to the D1 and D2 domains of rhesus macaque CD4 (rhFLSC) we systematically deleted the genes encoding early region 4 open reading frame 1 (E4orf1) through E4orf4. All the Ad-rhFLSC vectors produced similar levels of viral progeny. Cell cycle analysis of infected human and monkey cells revealed no differences in virus-host interaction. The parental and E4-deleted viruses expressed comparable levels of the transgene with kinetics similar to Ad late proteins. Similar levels of cellular immune responses and transgene-specific antibodies were elicited in vaccinated mice. However, differences in recognition of Ad proteins and induced antibody subtypes were observed, suggesting that the E4 gene products might modulate antibody responses by as yet unknown mechanisms. In short, we have improved the transgene carrying capacity by one thousand base pairs while preserving the replicability, levels of transgene expression, and immunogenicity critical to these vaccine vectors. This additional space allows for flexibility in vaccine design that could not be obtained with the current vector and as such should facilitate the goal of improving vaccine efficacy. To the best of our knowledge, this is the first report describing the effects of these E4 deletions on transgene expression and immunogenicity in a replicating Ad vector.

Effects of the Deletion of Early Region 4 (E4) Open Reading Frame 1 (orf1), orf1-2, orf1-3 and orf1-4 on Virus-Host Cell Interaction, Transgene Expression, and Immunogenicity of Replicating Adenovirus HIV Vaccine Vectors

PubMed Central

Thomas, Michael A.; Song, Rui; Demberg, Thorsten; Vargas-Inchaustegui, Diego A.; Venzon, David; Robert-Guroff, Marjorie

2013-01-01

The global health burden engendered by human immunodeficiency virus (HIV)-induced acquired immunodeficiency syndrome (AIDS) is a sobering reminder of the pressing need for a preventative vaccine. In non-human primate models replicating adenovirus (Ad)-HIV/SIV recombinant vaccine vectors have been shown to stimulate potent immune responses culminating in protection against challenge exposures. Nonetheless, an increase in the transgene carrying capacity of these Ad vectors, currently limited to approximately 3000 base pairs, would greatly enhance their utility. Using a replicating, E3-deleted Ad type 5 host range mutant (Ad5 hr) encoding full-length single-chain HIVBaLgp120 linked to the D1 and D2 domains of rhesus macaque CD4 (rhFLSC) we systematically deleted the genes encoding early region 4 open reading frame 1 (E4orf1) through E4orf4. All the Ad-rhFLSC vectors produced similar levels of viral progeny. Cell cycle analysis of infected human and monkey cells revealed no differences in virus-host interaction. The parental and E4-deleted viruses expressed comparable levels of the transgene with kinetics similar to Ad late proteins. Similar levels of cellular immune responses and transgene-specific antibodies were elicited in vaccinated mice. However, differences in recognition of Ad proteins and induced antibody subtypes were observed, suggesting that the E4 gene products might modulate antibody responses by as yet unknown mechanisms. In short, we have improved the transgene carrying capacity by one thousand base pairs while preserving the replicability, levels of transgene expression, and immunogenicity critical to these vaccine vectors. This additional space allows for flexibility in vaccine design that could not be obtained with the current vector and as such should facilitate the goal of improving vaccine efficacy. To the best of our knowledge, this is the first report describing the effects of these E4 deletions on transgene expression and immunogenicity in a replicating Ad vector. PMID:24143187

Using Multiorder Time-Correlation Functions (TCFs) To Elucidate Biomolecular Reaction Pathways from Microsecond Single-Molecule Fluorescence Experiments.

PubMed

Phelps, Carey; Israels, Brett; Marsh, Morgan C; von Hippel, Peter H; Marcus, Andrew H

2016-12-29

Recent advances in single-molecule fluorescence imaging have made it possible to perform measurements on microsecond time scales. Such experiments have the potential to reveal detailed information about the conformational changes in biological macromolecules, including the reaction pathways and dynamics of the rearrangements involved in processes, such as sequence-specific DNA "breathing" and the assembly of protein-nucleic acid complexes. Because microsecond-resolved single-molecule trajectories often involve "sparse" data, that is, they contain relatively few data points per unit time, they cannot be easily analyzed using the standard protocols that were developed for single-molecule experiments carried out with tens-of-millisecond time resolution and high "data density." Here, we describe a generalized approach, based on time-correlation functions, to obtain kinetic information from microsecond-resolved single-molecule fluorescence measurements. This approach can be used to identify short-lived intermediates that lie on reaction pathways connecting relatively long-lived reactant and product states. As a concrete illustration of the potential of this methodology for analyzing specific macromolecular systems, we accompany the theoretical presentation with the description of a specific biologically relevant example drawn from studies of reaction mechanisms of the assembly of the single-stranded DNA binding protein of the T4 bacteriophage replication complex onto a model DNA replication fork.

Induction of the tumor-suppressor p16(INK4a) within regenerative epithelial crypts in ulcerative colitis.

PubMed

Furth, Emma E; Gustafson, Karen S; Dai, Charlotte Y; Gibson, Steven L; Menard-Katcher, Paul; Chen, Tina; Koh, Jim; Enders, Greg H

2006-06-01

p16(INK4a) is a major tumor-suppressor protein, but its regulation and settings of fuction remain poorly understood. To explore the notion that p16 is induced in vivo in response to replicative stress, we examined p16 expression in tissues from human ulcerative colitis (UC; n = 25) and normal controls (n = 20). p16 was expressed strongly in UC-associated neoplasms (n = 17), as seen previously in sporadic colonic neoplasms. In non-neoplastic UC epithelium, p16 was expressed in 33% of crypts (the proliferative compartment) compared to < 1% of normal controls. p16 expression did not correlate with degree of inflammation but did correlate with the degree of crypt architecture distortion (P = .002)-a reflection of epithelial regeneration. In coimmunofluorescence studies with Ki67, p16 expression was associated with cell cycle arrest (P < .001). Both UC and normal crypts displayed evidence for the activation of the DNA damage checkpoint pathway, and p16 was induced in primary cultures of normal epithelial cells by ionizing irradiation (IR). However, induction by IR displayed delayed kinetics, implying that p16 is not an immediate target of the checkpoint pathway. These findings support a model in which p16 is induced as an "emergency brake" in cells experiencing sustained replicative stress.

Infection of neuroblastoma cells by rabies virus is modulated by the virus titer.

PubMed

Fuoco, Natalia Langenfeld; Dos Ramos Silva, Sandriana; Fernandes, Elaine Raniero; Luiz, Fernanda Guedes; Ribeiro, Orlando Garcia; Katz, Iana Suly Santos

2018-01-01

Rabies is a lethal viral infection that can affect almost all mammals, including humans. To better understand the replication of Rabies lyssavirus, we investigated if the viral load in brains naturally infected with rabies influences viral internalization and viral growth kinetics in neuroblastoma cells, and if the viral load affects mortality in mice after intradermal infection. We noted that high initial viral loads in brains (group II) were unfavourable for increasing viral titers during serial passages in neuroblastoma cells when compared to low initial viral loads in brains (group I). In addition, group I strains showed higher viral growth and enhanced internalization efficiency in neuroblastoma cells than group II strains. However, we observed that the dominant virus subpopulation in group II promoted efficient viral infection in the central nervous system in the new host, providing a selective advantage to the virus. Our data indicate that rabies infection in animal models depends on not only the virus strain but also the amount of virus. This study may serve as a basis for understanding the biologic proprieties of Rabies lyssavirus strains with respect to the effects on viral replication and the impact on pathogenesis, improving virus yields for use in vaccine development. Copyright © 2017 Elsevier B.V. All rights reserved.

Unbiased Rare Event Sampling in Spatial Stochastic Systems Biology Models Using a Weighted Ensemble of Trajectories

PubMed Central

Donovan, Rory M.; Tapia, Jose-Juan; Sullivan, Devin P.; Faeder, James R.; Murphy, Robert F.; Dittrich, Markus; Zuckerman, Daniel M.

2016-01-01

The long-term goal of connecting scales in biological simulation can be facilitated by scale-agnostic methods. We demonstrate that the weighted ensemble (WE) strategy, initially developed for molecular simulations, applies effectively to spatially resolved cell-scale simulations. The WE approach runs an ensemble of parallel trajectories with assigned weights and uses a statistical resampling strategy of replicating and pruning trajectories to focus computational effort on difficult-to-sample regions. The method can also generate unbiased estimates of non-equilibrium and equilibrium observables, sometimes with significantly less aggregate computing time than would be possible using standard parallelization. Here, we use WE to orchestrate particle-based kinetic Monte Carlo simulations, which include spatial geometry (e.g., of organelles, plasma membrane) and biochemical interactions among mobile molecular species. We study a series of models exhibiting spatial, temporal and biochemical complexity and show that although WE has important limitations, it can achieve performance significantly exceeding standard parallel simulation—by orders of magnitude for some observables. PMID:26845334

Next Generation Respiratory Viral Vaccine System: Advanced and Emerging Bioengineered Human Lung Epithelia Model (HLEM) Organoid Technology

NASA Technical Reports Server (NTRS)

Goodwin, Thomas J.; Schneider, Sandra L.; MacIntosh, Victor; Gibbons, Thomas F.

2010-01-01

Acute respiratory infections, including pneumonia and influenza, are the S t" leading cause of United States and worldwide deaths. Newly emerging pathogens signaled the need for an advanced generation of vaccine technology.. Human bronchial-tracheal epithelial tissue was bioengineered to detect, identify, host and study the pathogenesis of acute respiratory viral disease. The 3-dimensional (3D) human lung epithelio-mesechymal tissue-like assemblies (HLEM TLAs) share characteristics with human respiratory epithelium: tight junctions, desmosomes, microvilli, functional markers villin, keratins and production of tissue mucin. Respiratory Syntial Virus (RSV) studies demonstrate viral growth kinetics and membrane bound glycoproteins up to day 20 post infection in the human lung-orgainoid infected cell system. Peak replication of RSV occurred on day 10 at 7 log10 particles forming units per ml/day. HLEM is an advanced virus vaccine model and biosentinel system for emergent viral infectious diseases to support DoD global surveillance and military readiness.

Inferring the parameters of a Markov process from snapshots of the steady state

NASA Astrophysics Data System (ADS)

Dettmer, Simon L.; Berg, Johannes

2018-02-01

We seek to infer the parameters of an ergodic Markov process from samples taken independently from the steady state. Our focus is on non-equilibrium processes, where the steady state is not described by the Boltzmann measure, but is generally unknown and hard to compute, which prevents the application of established equilibrium inference methods. We propose a quantity we call propagator likelihood, which takes on the role of the likelihood in equilibrium processes. This propagator likelihood is based on fictitious transitions between those configurations of the system which occur in the samples. The propagator likelihood can be derived by minimising the relative entropy between the empirical distribution and a distribution generated by propagating the empirical distribution forward in time. Maximising the propagator likelihood leads to an efficient reconstruction of the parameters of the underlying model in different systems, both with discrete configurations and with continuous configurations. We apply the method to non-equilibrium models from statistical physics and theoretical biology, including the asymmetric simple exclusion process (ASEP), the kinetic Ising model, and replicator dynamics.

Kinetics of Respiratory Syncytial Virus (RSV) Memphis Strain 37 (M37) Infection in the Respiratory Tract of Newborn Lambs as an RSV Infection Model for Human Infants

PubMed Central

Larios Mora, Alejandro; Detalle, Laurent; Van Geelen, Albert; Davis, Michael S.; Stohr, Thomas; Gallup, Jack M.; Ackermann, Mark R.

2015-01-01

Rationale Respiratory syncytial virus (RSV) infection in preterm and newborn infants can result in severe bronchiolitis and hospitalization. The lamb lung has several key features conducive to modeling RSV infection in human infants, including susceptibility to human strains of RSV such as the A2, Long, and Memphis Strain 37 (M37). In this study, the kinetics of M37 infection was investigated in newborn lambs in order to better define clinical, viral, physiological, and immunological parameters as well as the pathology and lesions. Methods Newborn lambs were nebulized with M37 hRSV (6 mL of 1.27 x 107 FFU/mL), monitored daily for clinical responses, and respiratory tissues were collected from groups of lambs at days 1, 3, 4, 6, and 8 post-inoculation for the assessment of viral replication parameters, lesions and also cellular, immunologic and inflammatory responses. Results Lambs had increased expiratory effort (forced expiration) at days 4, 6, and 8 post-inoculation. Nasal wash lacked RSV titers at day 1, but titers were present at low levels at days 3 (peak), 4, and 8. Viral titers in bronchoalveolar lavage fluid (BALF) reached a plateau at day 3 (4.6 Log10 FFU/mL), which was maintained until day 6 (4.83 Log10 FFU/mL), and were markedly reduced or absent at day 8. Viral RNA levels (detected by RT-qPCR) in BALF were indistinguishable at days 3 (6.22 ± 0.08 Log10 M37 RNA copies/mL; mean ± se) and 4 (6.20 ± 0.16 Log10 M37 RNA copies/mL; mean ± se) and increased slightly on day 6 (7.15 ± 0.2 Log10 M37 RNA copies/mL; mean ± se). Viral antigen in lung tissue as detected by immunohistochemistry was not seen at day 1, was present at days 3 and 4 before reaching a peak by day 6, and was markedly reduced by day 8. Viral antigen was mainly present in airways (bronchi, bronchioles) at day 3 and was increasingly present in alveolar cells at days 4 and 6, with reduction at day 8. Histopathologic lesions such as bronchitis/bronchiolitis, epithelial necrosis and hyperplasia, peribronchial lymphocyte infiltration, and syncytial cells, were consistent with those described previously for lambs and infants. Conclusion This work demonstrates that M37 hRSV replication in the lower airways of newborn lambs is robust with peak replication on day 3 and sustained until day 6. These findings, along with the similarities of lamb lung to those of infants in terms of alveolar development, airway branching and epithelium, susceptibility to human RSV strains, lesion characteristics (bronchiolitis), lung size, clinical parameters, and immunity, further establish the neonatal lamb as a model with key features that mimic RSV infection in infants. PMID:26641081

Structure and Dynamics of the tRNA-like Structure Domain of Brome Mosaic Virus

NASA Astrophysics Data System (ADS)

Vieweger, Mario; Nesbitt, David

2014-03-01

Conformational switching is widely accepted as regulatory mechanism in gene expression in bacterial systems. More recently, similar regulation mechanisms are emerging for viral systems. One of the most abundant and best studied systems is the tRNA-like structure domain that is found in a number of plant viruses across eight genera. In this work, the folding dynamics of the tRNA-like structure domain of Brome Mosaic Virus are investigated using single-molecule Fluorescence Resonance Energy Transfer techniques. In particular, Burst fluorescence is applied to observe metal-ion induced folding in freely diffusing RNA constructs resembling the 3'-terminal 169nt of BMV RNA3. Histograms of EFRET probabilities reveal a complex equilibrium of three distinct populations. A step-wise kinetic model for TLS folding is developed in accord with the evolution of conformational populations and structural information in the literature. In this mechanism, formation of functional TLS domains from unfolded RNAs requires two consecutive steps; 1) hybridization of a long-range stem interaction followed by 2) formation of a 3' pseudoknot. This three-state equilibrium is well described by step-wise dissociation constants K1(328(30) μM) and K2(1092(183) μM) for [Mg2+] and K1(74(6) mM) and K2(243(52) mM) for [Na+]-induced folding. The kinetic model is validated by oligo competition with the STEM interaction. Implications of this conformational folding mechanism are discussed in regards to regulation of virus replication.

Diffusion in confinement: kinetic simulations of self- and collective diffusion behavior of adsorbed gases

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

Abouelnasr, MKF; Smit, B

2012-01-01

The self- and collective-diffusion behaviors of adsorbed methane, helium, and isobutane in zeolite frameworks LTA, MFI, AFI, and SAS were examined at various concentrations using a range of molecular simulation techniques including Molecular Dynamics (MD), Monte Carlo (MC), Bennett-Chandler (BC), and kinetic Monte Carlo (kMC). This paper has three main results. (1) A novel model for the process of adsorbate movement between two large cages was created, allowing the formulation of a mixing rule for the re-crossing coefficient between two cages of unequal loading. The predictions from this mixing rule were found to agree quantitatively with explicit simulations. (2) Amore » new approach to the dynamically corrected Transition State Theory method to analytically calculate self-diffusion properties was developed, explicitly accounting for nanoscale fluctuations in concentration. This approach was demonstrated to quantitatively agree with previous methods, but is uniquely suited to be adapted to a kMC simulation that can simulate the collective-diffusion behavior. (3) While at low and moderate loadings the self- and collective-diffusion behaviors in LTA are observed to coincide, at higher concentrations they diverge. A change in the adsorbate packing scheme was shown to cause this divergence, a trait which is replicated in a kMC simulation that explicitly models this behavior. These phenomena were further investigated for isobutane in zeolite MFI, where MD results showed a separation in self- and collective-diffusion behavior that was reproduced with kMC simulations.« less

Diffusion in confinement: kinetic simulations of self- and collective diffusion behavior of adsorbed gases.

PubMed

Abouelnasr, Mahmoud K F; Smit, Berend

2012-09-07

The self- and collective-diffusion behaviors of adsorbed methane, helium, and isobutane in zeolite frameworks LTA, MFI, AFI, and SAS were examined at various concentrations using a range of molecular simulation techniques including Molecular Dynamics (MD), Monte Carlo (MC), Bennett-Chandler (BC), and kinetic Monte Carlo (kMC). This paper has three main results. (1) A novel model for the process of adsorbate movement between two large cages was created, allowing the formulation of a mixing rule for the re-crossing coefficient between two cages of unequal loading. The predictions from this mixing rule were found to agree quantitatively with explicit simulations. (2) A new approach to the dynamically corrected Transition State Theory method to analytically calculate self-diffusion properties was developed, explicitly accounting for nanoscale fluctuations in concentration. This approach was demonstrated to quantitatively agree with previous methods, but is uniquely suited to be adapted to a kMC simulation that can simulate the collective-diffusion behavior. (3) While at low and moderate loadings the self- and collective-diffusion behaviors in LTA are observed to coincide, at higher concentrations they diverge. A change in the adsorbate packing scheme was shown to cause this divergence, a trait which is replicated in a kMC simulation that explicitly models this behavior. These phenomena were further investigated for isobutane in zeolite MFI, where MD results showed a separation in self- and collective- diffusion behavior that was reproduced with kMC simulations.

Presenting a new kinetic model for methanol to light olefins reactions over a hierarchical SAPO-34 catalyst using the Langmuir-Hinshelwood-Hougen-Watson mechanism

NASA Astrophysics Data System (ADS)

Javad Azarhoosh, Mohammad; Halladj, Rouein; Askari, Sima

2017-10-01

In this study, a new kinetic model for methanol to light olefins (MTO) reactions over a hierarchical SAPO-34 catalyst using the Langmuir-Hinshelwood-Hougen-Watson (LHHW) mechanism was presented and the kinetic parameters was obtained using a genetic algorithm (GA) and genetic programming (GP). Several kinetic models for the MTO reactions have been presented. However, due to the complexity of the reactions, most reactions are considered lumped and elementary, which cannot be deemed a completely accurate kinetic model of the process. Therefore, in this study, the LHHW mechanism is presented as kinetic models of MTO reactions. Because of the non-linearity of the kinetic models and existence of many local optimal points, evolutionary algorithms (GA and GP) are used in this study to estimate the kinetic parameters in the rate equations. Via the simultaneous connection of the code related to modelling the reactor and the GA and GP codes in the MATLAB R2013a software, optimization of the kinetic models parameters was performed such that the least difference between the results from the kinetic models and experiential results was obtained and the best kinetic parameters of MTO process reactions were achieved. A comparison of the results from the model with experiential results showed that the present model possesses good accuracy.

Evaluation of 793/B-like and Mass-like vaccine strain kinetics in experimental and field conditions by real-time RT-PCR quantification.

PubMed

Tucciarone, C M; Franzo, G; Berto, G; Drigo, M; Ramon, G; Koutoulis, K C; Catelli, E; Cecchinato, M

2018-01-01

Infectious bronchitis virus (IBV) is a great economic burden both for productive losses and costs of the control strategies. Many different vaccination protocols are applied in the same region and even in consecutive cycles on the same farm in order to find the perfect balance between costs and benefits. In Northern Italy, the usual second vaccination is more and more often moved up to the chick's first d of life. The second strain administration together with the common Mass priming by spray at the hatchery allows saving money and time and reducing animal stress. The present work compared the different vaccine strains (Mass-like or B48, and 1/96) kinetics both in field conditions and in a 21-day-long experimental trial in broilers, monitoring the viral replication by upper respiratory tract swabbing and vaccine specific real time reverse transcription PCR (RT-PCR) quantification. In both field and experimental conditions, titers for all the vaccines showed an increasing trend in the first 2 wk and then a decrease, though still remaining detectable during the whole monitored period. IBV field strain and avian Metapneumovirus (aMPV) presence also was also investigated by RT-PCR and sequencing, and by multiplex real-time RT-PCR, respectively, revealing a consistency in the pathogen introduction timing at around 30 d, in correspondence with the vaccine titer's main decrease. These findings suggest the need for an accurate knowledge of live vaccine kinetics, whose replication can compete with the other pathogen one, providing additional protection to be added to what is conferred by the adaptive immune response. © 2017 Poultry Science Association Inc.

Bioluminescence-based system for rapid detection of natural transformation.

PubMed

Santala, Ville; Karp, Matti; Santala, Suvi

2016-07-01

Horizontal gene transfer plays a significant role in bacterial evolution and has major clinical importance. Thus, it is vital to understand the mechanisms and kinetics of genetic transformations. Natural transformation is the driving mechanism for horizontal gene transfer in diverse genera of bacteria. Our study introduces a simple and rapid method for the investigation of natural transformation. This highly sensitive system allows the detection of a transformation event directly from a bacterial population without any separation step or selection of cells. The system is based on the bacterial luciferase operon from Photorhabdus luminescens The studied molecular tools consist of the functional modules luxCDE and luxAB, which involve a replicative plasmid and an integrative gene cassette. A well-established host for bacterial genetic investigations, Acinetobacter baylyi ADP1, is used as the model bacterium. We show that natural transformation followed by homologous recombination or plasmid recircularization can be readily detected in both actively growing and static biofilm-like cultures, including very rare transformation events. The system allows the detection of natural transformation within 1 h of introducing sample DNA into the culture. The introduced method provides a convenient means to study the kinetics of natural transformation under variable conditions and perturbations. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Incorporation of the catalytic domain of a hammerhead ribozyme into antisense RNA enhances its inhibitory effect on the replication of human immunodeficiency virus type 1.

PubMed Central

Homann, M; Tzortzakaki, S; Rittner, K; Sczakiel, G; Tabler, M

1993-01-01

The catalytic domain of a hammerhead ribozyme was incorporated into a 413 nucleotides long antisense RNA directed against the 5'-leader/gag region of the human immunodeficiency virus type 1 (HIV-1) (pos. +222 to +634). The resulting catalytic antisense RNA was shown to cleave its target RNA in vitro specifically at physiological ion strength and temperature. We compared the antiviral effectiveness of this catalytic antisense RNA with that of the corresponding unmodified antisense RNA and with a mutated catalytic antisense RNA, which did not cleave the substrate RNA in vitro. Each of these RNAs was co-transfected into human SW480 cells together with infectious complete proviral HIV-1 DNA, followed by analysis of HIV-1 replication. The presence of the catalytically active domain resulted in 4 to 7 fold stronger inhibition of HIV-1 replication as compared to the parental antisense RNA and the inactive mutant. Kinetic and structural studies performed in vitro indicated that the ability for double strand formation was not changed in catalytic antisense RNA versus parental antisense RNA. Together, these data suggest that the ability to cleave target RNA is a crucial prerequisite for the observed increase of inhibition of the replication of HIV-1. Images PMID:8332489

LAMP proteins account for the maturation delay during the establishment of the Coxiella burnetii-containing vacuole.

PubMed

Schulze-Luehrmann, Jan; Eckart, Rita A; Ölke, Martha; Saftig, Paul; Liebler-Tenorio, Elisabeth; Lührmann, Anja

2016-02-01

The obligate intracellular pathogen Coxiella burnetii replicates in a large phagolysosomal-like vacuole. Currently, both host and bacterial factors required for creating this replicative parasitophorous C. burnetii-containing vacuole (PV) are poorly defined. Here, we assessed the contributions of the most abundant proteins of the lysosomal membrane, LAMP-1 and LAMP-2, to the establishment and maintenance of the PV. Whereas these proteins were not critical for uptake of C. burnetii, they influenced the intracellular replication of C. burnetii. In LAMP-1/2 double-deficient fibroblasts as well as in LAMP-1/2 knock-down cells, C. burnetii establishes a significantly smaller, yet faster maturing vacuole, which harboured more bacteria. The accelerated maturation of PVs in LAMP double-deficient fibroblasts, which was partially or fully reversed by ectopic expression of LAMP-1 or LAMP-2, respectively, was characterized by an increased fusion rate with endosomes, lysosomes and bead-containing phagosomes, but not by different fusion kinetics with autophagy vesicles. These findings establish that LAMP proteins are critical for the maturation delay of PVs. Unexpectedly, neither the creation of the spacious vacuole nor the delay in maturation was found to be prerequisites for the intracellular replication of C. burnetii. © 2015 John Wiley & Sons Ltd.

Chicken Interferon-induced Protein with Tetratricopeptide Repeats 5 Antagonizes Replication of RNA Viruses.

PubMed

Santhakumar, Diwakar; Rohaim, Mohammed Abdel Mohsen Shahaat; Hussein, Hussein A; Hawes, Pippa; Ferreira, Helena Lage; Behboudi, Shahriar; Iqbal, Munir; Nair, Venugopal; Arns, Clarice W; Munir, Muhammad

2018-05-01

The intracellular actions of interferon (IFN)-regulated proteins, including IFN-induced proteins with tetratricopeptide repeats (IFITs), attribute a major component of the protective antiviral host defense. Here we applied genomics approaches to annotate the chicken IFIT locus and currently identified a single IFIT (chIFIT5) gene. The profound transcriptional level of this effector of innate immunity was mapped within its unique cis-acting elements. This highly virus- and IFN-responsive chIFIT5 protein interacted with negative sense viral RNA structures that carried a triphosphate group on its 5' terminus (ppp-RNA). This interaction reduced the replication of RNA viruses in lentivirus-mediated IFIT5-stable chicken fibroblasts whereas CRISPR/Cas9-edited chIFIT5 gene knockout fibroblasts supported the replication of RNA viruses. Finally, we generated mosaic transgenic chicken embryos stably expressing chIFIT5 protein or knocked-down for endogenous chIFIT5 gene. Replication kinetics of RNA viruses in these transgenic chicken embryos demonstrated the antiviral potential of chIFIT5 in ovo. Taken together, these findings propose that IFIT5 specifically antagonize RNA viruses by sequestering viral nucleic acids in chickens, which are unique in innate immune sensing and responses to viruses of both poultry and human health significance.

Mechanistic analysis of multi-omics datasets to generate kinetic parameters for constraint-based metabolic models.

PubMed

Cotten, Cameron; Reed, Jennifer L

2013-01-30

Constraint-based modeling uses mass balances, flux capacity, and reaction directionality constraints to predict fluxes through metabolism. Although transcriptional regulation and thermodynamic constraints have been integrated into constraint-based modeling, kinetic rate laws have not been extensively used. In this study, an in vivo kinetic parameter estimation problem was formulated and solved using multi-omic data sets for Escherichia coli. To narrow the confidence intervals for kinetic parameters, a series of kinetic model simplifications were made, resulting in fewer kinetic parameters than the full kinetic model. These new parameter values are able to account for flux and concentration data from 20 different experimental conditions used in our training dataset. Concentration estimates from the simplified kinetic model were within one standard deviation for 92.7% of the 790 experimental measurements in the training set. Gibbs free energy changes of reaction were calculated to identify reactions that were often operating close to or far from equilibrium. In addition, enzymes whose activities were positively or negatively influenced by metabolite concentrations were also identified. The kinetic model was then used to calculate the maximum and minimum possible flux values for individual reactions from independent metabolite and enzyme concentration data that were not used to estimate parameter values. Incorporating these kinetically-derived flux limits into the constraint-based metabolic model improved predictions for uptake and secretion rates and intracellular fluxes in constraint-based models of central metabolism. This study has produced a method for in vivo kinetic parameter estimation and identified strategies and outcomes of kinetic model simplification. We also have illustrated how kinetic constraints can be used to improve constraint-based model predictions for intracellular fluxes and biomass yield and identify potential metabolic limitations through the integrated analysis of multi-omics datasets.

Mechanistic analysis of multi-omics datasets to generate kinetic parameters for constraint-based metabolic models

PubMed Central

2013-01-01

Background Constraint-based modeling uses mass balances, flux capacity, and reaction directionality constraints to predict fluxes through metabolism. Although transcriptional regulation and thermodynamic constraints have been integrated into constraint-based modeling, kinetic rate laws have not been extensively used. Results In this study, an in vivo kinetic parameter estimation problem was formulated and solved using multi-omic data sets for Escherichia coli. To narrow the confidence intervals for kinetic parameters, a series of kinetic model simplifications were made, resulting in fewer kinetic parameters than the full kinetic model. These new parameter values are able to account for flux and concentration data from 20 different experimental conditions used in our training dataset. Concentration estimates from the simplified kinetic model were within one standard deviation for 92.7% of the 790 experimental measurements in the training set. Gibbs free energy changes of reaction were calculated to identify reactions that were often operating close to or far from equilibrium. In addition, enzymes whose activities were positively or negatively influenced by metabolite concentrations were also identified. The kinetic model was then used to calculate the maximum and minimum possible flux values for individual reactions from independent metabolite and enzyme concentration data that were not used to estimate parameter values. Incorporating these kinetically-derived flux limits into the constraint-based metabolic model improved predictions for uptake and secretion rates and intracellular fluxes in constraint-based models of central metabolism. Conclusions This study has produced a method for in vivo kinetic parameter estimation and identified strategies and outcomes of kinetic model simplification. We also have illustrated how kinetic constraints can be used to improve constraint-based model predictions for intracellular fluxes and biomass yield and identify potential metabolic limitations through the integrated analysis of multi-omics datasets. PMID:23360254

Possible Increased Pathogenicity of Pandemic (H1N1) 2009 Influenza Virus upon Reassortment

PubMed Central

Schrauwen, Eefje J.A.; Herfst, Sander; Chutinimitkul, Salin; Bestebroer, Theo M.; Rimmelzwaan, Guus F.; Osterhaus, Albert D.M.E.; Kuiken, Thijs

2011-01-01

Since emergence of the pandemic (H1N1) 2009 virus in April 2009, three influenza A viruses—seasonal (H3N2), seasonal (H1N1), and pandemic (H1N1) 2009—have circulated in humans. Genetic reassortment between these viruses could result in enhanced pathogenicity. We compared 4 reassortant viruses with favorable in vitro replication properties with the wild-type pandemic (H1N1) 2009 virus with respect to replication kinetics in vitro and pathogenicity and transmission in ferrets. Pandemic (H1N1) 2009 viruses containing basic polymerase 2 alone or in combination with acidic polymerase of seasonal (H1N1) virus were attenuated in ferrets. In contrast, pandemic (H1N1) 2009 with neuraminidase of seasonal (H3N2) virus resulted in increased virus replication and more severe pulmonary lesions. The data show that pandemic (H1N1) 2009 virus has the potential to reassort with seasonal influenza viruses, which may result in increased pathogenicity while it maintains the capacity of transmission through aerosols or respiratory droplets. PMID:21291589

Replication and Adaptive Mutations of Low Pathogenic Avian Influenza Viruses in Tracheal Organ Cultures of Different Avian Species

PubMed Central

Petersen, Henning; Matrosovich, Mikhail; Pleschka, Stephan; Rautenschlein, Silke

2012-01-01

Transmission of avian influenza viruses (AIV) between different avian species may require genome mutations that allow efficient virus replication in a new species and could increase virulence. To study the role of domestic poultry in the evolution of AIV we compared replication of low pathogenic (LP) AIV of subtypes H9N2, H7N7 and H6N8 in tracheal organ cultures (TOC) and primary embryo fibroblast cultures of chicken, turkey, Pekin duck and homing pigeon. Virus strain-dependent and avian species-related differences between LPAIV were observed in growth kinetics and induction of ciliostasis in TOC. In particular, our data demonstrate high susceptibility to LPAIV of turkey TOC contrasted with low susceptibility of homing pigeon TOC. Serial virus passages in the cells of heterologous host species resulted in adaptive mutations in the AIV genome, especially in the receptor-binding site and protease cleavage site of the hemagglutinin. Our data highlight differences in susceptibility of different birds to AIV viruses and emphasizes potential role of poultry in the emergence of new virus variants. PMID:22912693

Deciphering DNA replication dynamics in eukaryotic cell populations in relation with their averaged chromatin conformations

NASA Astrophysics Data System (ADS)

Goldar, A.; Arneodo, A.; Audit, B.; Argoul, F.; Rappailles, A.; Guilbaud, G.; Petryk, N.; Kahli, M.; Hyrien, O.

2016-03-01

We propose a non-local model of DNA replication that takes into account the observed uncertainty on the position and time of replication initiation in eukaryote cell populations. By picturing replication initiation as a two-state system and considering all possible transition configurations, and by taking into account the chromatin’s fractal dimension, we derive an analytical expression for the rate of replication initiation. This model predicts with no free parameter the temporal profiles of initiation rate, replication fork density and fraction of replicated DNA, in quantitative agreement with corresponding experimental data from both S. cerevisiae and human cells and provides a quantitative estimate of initiation site redundancy. This study shows that, to a large extent, the program that regulates the dynamics of eukaryotic DNA replication is a collective phenomenon that emerges from the stochastic nature of replication origins initiation.

Partial protection of SIV-infected rhesus monkeys against superinfection with a heterologous SIV isolate

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

Korber, Bette

2009-01-01

Although there is increasing evidence that individuals already infected with human immunodeficiency virus type 1 (HIV-1) can be infected with a heterologous strain of the virus, the extent of protection against superinfection conferred by the first infection and the biologic consequences of superinfection are not well understood. We explored these questions in the simian immunodeficiency virus (SIV)/rhesus monkey model of HIV-1/AIDS. We infected cohorts of rhesus monkeys with either SIVmac251 or SIVsmE660 and then exposed animals to the reciprocal virus through intrarectal inoculations. Employing a quantitative real-time PCR assay, we determined the replication kinetics of the two strains of virusmore » for 20 weeks. We found that primary infection with a replication-competent virus did not protect against acquisition of infection by a heterologous virus but did confer relative control of the superinfecting virus. In animals that became superinfected, there was a reduction in peak replication and rapid control of the second virus. The relative susceptibility to superinfection was not correlated with CD4(+) T-cell count, CD4(+) memory T-cell subsets, cytokine production by virus-specific CD8(+) or CD4(+) cells, or neutralizing antibodies at the time of exposure to the second virus. Although there were transient increases in viral loads of the primary virus and a modest decline in CD4(+) T-cell counts after superinfection, there was no evidence of disease acceleration. These findings indicate that an immunodeficiency virus infection confers partial protection against a second immunodeficiency virus infection, but this protection may be mediated by mechanisms other than classical adaptive immune responses.« less

IL-1β Signaling Promotes CNS-Intrinsic Immune Control of West Nile Virus Infection

PubMed Central

Ramos, Hilario J.; Lanteri, Marion C.; Blahnik, Gabriele; Negash, Amina; Suthar, Mehul S.; Brassil, Margaret M.; Sodhi, Khushbu; Treuting, Piper M.; Busch, Michael P.; Norris, Philip J.; Gale, Michael

2012-01-01

West Nile virus (WNV) is an emerging flavivirus capable of infecting the central nervous system (CNS) and mediating neuronal cell death and tissue destruction. The processes that promote inflammation and encephalitis within the CNS are important for control of WNV disease but, how inflammatory signaling pathways operate to control CNS infection is not defined. Here, we identify IL-1β signaling and the NLRP3 inflammasome as key host restriction factors involved in viral control and CNS disease associated with WNV infection. Individuals presenting with acute WNV infection displayed elevated levels of IL-1β in their plasma over the course of infection, suggesting a role for IL-1β in WNV immunity. Indeed, we found that in a mouse model of infection, WNV induced the acute production of IL-1β in vivo, and that animals lacking the IL-1 receptor or components involved in inflammasome signaling complex exhibited increased susceptibility to WNV pathogenesis. This outcome associated with increased accumulation of virus within the CNS but not peripheral tissues and was further associated with altered kinetics and magnitude of inflammation, reduced quality of the effector CD8+ T cell response and reduced anti-viral activity within the CNS. Importantly, we found that WNV infection triggers production of IL-1β from cortical neurons. Furthermore, we found that IL-1β signaling synergizes with type I IFN to suppress WNV replication in neurons, thus implicating antiviral activity of IL-1β within neurons and control of virus replication within the CNS. Our studies thus define the NLRP3 inflammasome pathway and IL-1β signaling as key features controlling WNV infection and immunity in the CNS, and reveal a novel role for IL-1β in antiviral action that restricts virus replication in neurons. PMID:23209411

Theoretical models for the regulation of DNA replication in fast-growing bacteria

NASA Astrophysics Data System (ADS)

Creutziger, Martin; Schmidt, Mischa; Lenz, Peter

2012-09-01

Growing in always changing environments, Escherichia coli cells are challenged by the task to coordinate growth and division. In particular, adaption of their growth program to the surrounding medium has to guarantee that the daughter cells obtain fully replicated chromosomes. Replication is therefore to be initiated at the right time, which is particularly challenging in media that support fast growth. Here, the mother cell initiates replication not only for the daughter but also for the granddaughter cells. This is possible only if replication occurs from several replication forks that all need to be correctly initiated. Despite considerable efforts during the last 40 years, regulation of this process is still unknown. Part of the difficulty arises from the fact that many details of the relevant molecular processes are not known. Here, we develop a novel theoretical strategy for dealing with this general problem: instead of analyzing a single model, we introduce a wide variety of 128 different models that make different assumptions about the unknown processes. By comparing the predictions of these models we are able to identify the key quantities that allow the experimental discrimination of the different models. Analysis of these quantities yields that out of the 128 models 94 are not consistent with available experimental data. From the remaining 34 models we are able to conclude that mass growth and DNA replication need either to be truly coupled, by coupling DNA replication initiation to the event of cell division, or to the amount of accumulated mass. Finally, we make suggestions for experiments to further reduce the number of possible regulation scenarios.

Aggregate and Individual Replication Probability within an Explicit Model of the Research Process

ERIC Educational Resources Information Center

Miller, Jeff; Schwarz, Wolf

2011-01-01

We study a model of the research process in which the true effect size, the replication jitter due to changes in experimental procedure, and the statistical error of effect size measurement are all normally distributed random variables. Within this model, we analyze the probability of successfully replicating an initial experimental result by…

Ecology and Evolution in the RNA World Dynamics and Stability of Prebiotic Replicator Systems.

PubMed

Szilágyi, András; Zachar, István; Scheuring, István; Kun, Ádám; Könnyű, Balázs; Czárán, Tamás

2017-11-27

As of today, the most credible scientific paradigm pertaining to the origin of life on Earth is undoubtedly the RNA World scenario. It is built on the assumption that catalytically active replicators (most probably RNA-like macromolecules) may have been responsible for booting up life almost four billion years ago. The many different incarnations of nucleotide sequence (string) replicator models proposed recently are all attempts to explain on this basis how the genetic information transfer and the functional diversity of prebiotic replicator systems may have emerged, persisted and evolved into the first living cell. We have postulated three necessary conditions for an RNA World model system to be a dynamically feasible representation of prebiotic chemical evolution: (1) it must maintain and transfer a sufficient diversity of information reliably and indefinitely, (2) it must be ecologically stable and (3) it must be evolutionarily stable. In this review, we discuss the best-known prebiotic scenarios and the corresponding models of string-replicator dynamics and assess them against these criteria. We suggest that the most popular of prebiotic replicator systems, the hypercycle, is probably the worst performer in almost all of these respects, whereas a few other model concepts (parabolic replicator, open chaotic flows, stochastic corrector, metabolically coupled replicator system) are promising candidates for development into coherent models that may become experimentally accessible in the future.

Ecology and Evolution in the RNA World Dynamics and Stability of Prebiotic Replicator Systems

PubMed Central

Szilágyi, András; Kun, Ádám; Könnyű, Balázs; Czárán, Tamás

2017-01-01

As of today, the most credible scientific paradigm pertaining to the origin of life on Earth is undoubtedly the RNA World scenario. It is built on the assumption that catalytically active replicators (most probably RNA-like macromolecules) may have been responsible for booting up life almost four billion years ago. The many different incarnations of nucleotide sequence (string) replicator models proposed recently are all attempts to explain on this basis how the genetic information transfer and the functional diversity of prebiotic replicator systems may have emerged, persisted and evolved into the first living cell. We have postulated three necessary conditions for an RNA World model system to be a dynamically feasible representation of prebiotic chemical evolution: (1) it must maintain and transfer a sufficient diversity of information reliably and indefinitely, (2) it must be ecologically stable and (3) it must be evolutionarily stable. In this review, we discuss the best-known prebiotic scenarios and the corresponding models of string-replicator dynamics and assess them against these criteria. We suggest that the most popular of prebiotic replicator systems, the hypercycle, is probably the worst performer in almost all of these respects, whereas a few other model concepts (parabolic replicator, open chaotic flows, stochastic corrector, metabolically coupled replicator system) are promising candidates for development into coherent models that may become experimentally accessible in the future. PMID:29186916

Kinetic parameter estimation model for anaerobic co-digestion of waste activated sludge and microalgae.

PubMed

Lee, Eunyoung; Cumberbatch, Jewel; Wang, Meng; Zhang, Qiong

2017-03-01

Anaerobic co-digestion has a potential to improve biogas production, but limited kinetic information is available for co-digestion. This study introduced regression-based models to estimate the kinetic parameters for the co-digestion of microalgae and Waste Activated Sludge (WAS). The models were developed using the ratios of co-substrates and the kinetic parameters for the single substrate as indicators. The models were applied to the modified first-order kinetics and Monod model to determine the rate of hydrolysis and methanogenesis for the co-digestion. The results showed that the model using a hyperbola function was better for the estimation of the first-order kinetic coefficients, while the model using inverse tangent function closely estimated the Monod kinetic parameters. The models can be used for estimating kinetic parameters for not only microalgae-WAS co-digestion but also other substrates' co-digestion such as microalgae-swine manure and WAS-aquatic plants. Copyright © 2016 Elsevier Ltd. All rights reserved.

Aggregate and individual replication probability within an explicit model of the research process.

PubMed

Miller, Jeff; Schwarz, Wolf

2011-09-01

We study a model of the research process in which the true effect size, the replication jitter due to changes in experimental procedure, and the statistical error of effect size measurement are all normally distributed random variables. Within this model, we analyze the probability of successfully replicating an initial experimental result by obtaining either a statistically significant result in the same direction or any effect in that direction. We analyze both the probability of successfully replicating a particular experimental effect (i.e., the individual replication probability) and the average probability of successful replication across different studies within some research context (i.e., the aggregate replication probability), and we identify the conditions under which the latter can be approximated using the formulas of Killeen (2005a, 2007). We show how both of these probabilities depend on parameters of the research context that would rarely be known in practice. In addition, we show that the statistical uncertainty associated with the size of an initial observed effect would often prevent accurate estimation of the desired individual replication probability even if these research context parameters were known exactly. We conclude that accurate estimates of replication probability are generally unattainable.

Controlled Chemical Patterns with ThermoChemical NanoLithography (TCNL)

NASA Astrophysics Data System (ADS)

Carroll, Keith; Giordano, Anthony; Wang, Debin; Kodali, Vamsi; King, W. P.; Marder, S. R.; Riedo, E.; Curtis, J. E.

2012-02-01

Many research areas, both fundamental and applied, rely upon the ability to organize non-trivial assemblies of molecules on surfaces. In this work, we introduce a significant extension of ThermoChemical NanoLithography (TCNL), a high throughput chemical patterning technique that uses temperature-driven chemical reactions localized near the tip of a thermal cantilever. By combining a chemical kinetics based model with experiments, we have developed a protocol for varying the concentration of surface bound molecules. The result is an unprecedented ability to fabricate extremely complex patterns comprised of varying chemical concentrations, as demonstrated by sinusoidal patterns of amine groups with varying pitches (˜5-15 μm) and the replication of Leonardo da Vinci's Mona Lisa with dimensions of ˜30 x 40 μm^2. Programmed control of the chemical reaction rate should have widespread applications for a technique which has already been shown to nanopattern various substrates including graphene nanowires, piezoelectric crystals, and optoelectronic materials.

Expanding Health Care Access Through Education: Dissemination and Implementation of the ECHO Model.

PubMed

Katzman, Joanna G; Galloway, Kevin; Olivas, Cynthia; McCoy-Stafford, Kimberly; Duhigg, Daniel; Comerci, George; Kalishman, Summers; Buckenmaier, Chester C; McGhee, Laura; Joltes, Kristin; Bradford, Andrea; Shelley, Brian; Hernandez, Jessica; Arora, Sanjeev

2016-03-01

Project ECHO (Extension for Community Healthcare Outcomes) is an evidence-based model that provides high-quality medical education for common and complex diseases through telementoring and comanagement of patients with primary care clinicians. In a one to many knowledge network, the ECHO model helps to bridge the gap between primary care clinicians and specialists by enhancing the knowledge, skills, confidence, and practice of primary care clinicians in their local communities. As a result, patients in rural and urban underserved areas are able to receive best practice care without long waits or having to travel long distances. The ECHO model has been replicated in 43 university hubs in the United States and five other countries. A new replication tool was developed by the Project ECHO Pain team and U.S. Army Medical Command to ensure a high-fidelity replication of the model. The adoption of the tool led to successful replication of ECHO in the Army Pain initiative. This replication tool has the potential to improve the fidelity of ECHO replication efforts around the world. Reprint & Copyright © 2016 Association of Military Surgeons of the U.S.

Cellular replication limits in the Luria-Delbrück mutation model

NASA Astrophysics Data System (ADS)

Rodriguez-Brenes, Ignacio A.; Wodarz, Dominik; Komarova, Natalia L.

2016-08-01

Originally developed to elucidate the mechanisms of natural selection in bacteria, the Luria-Delbrück model assumed that cells are intrinsically capable of dividing an unlimited number of times. This assumption however, is not true for human somatic cells which undergo replicative senescence. Replicative senescence is thought to act as a mechanism to protect against cancer and the escape from it is a rate-limiting step in cancer progression. Here we introduce a Luria-Delbrück model that explicitly takes into account cellular replication limits in the wild type cell population and models the emergence of mutants that escape replicative senescence. We present results on the mean, variance, distribution, and asymptotic behavior of the mutant population in terms of three classical formulations of the problem. More broadly the paper introduces the concept of incorporating replicative limits as part of the Luria-Delbrück mutational framework. Guidelines to extend the theory to include other types of mutations and possible applications to the modeling of telomere crisis and fluctuation analysis are also discussed.

Pattern of cell kinetics in colorectal mucosa of patients with different types of adenomatous polyps of the large bowel

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

Roncucci, L.; Scalmati, A.; Ponz de Leon, M.

1991-08-15

It is generally accepted that adenomatous polyps represent the natural precursor of many colorectal malignancies. The sequence, however, which leads from a normally appearing mucosa to cancer is complex and involves many steps, including a hyperproliferative mucosa with an upward expansion of the replicative compartment. The current study evaluates cell replication in normal colorectal mucosa of patients with adenomatous polyps of various types and relates the observed findings to the main clinical and morphologic features of adenomas. Forty-four patients with polyps and 27 controls entered the study. Samples of colorectal mucosa were taken at endoscopy and cell replication was evaluatedmore » with a standard autoradiographic procedure. Cell replication was expressed as labeling index (LI), in the whole crypt and in each of the five longitudinal compartments in which the crypts were divided. Total LI and LI per crypt compartment were significantly higher (P less than 0.02 and P less than 0.01, respectively) than in controls. There was no appreciable difference of LI values between patients with single or multiple, tubular or tubulovillous, small or large adenomas, but in all of these subgroups LI was significantly higher than in controls. In conclusion, in normally appearing colorectal mucosa of patients with adenomatous polyps there was a significant increase of cell replication and a marked upward expansion of the proliferative zone; these changes were more evident in the left colon and in the rectum. Finally, cell replication did not seem to be related to the number of polyps, to the most common histotypes, or to the pattern of recurrence.« less

Significant contribution of the 3′→5′ exonuclease activity to the high fidelity of nucleotide incorporation catalyzed by human DNA polymerase ϵ

PubMed Central

Zahurancik, Walter J.; Klein, Seth J.; Suo, Zucai

2014-01-01

Most eukaryotic DNA replication is performed by A- and B-family DNA polymerases which possess a faithful polymerase activity that preferentially incorporates correct over incorrect nucleotides. Additionally, many replicative polymerases have an efficient 3′→5′ exonuclease activity that excises misincorporated nucleotides. Together, these activities contribute to overall low polymerase error frequency (one error per 106–108 incorporations) and support faithful eukaryotic genome replication. Eukaryotic DNA polymerase ϵ (Polϵ) is one of three main replicative DNA polymerases for nuclear genomic replication and is responsible for leading strand synthesis. Here, we employed pre-steady-state kinetic methods and determined the overall fidelity of human Polϵ (hPolϵ) by measuring the individual contributions of its polymerase and 3′→5′ exonuclease activities. The polymerase activity of hPolϵ has a high base substitution fidelity (10−4–10−7) resulting from large decreases in both nucleotide incorporation rate constants and ground-state binding affinities for incorrect relative to correct nucleotides. The 3′→5′ exonuclease activity of hPolϵ further enhances polymerization fidelity by an unprecedented 3.5 × 102 to 1.2 × 104-fold. The resulting overall fidelity of hPolϵ (10−6–10−11) justifies hPolϵ to be a primary enzyme to replicate human nuclear genome (0.1–1.0 error per round). Consistently, somatic mutations in hPolϵ, which decrease its exonuclease activity, are connected with mutator phenotypes and cancer formation. PMID:25414327

De novo identification of replication-timing domains in the human genome by deep learning.

PubMed

Liu, Feng; Ren, Chao; Li, Hao; Zhou, Pingkun; Bo, Xiaochen; Shu, Wenjie

2016-03-01

The de novo identification of the initiation and termination zones-regions that replicate earlier or later than their upstream and downstream neighbours, respectively-remains a key challenge in DNA replication. Building on advances in deep learning, we developed a novel hybrid architecture combining a pre-trained, deep neural network and a hidden Markov model (DNN-HMM) for the de novo identification of replication domains using replication timing profiles. Our results demonstrate that DNN-HMM can significantly outperform strong, discriminatively trained Gaussian mixture model-HMM (GMM-HMM) systems and other six reported methods that can be applied to this challenge. We applied our trained DNN-HMM to identify distinct replication domain types, namely the early replication domain (ERD), the down transition zone (DTZ), the late replication domain (LRD) and the up transition zone (UTZ), using newly replicated DNA sequencing (Repli-Seq) data across 15 human cells. A subsequent integrative analysis revealed that these replication domains harbour unique genomic and epigenetic patterns, transcriptional activity and higher-order chromosomal structure. Our findings support the 'replication-domain' model, which states (1) that ERDs and LRDs, connected by UTZs and DTZs, are spatially compartmentalized structural and functional units of higher-order chromosomal structure, (2) that the adjacent DTZ-UTZ pairs form chromatin loops and (3) that intra-interactions within ERDs and LRDs tend to be short-range and long-range, respectively. Our model reveals an important chromatin organizational principle of the human genome and represents a critical step towards understanding the mechanisms regulating replication timing. Our DNN-HMM method and three additional algorithms can be freely accessed at https://github.com/wenjiegroup/DNN-HMM The replication domain regions identified in this study are available in GEO under the accession ID GSE53984. shuwj@bmi.ac.cn or boxc@bmi.ac.cn Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press.

The rolling-circle melting-pot model for porcine circovirus DNA replication

USDA-ARS?s Scientific Manuscript database

A stem-loop structure, formed by a pair of inverted repeats during DNA replication, is a conserved feature at the origin of DNA replication (Ori) among plant and animal viruses, bacteriophages and plasmids that replicate their genomes via the rolling-circle replication (RCR) mechanism. Porcine circo...

Predicting heavy metals' adsorption edges and adsorption isotherms on MnO2 with the parameters determined from Langmuir kinetics.

PubMed

Hu, Qinghai; Xiao, Zhongjin; Xiong, Xinmei; Zhou, Gongming; Guan, Xiaohong

2015-01-01

Although surface complexation models have been widely used to describe the adsorption of heavy metals, few studies have verified the feasibility of modeling the adsorption kinetics, edge, and isotherm data with one pH-independent parameter. A close inspection of the derivation process of Langmuir isotherm revealed that the equilibrium constant derived from the Langmuir kinetic model, KS-kinetic, is theoretically equivalent to the adsorption constant in Langmuir isotherm, KS-Langmuir. The modified Langmuir kinetic model (MLK model) and modified Langmuir isotherm model (MLI model) incorporating pH factor were developed. The MLK model was employed to simulate the adsorption kinetics of Cu(II), Co(II), Cd(II), Zn(II) and Ni(II) on MnO2 at pH3.2 or 3.3 to get the values of KS-kinetic. The adsorption edges of heavy metals could be modeled with the modified metal partitioning model (MMP model), and the values of KS-Langmuir were obtained. The values of KS-kinetic and KS-Langmuir are very close to each other, validating that the constants obtained by these two methods are basically the same. The MMP model with KS-kinetic constants could predict the adsorption edges of heavy metals on MnO2 very well at different adsorbent/adsorbate concentrations. Moreover, the adsorption isotherms of heavy metals on MnO2 at various pH levels could be predicted reasonably well by the MLI model with the KS-kinetic constants. Copyright © 2014. Published by Elsevier B.V.

Pre-steady-state kinetic investigation of bypass of a bulky guanine lesion by human Y-family DNA polymerases.

PubMed

Tokarsky, E John; Gadkari, Varun V; Zahurancik, Walter J; Malik, Chanchal K; Basu, Ashis K; Suo, Zucai

2016-10-01

3-Nitrobenzanthrone (3-NBA), a byproduct of diesel exhaust, is highly present in the environment and poses a significant health risk. Exposure to 3-NBA results in formation of N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone (dG C8- N -ABA ), a bulky DNA lesion that is of particular importance due to its mutagenic and carcinogenic potential. If not repaired or bypassed during genomic replication, dG C8- N -ABA can stall replication forks, leading to senescence and cell death. Here we used pre-steady-state kinetic methods to determine which of the four human Y-family DNA polymerases (hPolη, hPolκ, hPolι, or hRev1) are able to catalyze translesion synthesis of dG C8- N -ABA in vitro. Our studies demonstrated that hPolη and hPolκ most efficiently bypassed a site-specifically placed dG C8- N- ABA lesion, making them good candidates for catalyzing translesion synthesis (TLS) of this bulky lesion in vivo. Consistently, our publication (Biochemistry 53, 5323-31) in 2014 has shown that small interfering RNA-mediated knockdown of hPolη and hPolκ in HEK293T cells significantly reduces the efficiency of TLS of dG C8- N -ABA . In contrast, hPolι and hRev1 were severely stalled by dG C8- N -ABA and their potential role in vivo was discussed. Subsequently, we determined the kinetic parameters for correct and incorrect nucleotide incorporation catalyzed by hPolη at various positions upstream, opposite, and downstream from dG C8- N- ABA . Notably, nucleotide incorporation efficiency and fidelity both decreased significantly during dG C8- N -ABA bypass and the subsequent extension step, leading to polymerase pausing and error-prone DNA synthesis by hPolη. Furthermore, hPolη displayed nucleotide concentration-dependent biphasic kinetics at the two polymerase pause sites, suggesting that multiple enzyme•DNA complexes likely exist during nucleotide incorporation. Copyright © 2016 Elsevier B.V. All rights reserved.

Detection of cyprinid herpesvirus 2 in peripheral blood cells of silver crucian carp, Carassius auratus gibelio (Bloch), suggests its potential in viral diagnosis.

PubMed

Wang, H; Xu, Lj; Lu, Lq

2016-02-01

Epidemics caused by cyprinid herpesvirus 2 (CyHV-2) in domestic cyprinid species have been reported in both European and Asian countries. Although the mechanisms remain unknown, acute CyHV-2 infections generally result in high mortality, and the surviving carps become chronic carriers displaying no external clinical signs. In this study, in situ hybridization analysis showed that CyHV-2 tended to infect peripheral blood cells during either acute or chronic infections in silver crucian carp, Carassius auratus gibelio (Bloch). Laboratory challenge experiments coupled with real-time PCR quantification assays further indicated that steady-state levels of the viral genomic copy number in fish serum exhibited a typical 'one-step' growth curve post-viral challenge. Transcriptional expression of open reading frames (ORF) 121, which was selected due to its highest transcriptional levels in almost all tested tissues, was monitored to represent the replication kinetics of CyHV-2 in peripheral blood cells. Similar kinetic curve of active viral gene transcription in blood cells was obtained as that of serum viral load, indicating that CyHV-2 replicated in peripheral blood cells as well as in other well-characterized tissues. This study should pave the way for designing non-invasive and cost-effective serum diagnostic methods for quick detection of CyHV-2 infection. © 2015 John Wiley & Sons Ltd.

Induction of the Tumor-Suppressor p16INK4a within Regenerative Epithelial Crypts in Ulcerative Colitis1

PubMed Central

Furth, Emma E; Gustafson, Karen S; Dai, Charlotte Y; Gibson, Steven L; Menard-Katcher, Paul; Chen, Tina; Koh, Jim; Enders, Greg H

2006-01-01

Abstract p16INK4a is a major tumor-suppressor protein, but its regulation and settings of fuction remain poorly understood. To explore the notion that p16 is induced in vivo in response to replicative stress, we examined p16 expression in tissues from human ulcerative colitis (UC; n = 25) and normal controls (n = 20). p16 was expressed strongly in UC-associated neoplasms (n = 17), as seen previously in sporadic colonic neoplasms. In non-neoplastic UC epithelium, p16 was expressed in 33% of crypts (the proliferative compartment) compared to < 1% of normal controls. p16 expression did not correlate with degree of inflammation but did correlate with the degree of crypt architecture distortion (P = .002)—a reflection of epithelial regeneration. In coimmunofluorescence studies with Ki67, p16 expression was associated with cell cycle arrest (P < .001). Both UC and normal crypts displayed evidence for the activation of the DNA damage checkpoint pathway, and p16 was induced in primary cultures of normal epithelial cells by ionizing irradiation (IR). However, induction by IR displayed delayed kinetics, implying that p16 is not an immediate target of the checkpoint pathway. These findings support a model in which p16 is induced as an “emergency brake” in cells experiencing sustained replicative stress. PMID:16820088

Optical tweezers reveal how proteins alter replication

NASA Astrophysics Data System (ADS)

Chaurasiya, Kathy

Single molecule force spectroscopy is a powerful method that explores the DNA interaction properties of proteins involved in a wide range of fundamental biological processes such as DNA replication, transcription, and repair. We use optical tweezers to capture and stretch a single DNA molecule in the presence of proteins that bind DNA and alter its mechanical properties. We quantitatively characterize the DNA binding mechanisms of proteins in order to provide a detailed understanding of their function. In this work, we focus on proteins involved in replication of Escherichia coli (E. coli ), endogenous eukaryotic retrotransposons Ty3 and LINE-1, and human immunodeficiency virus (HIV). DNA polymerases replicate the entire genome of the cell, and bind both double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) during DNA replication. The replicative DNA polymerase in the widely-studied model system E. coli is the DNA polymerase III subunit alpha (DNA pol III alpha). We use optical tweezers to determine that UmuD, a protein that regulates bacterial mutagenesis through its interactions with DNA polymerases, specifically disrupts alpha binding to ssDNA. This suggests that UmuD removes alpha from its ssDNA template to allow DNA repair proteins access to the damaged DNA, and to facilitate exchange of the replicative polymerase for an error-prone translesion synthesis (TLS) polymerase that inserts nucleotides opposite the lesions, so that bacterial DNA replication may proceed. This work demonstrates a biophysical mechanism by which E. coli cells tolerate DNA damage. Retroviruses and retrotransposons reproduce by copying their RNA genome into the nuclear DNA of their eukaryotic hosts. Retroelements encode proteins called nucleic acid chaperones, which rearrange nucleic acid secondary structure and are therefore required for successful replication. The chaperone activity of these proteins requires strong binding affinity for both single- and double-stranded nucleic acids. We use single molecule DNA stretching to show that the nucleocapsid protein (NC) of the yeast retrotransposon Ty3, which is likely to be an ancestor of HIV NC, has optimal nucleic acid chaperone activity with only a single zinc finger. We also show that the chaperone activity of the ORF1 protein is responsible for successful replication of the mouse LINE-1 retrotransposon. LINE-1 is also 17% of the human genome, where it generates insertion mutations and alters gene expression. Retrotransposons such as LINE-1 and Ty3 are likely to be ancestors of retroviruses such as HIV. Human APOBEC3G (A3G) inhibits HIV-1 replication via cytidine deamination of the viral ssDNA genome, as well as via a distinct deamination-independent mechanism. Efficient deamination requires rapid on-off binding kinetics, but a slow dissociation rate is required for the proposed deaminase-independent mechanism. We resolve this apparent contradiction with a new quantitative single molecule method, which shows that A3G initially binds ssDNA with fast on-off rates and subsequently converts to a slow binding mode. This suggests that oligomerization transforms A3G from a fast enzyme to a slow binding protein, which is the biophysical mechanism that allows A3G to inhibit HIV replication. A complete understanding of the mechanism of A3G-mediated antiviral activity is required to design drugs that disrupt the viral response to A3G, enhance A3G packaging inside the viral core, and other potential strategies for long-term treatment of HIV infection. We use single molecule biophysics to explore the function of proteins involved in bacterial DNA replication, endogenous retrotransposition of retroelements in eukaryotic hosts such yeast and mice, and HIV replication in human cells. Our quantitative results provide insight into protein function in a range of complex biological systems and have wide-ranging implications for human health.

Problem solving during artificial selection of self-replicating loops

NASA Astrophysics Data System (ADS)

Chou, Hui-Hsien; Reggia, James A.

1998-05-01

Past cellular automata models of self-replication have generally done only one thing: replicate themselves. However, it has recently been demonstrated that such self-replicating structures can be programmed to also carry out a task during the replication process. Past models of this sort have been limited in that the “program” involved is copied unchanged from parent to child, so that each generation of replicants is executing exactly the same program on exactly the same data. Here we take a different approach in which each replicant receives a distinct partial solution that is modified during replication. Under artificial selection, replicants with promising solutions proliferate while those with failed solutions are lost. We show that this approach can be applied successfully to solve an NP-complete problem, the satisfiability problem. Bounds are given on the cellular space size and time needed to solve a given problem, and simulations demonstrate that this approach works effectively. These and other recent results raise the possibility of evolving self-replicating structures that have a simulated metabolism or that carry out useful tasks.

Kinetic modeling of cell metabolism for microbial production.

PubMed

Costa, Rafael S; Hartmann, Andras; Vinga, Susana

2016-02-10

Kinetic models of cellular metabolism are important tools for the rational design of metabolic engineering strategies and to explain properties of complex biological systems. The recent developments in high-throughput experimental data are leading to new computational approaches for building kinetic models of metabolism. Herein, we briefly survey the available databases, standards and software tools that can be applied for kinetic models of metabolism. In addition, we give an overview about recently developed ordinary differential equations (ODE)-based kinetic models of metabolism and some of the main applications of such models are illustrated in guiding metabolic engineering design. Finally, we review the kinetic modeling approaches of large-scale networks that are emerging, discussing their main advantages, challenges and limitations. Copyright © 2015 Elsevier B.V. All rights reserved.

Leveraging Genomic Annotations and Pleiotropic Enrichment for Improved Replication Rates in Schizophrenia GWAS

PubMed Central

Wang, Yunpeng; Thompson, Wesley K.; Schork, Andrew J.; Holland, Dominic; Chen, Chi-Hua; Bettella, Francesco; Desikan, Rahul S.; Li, Wen; Witoelar, Aree; Zuber, Verena; Devor, Anna; Nöthen, Markus M.; Rietschel, Marcella; Chen, Qiang; Werge, Thomas; Cichon, Sven; Weinberger, Daniel R.; Djurovic, Srdjan; O’Donovan, Michael; Visscher, Peter M.; Andreassen, Ole A.; Dale, Anders M.

2016-01-01

Most of the genetic architecture of schizophrenia (SCZ) has not yet been identified. Here, we apply a novel statistical algorithm called Covariate-Modulated Mixture Modeling (CM3), which incorporates auxiliary information (heterozygosity, total linkage disequilibrium, genomic annotations, pleiotropy) for each single nucleotide polymorphism (SNP) to enable more accurate estimation of replication probabilities, conditional on the observed test statistic (“z-score”) of the SNP. We use a multiple logistic regression on z-scores to combine information from auxiliary information to derive a “relative enrichment score” for each SNP. For each stratum of these relative enrichment scores, we obtain nonparametric estimates of posterior expected test statistics and replication probabilities as a function of discovery z-scores, using a resampling-based approach that repeatedly and randomly partitions meta-analysis sub-studies into training and replication samples. We fit a scale mixture of two Gaussians model to each stratum, obtaining parameter estimates that minimize the sum of squared differences of the scale-mixture model with the stratified nonparametric estimates. We apply this approach to the recent genome-wide association study (GWAS) of SCZ (n = 82,315), obtaining a good fit between the model-based and observed effect sizes and replication probabilities. We observed that SNPs with low enrichment scores replicate with a lower probability than SNPs with high enrichment scores even when both they are genome-wide significant (p < 5x10-8). There were 693 and 219 independent loci with model-based replication rates ≥80% and ≥90%, respectively. Compared to analyses not incorporating relative enrichment scores, CM3 increased out-of-sample yield for SNPs that replicate at a given rate. This demonstrates that replication probabilities can be more accurately estimated using prior enrichment information with CM3. PMID:26808560

Spectral method for a kinetic swarming model

DOE PAGES

Gamba, Irene M.; Haack, Jeffrey R.; Motsch, Sebastien

2015-04-28

Here we present the first numerical method for a kinetic description of the Vicsek swarming model. The kinetic model poses a unique challenge, as there is a distribution dependent collision invariant to satisfy when computing the interaction term. We use a spectral representation linked with a discrete constrained optimization to compute these interactions. To test the numerical scheme we investigate the kinetic model at different scales and compare the solution with the microscopic and macroscopic descriptions of the Vicsek model. Lastly, we observe that the kinetic model captures key features such as vortex formation and traveling waves.

Dynamic Model of Basic Oxygen Steelmaking Process Based on Multi-zone Reaction Kinetics: Model Derivation and Validation

NASA Astrophysics Data System (ADS)

Rout, Bapin Kumar; Brooks, Geoff; Rhamdhani, M. Akbar; Li, Zushu; Schrama, Frank N. H.; Sun, Jianjun

2018-04-01

A multi-zone kinetic model coupled with a dynamic slag generation model was developed for the simulation of hot metal and slag composition during the basic oxygen furnace (BOF) operation. The three reaction zones (i) jet impact zone, (ii) slag-bulk metal zone, (iii) slag-metal-gas emulsion zone were considered for the calculation of overall refining kinetics. In the rate equations, the transient rate parameters were mathematically described as a function of process variables. A micro and macroscopic rate calculation methodology (micro-kinetics and macro-kinetics) were developed to estimate the total refining contributed by the recirculating metal droplets through the slag-metal emulsion zone. The micro-kinetics involves developing the rate equation for individual droplets in the emulsion. The mathematical models for the size distribution of initial droplets, kinetics of simultaneous refining of elements, the residence time in the emulsion, and dynamic interfacial area change were established in the micro-kinetic model. In the macro-kinetics calculation, a droplet generation model was employed and the total amount of refining by emulsion was calculated by summing the refining from the entire population of returning droplets. A dynamic FetO generation model based on oxygen mass balance was developed and coupled with the multi-zone kinetic model. The effect of post-combustion on the evolution of slag and metal composition was investigated. The model was applied to a 200-ton top blowing converter and the simulated value of metal and slag was found to be in good agreement with the measured data. The post-combustion ratio was found to be an important factor in controlling FetO content in the slag and the kinetics of Mn and P in a BOF process.

The POLD3 subunit of DNA polymerase δ can promote translesion synthesis independently of DNA polymerase ζ

PubMed Central

Hirota, Kouji; Yoshikiyo, Kazunori; Guilbaud, Guillaume; Tsurimoto, Toshiki; Murai, Junko; Tsuda, Masataka; Phillips, Lara G.; Narita, Takeo; Nishihara, Kana; Kobayashi, Kaori; Yamada, Kouich; Nakamura, Jun; Pommier, Yves; Lehmann, Alan; Sale, Julian E.; Takeda, Shunichi

2015-01-01

The replicative DNA polymerase Polδ consists of a catalytic subunit POLD1/p125 and three regulatory subunits POLD2/p50, POLD3/p66 and POLD4/p12. The ortholog of POLD3 in Saccharomyces cerevisiae, Pol32, is required for a significant proportion of spontaneous and UV-induced mutagenesis through its additional role in translesion synthesis (TLS) as a subunit of DNA polymerase ζ. Remarkably, chicken DT40 B lymphocytes deficient in POLD3 are viable and able to replicate undamaged genomic DNA with normal kinetics. Like its counterpart in yeast, POLD3 is required for fully effective TLS, its loss resulting in hypersensitivity to a variety of DNA damaging agents, a diminished ability to maintain replication fork progression after UV irradiation and a significant decrease in abasic site-induced mutagenesis in the immunoglobulin loci. However, these defects appear to be largely independent of Polζ, suggesting that POLD3 makes a significant contribution to TLS independently of Polζ in DT40 cells. Indeed, combining polη, polζ and pold3 mutations results in synthetic lethality. Additionally, we show in vitro that POLD3 promotes extension beyond an abasic by the Polδ holoenzyme suggesting that while POLD3 is not required for normal replication, it may help Polδ to complete abasic site bypass independently of canonical TLS polymerases. PMID:25628356

Replication enhancer elements within the open reading frame of tick-borne encephalitis virus and their evolution within the Flavivirus genus

PubMed Central

Tuplin, A.; Evans, D. J.; Buckley, A.; Jones, I. M.; Gould, E. A.; Gritsun, T. S.

2011-01-01

We provide experimental evidence of a replication enhancer element (REE) within the capsid gene of tick-borne encephalitis virus (TBEV, genus Flavivirus). Thermodynamic and phylogenetic analyses predicted that the REE folds as a long stable stem–loop (designated SL6), conserved among all tick-borne flaviviruses (TBFV). Homologous sequences and potential base pairing were found in the corresponding regions of mosquito-borne flaviviruses, but not in more genetically distant flaviviruses. To investigate the role of SL6, nucleotide substitutions were introduced which changed a conserved hexanucleotide motif, the conformation of the terminal loop and the base-paired dsRNA stacking. Substitutions were made within a TBEV reverse genetic system and recovered mutants were compared for plaque morphology, single-step replication kinetics and cytopathic effect. The greatest phenotypic changes were observed in mutants with a destabilized stem. Point mutations in the conserved hexanucleotide motif of the terminal loop caused moderate virus attenuation. However, all mutants eventually reached the titre of wild-type virus late post-infection. Thus, although not essential for growth in tissue culture, the SL6 REE acts to up-regulate virus replication. We hypothesize that this modulatory role may be important for TBEV survival in nature, where the virus circulates by non-viraemic transmission between infected and non-infected ticks, during co-feeding on local rodents. PMID:21622960

Modeling the Population Dynamics of Antibiotic-Resistant Bacteria:. AN Agent-Based Approach

NASA Astrophysics Data System (ADS)

Murphy, James T.; Walshe, Ray; Devocelle, Marc

The response of bacterial populations to antibiotic treatment is often a function of a diverse range of interacting factors. In order to develop strategies to minimize the spread of antibiotic resistance in pathogenic bacteria, a sound theoretical understanding of the systems of interactions taking place within a colony must be developed. The agent-based approach to modeling bacterial populations is a useful tool for relating data obtained at the molecular and cellular level with the overall population dynamics. Here we demonstrate an agent-based model, called Micro-Gen, which has been developed to simulate the growth and development of bacterial colonies in culture. The model also incorporates biochemical rules and parameters describing the kinetic interactions of bacterial cells with antibiotic molecules. Simulations were carried out to replicate the development of methicillin-resistant S. aureus (MRSA) colonies growing in the presence of antibiotics. The model was explored to see how the properties of the system emerge from the interactions of the individual bacterial agents in order to achieve a better mechanistic understanding of the population dynamics taking place. Micro-Gen provides a good theoretical framework for investigating the effects of local environmental conditions and cellular properties on the response of bacterial populations to antibiotic exposure in the context of a simulated environment.

Accounting for inherent variability of growth in microbial risk assessment.

PubMed

Marks, H M; Coleman, M E

2005-04-15

Risk assessments of pathogens need to account for the growth of small number of cells under varying conditions. In order to determine the possible risks that occur when there are small numbers of cells, stochastic models of growth are needed that would capture the distribution of the number of cells over replicate trials of the same scenario or environmental conditions. This paper provides a simple stochastic growth model, accounting only for inherent cell-growth variability, assuming constant growth kinetic parameters, for an initial, small, numbers of cells assumed to be transforming from a stationary to an exponential phase. Two, basic, microbial sets of assumptions are considered: serial, where it is assume that cells transform through a lag phase before entering the exponential phase of growth; and parallel, where it is assumed that lag and exponential phases develop in parallel. The model is based on, first determining the distribution of the time when growth commences, and then modelling the conditional distribution of the number of cells. For the latter distribution, it is found that a Weibull distribution provides a simple approximation to the conditional distribution of the relative growth, so that the model developed in this paper can be easily implemented in risk assessments using commercial software packages.

Somatic stem cells and the kinetics of mutagenesis and carcinogenesis

PubMed Central

Cairns, John

2002-01-01

There is now strong experimental evidence that epithelial stem cells arrange their sister chromatids at mitosis such that the same template DNA strands stay together through successive divisions; DNA labeled with tritiated thymidine in infancy is still present in the stem cells of adult mice even though these cells are incorporating (and later losing) bromodeoxyuridine [Potten, C. S., Owen, G., Booth, D. & Booth, C. (2002) J. Cell Sci.115, 2381–2388]. But a cell that preserves “immortal strands” will avoid the accumulation of replication errors only if it inhibits those pathways for DNA repair that involve potentially error-prone resynthesis of damaged strands, and this appears to be a property of intestinal stem cells because they are extremely sensitive to the lethal effects of agents that damage DNA. It seems that the combination, in the stem cell, of immortal strands and the choice of death rather than error-prone repair makes epithelial stem cell systems resistant to short exposures to DNA-damaging agents, because the stem cell accumulates few if any errors, and any errors made by the daughters are destined to be discarded. This paper discusses these issues and shows that they lead to a model that explains the strange kinetics of mutagenesis and carcinogenesis in adult mammalian tissues. Coincidentally, the model also can explain why cancers arise even though the spontaneous mutation rate of differentiated mammalian cells is not high enough to generate the multiple mutations needed to form a cancer and why loss of nucleotide-excision repair does not significantly increase the frequency of the common internal cancers. PMID:12149477

Novel mechanism of antibodies to hepatitis B virus in blocking viral particle release from cells.

PubMed

Neumann, Avidan U; Phillips, Sandra; Levine, Idit; Ijaz, Samreen; Dahari, Harel; Eren, Rachel; Dagan, Shlomo; Naoumov, Nikolai V

2010-09-01

Antibodies are thought to exert antiviral activities by blocking viral entry into cells and/or accelerating viral clearance from circulation. In particular, antibodies to hepatitis B virus (HBV) surface antigen (HBsAg) confer protection, by binding circulating virus. Here, we used mathematical modeling to gain information about viral dynamics during and after single or multiple infusions of a combination of two human monoclonal anti-HBs (HepeX-B) antibodies in patients with chronic hepatitis B. The antibody HBV-17 recognizes a conformational epitope, whereas antibody HBV-19 recognizes a linear epitope on the HBsAg. The kinetic profiles of the decline of serum HBV DNA and HBsAg revealed partial blocking of virion release from infected cells as a new antiviral mechanism, in addition to acceleration of HBV clearance from the circulation. We then replicated this approach in vitro, using cells secreting HBsAg, and compared the prediction of the mathematical modeling obtained from the in vivo kinetics. In vitro, HepeX-B treatment of HBsAg-producing cells showed cellular uptake of antibodies, resulting in intracellular accumulation of viral particles. Blocking of HBsAg secretion also continued after HepeX-B was removed from the cell culture supernatants. These results identify a novel antiviral mechanism of antibodies to HBsAg (anti-HBs) involving prolonged blocking of the HBV and HBsAg subviral particles release from infected cells. This may have implications in designing new therapies for patients with chronic HBV infection and may also be relevant in other viral infections.

Inhibition of rotavirus replication by downregulation of fatty acid synthesis.

PubMed

Gaunt, Eleanor R; Cheung, Winsome; Richards, James E; Lever, Andrew; Desselberger, Ulrich

2013-06-01

Recently the recruitment of lipid droplets (LDs) to sites of rotavirus (RV) replication was reported. LDs are polymorphic organelles that store triacylglycerols, cholesterol and cholesterol esters. The neutral fats are derived from palmitoyl-CoA, synthesized via the fatty acid biosynthetic pathway. RV-infected cells were treated with chemical inhibitors of the fatty acid biosynthetic pathway, and the effects on viral replication kinetics were assessed. Treatment with compound C75, an inhibitor of the fatty acid synthase enzyme complex (FASN), reduced RV infectivity 3.2-fold (P = 0.07) and modestly reduced viral RNA synthesis (1.2-fold). Acting earlier in the fatty acid synthesis pathway, TOFA [5-(Tetradecyloxy)-2-furoic acid] inhibits the enzyme acetyl-CoA carboxylase 1 (ACC1). TOFA reduced the infectivity of progeny RV 31-fold and viral RNA production 6-fold. The effect of TOFA on RV infectivity and RNA replication was dose-dependent, and infectivity was reduced by administering TOFA up to 4 h post-infection. Co-treatment of RV-infected cells with C75 and TOFA synergistically reduced viral infectivity. Knockdown by siRNA of FASN and ACC1 produced findings similar to those observed by inhibiting these proteins with the chemical compounds. Inhibition of fatty acid synthesis using a range of approaches uniformly had a more marked impact on viral infectivity than on viral RNA yield, inferring a role for LDs in virus assembly and/or egress. Specific inhibitors of fatty acid metabolism may help pinpoint the critical structural and biochemical features of LDs that are essential for RV replication, and facilitate the development of antiviral therapies.

Enzyme catalysis in microgravity: steady-state kinetic analysis of the isocitrate lyase reaction.

PubMed

Ranaldi, Francesco; Vanni, Paolo; Giachetti, Eugenio

2003-01-21

Two decades of research in microgravity have shown that certain biochemical processes can be altered by weightlessness. Approximately 10 years ago, our team, supported by the European Space Agency (ESA) and the Agenzia Spaziale Italiana, started the Effect of Microgravity on Enzyme Catalysis project to test the possibility that the microgravity effect observed at cellular level could be mediated by enzyme reactions. An experiment to study the cleavage reaction catalyzed by isocitrate lyase was flown on the sounding rocket MASER 7, and we found that the kinetic parameters were not altered by microgravity. During the 28th ESA parabolic flight campaign, we had the opportunity to replicate the MASER 7 experiment and to perform a complete steady-state analysis of the isocitrate lyase reaction. This study showed that both in microgravity and in standard g controls the enzyme reaction obeyed the same kinetic mechanism and none of the kinetic parameters, nor the equilibrium constant of the overall reaction were altered. Our results contrast with those of a similar experiment, which was performed during the same parabolic flight campaign, and showed that microgravity increased the affinity of lipoxygenase-1 for linoleic acid. The hypotheses suggested to explain this change effect of the latter were here tested by computer simulation, and appeared to be inconsistent with the experimental outcome.

Rate-determining Step of Flap Endonuclease 1 (FEN1) Reflects a Kinetic Bias against Long Flaps and Trinucleotide Repeat Sequences.

PubMed

Tarantino, Mary E; Bilotti, Katharina; Huang, Ji; Delaney, Sarah

2015-08-21

Flap endonuclease 1 (FEN1) is a structure-specific nuclease responsible for removing 5'-flaps formed during Okazaki fragment maturation and long patch base excision repair. In this work, we use rapid quench flow techniques to examine the rates of 5'-flap removal on DNA substrates of varying length and sequence. Of particular interest are flaps containing trinucleotide repeats (TNR), which have been proposed to affect FEN1 activity and cause genetic instability. We report that FEN1 processes substrates containing flaps of 30 nucleotides or fewer at comparable single-turnover rates. However, for flaps longer than 30 nucleotides, FEN1 kinetically discriminates substrates based on flap length and flap sequence. In particular, FEN1 removes flaps containing TNR sequences at a rate slower than mixed sequence flaps of the same length. Furthermore, multiple-turnover kinetic analysis reveals that the rate-determining step of FEN1 switches as a function of flap length from product release to chemistry (or a step prior to chemistry). These results provide a kinetic perspective on the role of FEN1 in DNA replication and repair and contribute to our understanding of FEN1 in mediating genetic instability of TNR sequences. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Kinetics of bacterial phospholipase C activity at micellar interfaces: effect of substrate aggregate microstructure and a model for the kinetic parameters.

PubMed

Singh, Jasmeet; Ranganathan, Radha; Hajdu, Joseph

2008-12-25

Activity at micellar interfaces of bacterial phospholipase C from Bacillus cereus on phospholipids solubilized in micelles was investigated with the goal of elucidating the role of the interface microstructure and developing further an existing kinetic model. Enzyme kinetics and physicochemical characterization of model substrate aggregates were combined, thus enabling the interpretation of kinetics in the context of the interface. Substrates were diacylphosphatidylcholine of different acyl chain lengths in the form of mixed micelles with dodecyldimethylammoniopropanesulfonate. An early kinetic model, reformulated to reflect the interfacial nature of the kinetics, was applied to the kinetic data. A better method of data treatment is proposed, use of which makes the presence of microstructure effects quite transparent. Models for enzyme-micelle binding and enzyme-lipid binding are developed, and expressions incorporating the microstructural properties are derived for the enzyme-micelle dissociation constant K(s) and the interface Michaelis-Menten constant, K(M). Use of these expressions in the interface kinetic model brings excellent agreement between the kinetic data and the model. Numerical values for the thermodynamic and kinetic parameters are determined. Enzyme-lipid binding is found to be an activated process with an acyl chain length dependent free energy of activation that decreases with micelle lipid molar fraction with a coefficient of about -15RT and correlates with the tightness of molecular packing in the substrate aggregate. Thus, the physical insight obtained includes a model for the kinetic parameters that shows that these parameters depend on the substrate concentration and acyl chain length of the lipid. Enzyme-micelle binding is indicated to be hydrophobic and solvent mediated with a dissociation constant of 1.2 mM.

Replication: A "Model" Approach to the Healthy Development of Young Children

ERIC Educational Resources Information Center

Krieg, Iris; Lewis, Jan

2005-01-01

The authors, directors of the Chicago-based Pritzker Early Childhood Foundation, advocate "replication," the adaptation of a successful model program or practice to new locations or to new populations. Studies have shown that successful replications of early childhood programs that help at-risk children and their families can have long-term,…

DNA polymerases eta and kappa exchange with the polymerase delta holoenzyme to complete common fragile site synthesis.

PubMed

Barnes, Ryan P; Hile, Suzanne E; Lee, Marietta Y; Eckert, Kristin A

2017-09-01

Common fragile sites (CFSs) are inherently unstable genomic loci that are recurrently altered in human tumor cells. Despite their instability, CFS are ubiquitous throughout the human genome and associated with large tumor suppressor genes or oncogenes. CFSs are enriched with repetitive DNA sequences, one feature postulated to explain why these loci are inherently difficult to replicate, and sensitive to replication stress. We have shown that specialized DNA polymerases (Pols) η and κ replicate CFS-derived sequences more efficiently than the replicative Pol δ. However, we lacked an understanding of how these enzymes cooperate to ensure efficient CFS replication. Here, we designed a model of lagging strand replication with RFC loaded PCNA that allows for maximal activity of the four-subunit human Pol δ holoenzyme, Pol η, and Pol κ in polymerase mixing assays. We discovered that Pol η and κ are both able to exchange with Pol δ stalled at repetitive CFS sequences, enhancing Normalized Replication Efficiency. We used this model to test the impact of PCNA mono-ubiquitination on polymerase exchange, and found no change in polymerase cooperativity in CFS replication compared with unmodified PCNA. Finally, we modeled replication stress in vitro using aphidicolin and found that Pol δ holoenzyme synthesis was significantly inhibited in a dose-dependent manner, preventing any replication past the CFS. Importantly, Pol η and κ were still proficient in rescuing this stalled Pol δ synthesis, which may explain, in part, the CFS instability phenotype of aphidicolin-treated Pol η and Pol κ-deficient cells. In total, our data support a model wherein Pol δ stalling at CFSs allows for free exchange with a specialized polymerase that is not driven by PCNA. Copyright © 2017 Elsevier B.V. All rights reserved.

Lack of Norovirus Replication and Histo-Blood Group Antigen Expression in 3-Dimensional Intestinal Epithelial Cells

PubMed Central

Radtke, Andrea L.; Lay, Margarita K.; Hjelm, Brooke E.; Bolick, Alice N.; Sarker, Shameema S.; Atmar, Robert L.; Kingsley, David H.; Arntzen, Charles J.; Estes, Mary K.; Nickerson, Cheryl A.

2013-01-01

Noroviruses (NoVs) are a leading cause of gastroenteritis worldwide. An in vitro model for NoV replication remains elusive, making study of the virus difficult. A previous study, which used a 3-dimensional (3-D) intestinal model derived from INT-407 cells reported NoV replication and extensive cytopathic effects (CPE). Using the same 3-D model, but with highly purified Norwalk virus (NV), we attempted to replicate this study. Our results showed no evidence of NV replication by real-time PCR of viral RNA or by immunocytochemical detection of viral structural and nonstructural proteins. Immunocytochemical analysis of the 3-D cultures also showed no detectable presence of histo-blood group antigens that participate in NV binding and host tropism. To determine the potential cause of CPE observed in the previous study, we exposed 3-D cultures to lipopolysaccharide concentrations consistent with contaminated stool samples and observed morphologic features similar to CPE. We conclude that the 3-D INT-407 model does not support NV replication. PMID:23622517

Simple systems that exhibit self-directed replication

NASA Technical Reports Server (NTRS)

Reggia, James A.; Armentrout, Steven L.; Chou, Hui-Hsien; Peng, Yun

1993-01-01

Biological experience and intuition suggest that self-replication is an inherently complex phenomenon, and early cellular automata models support that conception. More recently, simpler computational models of self-directed replication called sheathed loops have been developed. It is shown here that 'unsheathing' these structures and altering certain assumptions about the symmetry of their components leads to a family of nontrivial self-replicating structures some substantially smaller and simpler than those previously reported. The dependence of replication time and transition function complexity on initial structure size, cell state symmetry, and neighborhood are examined. These results support the view that self-replication is not an inherently complex phenomenon but rather an emergent property arising from local interactions in systems that can be much simpler than is generally believed.

A kinetics database and scripts for PHREEQC

NASA Astrophysics Data System (ADS)

Hu, B.; Zhang, Y.; Teng, Y.; Zhu, C.

2017-12-01

Kinetics of geochemical reactions has been increasingly used in numerical models to simulate coupled flow, mass transport, and chemical reactions. However, the kinetic data are scattered in the literature. To assemble a kinetic dataset for a modeling project is an intimidating task for most. In order to facilitate the application of kinetics in geochemical modeling, we assembled kinetics parameters into a database for the geochemical simulation program, PHREEQC (version 3.0). Kinetics data were collected from the literature. Our database includes kinetic data for over 70 minerals. The rate equations are also programmed into scripts with the Basic language. Using the new kinetic database, we simulated reaction path during the albite dissolution process using various rate equations in the literature. The simulation results with three different rate equations gave difference reaction paths at different time scale. Another application involves a coupled reactive transport model simulating the advancement of an acid plume in an acid mine drainage site associated with Bear Creek Uranium tailings pond. Geochemical reactions including calcite, gypsum, and illite were simulated with PHREEQC using the new kinetic database. The simulation results successfully demonstrated the utility of new kinetic database.

Replicating annual North Atlantic hurricane activity 1878-2012 from environmental variables

NASA Astrophysics Data System (ADS)

Saunders, Mark A.; Klotzbach, Philip J.; Lea, Adam S. R.

2017-06-01

Statistical models can replicate annual North Atlantic hurricane activity from large-scale environmental field data for August and September, the months of peak hurricane activity. We assess how well the six environmental fields used most often in contemporary statistical modeling of seasonal hurricane activity replicate North Atlantic hurricane numbers and Accumulated Cyclone Energy (ACE) over the 135 year period from 1878 to 2012. We find that these fields replicate historical hurricane activity surprisingly well, showing that contemporary statistical models and their seasonal physical links have long-term robustness. We find that August-September zonal trade wind speed over the Caribbean Sea and the tropical North Atlantic is the environmental field which individually replicates long-term hurricane activity the best and that trade wind speed combined with the difference in sea surface temperature between the tropical Atlantic and the tropical mean is the best multi-predictor model. Comparing the performance of the best single-predictor and best multi-predictor models shows that they exhibit little difference in hindcast skill for predicting long-term ACE but that the best multipredictor model offers improved skill for predicting long-term hurricane numbers. We examine whether replicated real-time prediction skill 1983-2012 increases as the model training period lengthens and find evidence that this happens slowly. We identify a dropout in hurricane replication centered on the 1940s and show that this is likely due to a decrease in data quality which affects all data sets but Atlantic sea surface temperatures in particular. Finally, we offer insights on the implications of our findings for seasonal hurricane prediction.

Modelling dimercaptosuccinic acid (DMSA) plasma kinetics in humans.

PubMed

van Eijkeren, Jan C H; Olie, J Daniël N; Bradberry, Sally M; Vale, J Allister; de Vries, Irma; Meulenbelt, Jan; Hunault, Claudine C

2016-11-01

No kinetic models presently exist which simulate the effect of chelation therapy on lead blood concentrations in lead poisoning. Our aim was to develop a kinetic model that describes the kinetics of dimercaptosuccinic acid (DMSA; succimer), a commonly used chelating agent, that could be used in developing a lead chelating model. This was a kinetic modelling study. We used a two-compartment model, with a non-systemic gastrointestinal compartment (gut lumen) and the whole body as one systemic compartment. The only data available from the literature were used to calibrate the unknown model parameters. The calibrated model was then validated by comparing its predictions with measured data from three different experimental human studies. The model predicted total DMSA plasma and urine concentrations measured in three healthy volunteers after ingestion of DMSA 10 mg/kg. The model was then validated by using data from three other published studies; it predicted concentrations within a factor of two, representing inter-human variability. A simple kinetic model simulating the kinetics of DMSA in humans has been developed and validated. The interest of this model lies in the future potential to use it to predict blood lead concentrations in lead-poisoned patients treated with DMSA.

Modeling DNA Replication.

ERIC Educational Resources Information Center

Bennett, Joan

1998-01-01

Recommends the use of a model of DNA made out of Velcro to help students visualize the steps of DNA replication. Includes a materials list, construction directions, and details of the demonstration using the model parts. (DDR)

Kinetic Modeling of a Heterogeneous Fenton Oxidative Treatment of Petroleum Refining Wastewater

PubMed Central

Basheer Hasan, Diya'uddeen; Abdul Raman, Abdul Aziz; Wan Daud, Wan Mohd Ashri

2014-01-01

The mineralisation kinetics of petroleum refinery effluent (PRE) by Fenton oxidation were evaluated. Within the ambit of the experimental data generated, first-order kinetic model (FKM), generalised lumped kinetic model (GLKM), and generalized kinetic model (GKM) were tested. The obtained apparent kinetic rate constants for the initial oxidation step (k 2′), their final oxidation step (k 1′), and the direct conversion to endproducts step (k 3′) were 10.12, 3.78, and 0.24 min−1 for GKM; 0.98, 0.98, and nil min−1 for GLKM; and nil, nil, and >0.005 min−1 for FKM. The findings showed that GKM is superior in estimating the mineralization kinetics. PMID:24592152

Software reliability report

NASA Technical Reports Server (NTRS)

Wilson, Larry

1991-01-01

There are many software reliability models which try to predict future performance of software based on data generated by the debugging process. Unfortunately, the models appear to be unable to account for the random nature of the data. If the same code is debugged multiple times and one of the models is used to make predictions, intolerable variance is observed in the resulting reliability predictions. It is believed that data replication can remove this variance in lab type situations and that it is less than scientific to talk about validating a software reliability model without considering replication. It is also believed that data replication may prove to be cost effective in the real world, thus the research centered on verification of the need for replication and on methodologies for generating replicated data in a cost effective manner. The context of the debugging graph was pursued by simulation and experimentation. Simulation was done for the Basic model and the Log-Poisson model. Reasonable values of the parameters were assigned and used to generate simulated data which is then processed by the models in order to determine limitations on their accuracy. These experiments exploit the existing software and program specimens which are in AIR-LAB to measure the performance of reliability models.

In silico ribozyme evolution in a metabolically coupled RNA population.

PubMed

Könnyű, Balázs; Szilágyi, András; Czárán, Tamás

2015-05-27

The RNA World hypothesis offers a plausible bridge from no-life to life on prebiotic Earth, by assuming that RNA, the only known molecule type capable of playing genetic and catalytic roles at the same time, could have been the first evolvable entity on the evolutionary path to the first living cell. We have developed the Metabolically Coupled Replicator System (MCRS), a spatially explicit simulation modelling approach to prebiotic RNA-World evolution on mineral surfaces, in which we incorporate the most important experimental facts and theoretical considerations to comply with recent knowledge on RNA and prebiotic evolution. In this paper the MCRS model framework has been extended in order to investigate the dynamical and evolutionary consequences of adding an important physico-chemical detail, namely explicit replicator structure - nucleotide sequence and 2D folding calculated from thermodynamical criteria - and their possible mutational changes, to the assumptions of a previously less detailed toy model. For each mutable nucleotide sequence the corresponding 2D folded structure with minimum free energy is calculated, which in turn is used to determine the fitness components (degradation rate, replicability and metabolic enzyme activity) of the replicator. We show that the community of such replicators providing the monomer supply for their own replication by evolving metabolic enzyme activities features an improved propensity for stable coexistence and structural adaptation. These evolutionary advantages are due to the emergent uniformity of metabolic replicator fitnesses imposed on the community by local group selection and attained through replicator trait convergence, i.e., the tendency of replicator lengths, ribozyme activities and population sizes to become similar between the coevolving replicator species that are otherwise both structurally and functionally different. In the most general terms it is the surprisingly high extra viability of the metabolic replicator system that the present model adds to the MCRS concept of the origin of life. Surface-bound, metabolically coupled RNA replicators tend to evolve different, enzymatically active sites within thermodynamically stable secondary structures, and the system as a whole evolves towards the robust coexistence of a complete set of such ribozymes driving the metabolism producing monomers for their own replication.

Mark R. Nimlos | NREL

Science.gov Websites

, reaction kinetics, computational modeling, photochemistry, and molecular spectroscopy. Nimlos has served as Chemical reaction energetics and kinetics Biomass pyrolysis and gasification Heterogeneous catalysis in zeolites Quantum modeling and kinetic modeling of reaction Molecular dynamics modeling

Agent-based modeling and systems dynamics model reproduction.

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

North, M. J.; Macal, C. M.

2009-01-01

Reproducibility is a pillar of the scientific endeavour. We view computer simulations as laboratories for electronic experimentation and therefore as tools for science. Recent studies have addressed model reproduction and found it to be surprisingly difficult to replicate published findings. There have been enough failed simulation replications to raise the question, 'can computer models be fully replicated?' This paper answers in the affirmative by reporting on a successful reproduction study using Mathematica, Repast and Swarm for the Beer Game supply chain model. The reproduction process was valuable because it demonstrated the original result's robustness across modelling methodologies and implementation environments.

G4 motifs affect origin positioning and efficiency in two vertebrate replicators

PubMed Central

Valton, Anne-Laure; Hassan-Zadeh, Vahideh; Lema, Ingrid; Boggetto, Nicole; Alberti, Patrizia; Saintomé, Carole; Riou, Jean-François; Prioleau, Marie-Noëlle

2014-01-01

DNA replication ensures the accurate duplication of the genome at each cell cycle. It begins at specific sites called replication origins. Genome-wide studies in vertebrates have recently identified a consensus G-rich motif potentially able to form G-quadruplexes (G4) in most replication origins. However, there is no experimental evidence to demonstrate that G4 are actually required for replication initiation. We show here, with two model origins, that G4 motifs are required for replication initiation. Two G4 motifs cooperate in one of our model origins. The other contains only one critical G4, and its orientation determines the precise position of the replication start site. Point mutations affecting the stability of this G4 in vitro also impair origin function. Finally, this G4 is not sufficient for origin activity and must cooperate with a 200-bp cis-regulatory element. In conclusion, our study strongly supports the predicted essential role of G4 in replication initiation. PMID:24521668

Human Fanconi Anemia Complementation Group A Protein Stimulates the 5’ Flap Endonuclease Activity of FEN1

PubMed Central

Qian, Liangyue; Yuan, Fenghua; Rodriguez-Tello, Paola; Padgaonkar, Suyog; Zhang, Yanbin

2013-01-01

In eukaryotic cells, Flap endonuclease 1 (FEN1) is a major structure-specific endonuclease that processes 5’ flapped structures during maturation of lagging strand DNA synthesis, long patch base excision repair, and rescue of stalled replication forks. Here we report that fanconi anemia complementation group A protein (FANCA), a protein that recognizes 5’ flap structures and is involved in DNA repair and maintenance of replication forks, constantly stimulates FEN1-mediated incision of both DNA and RNA flaps. Kinetic analyses indicate that FANCA stimulates FEN1 by increasing the turnover rate of FEN1 and altering its substrate affinity. More importantly, six pathogenic FANCA mutants are significantly less efficient than the wild-type at stimulating FEN1 endonuclease activity, implicating that regulation of FEN1 by FANCA contributes to the maintenance of genomic stability. PMID:24349332

G0-G1 Transition and the Restriction Point in Pancreatic β-Cells In Vivo

PubMed Central

Hija, Ayat; Salpeter, Seth; Klochendler, Agnes; Grimsby, Joseph; Brandeis, Michael; Glaser, Benjamin; Dor, Yuval

2014-01-01

Most of our knowledge on cell kinetics stems from in vitro studies of continuously dividing cells. In this study, we determine in vivo cell-cycle parameters of pancreatic β-cells, a largely quiescent population, using drugs that mimic or prevent glucose-induced replication of β-cells in mice. Quiescent β-cells exposed to a mitogenic glucose stimulation require 8 h to enter the G1 phase of the cell cycle, and this time is prolonged in older age. The duration of G1, S, and G2/M is ∼5, 8, and 6 h, respectively. We further provide the first in vivo demonstration of the restriction point at the G0-G1 transition, discovered by Arthur Pardee 40 years ago. The findings may have pharmacodynamic implications in the design of regenerative therapies aimed at increasing β-cell replication and mass in patients with diabetes. PMID:24130333

Kinetics of liver macrophages (Kupffer cells) in SIV-infected macaques

PubMed Central

Ahsan, Muhammad H.; Gill, Amy F.; Alvarez, Xavier; Lackner, Andrew A.; Veazey, Ronald S.

2013-01-01

Since the liver drains antigens from the intestinal tract, and since the intestinal tract is a major site of viral replication, we examined the dynamics of liver macrophages (Kupffer cells) throughout SIV infection. Absolute numbers of Kupffer cells increased in the livers in acute infection, and in animals with AIDS. Significantly higher percentages of proliferating (BrdU+) Kupffer cells were detected in acute infection and in AIDS with similar trends in blood monocytes. Significantly higher percentages of apoptotic (AC3+) Kupffer cells were also found in acute and AIDS stages. However, productively infected cells were not detected in liver of 41/42 animals examined, despite abundant infected cells in gut and lymph nodes of all animals. Increased rates of Kupffer cell proliferation resulting in an increase in Kupffer cells without productive infection indicate SIV infection affects Kupffer cells, but the liver does not appear to be a major site of productive viral replication. PMID:24074569

Kinetics of Methylmercury Production Revisited

DOE PAGES

Olsen, Todd A.; Muller, Katherine A.; Painter, Scott L.; ...

2018-01-27

Laboratory measurements of the biologically mediated methylation of mercury (Hg) to the neurotoxin monomethylmercury (MMHg) often exhibit kinetics that are inconsistent with first-order kinetic models. Using time-resolved measurements of filter passing Hg and MMHg during methylation/demethylation assays, a multisite kinetic sorption model, and reanalyses of previous assays, we show in this paper that competing kinetic sorption reactions can lead to time-varying availability and apparent non-first-order kinetics in Hg methylation and MMHg demethylation. The new model employing a multisite kinetic sorption model for Hg and MMHg can describe the range of behaviors for time-resolved methylation/demethylation data reported in the literature includingmore » those that exhibit non-first-order kinetics. Additionally, we show that neglecting competing sorption processes can confound analyses of methylation/demethylation assays, resulting in rate constant estimates that are systematically biased low. Finally, simulations of MMHg production and transport in a hypothetical periphyton biofilm bed illustrate the implications of our new model and demonstrate that methylmercury production may be significantly different than projected by single-rate first-order models.« less

Correlation between lack of norovirus replication and histo-blood group antigen expression in 3D-intestinal epithelial cultures

USDA-ARS?s Scientific Manuscript database

Noroviruses (NoV) are a leading cause of gastroenteritis worldwide. An in vitro model for NoV replication remains elusive, making study of the virus difficult. One publication utilizing a 3-dimensional (3D) intestinal model derived from Int407 cells reported NoV replication and extensive cytopathi...

Replication of genetic associations as pseudoreplication due to shared genealogy.

PubMed

Rosenberg, Noah A; Vanliere, Jenna M

2009-09-01

The genotypes of individuals in replicate genetic association studies have some level of correlation due to shared descent in the complete pedigree of all living humans. As a result of this genealogical sharing, replicate studies that search for genotype-phenotype associations using linkage disequilibrium between marker loci and disease-susceptibility loci can be considered as "pseudoreplicates" rather than true replicates. We examine the size of the pseudoreplication effect in association studies simulated from evolutionary models of the history of a population, evaluating the excess probability that both of a pair of studies detect a disease association compared to the probability expected under the assumption that the two studies are independent. Each of nine combinations of a demographic model and a penetrance model leads to a detectable pseudoreplication effect, suggesting that the degree of support that can be attributed to a replicated genetic association result is less than that which can be attributed to a replicated result in a context of true independence.

Replication of genetic associations as pseudoreplication due to shared genealogy

PubMed Central

Rosenberg, Noah A.; VanLiere, Jenna M.

2009-01-01

The genotypes of individuals in replicate genetic association studies have some level of correlation due to shared descent in the complete pedigree of all living humans. As a result of this genealogical sharing, replicate studies that search for genotype-phenotype associations using linkage disequilibrium between marker loci and disease-susceptibility loci can be considered “pseudoreplicates” rather than true replicates. We examine the size of the pseudoreplication effect in association studies simulated from evolutionary models of the history of a population, evaluating the excess probability that both of a pair of studies detect a disease association compared to the probability expected under the assumption that the two studies are independent. Each of nine combinations of a demographic model and a penetrance model leads to a detectable pseudoreplication effect, suggesting that the degree of support that can be attributed to a replicated genetic association result is less than that which can be attributed to a replicated result in a context of true independence. PMID:19191270

Extracting surface diffusion coefficients from batch adsorption measurement data: application of the classic Langmuir kinetics model.

PubMed

Chu, Khim Hoong

2017-11-09

Surface diffusion coefficients may be estimated by fitting solutions of a diffusion model to batch kinetic data. For non-linear systems, a numerical solution of the diffusion model's governing equations is generally required. We report here the application of the classic Langmuir kinetics model to extract surface diffusion coefficients from batch kinetic data. The use of the Langmuir kinetics model in lieu of the conventional surface diffusion model allows derivation of an analytical expression. The parameter estimation procedure requires determining the Langmuir rate coefficient from which the pertinent surface diffusion coefficient is calculated. Surface diffusion coefficients within the 10 -9 to 10 -6  cm 2 /s range obtained by fitting the Langmuir kinetics model to experimental kinetic data taken from the literature are found to be consistent with the corresponding values obtained from the traditional surface diffusion model. The virtue of this simplified parameter estimation method is that it reduces the computational complexity as the analytical expression involves only an algebraic equation in closed form which is easily evaluated by spreadsheet computation.

NACSA Charter School Replication Guide: The Spectrum of Replication Options. Authorizing Matters. Replication Brief 1

ERIC Educational Resources Information Center

O'Neill, Paul

2010-01-01

One of the most important and high-profile issues in public education reform today is the replication of successful public charter school programs. With more than 5,000 failing public schools in the United States, there is a tremendous need for strong alternatives for parents and students. Replicating successful charter school models is an…

Comparison of the kinetic parameters of the truncated catalytic subunit and holoenzyme of human DNA polymerase ε

PubMed Central

Zahurancik, Walter J.; Baranovskiy, Andrey G.; Tahirov, Tahir H.; Suo, Zucai

2015-01-01

Numerous genetic studies have provided compelling evidence to establish DNA polymerase ε (Polε) as the primary DNA polymerase responsible for leading strand synthesis during eukaryotic nuclear genome replication. Polε is a heterotetramer consisting of a large catalytic subunit that contains the conserved polymerase core domain as well as a 3′ → 5′ exonuclease domain common to many replicative polymerases. In addition, Polε possesses three small subunits that lack a known catalytic activity but associate with components involved in a variety of DNA replication and maintenance processes. Previous enzymatic characterization of the Polε heterotetramer from budding yeast suggested that the small subunits slightly enhance DNA synthesis by Polε in vitro. However, similar studies of the human Polε heterote-tramer (hPolε) have been limited by the difficulty of obtaining hPolε in quantities suitable for thorough investigation of its catalytic activity. Utilization of a baculovirus expression system for overexpression and purification of hPolε from insect host cells has allowed for isolation of greater amounts of active hPolε, thus enabling a more detailed kinetic comparison between hPolε and an active N-terminal fragment of the hPolε catalytic subunit (p261N), which is readily overexpressed in Escherichia coli. Here, we report the first pre-steady-state studies of fully-assembled hPolε. We observe that the small subunits increase DNA binding by hPolε relative to p261N, but do not increase processivity during DNA synthesis on a single-stranded M13 template. Interestingly, the 3′ → 5′ exonuclease activity of hPolε is reduced relative to p261N on matched and mismatched DNA substrates, indicating that the presence of the small subunits may regulate the proofreading activity of hPolε and sway hPolε toward DNA synthesis rather than proofreading. PMID:25684708

Kinetics of liver macrophages (Kupffer cells) in SIV-infected macaques

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

Ahsan, Muhammad H.; Gill, Amy F.; Alvarez, Xavier

Since the liver drains antigens from the intestinal tract, and since the intestinal tract is a major site of viral replication, we examined the dynamics of liver macrophages (Kupffer cells) throughout SIV infection. Absolute numbers of Kupffer cells increased in the livers in acute infection, and in animals with AIDS. Significantly higher percentages of proliferating (BrdU+) Kupffer cells were detected in acute infection and in AIDS with similar trends in blood monocytes. Significantly higher percentages of apoptotic (AC3+) Kupffer cells were also found in acute and AIDS stages. However, productively infected cells were not detected in liver of 41/42 animalsmore » examined, despite abundant infected cells in gut and lymph nodes of all animals. Increased rates of Kupffer cell proliferation resulting in an increase in Kupffer cells without productive infection indicate SIV infection affects Kupffer cells, but the liver does not appear to be a major site of productive viral replication. - Highlights: • Kupffer cells increase in the liver of SIV-infected macaques. • Increased proliferation and apoptosis of Kupffer cells occurs in SIV infection. • Productively infected cells are rarely detected in the liver. • The liver is not a major site for SIV replication.« less

Template Directed Replication Supports the Maintenance of the Metabolically Coupled Replicator System

NASA Astrophysics Data System (ADS)

Könnyű, Balázs; Czárán, Tamás

2015-06-01

The RNA World scenario of prebiotic chemical evolution is among the most plausible conceptual framework available today for modelling the origin of life. RNA offers genetic and catalytic (metabolic) functionality embodied in a single chemical entity, and a metabolically cooperating community of RNA molecules would constitute a viable infrabiological subsystem with a potential to evolve into proto-cellular life. Our Metabolically Coupled Replicator System (MCRS) model is a spatially explicit computer simulation implementation of the RNA-World scenario, in which replicable ribozymes cooperate in supplying each other with monomers for their own replication. MCRS has been repeatedly demonstrated to be viable and evolvable, with different versions of the model improved in depth (chemical detail of metabolism) or in extension (additional functions of RNA molecules). One of the dynamically relevant extensions of the MCRS approach to prebiotic RNA evolution is the explicit inclusion of template replication into its assumptions, which we have studied in the present version. We found that this modification has not changed the behaviour of the system in the qualitative sense, just the range of the parameter space which is optimal for the coexistence of metabolically cooperating replicators has shifted in terms of metabolite mobility. The system also remains resistant and tolerant to parasitic replicators.

The replication domain model: regulating replicon firing in the context of large-scale chromosome architecture.

PubMed

Pope, Benjamin D; Gilbert, David M

2013-11-29

The "Replicon Theory" of Jacob, Brenner, and Cuzin has reliably served as the paradigm for regulating the sites where individual replicons initiate replication. Concurrent with the replicon model was Taylor's demonstration that plant and animal chromosomes replicate segmentally in a defined temporal sequence, via cytologically defined units too large to be accounted for by a single replicon. Instead, there seemed to be a program to choreograph when chromosome units replicate during S phase, executed by initiation at clusters of individual replicons within each segment. Here, we summarize recent molecular evidence for the existence of such units, now known as "replication domains", and discuss how the organization of large chromosomes into structural units has added additional layers of regulation to the original replicon model. Copyright © 2012 Elsevier Ltd. All rights reserved.

An evaluation of the Bayesian approach to fitting the N-mixture model for use with pseudo-replicated count data

USGS Publications Warehouse

Toribo, S.G.; Gray, B.R.; Liang, S.

2011-01-01

The N-mixture model proposed by Royle in 2004 may be used to approximate the abundance and detection probability of animal species in a given region. In 2006, Royle and Dorazio discussed the advantages of using a Bayesian approach in modelling animal abundance and occurrence using a hierarchical N-mixture model. N-mixture models assume replication on sampling sites, an assumption that may be violated when the site is not closed to changes in abundance during the survey period or when nominal replicates are defined spatially. In this paper, we studied the robustness of a Bayesian approach to fitting the N-mixture model for pseudo-replicated count data. Our simulation results showed that the Bayesian estimates for abundance and detection probability are slightly biased when the actual detection probability is small and are sensitive to the presence of extra variability within local sites.

A study of the kinetic energy generation with general circulation models

NASA Technical Reports Server (NTRS)

Chen, T.-C.; Lee, Y.-H.

1983-01-01

The history data of winter simulation by the GLAS climate model and the NCAR community climate model are used to examine the generation of atmospheric kinetic energy. The contrast between the geographic distributions of the generation of kinetic energy and divergence of kinetic energy flux shows that kinetic energy is generated in the upstream side of jets, transported to the downstream side and destroyed there. The contributions from the time-mean and transient modes to the counterbalance between generation of kinetic energy and divergence of kinetic energy flux are also investigated. It is observed that the kinetic energy generated by the time-mean mode is essentially redistributed by the time-mean flow, while that generated by the transient flow is mainly responsible for the maintenance of the kinetic energy of the entire atmospheric flow.

Genome-wide alterations of the DNA replication program during tumor progression

NASA Astrophysics Data System (ADS)

Arneodo, A.; Goldar, A.; Argoul, F.; Hyrien, O.; Audit, B.

2016-08-01

Oncogenic stress is a major driving force in the early stages of cancer development. Recent experimental findings reveal that, in precancerous lesions and cancers, activated oncogenes may induce stalling and dissociation of DNA replication forks resulting in DNA damage. Replication timing is emerging as an important epigenetic feature that recapitulates several genomic, epigenetic and functional specificities of even closely related cell types. There is increasing evidence that chromosome rearrangements, the hallmark of many cancer genomes, are intimately associated with the DNA replication program and that epigenetic replication timing changes often precede chromosomic rearrangements. The recent development of a novel methodology to map replication fork polarity using deep sequencing of Okazaki fragments has provided new and complementary genome-wide replication profiling data. We review the results of a wavelet-based multi-scale analysis of genomic and epigenetic data including replication profiles along human chromosomes. These results provide new insight into the spatio-temporal replication program and its dynamics during differentiation. Here our goal is to bring to cancer research, the experimental protocols and computational methodologies for replication program profiling, and also the modeling of the spatio-temporal replication program. To illustrate our purpose, we report very preliminary results obtained for the chronic myelogeneous leukemia, the archetype model of cancer. Finally, we discuss promising perspectives on using genome-wide DNA replication profiling as a novel efficient tool for cancer diagnosis, prognosis and personalized treatment.

MINIGENOMES, TRANSCRIPTION AND REPLICATION COMPETENT VIRUS-LIKE PARTICLES AND BEYOND: REVERSE GENETICS SYSTEMS FOR FILOVIRUSES AND OTHER NEGATIVE STRANDED HEMORRHAGIC FEVER VIRUSES

PubMed Central

Hoenen, Thomas; Groseth, Allison; de Kok-Mercado, Fabian; Kuhn, Jens H.; Wahl-Jensen, Victoria

2012-01-01

Reverse-genetics systems are powerful tools enabling researchers to study the replication cycle of RNA viruses, including filoviruses and other hemorrhagic fever viruses, as well as to discover new antivirals. They include full-length clone systems as well as a number of life cycle modeling systems. Full-length clone systems allow for the generation of infectious, recombinant viruses, and thus are an important tool for studying the virus replication cycle in its entirety. In contrast, life cycle modeling systems such as minigenome and transcription and replication competent virus-like particle systems can be used to simulate and dissect parts of the virus life cycle outside of containment facilities. Minigenome systems are used to model viral genome replication and transcription, whereas transcription and replication competent virus-like particle systems also model morphogenesis and budding as well as infection of target cells. As such, these modeling systems have tremendous potential to further the discovery and screening of new antivirals targeting hemorrhagic fever viruses. This review provides an overview of currently established reverse genetics systems for hemorrhagic fever-causing negative-sense RNA viruses, with a particular emphasis on filoviruses, and the potential application of these systems for antiviral research. PMID:21699921

Cardiac-gated parametric images from 82 Rb PET from dynamic frames and direct 4D reconstruction.

PubMed

Germino, Mary; Carson, Richard E

2018-02-01

Cardiac perfusion PET data can be reconstructed as a dynamic sequence and kinetic modeling performed to quantify myocardial blood flow, or reconstructed as static gated images to quantify function. Parametric images from dynamic PET are conventionally not gated, to allow use of all events with lower noise. An alternative method for dynamic PET is to incorporate the kinetic model into the reconstruction algorithm itself, bypassing the generation of a time series of emission images and directly producing parametric images. So-called "direct reconstruction" can produce parametric images with lower noise than the conventional method because the noise distribution is more easily modeled in projection space than in image space. In this work, we develop direct reconstruction of cardiac-gated parametric images for 82 Rb PET with an extension of the Parametric Motion compensation OSEM List mode Algorithm for Resolution-recovery reconstruction for the one tissue model (PMOLAR-1T). PMOLAR-1T was extended to accommodate model terms to account for spillover from the left and right ventricles into the myocardium. The algorithm was evaluated on a 4D simulated 82 Rb dataset, including a perfusion defect, as well as a human 82 Rb list mode acquisition. The simulated list mode was subsampled into replicates, each with counts comparable to one gate of a gated acquisition. Parametric images were produced by the indirect (separate reconstructions and modeling) and direct methods for each of eight low-count and eight normal-count replicates of the simulated data, and each of eight cardiac gates for the human data. For the direct method, two initialization schemes were tested: uniform initialization, and initialization with the filtered iteration 1 result of the indirect method. For the human dataset, event-by-event respiratory motion compensation was included. The indirect and direct methods were compared for the simulated dataset in terms of bias and coefficient of variation as a function of iteration. Convergence of direct reconstruction was slow with uniform initialization; lower bias was achieved in fewer iterations by initializing with the filtered indirect iteration 1 images. For most parameters and regions evaluated, the direct method achieved the same or lower absolute bias at matched iteration as the indirect method, with 23%-65% lower noise. Additionally, the direct method gave better contrast between the perfusion defect and surrounding normal tissue than the indirect method. Gated parametric images from the human dataset had comparable relative performance of indirect and direct, in terms of mean parameter values per iteration. Changes in myocardial wall thickness and blood pool size across gates were readily visible in the gated parametric images, with higher contrast between myocardium and left ventricle blood pool in parametric images than gated SUV images. Direct reconstruction can produce parametric images with less noise than the indirect method, opening the potential utility of gated parametric imaging for perfusion PET. © 2017 American Association of Physicists in Medicine.

Variance Estimation Using Replication Methods in Structural Equation Modeling with Complex Sample Data

ERIC Educational Resources Information Center

Stapleton, Laura M.

2008-01-01

This article discusses replication sampling variance estimation techniques that are often applied in analyses using data from complex sampling designs: jackknife repeated replication, balanced repeated replication, and bootstrapping. These techniques are used with traditional analyses such as regression, but are currently not used with structural…

DEIsoM: a hierarchical Bayesian model for identifying differentially expressed isoforms using biological replicates

PubMed Central

Peng, Hao; Yang, Yifan; Zhe, Shandian; Wang, Jian; Gribskov, Michael; Qi, Yuan

2017-01-01

Abstract Motivation High-throughput mRNA sequencing (RNA-Seq) is a powerful tool for quantifying gene expression. Identification of transcript isoforms that are differentially expressed in different conditions, such as in patients and healthy subjects, can provide insights into the molecular basis of diseases. Current transcript quantification approaches, however, do not take advantage of the shared information in the biological replicates, potentially decreasing sensitivity and accuracy. Results We present a novel hierarchical Bayesian model called Differentially Expressed Isoform detection from Multiple biological replicates (DEIsoM) for identifying differentially expressed (DE) isoforms from multiple biological replicates representing two conditions, e.g. multiple samples from healthy and diseased subjects. DEIsoM first estimates isoform expression within each condition by (1) capturing common patterns from sample replicates while allowing individual differences, and (2) modeling the uncertainty introduced by ambiguous read mapping in each replicate. Specifically, we introduce a Dirichlet prior distribution to capture the common expression pattern of replicates from the same condition, and treat the isoform expression of individual replicates as samples from this distribution. Ambiguous read mapping is modeled as a multinomial distribution, and ambiguous reads are assigned to the most probable isoform in each replicate. Additionally, DEIsoM couples an efficient variational inference and a post-analysis method to improve the accuracy and speed of identification of DE isoforms over alternative methods. Application of DEIsoM to an hepatocellular carcinoma (HCC) dataset identifies biologically relevant DE isoforms. The relevance of these genes/isoforms to HCC are supported by principal component analysis (PCA), read coverage visualization, and the biological literature. Availability and implementation The software is available at https://github.com/hao-peng/DEIsoM Contact pengh@alumni.purdue.edu Supplementary information Supplementary data are available at Bioinformatics online. PMID:28595376

Effect of Leflunomide, Cidofovir and Ciprofloxacin on replication of BKPyV in a salivary gland in vitro culture system.

PubMed

Jeffers-Francis, Liesl K; Burger-Calderon, Raquel; Webster-Cyriaque, Jennifer

2015-06-01

BK polyomavirus (BKPyV) is a known kidney tropic virus that has been detected at high levels in HIV-associated salivary gland disease (HIV-SGD), one of the most important AIDS associated oral lesions. BKPyV has been detected in HIV-SGD patient saliva and replicates in salivary gland cells in vitro. BKPyV antivirals are currently in wide use to guard against BKPyV mediated organ rejection in kidney transplant recipients. The goal of this study was to investigate the inhibitory effects of three such antiviral agents, Ciprofloxacin, Cidofovir, and Leflunomide in BKPyV infected salivary gland cells. Human salivary gland cells, and Vero cells, were infected with BKPyV, treated with antiviral drugs and assessed for BKPyV gene expression and viral replication for up to 5 days post infection. The kinetics of BKPyV replication were different in salivary gland cells compared to kidney cells. Ciprofloxacin and Cidofovir had minimal effect on metabolic activity and host cell DNA replication, however, cell toxicity was detected at the protein level with Leflunomide treatment. Ciprofloxacin decreased BKV T Ag and VP1 mRNA expression by at least 50% in both cell types, and decreased T Ag protein expression at days 3 and 4 post infection. A 2.5-4 log decrease in intracellular DNA replication and a 2-3 log decrease in progeny release were detected with Ciprofloxacin treatment. Cidofovir and Leflunomide also inhibited BKPyV gene expression and DNA replication. The three drugs diminished progeny release by 30-90% and 2- to 6-fold decreases in infectious virus were detected post drug treatment by fluorescence focus assay. Additionally, three clinical BKPyV isolates were assessed for their responses to these agents in vitro. Cidofovir and Leflunomide, but not Ciprofloxacin treatment resulted in statistically significant inhibition of BKPyV progeny release from salivary gland cells infected with HIVSGD BKPyV isolates. All three drugs decreased progeny release from cells infected with a transplant derived viral isolate. In conclusion, treatment of human salivary gland cells with each of the three drugs produced modest decreases in BKPyV genome replication. These data highlight the need for continued studies to discover more effective and less toxic drugs that inhibit BKPyV replication in salivary gland cells. Copyright © 2015 Elsevier B.V. All rights reserved.

Effect of sex steroid hormones on replication and transmission of major HIV subtypes.

PubMed

Ragupathy, Viswanath; Devadas, Krishnakumar; Tang, Shixing; Wood, Owen; Lee, Sherwin; Dastyer, Armeta; Wang, Xue; Dayton, Andrew; Hewlett, Indira

2013-11-01

The HIV epidemic is expanding worldwide with an increasing number of distinct viral subtypes and circulating recombinant forms (CRFs). Out of 34 million adults living with HIV and AIDS, women account for one half of all HIV-1 infections worldwide. These gender differences in HIV pathogenesis may be attributed to sex hormones. Little is known about the role of sex hormone effects on HIV Subtypes pathogenesis. The aim of our study was to determine sex hormone effects on replication and transmissibility of HIV subtypes. Peripheral blood mononuclear cells (PBMC) and monocyte derived dendritic cells (MDDC) from male and female donors were infected with HIV subtypes A-D and CRF02_AG, CRF01_AE, MN (lab adapted), Group-O, Group-N and HIV-2 at a concentration of 5ng/ml of p24 or p27. Virus production was evaluated by measuring p24 and p27 levels in culture supernatants. Similar experiments were carried out in the presence of physiological concentrations of sex steroid hormones. R5/X4 expressions measured by flow cytometry and transmissibility was evaluated by transfer of HIV from primary dendritic cells (DC) to autologous donor PBMC. Our results from primary PBMC and MDDC from male and female donors indicate in the absence of physiological concentrations of hormone treatment virus production was observed in three clusters; high replicating virus (subtype B and C), moderate replicative virus (subtype A, D, CRF01_AE, Group_N) and least replicative virus (strain MN). However, dose of sex steroid hormone treatment influenced HIV replication and transmission kinetics in PBMC, DCs and cell lines. Such effects were inconsistent between donors and HIV subtypes. Sex hormone effects on HIV entry receptors (CCR5/CXCR4) did not correlate with virus production. Subtypes B and C showed higher replication in PBMC from males and females and were transmitted more efficiently through DC to male and female PBMC compared with other HIV-1 subtypes, HIV-1 Group O and HIV-2. These findings are consistent with increased worldwide prevalence of subtype B and C compared to other subtypes. Sex steroid hormones had variable effect on replication or transmission of different subtypes. These findings suggest that subtype, gender and sex hormones may play a crucial role in the replication and transmission of HIV. Published by Elsevier Ltd.

Type I interferon inhibits varicella-zoster virus replication by interfering with the dynamic interaction between mediator and IE62 within replication compartments.

PubMed

Ku, Chia-Chi; Chang, Yi-Hsuan; Chien, Yun; Lee, Tsung-Lin

2016-01-01

Varicella-zoster virus (VZV) is the causative agent of varicella and zoster. The immediate-early protein, IE62 is the predominant VZ virion tegument protein, transactivating the expression of all kinetic classes of VZV genes. IE62 is localized to punctae that form DNA replication compartments in the nuclei of VZV infected cells. The morphological changes and the increase in the size of replication compartments that express IE62 are correlated with production of VZ virions. Mammalian Mediator serves as a coactivator of IE62 and functions by bridging DNA-binding transcription factors¸ RNA polymerase II (RNAP II) and their target DNAs for VZV replication. While VZV is highly sensitive to type I interferons (IFNs), how IFN-α inhibits early events during VZV replication is poorly understood. In this study, we performed in situ analysis to investigate the effects of IFN-α on the dynamic interactions of IE62 with the Mediator MED25 subunit and the RNAP II negative regulator cycle-dependent kinase 8 (CDK8) in VZV infected cells by confocal immunofluorescence. We found that in addition to dose-dependent inhibition of the yields of infectious virus by IFN treatment, IFN-α prominently impeded the development of large IE62(+) nuclear compartments and significantly decreased transcription of VZV genes. Both the expression level and stable recruitment of MED25 to IE62(+) replication compartments were inhibited by IFN-α. While IFN-α treatment upregulated CDK8 expression, redistribution and recruitment of CDK8 to IE62(+) replication compartments in infected cells was not affected by VZV. IFN-α exerts multiple inhibitory activities against virus infections. In this study, we provide visionary demonstration that continuous translocation of MED25 into VZV replication compartments ensures production of virions. IFN-α greatly impedes the formation of a stable complex between IE62 and the Mediator complex thereby suppresses VZV gene transcription. Our demonstration that IFN-α-induced antiviral effect against VZV infection is through inhibiting the reorganization of nuclear components uncovers a novel function of IFN-α. Targeting the interaction between IE62 and MED25 may offer a novel approach to the development of antiviral agents against VZV infection.

Phase-field Model for Interstitial Loop Growth Kinetics and Thermodynamic and Kinetic Models of Irradiated Fe-Cr Alloys

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

Li, Yulan; Hu, Shenyang Y.; Sun, Xin

2011-06-15

Microstructure evolution kinetics in irradiated materials has strongly spatial correlation. For example, void and second phases prefer to nucleate and grow at pre-existing defects such as dislocations, grain boundaries, and cracks. Inhomogeneous microstructure evolution results in inhomogeneity of microstructure and thermo-mechanical properties. Therefore, the simulation capability for predicting three dimensional (3-D) microstructure evolution kinetics and its subsequent impact on material properties and performance is crucial for scientific design of advanced nuclear materials and optimal operation conditions in order to reduce uncertainty in operational and safety margins. Very recently the meso-scale phase-field (PF) method has been used to predict gas bubblemore » evolution, void swelling, void lattice formation and void migration in irradiated materials,. Although most results of phase-field simulations are qualitative due to the lake of accurate thermodynamic and kinetic properties of defects, possible missing of important kinetic properties and processes, and the capability of current codes and computers for large time and length scale modeling, the simulations demonstrate that PF method is a promising simulation tool for predicting 3-D heterogeneous microstructure and property evolution, and providing microstructure evolution kinetics for higher scale level simulations of microstructure and property evolution such as mean field methods. This report consists of two parts. In part I, we will present a new phase-field model for predicting interstitial loop growth kinetics in irradiated materials. The effect of defect (vacancy/interstitial) generation, diffusion and recombination, sink strength, long-range elastic interaction, inhomogeneous and anisotropic mobility on microstructure evolution kinetics is taken into account in the model. The model is used to study the effect of elastic interaction on interstitial loop growth kinetics, the interstitial flux, and sink strength of interstitial loop for interstitials. In part II, we present a generic phase field model and discuss the thermodynamic and kinetic properties in phase-field models including the reaction kinetics of radiation defects and local free energy of irradiated materials. In particular, a two-sublattice thermodynamic model is suggested to describe the local free energy of alloys with irradiated defects. Fe-Cr alloy is taken as an example to explain the required thermodynamic and kinetic properties for quantitative phase-field modeling. Finally the great challenges in phase-field modeling will be discussed.« less

Impact of time-of-flight on indirect 3D and direct 4D parametric image reconstruction in the presence of inconsistent dynamic PET data.

PubMed

Kotasidis, F A; Mehranian, A; Zaidi, H

2016-05-07

Kinetic parameter estimation in dynamic PET suffers from reduced accuracy and precision when parametric maps are estimated using kinetic modelling following image reconstruction of the dynamic data. Direct approaches to parameter estimation attempt to directly estimate the kinetic parameters from the measured dynamic data within a unified framework. Such image reconstruction methods have been shown to generate parametric maps of improved precision and accuracy in dynamic PET. However, due to the interleaving between the tomographic and kinetic modelling steps, any tomographic or kinetic modelling errors in certain regions or frames, tend to spatially or temporally propagate. This results in biased kinetic parameters and thus limits the benefits of such direct methods. Kinetic modelling errors originate from the inability to construct a common single kinetic model for the entire field-of-view, and such errors in erroneously modelled regions could spatially propagate. Adaptive models have been used within 4D image reconstruction to mitigate the problem, though they are complex and difficult to optimize. Tomographic errors in dynamic imaging on the other hand, can originate from involuntary patient motion between dynamic frames, as well as from emission/transmission mismatch. Motion correction schemes can be used, however, if residual errors exist or motion correction is not included in the study protocol, errors in the affected dynamic frames could potentially propagate either temporally, to other frames during the kinetic modelling step or spatially, during the tomographic step. In this work, we demonstrate a new strategy to minimize such error propagation in direct 4D image reconstruction, focusing on the tomographic step rather than the kinetic modelling step, by incorporating time-of-flight (TOF) within a direct 4D reconstruction framework. Using ever improving TOF resolutions (580 ps, 440 ps, 300 ps and 160 ps), we demonstrate that direct 4D TOF image reconstruction can substantially prevent kinetic parameter error propagation either from erroneous kinetic modelling, inter-frame motion or emission/transmission mismatch. Furthermore, we demonstrate the benefits of TOF in parameter estimation when conventional post-reconstruction (3D) methods are used and compare the potential improvements to direct 4D methods. Further improvements could possibly be achieved in the future by combining TOF direct 4D image reconstruction with adaptive kinetic models and inter-frame motion correction schemes.

Impact of time-of-flight on indirect 3D and direct 4D parametric image reconstruction in the presence of inconsistent dynamic PET data

NASA Astrophysics Data System (ADS)

Kotasidis, F. A.; Mehranian, A.; Zaidi, H.

2016-05-01

Kinetic parameter estimation in dynamic PET suffers from reduced accuracy and precision when parametric maps are estimated using kinetic modelling following image reconstruction of the dynamic data. Direct approaches to parameter estimation attempt to directly estimate the kinetic parameters from the measured dynamic data within a unified framework. Such image reconstruction methods have been shown to generate parametric maps of improved precision and accuracy in dynamic PET. However, due to the interleaving between the tomographic and kinetic modelling steps, any tomographic or kinetic modelling errors in certain regions or frames, tend to spatially or temporally propagate. This results in biased kinetic parameters and thus limits the benefits of such direct methods. Kinetic modelling errors originate from the inability to construct a common single kinetic model for the entire field-of-view, and such errors in erroneously modelled regions could spatially propagate. Adaptive models have been used within 4D image reconstruction to mitigate the problem, though they are complex and difficult to optimize. Tomographic errors in dynamic imaging on the other hand, can originate from involuntary patient motion between dynamic frames, as well as from emission/transmission mismatch. Motion correction schemes can be used, however, if residual errors exist or motion correction is not included in the study protocol, errors in the affected dynamic frames could potentially propagate either temporally, to other frames during the kinetic modelling step or spatially, during the tomographic step. In this work, we demonstrate a new strategy to minimize such error propagation in direct 4D image reconstruction, focusing on the tomographic step rather than the kinetic modelling step, by incorporating time-of-flight (TOF) within a direct 4D reconstruction framework. Using ever improving TOF resolutions (580 ps, 440 ps, 300 ps and 160 ps), we demonstrate that direct 4D TOF image reconstruction can substantially prevent kinetic parameter error propagation either from erroneous kinetic modelling, inter-frame motion or emission/transmission mismatch. Furthermore, we demonstrate the benefits of TOF in parameter estimation when conventional post-reconstruction (3D) methods are used and compare the potential improvements to direct 4D methods. Further improvements could possibly be achieved in the future by combining TOF direct 4D image reconstruction with adaptive kinetic models and inter-frame motion correction schemes.

Establishment and characterization of a telomerase immortalized human gingival epithelial cell line.

PubMed

Moffatt-Jauregui, C E; Robinson, B; de Moya, A V; Brockman, R D; Roman, A V; Cash, M N; Culp, D J; Lamont, R J

2013-12-01

Gingival keratinocytes are used in model systems to investigate the interaction between periodontal bacteria and the epithelium in the initial stages of the periodontal disease process. Primary gingival epithelial cells (GECs) have a finite lifespan in culture before they enter senescence and cease to replicate, while epithelial cells immortalized with viral proteins can exhibit chromosomal rearrangements. The aim of this study was to generate a telomerase immortalized human gingival epithelial cell line and compare its in vitro behaviour to that of human GECs. Human primary GECs were immortalized with a bmi1/hTERT combination to prevent cell cycle triggers of senescence and telomere shortening. The resultant cell-line, telomerase immortalized gingival keratinocytes (TIGKs), were compared to GECs for cell morphology, karyotype, growth and cytokeratin expression, and further characterized for replicative lifespan, expression of toll-like receptors and invasion by P. gingivalis. TIGKs showed morphologies, karyotype, proliferation rates and expression of characteristic cytokeratin proteins comparable to GECs. TIGKs underwent 36 passages without signs of senescence and expressed transcripts for toll-like receptors 1-6, 8 and 9. A subpopulation of cells underwent stratification after extended time in culture. The cytokeratin profiles of TIGK monolayers were consistent with basal cells. When allowed to stratify, cytokeratin profiles of TIGKs were consistent with suprabasal cells of the junctional epithelium. Further, TIGKs were comparable to GECs in previously reported levels and kinetics of invasion by wild-type P. gingivalis and an invasion defective ΔserB mutant. Results confirm bmi1/hTERT immortalization of primary GECs generated a robust cell line with similar characteristics to the parental cell type. TIGKs represent a valuable model system for the study of oral bacteria interactions with host gingival cells. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

APOBEC3D and APOBEC3F potently promote HIV-1 diversification and evolution in humanized mouse model.

PubMed

Sato, Kei; Takeuchi, Junko S; Misawa, Naoko; Izumi, Taisuke; Kobayashi, Tomoko; Kimura, Yuichi; Iwami, Shingo; Takaori-Kondo, Akifumi; Hu, Wei-Shau; Aihara, Kazuyuki; Ito, Mamoru; An, Dong Sung; Pathak, Vinay K; Koyanagi, Yoshio

2014-10-01

Several APOBEC3 proteins, particularly APOBEC3D, APOBEC3F, and APOBEC3G, induce G-to-A hypermutations in HIV-1 genome, and abrogate viral replication in experimental systems, but their relative contributions to controlling viral replication and viral genetic variation in vivo have not been elucidated. On the other hand, an HIV-1-encoded protein, Vif, can degrade these APOBEC3 proteins via a ubiquitin/proteasome pathway. Although APOBEC3 proteins have been widely considered as potent restriction factors against HIV-1, it remains unclear which endogenous APOBEC3 protein(s) affect HIV-1 propagation in vivo. Here we use a humanized mouse model and HIV-1 with mutations in Vif motifs that are responsible for specific APOBEC3 interactions, DRMR/AAAA (4A) or YRHHY/AAAAA (5A), and demonstrate that endogenous APOBEC3D/F and APOBEC3G exert strong anti-HIV-1 activity in vivo. We also show that the growth kinetics of 4A HIV-1 negatively correlated with the expression level of APOBEC3F. Moreover, single genome sequencing analyses of viral RNA in plasma of infected mice reveal that 4A HIV-1 is specifically and significantly diversified. Furthermore, a mutated virus that is capable of using both CCR5 and CXCR4 as entry coreceptor is specifically detected in 4A HIV-1-infected mice. Taken together, our results demonstrate that APOBEC3D/F and APOBEC3G fundamentally work as restriction factors against HIV-1 in vivo, but at the same time, that APOBEC3D and APOBEC3F are capable of promoting viral diversification and evolution in vivo.

An Expanded Multi-scale Monte Carlo Simulation Method for Personalized Radiobiological Effect Estimation in Radiotherapy: a feasibility study

NASA Astrophysics Data System (ADS)

Zhang, Ying; Feng, Yuanming; Wang, Wei; Yang, Chengwen; Wang, Ping

2017-03-01

A novel and versatile “bottom-up” approach is developed to estimate the radiobiological effect of clinic radiotherapy. The model consists of multi-scale Monte Carlo simulations from organ to cell levels. At cellular level, accumulated damages are computed using a spectrum-based accumulation algorithm and predefined cellular damage database. The damage repair mechanism is modeled by an expanded reaction-rate two-lesion kinetic model, which were calibrated through replicating a radiobiological experiment. Multi-scale modeling is then performed on a lung cancer patient under conventional fractionated irradiation. The cell killing effects of two representative voxels (isocenter and peripheral voxel of the tumor) are computed and compared. At microscopic level, the nucleus dose and damage yields vary among all nucleuses within the voxels. Slightly larger percentage of cDSB yield is observed for the peripheral voxel (55.0%) compared to the isocenter one (52.5%). For isocenter voxel, survival fraction increase monotonically at reduced oxygen environment. Under an extreme anoxic condition (0.001%), survival fraction is calculated to be 80% and the hypoxia reduction factor reaches a maximum value of 2.24. In conclusion, with biological-related variations, the proposed multi-scale approach is more versatile than the existing approaches for evaluating personalized radiobiological effects in radiotherapy.

Experimental West Nile Virus Infection in Rabbits: An Alternative Model for Studying Induction of Disease and Virus Control

PubMed Central

Suen, Willy W.; Uddin, Muhammad J.; Wang, Wenqi; Brown, Vienna; Adney, Danielle R.; Broad, Nicole; Prow, Natalie A.; Bowen, Richard A.; Hall, Roy A.; Bielefeldt-Ohmann, Helle

2015-01-01

The economic impact of non-lethal human and equine West Nile virus (WNV) disease is substantial, since it is the most common presentation of the infection. Experimental infection with virulent WNV strains in the mouse and hamster models frequently results in severe neural infection and moderate to high mortality, both of which are not representative features of most human and equine infections. We have established a rabbit model for investigating pathogenesis and immune response of non-lethal WNV infection. Two species of rabbits, New Zealand White (Oryctolagus cuniculus) and North American cottontail (Sylvilagus sp.), were experimentally infected with virulent WNV and Murray Valley encephalitis virus strains. Infected rabbits exhibited a consistently resistant phenotype, with evidence of low viremia, minimal-absent neural infection, mild-moderate neuropathology, and the lack of mortality, even though productive virus replication occurred in the draining lymph node. The kinetics of anti-WNV neutralizing antibody response was comparable to that commonly seen in infected horses and humans. This may be explained by the early IFNα/β and/or γ response evident in the draining popliteal lymph node. Given this similarity to the human and equine disease, immunocompetent rabbits are, therefore, a valuable animal model for investigating various aspects of non-lethal WNV infections. PMID:26184326

Characterization of recent and minimally passaged Brazilian dengue viruses inducing robust infection in rhesus macaques.

PubMed

Borges, Maria Beatriz; Marchevsky, Renato Sergio; Mendes, Ygara S; Mendes, Luiz Gustavo; Duarte, Ana Claudia; Cruz, Michael; de Filippis, Ana Maria Bispo; Vasconcelos, Pedro Fernando C; Freire, Marcos; Homma, Akira; Mossman, Sally; Lepine, Edith; Vanloubbeeck, Yannick; Lorin, Clarisse; Malice, Marie-Pierre; Caride, Elena; Warter, Lucile

2018-01-01

The macaque is widely accepted as a suitable model for preclinical characterization of dengue vaccine candidates. However, the only vaccine for which both preclinical and clinical efficacy results were reported so far showed efficacy levels that were substantially different between macaques and humans. We hypothesized that this model's predictive capacity may be improved using recent and minimally passaged dengue virus isolates, and by assessing vaccine efficacy by characterizing not only the post-dengue virus challenge viremia/RNAemia but also the associated-cytokine profile. Ten recent and minimally passaged Brazilian clinical isolates from the four dengue virus serotypes were tested for their infectivity in rhesus macaques. For the strains showing robust replication capacity, the associated-changes in soluble mediator levels, and the elicited dengue virus-neutralizing antibody responses, were also characterized. Three isolates from dengue virus serotypes 1, 2 and 4 induced viremia of high magnitude and longer duration relative to previously reported viremia kinetics in this model, and robust dengue virus-neutralizing antibody responses. Consistent with observations in humans, increased MCP-1, IFN-γ and VEGF-A levels, and transiently decreased IL-8 levels were detected after infection with the selected isolates. These results may contribute to establishing a dengue macaque model showing a higher predictability for vaccine efficacy in humans.

Large scale structures in the kinetic gravity braiding model that can be unbraided

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

Kimura, Rampei; Yamamoto, Kazuhiro, E-mail: rampei@theo.phys.sci.hiroshima-u.ac.jp, E-mail: kazuhiro@hiroshima-u.ac.jp

2011-04-01

We study cosmological consequences of a kinetic gravity braiding model, which is proposed as an alternative to the dark energy model. The kinetic braiding model we study is characterized by a parameter n, which corresponds to the original galileon cosmological model for n = 1. We find that the background expansion of the universe of the kinetic braiding model is the same as the Dvali-Turner's model, which reduces to that of the standard cold dark matter model with a cosmological constant (ΛCDM model) for n equal to infinity. We also find that the evolution of the linear cosmological perturbation inmore » the kinetic braiding model reduces to that of the ΛCDM model for n = ∞. Then, we focus our study on the growth history of the linear density perturbation as well as the spherical collapse in the nonlinear regime of the density perturbations, which might be important in order to distinguish between the kinetic braiding model and the ΛCDM model when n is finite. The theoretical prediction for the large scale structure is confronted with the multipole power spectrum of the luminous red galaxy sample of the Sloan Digital Sky survey. We also discuss future prospects of constraining the kinetic braiding model using a future redshift survey like the WFMOS/SuMIRe PFS survey as well as the cluster redshift distribution in the South Pole Telescope survey.« less

β-adrenergic effects on cardiac myofilaments and contraction in an integrated rabbit ventricular myocyte model

PubMed Central

Negroni, Jorge A.; Morotti, Stefano; Lascano, Elena C.; Gomes, Aldrin V.; Grandi, Eleonora; Puglisi, José L; Bers, Donald M.

2015-01-01

A five-state model of myofilament contraction was integrated into a well-established rabbit ventricular myocyte model of ion channels, Ca2+ transporters and kinase signaling to analyze the relative contribution of different phosphorylation targets to the overall mechanical response driven by β-adrenergic stimulation (β-AS). β-AS effect on sarcoplasmic reticulum Ca2+ handling, Ca2+, K+ and Cl− currents, and Na+/K+-ATPase properties were included based on experimental data. The inotropic effect on the myofilaments was represented as reduced myofilament Ca2+ sensitivity (XBCa) and titin stiffness, and increased cross-bridge (XB) cycling rate (XBcy). Assuming independent roles of XBCa and XBcy, the model reproduced experimental β-AS responses on action potentials and Ca2+ transient amplitude and kinetics. It also replicated the behavior of force-Ca2+, release-restretch, length-step, stiffness-frequency and force-velocity relationships, and increased force and shortening in isometric and isotonic twitch contractions. The β-AS effect was then switched off from individual targets to analyze their relative impact on contractility. Preventing β-AS effects on L-type Ca2+ channels or phospholamban limited Ca2+ transients and contractile responses in parallel, while blocking phospholemman and K+ channel (IKs) effects enhanced Ca2+ and inotropy. Removal of β-AS effects from XBCa enhanced contractile force while decreasing peak Ca2+ (due to greater Ca2+ buffering), but had less effect on shortening. Conversely, preventing β-AS effects on XBcy preserved Ca2+ transient effects, but blunted inotropy (both isometric force and especially shortening). Removal of titin effects had little impact on contraction. Finally, exclusion of β-AS from XBCa and XBcy while preserving effects on other targets resulted in preserved peak isometric force response (with slower kinetics) but nearly abolished enhanced shortening. β-AS effects on XBCa vs. XBcy have greater impact on isometric vs. isotonic contraction, respectively. PMID:25724724

Mechanistic Basis for the Bypass of a Bulky DNA Adduct Catalyzed by a Y-Family DNA Polymerase

PubMed Central

Vyas, Rajan; Efthimiopoulos, Georgia; Tokarsky, E. John; Malik, Chanchal K.; Basu, Ashis K.; Suo, Zucai

2015-01-01

1-Nitropyrene (1-NP), an environmental pollutant, induces DNA damage in vivo and is considered to be carcinogenic. The DNA adducts formed by the 1-NP metabolites stall replicative DNA polymerases but are presumably bypassed by error-prone Y-family DNA polymerases at the expense of replication fidelity and efficiency in vivo. Our running start assays confirmed that a site-specifically placed 8-(deoxyguanosin-N2-yl)-1-aminopyrene (dG1,8), one of the DNA adducts derived from 1-NP, can be bypassed by Sulfolobus solfataricus DNA polymerase IV (Dpo4), although this representative Y-family enzyme was paused strongly by the lesion. Pre-steady-state kinetic assays were employed to determine the low nucleotide incorporation fidelity and establish a minimal kinetic mechanism for the dG1,8 bypass by Dpo4. To reveal a structural basis for dCTP incorporation opposite dG1,8, we solved the crystal structures of the complexes of Dpo4 and DNA containing a templating dG1,8 lesion in the absence or presence of dCTP. The Dpo4·DNA-dG1,8 binary structure shows that the aminopyrene moiety of the lesion stacks against the primer/template junction pair, while its dG moiety projected into the cleft between the Finger and Little Finger domains of Dpo4. In the Dpo4·DNA-dG1,8·dCTP ternary structure, the aminopyrene moiety of the dG1,8 lesion, is sandwiched between the nascent and junction base pairs, while its base is present in the major groove. Moreover, dCTP forms a Watson–Crick base pair with dG, two nucleotides upstream from the dG1,8 site, creating a complex for “-2” frameshift mutation. Mechanistically, these crystal structures provide additional insight into the aforementioned minimal kinetic mechanism. PMID:26327169

A tracer kinetic model for 18F-FHBG for quantitating herpes simplex virus type 1 thymidine kinase reporter gene expression in living animals using PET.

PubMed

Green, Leeta Alison; Nguyen, Khoi; Berenji, Bijan; Iyer, Meera; Bauer, Eileen; Barrio, Jorge R; Namavari, Mohammad; Satyamurthy, Nagichettiar; Gambhir, Sanjiv S

2004-09-01

Reporter probe 9-(4-18F-fluoro-3-[hydroxymethyl]butyl)guanine (18F-FHBG) and reporter gene mutant herpes simplex virus type 1 thymidine kinase (HSV1-sr39tk) have been used for imaging reporter gene expression with PET. Current methods for quantitating the images using the percentage injected dose per gram of tissue do not distinguish between the effects of probe transport and subsequent phosphorylation. We therefore investigated tracer kinetic models for 18F-FHBG dynamic microPET data and noninvasive methods for determining blood time-activity curves in an adenoviral gene delivery model in mice. 18F-FHBG (approximately 7.4 MBq [approximately 200 microCi]) was injected into 4 mice; 18F-FHBG concentrations in plasma and whole blood were measured from mouse heart left ventricle (LV) direct sampling. Replication-incompetent adenovirus (0-2 x 10(9) plaque-forming units) with the E1 region deleted (n = 8) or replaced by HSV1-sr39tk (n = 18) was tail-vein injected into mice. Mice were dynamically scanned using microPET (approximately 7.4 MBq [approximately 200 microCi] 18F-FHBG) over 1 h; regions of interest were drawn on images of the heart and liver. Serial whole blood 18F-FHBG concentrations were measured in 6 of the mice by LV sampling, and 1 least-squares ratio of the heart image to the LV time-activity curve was calculated for all 6 mice. For 2 control mice and 9 mice expressing HSV1-sr39tk, heart image (input function) and liver image time-activity curves (tissue curves) were fit to 2- and 3-compartment models using Levenberg-Marquardt nonlinear regression. The models were compared using an F statistic. HSV1-sr39TK enzyme activity was determined from liver samples and compared with model parameter estimates. For another 3 control mice and 6 HSV1-sr39TK-positive mice, the model-predicted relative percentage of metabolites was compared with high-performance liquid chromatography analysis. The ratio of 18F-FHBG in plasma to whole blood was 0.84 +/- 0.05 (mean +/- SE) by 30 s after injection. The least-squares ratio of the heart image time-activity curve to the LV time-activity curve was 0.83 +/- 0.02, consistent with the recovery coefficient for the partial-volume effect (0.81) based on independent measures of heart geometry. A 3-compartment model best described 18F-FHBG kinetics in mice expressing HSV1-sr39tk in the liver; a 2-compartment model best described the kinetics in control mice. The 3-compartment model parameter, k3, correlated well with the HSV1-sr39TK enzyme activity (r2 = 0.88). 18F-FHBG equilibrates rapidly between plasma and whole blood in mice. Heart image time-activity curves corrected for partial-volume effects well approximate LV time-activity curves and can be used as input functions for 2- and 3-compartment models. The model parameter k3 from the 3-compartment model can be used as a noninvasive estimate for HSV1-sr39TK reporter protein activity and can predict the relative percentage of metabolites.

Modeling of autocatalytic hydrolysis of adefovir dipivoxil in solid formulations.

PubMed

Dong, Ying; Zhang, Yan; Xiang, Bingren; Deng, Haishan; Wu, Jingfang

2011-04-01

The stability and hydrolysis kinetics of a phosphate prodrug, adefovir dipivoxil, in solid formulations were studied. The stability relationship between five solid formulations was explored. An autocatalytic mechanism for hydrolysis could be proposed according to the kinetic behavior which fits the Prout-Tompkins model well. For the classical kinetic models could hardly describe and predict the hydrolysis kinetics of adefovir dipivoxil in solid formulations accurately when the temperature is high, a feedforward multilayer perceptron (MLP) neural network was constructed to model the hydrolysis kinetics. The build-in approaches in Weka, such as lazy classifiers and rule-based learners (IBk, KStar, DecisionTable and M5Rules), were used to verify the performance of MLP. The predictability of the models was evaluated by 10-fold cross-validation and an external test set. It reveals that MLP should be of general applicability proposing an alternative efficient way to model and predict autocatalytic hydrolysis kinetics for phosphate prodrugs.

A Study of the Optimal Model of the Flotation Kinetics of Copper Slag from Copper Mine BOR

NASA Astrophysics Data System (ADS)

Stanojlović, Rodoljub D.; Sokolović, Jovica M.

2014-10-01

In this study the effect of mixtures of copper slag and flotation tailings from copper mine Bor, Serbia on the flotation results of copper recovery and flotation kinetics parameters in a batch flotation cell has been investigated. By simultaneous adding old flotation tailings in the ball mill at the rate of 9%, it is possible to increase copper recovery for about 20%. These results are compared with obtained copper recovery of pure copper slag. The results of batch flotation test were fitted by MatLab software for modeling the first-order flotation kinetics in order to determine kinetics parameters and define an optimal model of the flotation kinetics. Six kinetic models are tested on the batch flotation copper recovery against flotation time. All models showed good correlation, however the modified Kelsall model provided the best fit.

The origin of replicators and reproducers

PubMed Central

Szathmáry, Eörs

2006-01-01

Replicators are fundamental to the origin of life and evolvability. Their survival depends on the accuracy of replication and the efficiency of growth relative to spontaneous decay. Infrabiological systems are built of two coupled autocatalytic systems, in contrast to minimal living systems that must comprise at least a metabolic subsystem, a hereditary subsystem and a boundary, serving respective functions. Some scenarios prefer to unite all these functions into one primordial system, as illustrated in the lipid world scenario, which is considered as a didactic example in detail. Experimentally produced chemical replicators grow parabolically owing to product inhibition. A selection consequence is survival of everybody. The chromatographized replicator model predicts that such replicators spreading on surfaces can be selected for higher replication rate because double strands are washed away slower than single strands from the surface. Analysis of real ribozymes suggests that the error threshold of replication is less severe by about one order of magnitude than thought previously. Surface-bound dynamics is predicted to play a crucial role also for exponential replicators: unlinked genes belonging to the same genome do not displace each other by competition, and efficient and accurate replicases can spread. The most efficient form of such useful population structure is encapsulation by reproducing vesicles. The stochastic corrector model shows how such a bag of genes can survive, and what the role of chromosome formation and intragenic recombination could be. Prebiotic and early evolution cannot be understood without the models of dynamics. PMID:17008217

Multiseason occupancy models for correlated replicate surveys

USGS Publications Warehouse

Hines, James; Nichols, James D.; Collazo, Jaime

2014-01-01

Occupancy surveys collecting data from adjacent (sometimes correlated) spatial replicates have become relatively popular for logistical reasons. Hines et al. (2010) presented one approach to modelling such data for single-season occupancy surveys. Here, we present a multiseason analogue of this model (with corresponding software) for inferences about occupancy dynamics. We include a new parameter to deal with the uncertainty associated with the first spatial replicate for both single-season and multiseason models. We use a case study, based on the brown-headed nuthatch, to assess the need for these models when analysing data from the North American Breeding Bird Survey (BBS), and we test various hypotheses about occupancy dynamics for this species in the south-eastern United States. The new model permits inference about local probabilities of extinction, colonization and occupancy for sampling conducted over multiple seasons. The model performs adequately, based on a small simulation study and on results of the case study analysis. The new model incorporating correlated replicates was strongly favoured by model selection for the BBS data for brown-headed nuthatch (Sitta pusilla). Latitude was found to be an important source of variation in local colonization and occupancy probabilities for brown-headed nuthatch, with both probabilities being higher near the centre of the species range, as opposed to more northern and southern areas. We recommend this new occupancy model for detection–nondetection studies that use potentially correlated replicates.

Replication fork collapse is a major cause of the high mutation frequency at three-base lesion clusters

PubMed Central

Sedletska, Yuliya; Radicella, J. Pablo; Sage, Evelyne

2013-01-01

Unresolved repair of clustered DNA lesions can lead to the formation of deleterious double strand breaks (DSB) or to mutation induction. Here, we investigated the outcome of clusters composed of base lesions for which base excision repair enzymes have different kinetics of excision/incision. We designed multiply damaged sites (MDS) composed of a rapidly excised uracil (U) and two oxidized bases, 5-hydroxyuracil (hU) and 8-oxoguanine (oG), excised more slowly. Plasmids harboring these U-oG/hU MDS-carrying duplexes were introduced into Escherichia coli cells either wild type or deficient for DNA n-glycosylases. Induction of DSB was estimated from plasmid survival and mutagenesis determined by sequencing of surviving clones. We show that a large majority of MDS is converted to DSB, whereas almost all surviving clones are mutated at hU. We demonstrate that mutagenesis at hU is correlated with excision of the U placed on the opposite strand. We propose that excision of U by Ung initiates the loss of U-oG-carrying strand, resulting in enhanced mutagenesis at the lesion present on the opposite strand. Our results highlight the importance of the kinetics of excision by base excision repair DNA n-glycosylases in the processing and fate of MDS and provide evidence for the role of strand loss/replication fork collapse during the processing of MDS on their mutational consequences. PMID:23945941

A new model for CD8+ T cell memory inflation based upon a recombinant adenoviral vector1

PubMed Central

Bolinger, Beatrice; Sims, Stuart; O’Hara, Geraldine; de Lara, Catherine; Tchilian, Elma; Firner, Sonja; Engeler, Daniel; Ludewig, Burkhard; Klenerman, Paul

2013-01-01

CD8+ T cell memory inflation, first described in murine cytomegalovirus (MCMV) infection, is characterized by the accumulation of high-frequency, functional antigen-specific CD8+ T cell pools with an effector-memory phenotype and enrichment in peripheral organs. Although persistence of antigen is considered essential, the rules underpinning memory inflation are still unclear. The MCMV model is, however, complicated by the virus’s low-level persistence, and stochastic reactivation. We developed a new model of memory inflation based upon a βgal-recombinant adenovirus vector (Ad-LacZ). After i.v. administration in C57BL/6 mice we observe marked memory inflation in the βgal96 epitope, while a second epitope, βgal497, undergoes classical memory formation. The inflationary T cell responses show kinetics, distribution, phenotype and functions similar to those seen in MCMV and are reproduced using alternative routes of administration. Memory inflation in this model is dependent on MHC Class II. As in MCMV, only the inflating epitope showed immunoproteasome-independence. These data define a new model for memory inflation, which is fully replication-independent, internally controlled and reproduces the key immunologic features of the CD8+ T cell response. This model provides insight into the mechanisms responsible for memory inflation, and since it is based on a vaccine vector, also is relevant to novel T cell-inducing vaccines in humans. PMID:23509359

Self-Assembly of Measles Virus Nucleocapsid-like Particles: Kinetics and RNA Sequence Dependence.

PubMed

Milles, Sigrid; Jensen, Malene Ringkjøbing; Communie, Guillaume; Maurin, Damien; Schoehn, Guy; Ruigrok, Rob W H; Blackledge, Martin

2016-08-01

Measles virus RNA genomes are packaged into helical nucleocapsids (NCs), comprising thousands of nucleo-proteins (N) that bind the entire genome. N-RNA provides the template for replication and transcription by the viral polymerase and is a promising target for viral inhibition. Elucidation of mechanisms regulating this process has been severely hampered by the inability to controllably assemble NCs. Here, we demonstrate self-organization of N into NC-like particles in vitro upon addition of RNA, providing a simple and versatile tool for investigating assembly. Real-time NMR and fluorescence spectroscopy reveals biphasic assembly kinetics. Remarkably, assembly depends strongly on the RNA-sequence, with the genomic 5' end and poly-Adenine sequences assembling efficiently, while sequences such as poly-Uracil are incompetent for NC formation. This observation has important consequences for understanding the assembly process. © 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

Synthesis of novel ICIE16/BSG and ICIE16/BSG-NITRI bioglasses and description of ionic release kinetics upon immersion in SBF fluid: Effect of nitridation

PubMed Central

Orgaz, Felipe; Amat, Daniel; Szycht, Olga; Dzika, Aleksandra; Barba, Flora; Becerra, José; Santos-Ruiz, Leonor

2015-01-01

A novel bioactive glass scaffold ICIE16/BSG has been prepared from a mixture of two different melt-derived glasses: a silicate bioglass (ICIE16) and a borosilicate bioglass (BSG). Combined processing techniques (gel casting and foam replication) were used to form three-dimensional, interconnected porous monolith scaffolds (Orgaz et al., 2016) [1]. They were then nitrided with a hot ammonia flow as described in (Aleixandre et al., 1973) [3] and (Nieto, 1984) [4] to synthesize the ICIE16/BSG-NITRI bioglass (Orgaz et al., 2016) [1]. Herein we present a flow chart summarizing the forming process, plus images of the resulting scaffold after sintering and drying. Bioactivity was characterized in vitro by immersion in simulated body fluid (SBF) for up to seven days. Data of ionic release kinetics upon SBF immersion are presented. PMID:26858981

Synthesis of novel ICIE16/BSG and ICIE16/BSG-NITRI bioglasses and description of ionic release kinetics upon immersion in SBF fluid: Effect of nitridation.

PubMed

Orgaz, Felipe; Amat, Daniel; Szycht, Olga; Dzika, Aleksandra; Barba, Flora; Becerra, José; Santos-Ruiz, Leonor

2016-03-01

A novel bioactive glass scaffold ICIE16/BSG has been prepared from a mixture of two different melt-derived glasses: a silicate bioglass (ICIE16) and a borosilicate bioglass (BSG). Combined processing techniques (gel casting and foam replication) were used to form three-dimensional, interconnected porous monolith scaffolds (Orgaz et al., 2016) [1]. They were then nitrided with a hot ammonia flow as described in (Aleixandre et al., 1973) [3] and (Nieto, 1984) [4] to synthesize the ICIE16/BSG-NITRI bioglass (Orgaz et al., 2016) [1]. Herein we present a flow chart summarizing the forming process, plus images of the resulting scaffold after sintering and drying. Bioactivity was characterized in vitro by immersion in simulated body fluid (SBF) for up to seven days. Data of ionic release kinetics upon SBF immersion are presented.

Single-Molecule FISH Reveals Non-selective Packaging of Rift Valley Fever Virus Genome Segments

PubMed Central

Wichgers Schreur, Paul J.; Kortekaas, Jeroen

2016-01-01

The bunyavirus genome comprises a small (S), medium (M), and large (L) RNA segment of negative polarity. Although genome segmentation confers evolutionary advantages by enabling genome reassortment events with related viruses, genome segmentation also complicates genome replication and packaging. Accumulating evidence suggests that genomes of viruses with eight or more genome segments are incorporated into virions by highly selective processes. Remarkably, little is known about the genome packaging process of the tri-segmented bunyaviruses. Here, we evaluated, by single-molecule RNA fluorescence in situ hybridization (FISH), the intracellular spatio-temporal distribution and replication kinetics of the Rift Valley fever virus (RVFV) genome and determined the segment composition of mature virions. The results reveal that the RVFV genome segments start to replicate near the site of infection before spreading and replicating throughout the cytoplasm followed by translocation to the virion assembly site at the Golgi network. Despite the average intracellular S, M and L genome segments approached a 1:1:1 ratio, major differences in genome segment ratios were observed among cells. We also observed a significant amount of cells lacking evidence of M-segment replication. Analysis of two-segmented replicons and four-segmented viruses subsequently confirmed the previous notion that Golgi recruitment is mediated by the Gn glycoprotein. The absence of colocalization of the different segments in the cytoplasm and the successful rescue of a tri-segmented variant with a codon shuffled M-segment suggested that inter-segment interactions are unlikely to drive the copackaging of the different segments into a single virion. The latter was confirmed by direct visualization of RNPs inside mature virions which showed that the majority of virions lack one or more genome segments. Altogether, this study suggests that RVFV genome packaging is a non-selective process. PMID:27548280

CMV-Specific T Cell Monitoring Offers Superior Risk Stratification of CMV-Seronegative Kidney Transplant Recipients of a CMV-Seropositive Donor.

PubMed

Schachtner, Thomas; Stein, Maik; Reinke, Petra

2017-10-01

Detectable cytomegalovirus (CMV)-specific T cells in CMV-seronegative kidney transplant recipients (KTRs) have been attributed to an absence of circulating antibodies despite CMV sensitization. The diagnostic value of CMV-specific T cells, however, needs to be implemented in risk stratification for CMV replication. Three hundred twenty-six KTRs were studied and classified with respect to CMV serostatus and presence of CMV-specific T cells. Samples were collected pretransplantation, at +1, +2, and +3 months posttransplantation. CMV-specific T cells directed to CMV-IE1 and CMV-pp65 were measured by interferon-γ Elispot assay. Nineteen (28%) of 67 D+R- KTRs showed pretransplant CMV-specific T cells. Although no differences were observed for CMV replication, KTRs with CMV-specific T cells presented with lower initial and peak CMV loads (P < 0.05). KTRs with decreasing/undetectable CMV-IE1-specific T cells pretransplantation and posttransplantation were at greatest risk of CMV replication. KTRs with stable/increasing CMV-IE1-specific T cells from pretransplantation to posttransplantation, however, showed low risk of CMV replication (P < 0.001). One hundred sixty-two (80%) of 203 R+ KTRs showed pretransplant CMV-specific T cells. Decreasing/undetectable CMV-IE1-specific T cells from pretransplantation and posttransplantation identified those R+ KTRs at increased risk of CMV replication (65/80 KTRs; 81%; P < 0.001). Despite CMV prophylaxis, D+R- KTRs are at greatest risk of CMV disease. Our data suggest that monitoring CMV-specific T cell kinetics from pretransplantation to posttransplantation, particularly directed to CMV-IE1, offers superior risk stratification compared with CMV serostatus alone.

Viral FGARAT ORF75A promotes early events in lytic infection and gammaherpesvirus pathogenesis in mice

PubMed Central

Hogan, Chad H.; Oldenburg, Darby G.; Kara, Mehmet

2018-01-01

Gammaherpesviruses encode proteins with homology to the cellular purine metabolic enzyme formyl-glycinamide-phosphoribosyl-amidotransferase (FGARAT), but the role of these viral FGARATs (vFGARATs) in the pathogenesis of a natural host has not been investigated. We report a novel role for the ORF75A vFGARAT of murine gammaherpesvirus 68 (MHV68) in infectious virion production and colonization of mice. MHV68 mutants with premature stop codons in orf75A exhibited a log reduction in acute replication in the lungs after intranasal infection, which preceded a defect in colonization of multiple host reservoirs including the mediastinal lymph nodes, peripheral blood mononuclear cells, and the spleen. Intraperitoneal infection rescued splenic latency, but not reactivation. The 75A.stop virus also exhibited defective replication in primary fibroblast and macrophage cells. Viruses produced in the absence of ORF75A were characterized by an increase in the ratio of particles to PFU. In the next round of infection this led to the alteration of early events in lytic replication including the deposition of the ORF75C tegument protein, the accelerated kinetics of viral gene expression, and induction of TNFα release and cell death. Infecting cells to deliver equivalent genomes revealed that ORF75A was required for initiating early events in infection. In contrast with the numerous phenotypes observed in the absence of ORF75A, ORF75B was dispensable for replication and pathogenesis. These studies reveal that murine rhadinovirus vFGARAT family members ORF75A and ORF75C have evolved to perform divergent functions that promote replication and colonization of the host. PMID:29390024

Viral FGARAT ORF75A promotes early events in lytic infection and gammaherpesvirus pathogenesis in mice.

PubMed

Van Skike, Nick D; Minkah, Nana K; Hogan, Chad H; Wu, Gary; Benziger, Peter T; Oldenburg, Darby G; Kara, Mehmet; Kim-Holzapfel, Deborah M; White, Douglas W; Tibbetts, Scott A; French, Jarrod B; Krug, Laurie T

2018-02-01

Gammaherpesviruses encode proteins with homology to the cellular purine metabolic enzyme formyl-glycinamide-phosphoribosyl-amidotransferase (FGARAT), but the role of these viral FGARATs (vFGARATs) in the pathogenesis of a natural host has not been investigated. We report a novel role for the ORF75A vFGARAT of murine gammaherpesvirus 68 (MHV68) in infectious virion production and colonization of mice. MHV68 mutants with premature stop codons in orf75A exhibited a log reduction in acute replication in the lungs after intranasal infection, which preceded a defect in colonization of multiple host reservoirs including the mediastinal lymph nodes, peripheral blood mononuclear cells, and the spleen. Intraperitoneal infection rescued splenic latency, but not reactivation. The 75A.stop virus also exhibited defective replication in primary fibroblast and macrophage cells. Viruses produced in the absence of ORF75A were characterized by an increase in the ratio of particles to PFU. In the next round of infection this led to the alteration of early events in lytic replication including the deposition of the ORF75C tegument protein, the accelerated kinetics of viral gene expression, and induction of TNFα release and cell death. Infecting cells to deliver equivalent genomes revealed that ORF75A was required for initiating early events in infection. In contrast with the numerous phenotypes observed in the absence of ORF75A, ORF75B was dispensable for replication and pathogenesis. These studies reveal that murine rhadinovirus vFGARAT family members ORF75A and ORF75C have evolved to perform divergent functions that promote replication and colonization of the host.

Novel Chromosome Organization Pattern in Actinomycetales-Overlapping Replication Cycles Combined with Diploidy.

PubMed

Böhm, Kati; Meyer, Fabian; Rhomberg, Agata; Kalinowski, Jörn; Donovan, Catriona; Bramkamp, Marc

2017-06-06

Bacteria regulate chromosome replication and segregation tightly with cell division to ensure faithful segregation of DNA to daughter generations. The underlying mechanisms have been addressed in several model species. It became apparent that bacteria have evolved quite different strategies to regulate DNA segregation and chromosomal organization. We have investigated here how the actinobacterium Corynebacterium glutamicum organizes chromosome segregation and DNA replication. Unexpectedly, we found that C. glutamicum cells are at least diploid under all of the conditions tested and that these organisms have overlapping C periods during replication, with both origins initiating replication simultaneously. On the basis of experimental data, we propose growth rate-dependent cell cycle models for C. glutamicum IMPORTANCE Bacterial cell cycles are known for few model organisms and can vary significantly between species. Here, we studied the cell cycle of Corynebacterium glutamicum , an emerging cell biological model organism for mycolic acid-containing bacteria, including mycobacteria. Our data suggest that C. glutamicum carries two pole-attached chromosomes that replicate with overlapping C periods, thus initiating a new round of DNA replication before the previous one is terminated. The newly replicated origins segregate to midcell positions, where cell division occurs between the two new origins. Even after long starvation or under extremely slow-growth conditions, C. glutamicum cells are at least diploid, likely as an adaptation to environmental stress that may cause DNA damage. The cell cycle of C. glutamicum combines features of slow-growing organisms, such as polar origin localization, and fast-growing organisms, such as overlapping C periods. Copyright © 2017 Böhm et al.

Kinetic Model of Growth of Arthropoda Populations

NASA Astrophysics Data System (ADS)

Ershov, Yu. A.; Kuznetsov, M. A.

2018-05-01

Kinetic equations were derived for calculating the growth of crustacean populations ( Crustacea) based on the biological growth model suggested earlier using shrimp ( Caridea) populations as an example. The development cycle of successive stages for populations can be represented in the form of quasi-chemical equations. The kinetic equations that describe the development cycle of crustaceans allow quantitative prediction of the development of populations depending on conditions. In contrast to extrapolation-simulation models, in the developed kinetic model of biological growth the kinetic parameters are the experimental characteristics of population growth. Verification and parametric identification of the developed model on the basis of the experimental data showed agreement with experiment within the error of the measurement technique.

Automated chemical kinetic modeling via hybrid reactive molecular dynamics and quantum chemistry simulations.

PubMed

Döntgen, Malte; Schmalz, Felix; Kopp, Wassja A; Kröger, Leif C; Leonhard, Kai

2018-06-13

An automated scheme for obtaining chemical kinetic models from scratch using reactive molecular dynamics and quantum chemistry simulations is presented. This methodology combines the phase space sampling of reactive molecular dynamics with the thermochemistry and kinetics prediction capabilities of quantum mechanics. This scheme provides the NASA polynomial and modified Arrhenius equation parameters for all species and reactions that are observed during the simulation and supplies them in the ChemKin format. The ab initio level of theory for predictions is easily exchangeable and the presently used G3MP2 level of theory is found to reliably reproduce hydrogen and methane oxidation thermochemistry and kinetics data. Chemical kinetic models obtained with this approach are ready-to-use for, e.g., ignition delay time simulations, as shown for hydrogen combustion. The presented extension of the ChemTraYzer approach can be used as a basis for methodologically advancing chemical kinetic modeling schemes and as a black-box approach to generate chemical kinetic models.

Competitive replication kinetics and pathogenicity in pigs co-infected with historical and newly invading classical swine fever viruses.

PubMed

Huang, Yu-Liang; Deng, Ming-Chung; Tsai, Kuo-Jung; Liu, Hsin-Meng; Huang, Chin-Cheng; Wang, Fun-In; Chang, Chia-Yi

2017-01-15

Classical swine fever (CSF), an economically important and highly contagious disease of pigs, is caused by classical swine fever virus (CSFV). In Taiwan, CSFVs from field outbreaks belong to two distinct genotypes. The historical genotype 3.4 dominated from the 1920s to 1996, and since 1996, the newly invading genotype 2.1 has dominated. To explain the phenomenon of this virus shift in the field, representative viruses belonging to genotypes 2.1 and 3.4 were either inoculated alone (single infection) or co-inoculated (co-infection), both in vivo and in vitro, to compare the virus replication and pathogenesis. In pigs co-infected with the genotype 2.1 TD/96/TWN strain and the genotype 3.4 94.4/IL/94/TWN strain, the newly invading genotype 2.1 was detected earlier in the blood, oral fluid, and feces, and the viral loads were consistently and significantly higher than that of the historical genotype 3.4. In cell cultures, the ratio of secreted virus to cell-associated virus of the genotype 2.1 strain was higher than that of the genotype 3.4 strain. This study is the first to demonstrate a possible explanation of virus shift in the field, wherein the newly invading genotype 2.1 replicates more efficiently than did genotype 3.4 and outcompetes the replication and pathogenicity of genotype 3.4 in pigs in the field. Copyright © 2016 Elsevier B.V. All rights reserved.

Differences in replication kinetics and cell tropism between neurovirulent and non-neurovirulent EHV1 strains during the acute phase of infection in horses.

PubMed

Gryspeerdt, Annick C; Vandekerckhove, A P; Garré, B; Barbé, F; Van de Walle, G R; Nauwynck, H J

2010-05-19

Equine herpesvirus 1 (EHV1) replicates in the respiratory tract of horses, after which infected leukocytes transport virus throughout the body, resulting in abortion or nervous system disorders. Two EHV1 strains circulate in the field: neurovirulent and non-neurovirulent. To investigate differences in replication in the upper respiratory tract (URT), an experimental inoculation study in ponies was performed with both strains. Two groups of six ponies, were inoculated intranasally with 10(6.5) TCID(50) of either strain. Clinical signs, nasal shedding and viremia were evaluated. At early time points post-inoculation (pi), one pony of each group was euthanized. Tissues were collected for titration and immunostainings. Number and size of EHV1-induced plaques were calculated, and individual EHV1-infected cells were quantified and characterized. Inoculation with either strain resulted in nasal shedding and replication in several tissues of the URT. Both strains replicated in a plaquewise manner in epithelium of the nasal mucosa, but replication in epithelium of the nasopharynx was largely limited to non-neurovirulent EHV1. Plaques were never able to cross the basement membrane, but individual infected cells were noticed in the connective tissue of all examined tissues for both strains. The total number of these cells however, was 3-7 times lower with non-neurovirulent EHV1 compared to neurovirulent EHV1. CD172a(+) cells and CD5(+) lymphocytes were important target cells for both strains. Interestingly, in lymph nodes, B-lymphocytes were also important target cells for EHV1, irrespective of the strain. Viremia was detected very early pi and infected cells were mainly CD172a(+) for both strains. In summary, these results are valuable for understanding EHV1 pathogenesis at the port of entry, the URT. Copyright 2009 Elsevier B.V. All rights reserved.

Translesion synthesis DNA polymerases promote error-free replication through the minor-groove DNA adduct 3-deaza-3-methyladenine.

PubMed

Yoon, Jung-Hoon; Roy Choudhury, Jayati; Park, Jeseong; Prakash, Satya; Prakash, Louise

2017-11-10

N3-Methyladenine (3-MeA) is formed in DNA by reaction with S -adenosylmethionine, the reactive methyl donor, and by reaction with alkylating agents. 3-MeA protrudes into the DNA minor groove and strongly blocks synthesis by replicative DNA polymerases (Pols). However, the mechanisms for replicating through this lesion in human cells remain unidentified. Here we analyzed the roles of translesion synthesis (TLS) Pols in the replication of 3-MeA-damaged DNA in human cells. Because 3-MeA has a short half-life in vitro , we used the stable 3-deaza analog, 3-deaza-3-methyladenine (3-dMeA), which blocks the DNA minor groove similarly to 3-MeA. We found that replication through the 3-dMeA adduct is mediated via three different pathways, dependent upon Polι/Polκ, Polθ, and Polζ. As inferred from biochemical studies, in the Polι/Polκ pathway, Polι inserts a nucleotide (nt) opposite 3-dMeA and Polκ extends synthesis from the inserted nt. In the Polθ pathway, Polθ carries out both the insertion and extension steps of TLS opposite 3-dMeA, and in the Polζ pathway, Polζ extends synthesis following nt insertion by an as yet unidentified Pol. Steady-state kinetic analyses indicated that Polι and Polθ insert the correct nt T opposite 3-dMeA with a much reduced catalytic efficiency and that both Pols exhibit a high propensity for inserting a wrong nt opposite this adduct. However, despite their low fidelity of synthesis opposite 3-dMeA, TLS opposite this lesion replicates DNA in a highly error-free manner in human cells. We discuss the implications of these observations for TLS mechanisms in human cells. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Carotene Degradation and Isomerization during Thermal Processing: A Review on the Kinetic Aspects.

PubMed

Colle, Ines J P; Lemmens, Lien; Knockaert, Griet; Van Loey, Ann; Hendrickx, Marc

2016-08-17

Kinetic models are important tools for process design and optimization to balance desired and undesired reactions taking place in complex food systems during food processing and preservation. This review covers the state of the art on kinetic models available to describe heat-induced conversion of carotenoids, in particular lycopene and β-carotene. First, relevant properties of these carotenoids are discussed. Second, some general aspects of kinetic modeling are introduced, including both empirical single-response modeling and mechanism-based multi-response modeling. The merits of multi-response modeling to simultaneously describe carotene degradation and isomerization are demonstrated. The future challenge in this research field lies in the extension of the current multi-response models to better approach the real reaction pathway and in the integration of kinetic models with mass transfer models in case of reaction in multi-phase food systems.

Novel Chromosome Organization Pattern in Actinomycetales—Overlapping Replication Cycles Combined with Diploidy

PubMed Central

Böhm, Kati; Meyer, Fabian; Rhomberg, Agata; Kalinowski, Jörn; Donovan, Catriona

2017-01-01

ABSTRACT Bacteria regulate chromosome replication and segregation tightly with cell division to ensure faithful segregation of DNA to daughter generations. The underlying mechanisms have been addressed in several model species. It became apparent that bacteria have evolved quite different strategies to regulate DNA segregation and chromosomal organization. We have investigated here how the actinobacterium Corynebacterium glutamicum organizes chromosome segregation and DNA replication. Unexpectedly, we found that C. glutamicum cells are at least diploid under all of the conditions tested and that these organisms have overlapping C periods during replication, with both origins initiating replication simultaneously. On the basis of experimental data, we propose growth rate-dependent cell cycle models for C. glutamicum. PMID:28588128

Detonation initiation in a model of explosive: Comparative atomistic and hydrodynamics simulations

NASA Astrophysics Data System (ADS)

Murzov, S. A.; Sergeev, O. V.; Dyachkov, S. A.; Egorova, M. S.; Parshikov, A. N.; Zhakhovsky, V. V.

2016-11-01

Here we extend consistent simulations to reactive materials by the example of AB model explosive. The kinetic model of chemical reactions observed in a molecular dynamics (MD) simulation of self-sustained detonation wave can be used in hydrodynamic simulation of detonation initiation. Kinetic coefficients are obtained by minimization of difference between profiles of species calculated from the kinetic model and observed in MD simulations of isochoric thermal decomposition with a help of downhill simplex method combined with random walk in multidimensional space of fitting kinetic model parameters.

Immunocompetent syngeneic cotton rat tumor models for the assessment of replication-competent oncolytic adenovirus

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

Steel, Jason C.; Morrison, Brian J.; Mannan, Poonam

Oncolytic adenoviruses as a treatment for cancer have demonstrated limited clinical activity. Contributing to this may be the relevance of preclinical animal models used to study these agents. Syngeneic mouse tumor models are generally non-permissive for adenoviral replication, whereas human tumor xenograft models exhibit attenuated immune responses to the vector. The cotton rat (Sigmodon hispidus) is susceptible to human adenovirus infection, permissive for viral replication and exhibits similar inflammatory pathology to humans with adenovirus replicating in the lungs, respiratory passages and cornea. We evaluated three transplantable tumorigenic cotton rat cell lines, CCRT, LCRT and VCRT as models for the studymore » of oncolytic adenoviruses. All three cells lines were readily infected with adenovirus type-5-based vectors and exhibited high levels of transgene expression. The cell lines supported viral replication demonstrated by the induction of cytopathogenic effect (CPE) in tissue culture, increase in virus particle numbers and assembly of virions seen on transmission electron microscopy. In vivo, LCRT and VCRT tumors demonstrated delayed growth after injection with replicating adenovirus. No in vivo antitumor activity was seen in CCRT tumors despite in vitro oncolysis. Adenovirus was also rapidly cleared from the CCRT tumors compared to LCRT and VCRT tumors. The effect observed with the different cotton rat tumor cell lines mimics the variable results of human clinical trials highlighting the potential relevance of this model for assessing the activity and toxicity of oncolytic adenoviruses.« less

Kinetic Description of the Impedance Probe

NASA Astrophysics Data System (ADS)

Oberrath, Jens; Lapke, Martin; Mussenbrock, Thomas; Brinkmann, Ralf

2011-10-01

Active plasma resonance spectroscopy is a well known diagnostic method. Many concepts of this method are theoretically investigated and realized as a diagnostic tool, one of which is the impedance probe (IP). The application of such a probe in plasmas with pressures of a few Pa raises the question whether kinetic effects have to be taken into account or not. To address this question a kinetic model is necessary. A general kinetic model for an electrostatic concept of active plasma spectroscopy was presented by R.P. Brinkmann and can be used to describe the multipole resonance probe (MRP). In principle the IP is interpretable as a special case of the MRP in lower order. Thus, we are able to describe the IP by the kinetic model of the MRP. Based on this model we derive a solution to investigate the influence of kinetic effects to the resonance behavior of the IP. Active plasma resonance spectroscopy is a well known diagnostic method. Many concepts of this method are theoretically investigated and realized as a diagnostic tool, one of which is the impedance probe (IP). The application of such a probe in plasmas with pressures of a few Pa raises the question whether kinetic effects have to be taken into account or not. To address this question a kinetic model is necessary. A general kinetic model for an electrostatic concept of active plasma spectroscopy was presented by R.P. Brinkmann and can be used to describe the multipole resonance probe (MRP). In principle the IP is interpretable as a special case of the MRP in lower order. Thus, we are able to describe the IP by the kinetic model of the MRP. Based on this model we derive a solution to investigate the influence of kinetic effects to the resonance behavior of the IP. The authors acknowledge the support by the Deutsche Forschungsgemeinschaft (DFG) via the Ruhr University Research School and the Federal Ministry of Education and Research in frame of the PluTO project.

New reassortant and enzootic European swine influenza viruses transmit efficiently through direct contact in the ferret model.

PubMed

Fobian, Kristina; Fabrizio, Thomas P; Yoon, Sun-Woo; Hansen, Mette Sif; Webby, Richard J; Larsen, Lars E

2015-07-01

The reverse zoonotic events that introduced the 2009 pandemic influenza virus into pigs have drastically increased the diversity of swine influenza viruses in Europe. The pandemic potential of these novel reassortments is still unclear, necessitating enhanced surveillance of European pigs with additional focus on risk assessment of these new viruses. In this study, four European swine influenza viruses were assessed for their zoonotic potential. Two of the four viruses were enzootic viruses of subtype H1N2 (with avian-like H1) and H3N2, and two were new reassortants, one with avian-like H1 and human-like N2 and one with 2009 pandemic H1 and swine-like N2. All viruses replicated to high titres in nasal wash and nasal turbinate samples from inoculated ferrets and transmitted efficiently by direct contact. Only the H3N2 virus transmitted to naïve ferrets via the airborne route. Growth kinetics using a differentiated human bronchial epithelial cell line showed that all four viruses were able to replicate to high titres. Further, the viruses revealed preferential binding to the 2,6-α-silalylated glycans and investigation of the antiviral susceptibility of the viruses revealed that all were sensitive to neuraminidase inhibitors. These findings suggested that these viruses have the potential to infect humans and further underline the need for continued surveillance as well as biological characterization of new influenza A viruses.

rMATS: robust and flexible detection of differential alternative splicing from replicate RNA-Seq data.

PubMed

Shen, Shihao; Park, Juw Won; Lu, Zhi-xiang; Lin, Lan; Henry, Michael D; Wu, Ying Nian; Zhou, Qing; Xing, Yi

2014-12-23

Ultra-deep RNA sequencing (RNA-Seq) has become a powerful approach for genome-wide analysis of pre-mRNA alternative splicing. We previously developed multivariate analysis of transcript splicing (MATS), a statistical method for detecting differential alternative splicing between two RNA-Seq samples. Here we describe a new statistical model and computer program, replicate MATS (rMATS), designed for detection of differential alternative splicing from replicate RNA-Seq data. rMATS uses a hierarchical model to simultaneously account for sampling uncertainty in individual replicates and variability among replicates. In addition to the analysis of unpaired replicates, rMATS also includes a model specifically designed for paired replicates between sample groups. The hypothesis-testing framework of rMATS is flexible and can assess the statistical significance over any user-defined magnitude of splicing change. The performance of rMATS is evaluated by the analysis of simulated and real RNA-Seq data. rMATS outperformed two existing methods for replicate RNA-Seq data in all simulation settings, and RT-PCR yielded a high validation rate (94%) in an RNA-Seq dataset of prostate cancer cell lines. Our data also provide guiding principles for designing RNA-Seq studies of alternative splicing. We demonstrate that it is essential to incorporate biological replicates in the study design. Of note, pooling RNAs or merging RNA-Seq data from multiple replicates is not an effective approach to account for variability, and the result is particularly sensitive to outliers. The rMATS source code is freely available at rnaseq-mats.sourceforge.net/. As the popularity of RNA-Seq continues to grow, we expect rMATS will be useful for studies of alternative splicing in diverse RNA-Seq projects.

Structure-Function Aspects of Membrane Associated Prokaryotic DNA replication

DTIC Science & Technology

1994-09-01

Membrane associated DNA replication in prokaryotes has been studied intensively using two model systems, Bacillus subtilis and plasmid RK2 cultured...in its Escherichia coli host. In the former a new membrane protein that had previously been found to act as an inhibitor of DNA replication was...prior to a round of DNA replication . In the latter, plasmid DNA replication has been found to be associated with the inner but not outer membrane of

A Self Consistent RF Discharge, Plasma Chemistry and Surface Model for Plasma Enhanced Chemical Vapor Deposition

DTIC Science & Technology

1988-06-30

consists of three submodels for the electron kinetics, plasma chemistry , and surface deposition kinetics for a-Si:H deposited from radio frequency...properties. Plasma enhanced, Chemical vapor deposition, amorphous silicon, Modeling, Electron kinetics, Plasma chemistry , Deposition kinetics, Rf discharge, Silane, Film properties, Silicon.

LHCb experience with LFC replication

NASA Astrophysics Data System (ADS)

Bonifazi, F.; Carbone, A.; Perez, E. D.; D'Apice, A.; dell'Agnello, L.; Duellmann, D.; Girone, M.; Re, G. L.; Martelli, B.; Peco, G.; Ricci, P. P.; Sapunenko, V.; Vagnoni, V.; Vitlacil, D.

2008-07-01

Database replication is a key topic in the framework of the LHC Computing Grid to allow processing of data in a distributed environment. In particular, the LHCb computing model relies on the LHC File Catalog, i.e. a database which stores information about files spread across the GRID, their logical names and the physical locations of all the replicas. The LHCb computing model requires the LFC to be replicated at Tier-1s. The LCG 3D project deals with the database replication issue and provides a replication service based on Oracle Streams technology. This paper describes the deployment of the LHC File Catalog replication to the INFN National Center for Telematics and Informatics (CNAF) and to other LHCb Tier-1 sites. We performed stress tests designed to evaluate any delay in the propagation of the streams and the scalability of the system. The tests show the robustness of the replica implementation with performance going much beyond the LHCb requirements.

A note on the maintenance of the atmospheric kinetic energy

NASA Technical Reports Server (NTRS)

Chen, T.-C.; Lee, Y.-H.

1982-01-01

The winter simulations of the GLAS climate model and the NCAR community climate model are used to examine the maintenance of the atmospheric kinetic energy. It is found that the kinetic energy is generated in the lower latitudes south of the maximum westerlies, transported northward and then, destroyed in the midlatitudes north of the maximum westerlies. Therefore, the atmospheric kinetic energy is maintained by the counterbalance between the divergence (convergence) of kinetic energy flux and generation (destruction) of kinetic energy in lower (middle) latitudes.

A large column analog experiment of stable isotope variations during reactive transport: I. A comprehensive model of sulfur cycling and δ34S fractionation

NASA Astrophysics Data System (ADS)

Druhan, Jennifer L.; Steefel, Carl I.; Conrad, Mark E.; DePaolo, Donald J.

2014-01-01

This study demonstrates a mechanistic incorporation of the stable isotopes of sulfur within the CrunchFlow reactive transport code to model the range of microbially-mediated redox processes affecting kinetic isotope fractionation. Previous numerical models of microbially mediated sulfate reduction using Monod-type rate expressions have lacked rigorous coupling of individual sulfur isotopologue rates, with the result that they cannot accurately simulate sulfur isotope fractionation over a wide range of substrate concentrations using a constant fractionation factor. Here, we derive a modified version of the dual-Monod or Michaelis-Menten formulation (Maggi and Riley, 2009, 2010) that successfully captures the behavior of the 32S and 34S isotopes over a broad range from high sulfate and organic carbon availability to substrate limitation using a constant fractionation factor. The new model developments are used to simulate a large-scale column study designed to replicate field scale conditions of an organic carbon (acetate) amended biostimulation experiment at the Old Rifle site in western Colorado. Results demonstrate an initial period of iron reduction that transitions to sulfate reduction, in agreement with field-scale behavior observed at the Old Rifle site. At the height of sulfate reduction, effluent sulfate concentrations decreased to 0.5 mM from an influent value of 8.8 mM over the 100 cm flow path, and thus were enriched in sulfate δ34S from 6.3‰ to 39.5‰. The reactive transport model accurately reproduced the measured enrichment in δ34S of both the reactant (sulfate) and product (sulfide) species of the reduction reaction using a single fractionation factor of 0.987 obtained independently from field-scale measurements. The model also accurately simulated the accumulation and δ34S signature of solid phase elemental sulfur over the duration of the experiment, providing a new tool to predict the isotopic signatures associated with reduced mineral pools. To our knowledge, this is the first rigorous treatment of sulfur isotope fractionation subject to Monod kinetics in a mechanistic reactive transport model that considers the isotopic spatial distribution of both dissolved and solid phase sulfur species during microbially-mediated sulfate reduction. describe the design and results of the large-scale column experiment; demonstrate incorporation of the stable isotopes of sulfur in a dual-Monod kinetic expression such that fractionation is accurately modeled at both high and low substrate availability; verify accurate simulation of the chemical and isotopic gradients in reactant and product sulfur species using a kinetic fractionation factor obtained from field-scale analysis (Druhan et al., 2012); utilize the model to predict the final δ34S values of secondary sulfur minerals accumulated in the sediment over the course of the experiment. The development of rigorous isotope-specific Monod-type rate expressions are presented here in application to sulfur cycling during amended biostimulation, but are readily applicable to a variety of stable isotope systems associated with both steady state and transient biogenic redox environments. In other words, the association of this model with a uranium remediation experiment does not limit its applicability to more general redox systems. Furthermore, the ability of this model treatment to predict the isotopic composition of secondary minerals accumulated as a result of fractionating processes (item 4) offers an important means of interpreting solid phase isotopic compositions and tracking long-term stability of precipitates.

Seven Activities for Enhancing the Replicability of Evidence-Based Practices. Research-to-Results Brief. Publication #2007-30

ERIC Educational Resources Information Center

Metz, Allison J. R.; Bowie, Lillian; Blase, Karen

2007-01-01

This brief will define program replication, describe the critical role of "core components" in program replication, and outline seven activities that program developers and researchers can conduct to enhance the replicability of effective program models and facilitate their adoption by other organizations and programs. Outlined is seven specific…

A Paper Model of DNA Structure and Replication.

ERIC Educational Resources Information Center

Sigismondi, Linda A.

1989-01-01

A paper model which is designed to give students a hands-on experience during lecture and blackboard instruction on DNA structure is provided. A list of materials, paper patterns, and procedures for using the models to teach DNA structure and replication are given. (CW)

Kinetic analysis of overlapping multistep thermal decomposition comprising exothermic and endothermic processes: thermolysis of ammonium dinitramide.

PubMed

Muravyev, Nikita V; Koga, Nobuyoshi; Meerov, Dmitry B; Pivkina, Alla N

2017-01-25

This study focused on kinetic modeling of a specific type of multistep heterogeneous reaction comprising exothermic and endothermic reaction steps, as exemplified by the practical kinetic analysis of the experimental kinetic curves for the thermal decomposition of molten ammonium dinitramide (ADN). It is known that the thermal decomposition of ADN occurs as a consecutive two step mass-loss process comprising the decomposition of ADN and subsequent evaporation/decomposition of in situ generated ammonium nitrate. These reaction steps provide exothermic and endothermic contributions, respectively, to the overall thermal effect. The overall reaction process was deconvoluted into two reaction steps using simultaneously recorded thermogravimetry and differential scanning calorimetry (TG-DSC) curves by considering the different physical meanings of the kinetic data derived from TG and DSC by P value analysis. The kinetic data thus separated into exothermic and endothermic reaction steps were kinetically characterized using kinetic computation methods including isoconversional method, combined kinetic analysis, and master plot method. The overall kinetic behavior was reproduced as the sum of the kinetic equations for each reaction step considering the contributions to the rate data derived from TG and DSC. During reproduction of the kinetic behavior, the kinetic parameters and contributions of each reaction step were optimized using kinetic deconvolution analysis. As a result, the thermal decomposition of ADN was successfully modeled as partially overlapping exothermic and endothermic reaction steps. The logic of the kinetic modeling was critically examined, and the practical usefulness of phenomenological modeling for the thermal decomposition of ADN was illustrated to demonstrate the validity of the methodology and its applicability to similar complex reaction processes.

Preparation, characterization and application of Saussurea tridactyla Sch-Bip as green adsorbents for preconcentration of rare earth elements in environmental water samples

NASA Astrophysics Data System (ADS)

Zhang, Qiangying; He, Man; Chen, Beibei; Hu, Bin

2016-07-01

This paper deals with preparation, characterization and application of the Saussurea tridactyla Sch-Bip (STSB) as a new green adsorbent for separation of matrix elements and preconcentration of rare earth elements (REEs) in environmental water samples. The pretreated STSB adsorbent with 2 mol L- 1 NaOH is characterized with higher surface area and adsorption capacities in comparison with a raw STSB material. The new adsorbent was used for the development of on-line solid phase extraction (SPE) for the determination of REEs by radial viewing 27 MHz inductively coupled plasma optical emission spectrometry (ICP-OES). Various parameters affecting the adsorption/desorption procedure were optimized. The adsorption capacities for the STSB were found to be 62.2 (Y)-153 mg g- 1 (Tm). Under the optimized conditions, the limits of detection (LODs, 3σ) for REEs were in the range of 0.06 (Yb)-8.77 (Sm) ng mL- 1. The relative standard deviations (RSDs) for 7 replicate determinations of target REEs at low concentration level ranged from 2.4 (Yb) to 8.9 (Sm)%. The adsorption isotherm fitted Langmuir model and the adsorption kinetics fitted well with both Pseudo-first order and Pseudo-second order models. The predominant adsorption mechanism is ion exchange. The STSB pretreated with 2 mol L- 1 NaOH has been demonstrated to be low cost, green and environment friendly adsorbent, featuring with high adsorption capacity, wide pH range, and fast adsorption/desorption kinetics for target REEs with long lifetime. The proposed method was applied to the determination of REEs in East Lake, Yangtze River and rain water samples.

Heat shock protein 72 expression allows permissive replication of oncolytic adenovirus dl1520 (ONYX-015) in rat glioblastoma cells

PubMed Central

Madara, Jonathan; Krewet, James A; Shah, Maulik

2005-01-01

In this study we have made novel observations with regards to potentiation of the tumoricidal activity of the oncolytic adenovirus, dl1520 (ONYX-015) in rat glioblastoma cell lines expressing heat shock protein 72 (HSP72) due to permissive virus replication. ONYX-015 is a conditionally replicating adenovirus that is deleted for the E1B 55 kDA gene product whose normal function is to interact with cell-cycle regulatory proteins to permit virus replication. However, many murine and rodent cell lines are not permissive for adenovirus replication. Previously, it has been reported that the heat shock response is necessary for adenovirus replication and that induction of heat shock proteins is mediated by E1 region gene products. Therefore, we hypothesized that HSP72 expression may allow for permissive replication of ONYX-015 in previously non-permissive cells. Rat glioma cell lines 9L and RT2 were transfected with a plasmids expressing HSP72 or GFP. After infection with ONYX-015, no tumoricidal activity is observed in GFP expressing cell lines despite adequate transduction. In contrast, HSP72 transfected cells show cytopathic effects by 72 hours and greater than 75% loss of viability by 96 hours. Burst assays show active virus replication in the HSP72 expressing cell lines. Therefore, 9L-HSP72 and RT2-HSP72 are ideal models to evaluate the efficacy of ONYX-015 in an immunocompetent rat model. Our study has implications for creating rodent tumor models for pre-clinical studies with E1 region deleted conditionally replicating adenovirus. PMID:15762988

Statistical models for RNA-seq data derived from a two-condition 48-replicate experiment.

PubMed

Gierliński, Marek; Cole, Christian; Schofield, Pietà; Schurch, Nicholas J; Sherstnev, Alexander; Singh, Vijender; Wrobel, Nicola; Gharbi, Karim; Simpson, Gordon; Owen-Hughes, Tom; Blaxter, Mark; Barton, Geoffrey J

2015-11-15

High-throughput RNA sequencing (RNA-seq) is now the standard method to determine differential gene expression. Identifying differentially expressed genes crucially depends on estimates of read-count variability. These estimates are typically based on statistical models such as the negative binomial distribution, which is employed by the tools edgeR, DESeq and cuffdiff. Until now, the validity of these models has usually been tested on either low-replicate RNA-seq data or simulations. A 48-replicate RNA-seq experiment in yeast was performed and data tested against theoretical models. The observed gene read counts were consistent with both log-normal and negative binomial distributions, while the mean-variance relation followed the line of constant dispersion parameter of ∼0.01. The high-replicate data also allowed for strict quality control and screening of 'bad' replicates, which can drastically affect the gene read-count distribution. RNA-seq data have been submitted to ENA archive with project ID PRJEB5348. g.j.barton@dundee.ac.uk. © The Author 2015. Published by Oxford University Press.

Statistical models for RNA-seq data derived from a two-condition 48-replicate experiment

PubMed Central

Cole, Christian; Schofield, Pietà; Schurch, Nicholas J.; Sherstnev, Alexander; Singh, Vijender; Wrobel, Nicola; Gharbi, Karim; Simpson, Gordon; Owen-Hughes, Tom; Blaxter, Mark; Barton, Geoffrey J.

2015-01-01

Motivation: High-throughput RNA sequencing (RNA-seq) is now the standard method to determine differential gene expression. Identifying differentially expressed genes crucially depends on estimates of read-count variability. These estimates are typically based on statistical models such as the negative binomial distribution, which is employed by the tools edgeR, DESeq and cuffdiff. Until now, the validity of these models has usually been tested on either low-replicate RNA-seq data or simulations. Results: A 48-replicate RNA-seq experiment in yeast was performed and data tested against theoretical models. The observed gene read counts were consistent with both log-normal and negative binomial distributions, while the mean-variance relation followed the line of constant dispersion parameter of ∼0.01. The high-replicate data also allowed for strict quality control and screening of ‘bad’ replicates, which can drastically affect the gene read-count distribution. Availability and implementation: RNA-seq data have been submitted to ENA archive with project ID PRJEB5348. Contact: g.j.barton@dundee.ac.uk PMID:26206307

Microbial Kinetic Model for the Degradation of Poorly Soluble Organic Materials

EPA Science Inventory

A novel mechanistic model is presented that describes the aerobic biodegradation kinetics of soybean biodiesel and petroleum diesel in batch experiments. The model was built on the assumptions that biodegradation takes place in the aqueous phase according to Monod kinetics, and ...

Kinetics of Cd(ii) adsorption and desorption on ferrihydrite: experiments and modeling.

PubMed

Liang, Yuzhen; Tian, Lei; Lu, Yang; Peng, Lanfang; Wang, Pei; Lin, Jingyi; Cheng, Tao; Dang, Zhi; Shi, Zhenqing

2018-05-15

The kinetics of Cd(ii) adsorption/desorption on ferrihydrite is an important process affecting the fate, transport, and bioavailability of Cd(ii) in the environment, which was rarely systematically studied and understood at quantitative levels. In this work, a combination of stirred-flow kinetic experiments, batch adsorption equilibrium experiments, high-resolution transmission electron microscopy (HR-TEM), and mechanistic kinetic modeling were used to study the kinetic behaviors of Cd(ii) adsorption/desorption on ferrihydrite. HR-TEM images showed the open, loose, and sponge-like structure of ferrihydrite. The batch adsorption equilibrium experiments revealed that higher pH and initial metal concentration increased Cd(ii) adsorption on ferrihydrite. The stirred-flow kinetic results demonstrated the increased adsorption rate and capacity as a result of the increased pH, influent concentration, and ferrihydrite concentration. The mechanistic kinetic model successfully described the kinetic behaviors of Cd(ii) during the adsorption and desorption stages under various chemistry conditions. The model calculations showed that the adsorption rate coefficients varied as a function of solution chemistry, and the relative contributions of the weak and strong ferrihydrite sites for Cd(ii) binding varied with time at different pH and initial metal concentrations. Our model is able to quantitatively assess the contributions of each individual ferrihydrite binding site to the overall Cd(ii) adsorption/desorption kinetics. This study provided insights into the dynamic behavior of Cd(ii) and a predictive modeling tool for Cd(ii) adsorption/desorption kinetics when ferrihydrite is present, which may be helpful for the risk assessment and management of Cd contaminated sites.

Topological and kinetic determinants of the modal matrices of dynamic models of metabolism

PubMed Central

2017-01-01

Large-scale kinetic models of metabolism are becoming increasingly comprehensive and accurate. A key challenge is to understand the biochemical basis of the dynamic properties of these models. Linear analysis methods are well-established as useful tools for characterizing the dynamic response of metabolic networks. Central to linear analysis methods are two key matrices: the Jacobian matrix (J) and the modal matrix (M-1) arising from its eigendecomposition. The modal matrix M-1 contains dynamically independent motions of the kinetic model near a reference state, and it is sparse in practice for metabolic networks. However, connecting the structure of M-1 to the kinetic properties of the underlying reactions is non-trivial. In this study, we analyze the relationship between J, M-1, and the kinetic properties of the underlying network for kinetic models of metabolism. Specifically, we describe the origin of mode sparsity structure based on features of the network stoichiometric matrix S and the reaction kinetic gradient matrix G. First, we show that due to the scaling of kinetic parameters in real networks, diagonal dominance occurs in a substantial fraction of the rows of J, resulting in simple modal structures with clear biological interpretations. Then, we show that more complicated modes originate from topologically-connected reactions that have similar reaction elasticities in G. These elasticities represent dynamic equilibrium balances within reactions and are key determinants of modal structure. The work presented should prove useful towards obtaining an understanding of the dynamics of kinetic models of metabolism, which are rooted in the network structure and the kinetic properties of reactions. PMID:29267329

Second-order kinetic model for the sorption of cadmium onto tree fern: a comparison of linear and non-linear methods.

PubMed

Ho, Yuh-Shan

2006-01-01

A comparison was made of the linear least-squares method and a trial-and-error non-linear method of the widely used pseudo-second-order kinetic model for the sorption of cadmium onto ground-up tree fern. Four pseudo-second-order kinetic linear equations are discussed. Kinetic parameters obtained from the four kinetic linear equations using the linear method differed but they were the same when using the non-linear method. A type 1 pseudo-second-order linear kinetic model has the highest coefficient of determination. Results show that the non-linear method may be a better way to obtain the desired parameters.

Computational Analyses of Complex Flows with Chemical Reactions

NASA Astrophysics Data System (ADS)

Bae, Kang-Sik

The heat and mass transfer phenomena in micro-scale for the mass transfer phenomena on drug in cylindrical matrix system, the simulation of oxygen/drug diffusion in a three dimensional capillary network, and a reduced chemical kinetic modeling of gas turbine combustion for Jet propellant-10 have been studied numerically. For the numerical analysis of the mass transfer phenomena on drug in cylindrical matrix system, the governing equations are derived from the cylindrical matrix systems, Krogh cylinder model, which modeling system is comprised of a capillary to a surrounding cylinder tissue along with the arterial distance to veins. ADI (Alternative Direction Implicit) scheme and Thomas algorithm are applied to solve the nonlinear partial differential equations (PDEs). This study shows that the important factors which have an effect on the drug penetration depth to the tissue are the mass diffusivity and the consumption of relevant species during the time allowed for diffusion to the brain tissue. Also, a computational fluid dynamics (CFD) model has been developed to simulate the blood flow and oxygen/drug diffusion in a three dimensional capillary network, which are satisfied in the physiological range of a typical capillary. A three dimensional geometry has been constructed to replicate the one studied by Secomb et al. (2000), and the computational framework features a non-Newtonian viscosity model for blood, the oxygen transport model including in oxygen-hemoglobin dissociation and wall flux due to tissue absorption, as well as an ability to study the diffusion of drugs and other materials in the capillary streams. Finally, a chemical kinetic mechanism of JP-10 has been compiled and validated for a wide range of combustion regimes, covering pressures of 1atm to 40atm with temperature ranges of 1,200 K--1,700 K, which is being studied as a possible Jet propellant for the Pulse Detonation Engine (PDE) and other high-speed flight applications such as hypersonic missiles. The comprehensive skeletal mechanism consists of 58 species and 315 reactions including in CPD, Benzene formation process by the theory for polycyclic aromatic hydrocarbons (PAH) and soot formation process on the constant volume combustor, premixed flame characteristics.

Analysis of host response to bacterial infection using error model based gene expression microarray experiments

PubMed Central

Stekel, Dov J.; Sarti, Donatella; Trevino, Victor; Zhang, Lihong; Salmon, Mike; Buckley, Chris D.; Stevens, Mark; Pallen, Mark J.; Penn, Charles; Falciani, Francesco

2005-01-01

A key step in the analysis of microarray data is the selection of genes that are differentially expressed. Ideally, such experiments should be properly replicated in order to infer both technical and biological variability, and the data should be subjected to rigorous hypothesis tests to identify the differentially expressed genes. However, in microarray experiments involving the analysis of very large numbers of biological samples, replication is not always practical. Therefore, there is a need for a method to select differentially expressed genes in a rational way from insufficiently replicated data. In this paper, we describe a simple method that uses bootstrapping to generate an error model from a replicated pilot study that can be used to identify differentially expressed genes in subsequent large-scale studies on the same platform, but in which there may be no replicated arrays. The method builds a stratified error model that includes array-to-array variability, feature-to-feature variability and the dependence of error on signal intensity. We apply this model to the characterization of the host response in a model of bacterial infection of human intestinal epithelial cells. We demonstrate the effectiveness of error model based microarray experiments and propose this as a general strategy for a microarray-based screening of large collections of biological samples. PMID:15800204

Spatial-pattern-induced evolution of a self-replicating loop network.

PubMed

Suzuki, Keisuke; Ikegami, Takashi

2006-01-01

We study a system of self-replicating loops in which interaction rules between individuals allow competition that leads to the formation of a hypercycle-like network. The main feature of the model is the multiple layers of interaction between loops, which lead to both global spatial patterns and local replication. The network of loops manifests itself as a spiral structure from which new kinds of self-replicating loops emerge at the boundaries between different species. In these regions, larger and more complex self-replicating loops live for longer periods of time, managing to self-replicate in spite of their slower replication. Of particular interest is how micro-scale interactions between replicators lead to macro-scale spatial pattern formation, and how these macro-scale patterns in turn perturb the micro-scale replication dynamics.

Kinetics, bioavailability, and metabolism of RRR-alpha-tocopherol in humans supports lower requirement for vitamin E

USDA-ARS?s Scientific Manuscript database

Kinetic models enable nutrient needs and kinetic behaviors to be quantified and provide mechanistic insights into metabolism. Therefore, we modeled and quantified the kinetics, bioavailability and metabolism of RRR-alpha-tocopherol in 12 healthy adults. Six men and six women, aged 27 ± 6 y, each i...

Tigers on trails: occupancy modeling for cluster sampling.

PubMed

Hines, J E; Nichols, J D; Royle, J A; MacKenzie, D I; Gopalaswamy, A M; Kumar, N Samba; Karanth, K U

2010-07-01

Occupancy modeling focuses on inference about the distribution of organisms over space, using temporal or spatial replication to allow inference about the detection process. Inference based on spatial replication strictly requires that replicates be selected randomly and with replacement, but the importance of these design requirements is not well understood. This paper focuses on an increasingly popular sampling design based on spatial replicates that are not selected randomly and that are expected to exhibit Markovian dependence. We develop two new occupancy models for data collected under this sort of design, one based on an underlying Markov model for spatial dependence and the other based on a trap response model with Markovian detections. We then simulated data under the model for Markovian spatial dependence and fit the data to standard occupancy models and to the two new models. Bias of occupancy estimates was substantial for the standard models, smaller for the new trap response model, and negligible for the new spatial process model. We also fit these models to data from a large-scale tiger occupancy survey recently conducted in Karnataka State, southwestern India. In addition to providing evidence of a positive relationship between tiger occupancy and habitat, model selection statistics and estimates strongly supported the use of the model with Markovian spatial dependence. This new model provides another tool for the decomposition of the detection process, which is sometimes needed for proper estimation and which may also permit interesting biological inferences. In addition to designs employing spatial replication, we note the likely existence of temporal Markovian dependence in many designs using temporal replication. The models developed here will be useful either directly, or with minor extensions, for these designs as well. We believe that these new models represent important additions to the suite of modeling tools now available for occupancy estimation in conservation monitoring. More generally, this work represents a contribution to the topic of cluster sampling for situations in which there is a need for specific modeling (e.g., reflecting dependence) for the distribution of the variable(s) of interest among subunits.

Dynamic Metabolic Model Building Based on the Ensemble Modeling Approach

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

Liao, James C.

2016-10-01

Ensemble modeling of kinetic systems addresses the challenges of kinetic model construction, with respect to parameter value selection, and still allows for the rich insights possible from kinetic models. This project aimed to show that constructing, implementing, and analyzing such models is a useful tool for the metabolic engineering toolkit, and that they can result in actionable insights from models. Key concepts are developed and deliverable publications and results are presented.

An improved kinetics approach to describe the physical stability of amorphous solid dispersions.

PubMed

Yang, Jiao; Grey, Kristin; Doney, John

2010-01-15

The recrystallization of amorphous solid dispersions may lead to a loss in the dissolution rate, and consequently reduce bioavailability. The purpose of this work is to understand factors governing the recrystallization of amorphous drug-polymer solid dispersions, and develop a kinetics model capable of accurately predicting their physical stability. Recrystallization kinetics was measured using differential scanning calorimetry for initially amorphous efavirenz-polyvinylpyrrolidone solid dispersions stored at controlled temperature and relative humidity. The experimental measurements were fitted by a new kinetic model to estimate the recrystallization rate constant and microscopic geometry of crystal growth. The new kinetics model was used to illustrate the governing factors of amorphous solid dispersions stability. Temperature was found to affect efavirenz recrystallization in an Arrhenius manner, while recrystallization rate constant was shown to increase linearly with relative humidity. Polymer content tremendously inhibited the recrystallization process by increasing the crystallization activation energy and decreasing the equilibrium crystallinity. The new kinetic model was validated by the good agreement between model fits and experiment measurements. A small increase in polyvinylpyrrolidone resulted in substantial stability enhancements of efavirenz amorphous solid dispersion. The new established kinetics model provided more accurate predictions than the Avrami equation.

Assessment of kinetic models on Fe adsorption in groundwater using high-quality limestone

NASA Astrophysics Data System (ADS)

Akbar, N. A.; Kamil, N. A. F. Mohd; Zin, N. S. Md; Adlan, M. N.; Aziz, H. A.

2018-04-01

During the groundwater pumping process, dissolved Fe2+ is oxidized into Fe3+ and produce rust-coloured iron mineral. Adsorption kinetic models are used to evaluate the performance of limestone adsorbent and describe the mechanism of adsorption and the diffusion processes of Fe adsorption in groundwater. This work presents the best kinetic model of Fe adsorption, which was chosen based on a higher value of coefficient correlation, R2. A batch adsorption experiment was conducted for various contact times ranging from 0 to 135 minutes. From the results of the batch study, three kinetic models were analyzed for Fe removal onto limestone sorbent, including the pseudo-first order (PFO), pseudo-second order (PSO) and intra-particle diffusion (IPD) models. Results show that the adsorption kinetic models follow the sequence: PSO > PFO > IPD, where the values of R2 are 0.997 > 0.919 > 0.918. A high value of R2 (0.997) reveals better fitted experimental data. Furthermore, the value of qe cal in the PSO kinetic model is very near to qe exp rather than that in other models. This finding therefore suggests that the PSO kinetic model has the good fitted with the experimental data which involved chemisorption process of divalent Fe removal in groundwater solution. Thus, limestone adsorbent media found to be an alternative and effective treatment of Fe removal from groundwater.

Successful Establishment of Plasmids R1 and pMV158 in a New Host Requires the Relief of the Transcriptional Repression of Their Essential rep Genes

PubMed Central

Ruiz-Masó, José Á.; Luengo, Luis M.; Moreno-Córdoba, Inmaculada; Díaz-Orejas, Ramón; del Solar, Gloria

2017-01-01

Although differing in size, encoded traits, host range, and replication mechanism, both narrow-host-range theta-type conjugative enterobacterial plasmid R1 and promiscuous rolling-circle-type mobilizable streptococcal plasmid pMV158 encode a transcriptional repressor protein, namely CopB in R1 and CopG in pMV158, involved in replication control. The gene encoding CopB or CopG is cotranscribed with a downstream gene that encodes the replication initiator Rep protein of the corresponding plasmid. However, whereas CopG is an auto-repressor that inhibits transcription of the entire copG-repB operon, CopB is expressed constitutively and represses a second, downstream promoter that directs transcription of repA. As a consequence of the distinct regulatory pathways implied by CopB and CopG, these repressor proteins play a different role in control of plasmid replication during the steady state: while CopB has an auxiliary role by keeping repressed the regulated promoter whenever the plasmid copy number is above a low threshold, CopG plays a primary role by acting coordinately with RNAII. Here, we have studied the role of the regulatory circuit mediated by these transcriptional repressors during the establishment of these two plasmids in a new host cell, and found that excess Cop repressor molecules in the recipient cell result in a severe decrease in the frequency and/or the velocity of appearance of transformant colonies for the cognate plasmid but not for unrelated plasmids. Using the pMV158 replicon as a model system, together with highly sensitive real-time qPCR and inverse PCR methods, we have also analyzed the effect of CopG on the kinetics of repopulation of the plasmid in Streptococcus pneumoniae. We show that, whereas in the absence of CopG pMV158 repopulation occurs mainly during the first 45 min following plasmid transfer, the presence of the transcriptional repressor in the recipient cell severely impairs the replicon repopulation and makes the plasmid replicate at approximately the same rate as the chromosome at any time after transformation, which results in maximal plasmid loss rate in the absence of selection. Overall, these findings indicate that unrepressed activity of the Cop-regulated promoter is crucial for the successful colonization of the recipient bacterial cells by the plasmid. PMID:29250051

Kinetic operational models of agonism for G-protein-coupled receptors.

PubMed

Hoare, Samuel R J; Pierre, Nicolas; Moya, Arturo Gonzalez; Larson, Brad

2018-06-07

The application of kinetics to research and therapeutic development of G-protein-coupled receptors has become increasingly valuable. Pharmacological models provide the foundation of pharmacology, providing concepts and measurable parameters such as efficacy and potency that have underlain decades of successful drug discovery. Currently there are few pharmacological models that incorporate kinetic activity in such a way as to yield experimentally-accessible drug parameters. In this study, a kinetic model of pharmacological response was developed that provides a kinetic descriptor of efficacy (the transduction rate constant, k τ ) and allows measurement of receptor-ligand binding kinetics from functional data. The model assumes: (1) receptor interacts with a precursor of the response ("Transduction potential") and converts it to the response. (2) The response can decay. Familiar response vs time plots emerge, depending on whether transduction potential is depleted and/or response decays. These are the straight line, the "association" exponential curve, and the rise-and-fall curve. Convenient, familiar methods are described for measuring the model parameters and files are provided for the curve-fitting program Prism (GraphPad Software) that can be used as a guide. The efficacy parameter k τ is straightforward to measure and accounts for receptor reserve; all that is required is measurement of response over time at a maximally-stimulating concentration of agonist. The modular nature of the model framework allows it to be extended. Here this is done to incorporate antagonist-receptor binding kinetics and slow agonist-receptor equilibration. In principle, the modular framework can incorporate other cellular processes, such as receptor desensitization. The kinetic response model described here can be applied to measure kinetic pharmacological parameters than can be used to advance the understanding of GPCR pharmacology and optimize new and improved therapeutics. Copyright © 2018 Elsevier Ltd. All rights reserved.

Kinetics of heavy metal adsorption and desorption in soil: Developing a unified model based on chemical speciation

NASA Astrophysics Data System (ADS)

Peng, Lanfang; Liu, Paiyu; Feng, Xionghan; Wang, Zimeng; Cheng, Tao; Liang, Yuzhen; Lin, Zhang; Shi, Zhenqing

2018-03-01

Predicting the kinetics of heavy metal adsorption and desorption in soil requires consideration of multiple heterogeneous soil binding sites and variations of reaction chemistry conditions. Although chemical speciation models have been developed for predicting the equilibrium of metal adsorption on soil organic matter (SOM) and important mineral phases (e.g. Fe and Al (hydr)oxides), there is still a lack of modeling tools for predicting the kinetics of metal adsorption and desorption reactions in soil. In this study, we developed a unified model for the kinetics of heavy metal adsorption and desorption in soil based on the equilibrium models WHAM 7 and CD-MUSIC, which specifically consider metal kinetic reactions with multiple binding sites of SOM and soil minerals simultaneously. For each specific binding site, metal adsorption and desorption rate coefficients were constrained by the local equilibrium partition coefficients predicted by WHAM 7 or CD-MUSIC, and, for each metal, the desorption rate coefficients of various binding sites were constrained by their metal binding constants with those sites. The model had only one fitting parameter for each soil binding phase, and all other parameters were derived from WHAM 7 and CD-MUSIC. A stirred-flow method was used to study the kinetics of Cd, Cu, Ni, Pb, and Zn adsorption and desorption in multiple soils under various pH and metal concentrations, and the model successfully reproduced most of the kinetic data. We quantitatively elucidated the significance of different soil components and important soil binding sites during the adsorption and desorption kinetic processes. Our model has provided a theoretical framework to predict metal adsorption and desorption kinetics, which can be further used to predict the dynamic behavior of heavy metals in soil under various natural conditions by coupling other important soil processes.

Kinetic Framework for the Magnetosphere-Ionosphere-Plasmasphere-Polar Wind System: Modeling Ion Outflow

NASA Astrophysics Data System (ADS)

Schunk, R. W.; Barakat, A. R.; Eccles, V.; Karimabadi, H.; Omelchenko, Y.; Khazanov, G. V.; Glocer, A.; Kistler, L. M.

2014-12-01

A Kinetic Framework for the Magnetosphere-Ionosphere-Plasmasphere-Polar Wind System is being developed in order to provide a rigorous approach to modeling the interaction of hot and cold particle interactions. The framework will include ion and electron kinetic species in the ionosphere, plasmasphere and polar wind, and kinetic ion, super-thermal electron and fluid electron species in the magnetosphere. The framework is ideally suited to modeling ion outflow from the ionosphere and plasmasphere, where a wide range for fluid and kinetic processes are important. These include escaping ion interactions with (1) photoelectrons, (2) cusp/auroral waves, double layers, and field-aligned currents, (3) double layers in the polar cap due to the interaction of cold ionospheric and hot magnetospheric electrons, (4) counter-streaming ions, and (5) electromagnetic wave turbulence. The kinetic ion interactions are particularly strong during geomagnetic storms and substorms. The presentation will provide a brief description of the models involved and discuss the effect that kinetic processes have on the ion outflow.

Virus Neutralisation: New Insights from Kinetic Neutralisation Curves

PubMed Central

Magnus, Carsten

2013-01-01

Antibodies binding to the surface of virions can lead to virus neutralisation. Different theories have been proposed to determine the number of antibodies that must bind to a virion for neutralisation. Early models are based on chemical binding kinetics. Applying these models lead to very low estimates of the number of antibodies needed for neutralisation. In contrast, according to the more conceptual approach of stoichiometries in virology a much higher number of antibodies is required for virus neutralisation by antibodies. Here, we combine chemical binding kinetics with (virological) stoichiometries to better explain virus neutralisation by antibody binding. This framework is in agreement with published data on the neutralisation of the human immunodeficiency virus. Knowing antibody reaction constants, our model allows us to estimate stoichiometrical parameters from kinetic neutralisation curves. In addition, we can identify important parameters that will make further analysis of kinetic neutralisation curves more valuable in the context of estimating stoichiometries. Our model gives a more subtle explanation of kinetic neutralisation curves in terms of single-hit and multi-hit kinetics. PMID:23468602

Cytosine methylation inhibits replication of African cassava mosaic virus by two distinct mechanisms.

PubMed Central

Ermak, G; Paszkowski, U; Wohlmuth, M; Scheid, O M; Paszkowski, J

1993-01-01

Extrachromosomally replicating viral DNA is usually free of cytosine methylation and viral templates methylated in vitro are poor substrates when used in replication assays. We have investigated the mechanism of inhibition of viral replication by DNA methylation using as a model the DNA A of African cassava mosaic virus. We have constructed two component helper systems which allow for separation of the transcriptional inhibition of viral genes necessary for replication from replication inhibition due to altered interaction between the replication complex and methylated viral DNA. Our results suggest that methylation-mediated reduction of viral replication is due to both repression mechanisms and that this provides two independent selection pressures for the maintenance of methylation-free replicons in infected cells. Images PMID:7688453

Parameter Balancing in Kinetic Models of Cell Metabolism†

PubMed Central

2010-01-01

Kinetic modeling of metabolic pathways has become a major field of systems biology. It combines structural information about metabolic pathways with quantitative enzymatic rate laws. Some of the kinetic constants needed for a model could be collected from ever-growing literature and public web resources, but they are often incomplete, incompatible, or simply not available. We address this lack of information by parameter balancing, a method to complete given sets of kinetic constants. Based on Bayesian parameter estimation, it exploits the thermodynamic dependencies among different biochemical quantities to guess realistic model parameters from available kinetic data. Our algorithm accounts for varying measurement conditions in the input data (pH value and temperature). It can process kinetic constants and state-dependent quantities such as metabolite concentrations or chemical potentials, and uses prior distributions and data augmentation to keep the estimated quantities within plausible ranges. An online service and free software for parameter balancing with models provided in SBML format (Systems Biology Markup Language) is accessible at www.semanticsbml.org. We demonstrate its practical use with a small model of the phosphofructokinase reaction and discuss its possible applications and limitations. In the future, parameter balancing could become an important routine step in the kinetic modeling of large metabolic networks. PMID:21038890

Cognitive Mediation of Rape's Mental Health Impact: Constructive Replication of a Cross-Sectional Model in Longitudinal Data

ERIC Educational Resources Information Center

Koss, Mary P.; Figueredo, Aurelio Jose

2004-01-01

The constructive replication of a prespecified, cognitively mediated model of rape's impact on psychosocial health is reported using longitudinal data (see Koss, Figueredo, & Prince, 2002, for a summary of model development). Rape survivors (n= 59) were assessed four times, 3 to 24 months postrape. Structural equations modeling of baseline data…

Exogenous ACE2 Expression Allows Refractory Cell Lines To Support Severe Acute Respiratory Syndrome Coronavirus Replication

PubMed Central

Mossel, Eric C.; Huang, Cheng; Narayanan, Krishna; Makino, Shinji; Tesh, Robert B.; Peters, C. J.

2005-01-01

Of 30 cell lines and primary cells examined, productive severe acute respiratory syndrome coronavirus (Urbani strain) (SARS-CoV) infection after low-multiplicity inoculation was detected in only six: three African green monkey kidney epithelial cell lines (Vero, Vero E6, and MA104), a human colon epithelial line (CaCo-2), a porcine kidney epithelial line [PK(15)], and mink lung epithelial cells (Mv 1 Lu). SARS-CoV produced a lytic infection in Vero, Vero E6, and MA104 cells, but there was no visible cytopathic effect in Caco-2, Mv 1 Lu, or PK(15) cells. Multistep growth kinetics were identical in Vero E6 and MA104 cells, with maximum titer reached 24 h postinoculation (hpi). Virus titer was maximal 96 hpi in CaCo-2 cells, and virus was continually produced from infected CaCo-2 cells for at least 6 weeks after infection. CaCo-2 was the only human cell type of 13 tested that supported efficient SARS-CoV replication. Expression of the SARS-CoV receptor, angiotensin-converting enzyme 2 (ACE2), resulted in SARS-CoV replication in all refractory cell lines examined. Titers achieved were variable and dependent upon the method of ACE2 expression. PMID:15731278

Accelerated telomere shortening and replicative senescence in human fibroblasts overexpressing mutant and wild-type lamin A

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

Huang Shurong; Risques, Rosa Ana; Martin, George M.

2008-01-01

LMNA mutations are responsible for a variety of genetic disorders, including muscular dystrophy, lipodystrophy, and certain progeroid syndromes, notably Hutchinson-Gilford Progeria. Although a number of clinical features of these disorders are suggestive of accelerated aging, it is not known whether cells derived from these patients exhibit cellular phenotypes associated with accelerated aging. We examined a series of isogenic skin fibroblast lines transfected with LMNA constructs bearing known pathogenic point mutations or deletion mutations found in progeroid syndromes. Fibroblasts overexpressing mutant lamin A exhibited accelerated rates of loss of telomeres and shortened replicative lifespans, in addition to abnormal nuclear morphology. Tomore » our surprise, these abnormalities were also observed in lines overexpressing wild-type lamin A. Copy number variants are common in human populations; those involving LMNA, whether arising meiotically or mitotically, might lead to progeroid phenotypes. In an initial pilot study of 23 progeroid cases without detectable WRN or LMNA mutations, however, no cases of altered LMNA copy number were detected. Nevertheless, our findings raise a hypothesis that changes in lamina organization may cause accelerated telomere attrition, with different kinetics for overexpession of wild-type and mutant lamin A, which leads to rapid replicative senescence and progroid phenotypes.« less

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

Rudneva, Irina A.; Timofeeva, Tatiana A.; Ignatieva, Anna V.

In the present study we assessed pleiotropic characteristics of the antibody-selected mutations. We examined pH optimum of fusion, temperatures of HA heat inactivation, and in vitro and in vivo replication kinetics of the previously obtained influenza H5 escape mutants. Our results showed that HA1 N142K mutation significantly lowered the pH of fusion optimum. Mutations of the escape mutants located in the HA lateral loop significantly affected H5 HA thermostability (P<0.05). HA changes at positions 131, 144, 145, and 156 and substitutions at positions 131, 142, 145, and 156 affected the replicative ability of H5 escape mutants in vitro and inmore » vivo, respectively. Overall, a co-variation between antigenic specificity and different HA phenotypic properties has been demonstrated. We believe that the monitoring of pleiotropic effects of the HA mutations found in H5 escape mutants is essential for accurate prediction of mutants with pandemic potential. - Highlights: • HA1 N142K mutation significantly lowered the pH of fusion optimum. • Mutations located in the HA lateral loop significantly affected H5 HA thermostability. • HA changes at positions 131, 142, 144, 145, and 156 affected the replicative ability of H5 mutants. • Acquisition of glycosylation site could lead to the emergence of multiple pleiotropic effects.« less

Amyloid Oligomers and Protofibrils, but Not Filaments, Self-Replicate from Native Lysozyme

PubMed Central

2015-01-01

Self-assembly of amyloid fibrils is the molecular mechanism best known for its connection with debilitating human disorders such as Alzheimer’s disease but is also associated with various functional cellular responses. There is increasing evidence that amyloid formation proceeds along two distinct assembly pathways involving either globular oligomers and protofibrils or rigid monomeric filaments. Oligomers, in particular, have been implicated as the dominant molecular species responsible for pathogenesis. Yet the molecular mechanisms regulating their self-assembly have remained elusive. Here we show that oligomers/protofibrils and monomeric filaments, formed along distinct assembly pathways, display critical differences in their ability to template amyloid growth at physiological vs denaturing temperatures. At physiological temperatures, amyloid filaments remained stable but could not seed growth of native monomers. In contrast, oligomers and protofibrils not only remained intact but were capable of self-replication using native monomers as the substrate. Kinetic data further suggested that this prion-like growth mode of oligomers/protofibrils involved two distinct activities operating orthogonal from each other: autocatalytic self-replication of oligomers from native monomers and nucleated polymerization of oligomers into protofibrils. The environmental changes to stability and templating competence of these different amyloid species in different environments are likely to be important for understanding the molecular mechanisms underlying both pathogenic and functional amyloid self-assembly. PMID:24884889

Amyloid oligomers and protofibrils, but not filaments, self-replicate from native lysozyme.

PubMed

Mulaj, Mentor; Foley, Joseph; Muschol, Martin

2014-06-25

Self-assembly of amyloid fibrils is the molecular mechanism best known for its connection with debilitating human disorders such as Alzheimer's disease but is also associated with various functional cellular responses. There is increasing evidence that amyloid formation proceeds along two distinct assembly pathways involving either globular oligomers and protofibrils or rigid monomeric filaments. Oligomers, in particular, have been implicated as the dominant molecular species responsible for pathogenesis. Yet the molecular mechanisms regulating their self-assembly have remained elusive. Here we show that oligomers/protofibrils and monomeric filaments, formed along distinct assembly pathways, display critical differences in their ability to template amyloid growth at physiological vs denaturing temperatures. At physiological temperatures, amyloid filaments remained stable but could not seed growth of native monomers. In contrast, oligomers and protofibrils not only remained intact but were capable of self-replication using native monomers as the substrate. Kinetic data further suggested that this prion-like growth mode of oligomers/protofibrils involved two distinct activities operating orthogonal from each other: autocatalytic self-replication of oligomers from native monomers and nucleated polymerization of oligomers into protofibrils. The environmental changes to stability and templating competence of these different amyloid species in different environments are likely to be important for understanding the molecular mechanisms underlying both pathogenic and functional amyloid self-assembly.

Replication of urban innovations - prioritization of strategies for the replication of Dhaka's community-based decentralized composting model.

PubMed

Yedla, Sudhakar

2012-01-01

Dhaka's community-based decentralized composting (DCDC) is a successful demonstration of solid waste management by adopting low-cost technology, local resources community participation and partnerships among the various actors involved. This paper attempts to understand the model, necessary conditions, strategies and their priorities to replicate DCDC in the other developing cities of Asia. Thirteen strategies required for its replication are identified and assessed based on various criteria, namely transferability, longevity, economic viability, adaptation and also overall replication. Priority setting by multi-criteria analysis by applying analytic hierarchy process revealed that immediate transferability without long-term and economic viability consideration is not advisable as this would result in unsustainable replication of DCDC. Based on the analysis, measures to ensure the product quality control; partnership among stakeholders (public-private-community); strategies to achieve better involvement of the private sector in solid waste management (entrepreneurship in approach); simple and low-cost technology; and strategies to provide an effective interface among the complementing sectors are identified as important strategies for its replication.

Derivation and Characterization of Pathogenic Transmitted/Founder Molecular Clones from Simian Immunodeficiency Virus SIVsmE660 and SIVmac251 following Mucosal Infection

PubMed Central

Lopker, Michael J.; Del Prete, Gregory Q.; Estes, Jacob D.; Li, Hui; Reid, Carolyn; Newman, Laura; Lipkey, Leslie; Camus, Celine; Easlick, Juliet L.; Wang, Shuyi; Decker, Julie M.; Bar, Katharine J.; Learn, Gerald; Pal, Ranajit; Weiss, Deborah E.; Hahn, Beatrice H.; Lifson, Jeffrey D.; Shaw, George M.

2016-01-01

ABSTRACT Currently available simian immunodeficiency virus (SIV) infectious molecular clones (IMCs) and isolates used in nonhuman primate (NHP) models of AIDS were originally derived from infected macaques during chronic infection or end stage disease and may not authentically recapitulate features of transmitted/founder (T/F) genomes that are of particular interest in transmission, pathogenesis, prevention, and treatment studies. We therefore generated and characterized T/F IMCs from genetically and biologically heterogeneous challenge stocks of SIVmac251 and SIVsmE660. Single-genome amplification (SGA) was used to identify full-length T/F genomes present in plasma during acute infection resulting from atraumatic rectal inoculation of Indian rhesus macaques with low doses of SIVmac251 or SIVsmE660. All 8 T/F clones yielded viruses that were infectious and replication competent in vitro, with replication kinetics similar to those of the widely used chronic-infection-derived IMCs SIVmac239 and SIVsmE543. Phenotypically, the new T/F virus strains exhibited a range of neutralization sensitivity profiles. Four T/F virus strains were inoculated into rhesus macaques, and each exhibited typical SIV replication kinetics. The SIVsm T/F viruses were sensitive to TRIM5α restriction. All T/F viruses were pathogenic in rhesus macaques, resulting in progressive CD4+ T cell loss in gastrointestinal tissues, peripheral blood, and lymphatic tissues. The animals developed pathological immune activation; lymphoid tissue damage, including fibrosis; and clinically significant immunodeficiency leading to AIDS-defining clinical endpoints. These T/F clones represent a new molecular platform for the analysis of virus transmission and immunopathogenesis and for the generation of novel “bar-coded” challenge viruses and next-generation simian-human immunodeficiency viruses that may advance the HIV/AIDS vaccine agenda. IMPORTANCE Nonhuman primate research has relied on only a few infectious molecular clones for a myriad of diverse research projects, including pathogenesis, preclinical vaccine evaluations, transmission, and host-versus-pathogen interactions. With new data suggesting a selected phenotype of the virus that causes infection (i.e., the transmitted/founder virus), we sought to generate and characterize infectious molecular clones from two widely used simian immunodeficiency virus lineages (SIVmac251 and SIVsmE660). Although the exact requirements necessary to be a T/F virus are not yet fully understood, we generated cloned viruses with all the necessary characteristic of a successful T/F virus. The cloned viruses revealed typical acute and set point viral-load dynamics with pathological immune activation, lymphoid tissue damage progressing to significant immunodeficiency, and AIDS-defining clinical endpoints in some animals. These T/F clones represent a new molecular platform for studies requiring authentic T/F viruses. PMID:27412591

The DIRT on Q10: In situ depletion of labile-inputs does not increase temperature sensitivity in a laboratory incubation (Invited)

NASA Astrophysics Data System (ADS)

Reynolds, L. L.; Lajtha, K.; Bowden, R.; Johnson, B. R.; Bridgham, S. D.

2013-12-01

The decomposition of soil organic matter is expected to increase with global warming and has been commonly described by kinetic models with at least two pools with differing turnover times. Pools characterized by rapid turnover are thought to consist of labile substrates. Meanwhile, slower turnover is attributed, in part, to greater chemical complexity and a necessarily higher activation energy which should in turn lead to a higher sensitivity (Q10) to temperature and a proportionally larger response to warming. Experimental tests of the relative Q10 of these pools have been inconclusive and contradictory in part due the fact that all pools are decomposing simultaneously and soils kept under differing conditions over long periods of time diverge in more than the Q10 response making them less comparable over time. We present here a test of the temperature response on soils from a 20 yr litter manipulation experiment incubated under an experimental regime that minimizes divergence among the soils. We hypothesize that 1) if exclusion of inputs has depleted labile substrates and 2) the remaining carbon is more chemically complex, then the input exclusion treatments should show a higher Q10 compared to the ambient or increased input treatments. The soils are taken from the Detritus Input and Removal Treatment (DIRT) plots in the Bousson Forest, Pennsylvania, US. The DIRT treatments consist of litter and root exclusion (no inputs = NI), no roots (NR), no litter (NL), double litter (DL), and ambient conditions (C). Soils were incubated at 25oC for 525 days. Periodically, replicate sets were rotated into 15oC, 35oC or remained at 25oC for 24 hr. The headspace CO2 concentration was measured before and after the 24 hr temperature treatments, and then all replicate sets were returned to 25oC. Twenty years of input exclusion decreased respiration rate, with NI < NR = NL < C = DL, and total carbon content, and thus, we conclude, labile substrates. The respiration rate at 25oC was the same for all replicate sets throughout, indicating no divergence due to the temperature rotations. Contrary to our hypothesis, our data indicates that Q10 was similar among the DIRT treatments, despite the clear differences in their carbon pools. Similar studies have examined the temperature response due to depletion labile substrate through laboratory incubation, rather beginning with presumably very different initial labile pools. Our results would suggest that soils with differing soil carbon content and presumably differing carbon quality have the same relative temperature responses. Recent studies have questioned the putative importance of chemical recalcitrance in soils, which would explain our results relative to the predictions of enzymatic kinetic theory.

Termination of DNA replication forks: "Breaking up is hard to do".

PubMed

Bailey, Rachael; Priego Moreno, Sara; Gambus, Agnieszka

2015-01-01

To ensure duplication of the entire genome, eukaryotic DNA replication initiates from thousands of replication origins. The replication forks move through the chromatin until they encounter forks from neighboring origins. During replication fork termination forks converge, the replisomes disassemble and topoisomerase II resolves the daughter DNA molecules. If not resolved efficiently, terminating forks result in genomic instability through the formation of pathogenic structures. Our recent findings shed light onto the mechanism of replisome disassembly upon replication fork termination. We have shown that termination-specific polyubiquitylation of the replicative helicase component - Mcm7, leads to dissolution of the active helicase in a process dependent on the p97/VCP/Cdc48 segregase. The inhibition of terminating helicase disassembly resulted in a replication termination defect. In this extended view we present hypothetical models of replication fork termination and discuss remaining and emerging questions in the DNA replication termination field.

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

PubMed Central

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

2017-01-01

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

Spatio-temporal organization of replication in bacteria and eukaryotes (nucleoids and nuclei).

PubMed

Jackson, Dean; Wang, Xindan; Rudner, David Z

2012-08-01

Here we discuss the spatio-temporal organization of replication in eubacteria and eukaryotes. Although there are significant differences in how replication is organized in cells that contain nuclei from those that do not, you will see that organization of replication in all organisms is principally dictated by the structured arrangement of the chromosome. We will begin with how replication is organized in eubacteria with particular emphasis on three well studied model organisms. We will then discuss spatial and temporal organization of replication in eukaryotes highlighting the similarities and differences between these two domains of life.

Spatio-Temporal Organization of Replication in Bacteria and Eukaryotes (Nucleoids and Nuclei)

PubMed Central

Jackson, Dean; Wang, Xindan; Rudner, David Z.

2012-01-01

Here we discuss the spatio-temporal organization of replication in eubacteria and eukaryotes. Although there are significant differences in how replication is organized in cells that contain nuclei from those that do not, you will see that organization of replication in all organisms is principally dictated by the structured arrangement of the chromosome. We will begin with how replication is organized in eubacteria with particular emphasis on three well studied model organisms. We will then discuss spatial and temporal organization of replication in eukaryotes highlighting the similarities and differences between these two domains of life. PMID:22855726

Minireview: DNA Replication in Plant Mitochondria

PubMed Central

Cupp, John D.; Nielsen, Brent L.

2014-01-01

Higher plant mitochondrial genomes exhibit much greater structural complexity as compared to most other organisms. Unlike well-characterized metazoan mitochondrial DNA (mtDNA) replication, an understanding of the mechanism(s) and proteins involved in plant mtDNA replication remains unclear. Several plant mtDNA replication proteins, including DNA polymerases, DNA primase/helicase, and accessory proteins have been identified. Mitochondrial dynamics, genome structure, and the complexity of dual-targeted and dual-function proteins that provide at least partial redundancy suggest that plants have a unique model for maintaining and replicating mtDNA when compared to the replication mechanism utilized by most metazoan organisms. PMID:24681310

Characterization of recent and minimally passaged Brazilian dengue viruses inducing robust infection in rhesus macaques

PubMed Central

Borges, Maria Beatriz; Marchevsky, Renato Sergio; Mendes, Ygara S.; Mendes, Luiz Gustavo; Duarte, Ana Claudia; Cruz, Michael; de Filippis, Ana Maria Bispo; Vasconcelos, Pedro Fernando C.; Freire, Marcos; Homma, Akira; Mossman, Sally; Lepine, Edith; Vanloubbeeck, Yannick; Lorin, Clarisse; Malice, Marie-Pierre; Caride, Elena

2018-01-01

The macaque is widely accepted as a suitable model for preclinical characterization of dengue vaccine candidates. However, the only vaccine for which both preclinical and clinical efficacy results were reported so far showed efficacy levels that were substantially different between macaques and humans. We hypothesized that this model’s predictive capacity may be improved using recent and minimally passaged dengue virus isolates, and by assessing vaccine efficacy by characterizing not only the post-dengue virus challenge viremia/RNAemia but also the associated-cytokine profile. Ten recent and minimally passaged Brazilian clinical isolates from the four dengue virus serotypes were tested for their infectivity in rhesus macaques. For the strains showing robust replication capacity, the associated-changes in soluble mediator levels, and the elicited dengue virus-neutralizing antibody responses, were also characterized. Three isolates from dengue virus serotypes 1, 2 and 4 induced viremia of high magnitude and longer duration relative to previously reported viremia kinetics in this model, and robust dengue virus-neutralizing antibody responses. Consistent with observations in humans, increased MCP-1, IFN-γ and VEGF-A levels, and transiently decreased IL-8 levels were detected after infection with the selected isolates. These results may contribute to establishing a dengue macaque model showing a higher predictability for vaccine efficacy in humans. PMID:29694440

Cell survival fraction estimation based on the probability densities of domain and cell nucleus specific energies using improved microdosimetric kinetic models.

PubMed

Sato, Tatsuhiko; Furusawa, Yoshiya

2012-10-01

Estimation of the survival fractions of cells irradiated with various particles over a wide linear energy transfer (LET) range is of great importance in the treatment planning of charged-particle therapy. Two computational models were developed for estimating survival fractions based on the concept of the microdosimetric kinetic model. They were designated as the double-stochastic microdosimetric kinetic and stochastic microdosimetric kinetic models. The former model takes into account the stochastic natures of both domain and cell nucleus specific energies, whereas the latter model represents the stochastic nature of domain specific energy by its approximated mean value and variance to reduce the computational time. The probability densities of the domain and cell nucleus specific energies are the fundamental quantities for expressing survival fractions in these models. These densities are calculated using the microdosimetric and LET-estimator functions implemented in the Particle and Heavy Ion Transport code System (PHITS) in combination with the convolution or database method. Both the double-stochastic microdosimetric kinetic and stochastic microdosimetric kinetic models can reproduce the measured survival fractions for high-LET and high-dose irradiations, whereas a previously proposed microdosimetric kinetic model predicts lower values for these fractions, mainly due to intrinsic ignorance of the stochastic nature of cell nucleus specific energies in the calculation. The models we developed should contribute to a better understanding of the mechanism of cell inactivation, as well as improve the accuracy of treatment planning of charged-particle therapy.

African Green Monkeys Recapitulate the Clinical Experience with Replication of Live Attenuated Pandemic Influenza Virus Vaccine Candidates

PubMed Central

Matsuoka, Yumiko; Suguitan, Amorsolo; Orandle, Marlene; Paskel, Myeisha; Boonnak, Kobporn; Gardner, Donald J.; Feldmann, Friederike; Feldmann, Heinz; Marino, Michael; Jin, Hong; Kemble, George

2014-01-01

ABSTRACT Live attenuated cold-adapted (ca) H5N1, H7N3, H6N1, and H9N2 influenza vaccine viruses replicated in the respiratory tract of mice and ferrets, and 2 doses of vaccines were immunogenic and protected these animals from challenge infection with homologous and heterologous wild-type (wt) viruses of the corresponding subtypes. However, when these vaccine candidates were evaluated in phase I clinical trials, there were inconsistencies between the observations in animal models and in humans. The vaccine viruses did not replicate well and immune responses were variable in humans, even though the study subjects were seronegative with respect to the vaccine viruses before vaccination. Therefore, we sought a model that would better reflect the findings in humans and evaluated African green monkeys (AGMs) as a nonhuman primate model. The distribution of sialic acid (SA) receptors in the respiratory tract of AGMs was similar to that in humans. We evaluated the replication of wt and ca viruses of avian influenza (AI) virus subtypes H5N1, H6N1, H7N3, and H9N2 in the respiratory tract of AGMs. All of the wt viruses replicated efficiently, while replication of the ca vaccine viruses was restricted to the upper respiratory tract. Interestingly, the patterns and sites of virus replication differed among the different subtypes. We also evaluated the immunogenicity and protective efficacy of H5N1, H6N1, H7N3, and H9N2 ca vaccines. Protection from wt virus challenge correlated well with the level of serum neutralizing antibodies. Immune responses were slightly better when vaccine was delivered by both intranasal and intratracheal delivery than when it was delivered intranasally by sprayer. We conclude that live attenuated pandemic influenza virus vaccines replicate similarly in AGMs and human subjects and that AGMs may be a useful model to evaluate the replication of ca vaccine candidates. IMPORTANCE Ferrets and mice are commonly used for preclinical evaluation of influenza vaccines. However, we observed significant inconsistencies between observations in humans and in these animal models. We used African green monkeys (AGMs) as a nonhuman primate (NHP) model for a comprehensive and comparative evaluation of pairs of wild-type and pandemic live attenuated influenza virus vaccines (pLAIV) representing four subtypes of avian influenza viruses and found that pLAIVs replicate similarly in AGMs and humans and that AGMs can be useful for evaluation of the protective efficacy of pLAIV. PMID:24807726

Kinetics of Cation and Oxyanion Adsorption and Desorption on Ferrihydrite: Roles of Ferrihydrite Binding Sites and a Unified Model.

PubMed

Tian, Lei; Shi, Zhenqing; Lu, Yang; Dohnalkova, Alice C; Lin, Zhang; Dang, Zhi

2017-09-19

Quantitative understanding the kinetics of toxic ion reactions with various heterogeneous ferrihydrite binding sites is crucial for accurately predicting the dynamic behavior of contaminants in environment. In this study, kinetics of As(V), Cr(VI), Cu(II), and Pb(II) adsorption and desorption on ferrihydrite was studied using a stirred-flow method, which showed that metal adsorption/desorption kinetics was highly dependent on the reaction conditions and varied significantly among four metals. High resolution scanning transmission electron microscopy coupled with energy-dispersive X-ray spectroscopy showed that all four metals were distributed within the ferrihydrite aggregates homogeneously after adsorption reactions. Based on the equilibrium model CD-MUSIC, we developed a novel unified kinetics model applicable for both cation and oxyanion adsorption and desorption on ferrihydrite, which is able to account for the heterogeneity of ferrihydrite binding sites, different binding properties of cations and oxyanions, and variations of solution chemistry. The model described the kinetic results well. We quantitatively elucidated how the equilibrium properties of the cation and oxyanion binding to various ferrihydrite sites and the formation of various surface complexes controlled the adsorption and desorption kinetics at different reaction conditions and time scales. Our study provided a unified modeling method for the kinetics of ion adsorption/desorption on ferrihydrite.

Noncollisional kinetic model for non-neutral plasmas in a Penning trap: General properties and stationary solutions

NASA Astrophysics Data System (ADS)

Coppa, G. G.; Ricci, Paolo

2002-10-01

This work deals with a noncollisional kinetic model for non-neutral plasmas in a Penning trap. Using the spatial coordinates r, θ, z and the axial velocity vz as phase-space variables, a kinetic model is developed starting from the kinetic equation for the distribution function f(r,θ,z,vz,t). In order to reduce the complexity of the model, the kinetic equations are integrated along the axial direction by assuming an ergodic distribution in the phase space (z,vz) for particles of the same axial energy ɛ and the same planar position. In this way, a kinetic equation for the z-integrated electron distribution F(r,θ,ɛ,t) is obtained taking into account implicitly the three-dimensionality of the problem. The general properties of the model are discussed, in particular the conservation laws. The model is also related to the fluid model that was introduced by Finn et al. [Phys. Plasmas 6, 3744 (1999); Phys. Rev. Lett. 84, 2401 (2000)] and developed by Coppa et al. [Phys. Plasmas 8, 1133 (2001)]. Finally, numerical investigations are presented regarding the stationary solutions of the model.

WESTPA: An interoperable, highly scalable software package for weighted ensemble simulation and analysis

PubMed Central

Zwier, Matthew C.; Adelman, Joshua L.; Kaus, Joseph W.; Pratt, Adam J.; Wong, Kim F.; Rego, Nicholas B.; Suárez, Ernesto; Lettieri, Steven; Wang, David W.; Grabe, Michael; Zuckerman, Daniel M.; Chong, Lillian T.

2015-01-01

The weighted ensemble (WE) path sampling approach orchestrates an ensemble of parallel calculations with intermittent communication to enhance the sampling of rare events, such as molecular associations or conformational changes in proteins or peptides. Trajectories are replicated and pruned in a way that focuses computational effort on under-explored regions of configuration space while maintaining rigorous kinetics. To enable the simulation of rare events at any scale (e.g. atomistic, cellular), we have developed an open-source, interoperable, and highly scalable software package for the execution and analysis of WE simulations: WESTPA (The Weighted Ensemble Simulation Toolkit with Parallelization and Analysis). WESTPA scales to thousands of CPU cores and includes a suite of analysis tools that have been implemented in a massively parallel fashion. The software has been designed to interface conveniently with any dynamics engine and has already been used with a variety of molecular dynamics (e.g. GROMACS, NAMD, OpenMM, AMBER) and cell-modeling packages (e.g. BioNetGen, MCell). WESTPA has been in production use for over a year, and its utility has been demonstrated for a broad set of problems, ranging from atomically detailed host-guest associations to non-spatial chemical kinetics of cellular signaling networks. The following describes the design and features of WESTPA, including the facilities it provides for running WE simulations, storing and analyzing WE simulation data, as well as examples of input and output. PMID:26392815

Albumin nanoparticles coated with polysorbate 80 as a novel drug carrier for the delivery of antiretroviral drug—Efavirenz

PubMed Central

Jenita, Josephine Leno; Chocalingam, Vijaya; Wilson, Barnabas

2014-01-01

Purpose of the study: The antiretroviral therapy (ART) has dramatically improved human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) treatment, prevention and also has been found to increase the lifespan of HIV/AIDS patients by providing durable control of the HIV replication in patients. Efavirenz is a non-nucleoside reverse transcriptase inhibitor of HIV-1. The purpose of this study is to formulate efavirenz-loaded bovine serum albumin nanoparticles to improve efavirenz delivery into various organs. Materials and Methods: Nanoparticles were prepared by desolvation technique and coated with polysorbate 80. Ethanol, glutaraldehyde, and mannitol were used as desolvating, cross linking agent, and cryoprotectant, respectively. Drug to polymer ratio was chosen at five levels from 1:2, 1:3, 1:4, 1:5, and 1:6 (by weight). The formulated nanoparticles were characterized for Fourier Transform Infrared (FT-IR) spectroscopy, differential scanning calorimetry (DSC) studies, entrapment efficiency, particle size, surface charge, surface morphology, in vitro drug release, release kinetics, stability studies, and biodistribution studies. Results and Major Conclusion: The particle size of the prepared formulations was found below 250nm with narrow size distribution, spherical in shape and showed good entrapment efficiency (45.62-72.49%). The in vitro drug release indicated biphasic release and its data were fitted to release kinetics models and release pattern was Fickian diffusion controlled release profile. The prepared nanoparticles increased efavirenz delivery into various organs by several fold in comparison with the free drug. PMID:25126528

Cytology of DNA Replication Reveals Dynamic Plasticity of Large-Scale Chromatin Fibers.

PubMed

Deng, Xiang; Zhironkina, Oxana A; Cherepanynets, Varvara D; Strelkova, Olga S; Kireev, Igor I; Belmont, Andrew S

2016-09-26

In higher eukaryotic interphase nuclei, the 100- to >1,000-fold linear compaction of chromatin is difficult to reconcile with its function as a template for transcription, replication, and repair. It is challenging to imagine how DNA and RNA polymerases with their associated molecular machinery would move along the DNA template without transient decondensation of observed large-scale chromatin "chromonema" fibers [1]. Transcription or "replication factory" models [2], in which polymerases remain fixed while DNA is reeled through, are similarly difficult to conceptualize without transient decondensation of these chromonema fibers. Here, we show how a dynamic plasticity of chromatin folding within large-scale chromatin fibers allows DNA replication to take place without significant changes in the global large-scale chromatin compaction or shape of these large-scale chromatin fibers. Time-lapse imaging of lac-operator-tagged chromosome regions shows no major change in the overall compaction of these chromosome regions during their DNA replication. Improved pulse-chase labeling of endogenous interphase chromosomes yields a model in which the global compaction and shape of large-Mbp chromatin domains remains largely invariant during DNA replication, with DNA within these domains undergoing significant movements and redistribution as they move into and then out of adjacent replication foci. In contrast to hierarchical folding models, this dynamic plasticity of large-scale chromatin organization explains how localized changes in DNA topology allow DNA replication to take place without an accompanying global unfolding of large-scale chromatin fibers while suggesting a possible mechanism for maintaining epigenetic programming of large-scale chromatin domains throughout DNA replication. Copyright © 2016 Elsevier Ltd. All rights reserved.

Dynamics of Tissue-Specific CD8+ T Cell Responses during West Nile Virus Infection.

PubMed

Aguilar-Valenzuela, Renan; Netland, Jason; Seo, Young-Jin; Bevan, Michael J; Grakoui, Arash; Suthar, Mehul S

2018-05-15

The mouse model of West Nile virus (WNV), which is a leading cause of mosquito-borne encephalitis worldwide, has provided fundamental insights into the host and viral factors that regulate viral pathogenesis and infection outcome. In particular, CD8 + T cells are critical for controlling WNV replication and promoting protection against infection. Here, we present the characterization of a T cell receptor (TCR)-transgenic mouse with specificity for the immunodominant epitope in the WNV NS4B protein (here referred to as transgenic WNV-I mice). Using an adoptive-transfer model, we found that WNV-I CD8 + T cells behave similarly to endogenous CD8 + T cell responses, with an expansion phase in the periphery beginning around day 7 postinfection (p.i.) followed by a contraction phase through day 15 p.i. Through the use of in vivo intravascular immune cell staining, we determined the kinetics, expansion, and differentiation into effector and memory subsets of WNV-I CD8 + T cells within the spleen and brain. We found that red-pulp WNV-I CD8 + T cells were more effector-like than white-pulp WNV-I CD8 + T cells, which displayed increased differentiation into memory precursor cells. Within the central nervous system (CNS), we found that WNV-I CD8 + T cells were polyfunctional (gamma interferon [IFN-γ] and tumor necrosis factor alpha [TNF-α]), displayed tissue-resident characteristics (CD69 + and CD103 + ), persisted in the brain through day 15 p.i., and reduced the viral burden within the brain. The use of these TCR-transgenic WNV-I mice provides a new resource to dissect the immunological mechanisms of CD8 + T cell-mediated protection during WNV infection. IMPORTANCE West Nile Virus (WNV) is the leading cause of mosquito-borne encephalitis worldwide. There are currently no approved therapeutics or vaccines for use in humans to treat or prevent WNV infection. CD8 + T cells are critical for controlling WNV replication and protecting against infection. Here, we present a comprehensive characterization of a novel TCR-transgenic mouse with specificity for the immunodominant epitope in the WNV NS4B protein. In this study, we determine the kinetics, proliferation, differentiation into effector and memory subsets, homing, and clearance of WNV in the CNS. Our findings provide a new resource to dissect the immunological mechanisms of CD8 + T cell-mediated protection during WNV infection. Copyright © 2018 American Society for Microbiology.

Multivariate curve resolution-alternating least squares and kinetic modeling applied to near-infrared data from curing reactions of epoxy resins: mechanistic approach and estimation of kinetic rate constants.

PubMed

Garrido, M; Larrechi, M S; Rius, F X

2006-02-01

This study describes the combination of multivariate curve resolution-alternating least squares with a kinetic modeling strategy for obtaining the kinetic rate constants of a curing reaction of epoxy resins. The reaction between phenyl glycidyl ether and aniline is monitored by near-infrared spectroscopy under isothermal conditions for several initial molar ratios of the reagents. The data for all experiments, arranged in a column-wise augmented data matrix, are analyzed using multivariate curve resolution-alternating least squares. The concentration profiles recovered are fitted to a chemical model proposed for the reaction. The selection of the kinetic model is assisted by the information contained in the recovered concentration profiles. The nonlinear fitting provides the kinetic rate constants. The optimized rate constants are in agreement with values reported in the literature.

Protection of Chickens against Avian Influenza with Non-Replicating Adenovirus-Vectored Vaccine

PubMed Central

Toro, Haroldo; Tang, De-chu C.; Suarez, David L.; Shi, Z.

2009-01-01

Protective immunity against avian influenza (AI) virus was elicited in chickens by single-dose vaccination with a replication competent adenovirus (RCA) -free human adenovirus (Ad) vector encoding an H7 AI hemagglutinin (AdChNY94.H7). Chickens vaccinated in ovo with an Ad vector encoding an AI H5 (AdTW68.H5) previously described, which were subsequently vaccinated intramuscularly with AdChNY94.H7 post-hatch, responded with robust antibody titers against both the H5 and H7 AI proteins. Antibody responses to Ad vector in ovo vaccination follow a dose-response kinetic. The use of a synthetic AI H5 gene codon optimized to match the chicken cell tRNA pool was more potent than the cognate H5 gene. The use of Ad-vectored vaccines to increase resistance of chicken populations against multiple AI strains could reduce the risk of an avian-originating influenza pandemic in humans. PMID:18384919

The Replication Focus Targeting Sequence (RFTS) Domain Is a DNA-competitive Inhibitor of Dnmt1

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

Syeda, Farisa; Fagan, Rebecca L.; Wean, Matthew

Dnmt1 (DNA methyltransferase 1) is the principal enzyme responsible for maintenance of cytosine methylation at CpG dinucleotides in the mammalian genome. The N-terminal replication focus targeting sequence (RFTS) domain of Dnmt1 has been implicated in subcellular localization, protein association, and catalytic function. However, progress in understanding its function has been limited by the lack of assays for and a structure of this domain. Here, we show that the naked DNA- and polynucleosome-binding activities of Dnmt1 are inhibited by the RFTS domain, which functions by virtue of binding the catalytic domain to the exclusion of DNA. Kinetic analysis with a fluorogenicmore » DNA substrate established the RFTS domain as a 600-fold inhibitor of Dnmt1 enzymatic activity. The crystal structure of the RFTS domain reveals a novel fold and supports a mechanism in which an RFTS-targeted Dnmt1-binding protein, such as Uhrf1, may activate Dnmt1 for DNA binding.« less

Gene-Based Detection of Microorganisms in Environmental Samples Using PCR

NASA Technical Reports Server (NTRS)

Glass, John I.; Lefkowitz, Elliot J.; Cassell, Gail H.; Wechser, Mark; Taylor, Theresa B.; Albin, Michael; Paszko-Kolva, Christine; Roman, Monsi C.

1997-01-01

Contaminating microorganisms pose a serious potential risk to the crew's well being and water system integrity aboard the International Space Station (ISS). We are developing a gene-based microbial monitor that functions by replicating specific segments of DNA as much as 10(exp 12) x. Thus a single molecule of DNA can be replicated to detectable levels, and the kinetics of that molecule's accumulation can be used to determine the original concentration of specific microorganisms in a sample. Referred to as the polymerase chain reaction (PCR), this enzymatic amplification of specific segments of the DNA or RNA from contaminating microbes offers the promise of rapid, sensitive, quantitative detection and identification of bacteria, fungi, viruses, and parasites. We envision a small instrument capable of assaying an ISS water sample for 48 different microbes in a 24 hour period. We will report on both the developments in the chemistry necessary for the PCR assays to detect microbial contaminants in ISS water, and on progress towards the miniaturization and automation of the instrumentation.

Kinetics of liver macrophages (Kupffer cells) in SIV-infected macaques.

PubMed

Ahsan, Muhammad H; Gill, Amy F; Alvarez, Xavier; Lackner, Andrew A; Veazey, Ronald S

2013-11-01

Since the liver drains antigens from the intestinal tract, and since the intestinal tract is a major site of viral replication, we examined the dynamics of liver macrophages (Kupffer cells) throughout SIV infection. Absolute numbers of Kupffer cells increased in the livers in acute infection, and in animals with AIDS. Significantly higher percentages of proliferating (BrdU+) Kupffer cells were detected in acute infection and in AIDS with similar trends in blood monocytes. Significantly higher percentages of apoptotic (AC3+) Kupffer cells were also found in acute and AIDS stages. However, productively infected cells were not detected in liver of 41/42 animals examined, despite abundant infected cells in gut and lymph nodes of all animals. Increased rates of Kupffer cell proliferation resulting in an increase in Kupffer cells without productive infection indicate SIV infection affects Kupffer cells, but the liver does not appear to be a major site of productive viral replication. © 2013 Elsevier Inc. All rights reserved.

Global analysis of host-pathogen interactions that regulate early stage HIV-1 replication

PubMed Central

König, Renate; Zhou, Yingyao; Elleder, Daniel; Diamond, Tracy L.; Bonamy, Ghislain M.C.; Irelan, Jeffrey T.; Chiang, Chih-yuan; Tu, Buu P.; De Jesus, Paul D.; Lilley, Caroline E.; Seidel, Shannon; Opaluch, Amanda M.; Caldwell, Jeremy S.; Weitzman, Matthew D.; Kuhen, Kelli L.; Bandyopadhyay, Sourav; Ideker, Trey; Orth, Anthony P.; Miraglia, Loren J.; Bushman, Frederic D.; Young, John A.; Chanda, Sumit K.

2008-01-01

Human Immunodeficiency Viruses (HIV-1 and HIV-2) rely upon host-encoded proteins to facilitate their replication. Here we combined genome-wide siRNA analyses with interrogation of human interactome databases to assemble a host-pathogen biochemical network containing 213 confirmed host cellular factors and 11 HIV-1-encoded proteins. Protein complexes that regulate ubiquitin conjugation, proteolysis, DNA damage response and RNA splicing were identified as important modulators of early stage HIV-1 infection. Additionally, over 40 new factors were shown to specifically influence initiation and/or kinetics of HIV-1 DNA synthesis, including cytoskeletal regulatory proteins, modulators of post-translational modification, and nucleic acid binding proteins. Finally, fifteen proteins with diverse functional roles, including nuclear transport, prostaglandin synthesis, ubiquitination, and transcription, were found to influence nuclear import or viral DNA integration. Taken together, the multi-scale approach described here has uncovered multiprotein virus-host interactions that likely act in concert to facilitate early steps of HIV-1 infection. PMID:18854154

The initial value problem in Lagrangian drift kinetic theory

NASA Astrophysics Data System (ADS)

Burby, J. W.

2016-06-01

> Existing high-order variational drift kinetic theories contain unphysical rapidly varying modes that are not seen at low orders. These unphysical modes, which may be rapidly oscillating, damped or growing, are ushered in by a failure of conventional high-order drift kinetic theory to preserve the structure of its parent model's initial value problem. In short, the (infinite dimensional) system phase space is unphysically enlarged in conventional high-order variational drift kinetic theory. I present an alternative, `renormalized' variational approach to drift kinetic theory that manifestly respects the parent model's initial value problem. The basic philosophy underlying this alternate approach is that high-order drift kinetic theory ought to be derived by truncating the all-orders system phase-space Lagrangian instead of the usual `field particle' Lagrangian. For the sake of clarity, this story is told first through the lens of a finite-dimensional toy model of high-order variational drift kinetics; the analogous full-on drift kinetic story is discussed subsequently. The renormalized drift kinetic system, while variational and just as formally accurate as conventional formulations, does not support the troublesome rapidly varying modes.

Spatiotemporal structure of wind farm-atmospheric boundary layer interactions

NASA Astrophysics Data System (ADS)

Cervarich, Matthew; Baidya Roy, Somnath; Zhou, Liming

2013-04-01

Wind power is currently one of the fastest growing energy sources in the world. Most of the growth is in the utility sector consisting of large wind farms with numerous industrial-scale wind turbines. Wind turbines act as a sink of mean kinetic energy and a source of turbulent kinetic energy in the atmospheric boundary layer (ABL). In doing so, they modify the ABL profiles and land-atmosphere exchanges of energy, momentum, mass and moisture. This project explores theses interactions using remote sensing data and numerical model simulations. The domain is central Texas where 4 of the world's largest wind farms are located. A companion study of seasonally-averaged Land Surface Temperature data from the Moderate Resolution Imaging Spectroradiometer (MODIS) on TERRA and AQUA satellites shows a warming signal at night and a mixed cooling/warming signal during the daytime within the wind farms. In the present study, wind farm-ABL interactions are simulated with the Weather Research and Forecasting (WRF) model. The simulations show that the model is capable of replicating the observed signal in land surface temperature. Moreover, similar warming/cooling effect, up to 1C, was observed in seasonal mean 2m air temperature as well. Further analysis show that enhanced turbulent mixing in the rotor wakes is responsible for the impacts on 2m and surface air temperatures. The mixing is due to 2 reasons: (i) turbulent momentum transport to compensate the momentum deficit in the wakes of the turbines and (ii) turbulence generated due to motion of turbine rotors. Turbulent mixing also alters vertical profiles of moisture. Changes in land-atmosphere temperature and moisture gradient and increase in turbulent mixing leads to more than 10% change in seasonal mean surface sensible and latent heat flux. Given the current installed capacity and the projected installation across the world, wind farms are likely becoming a major driver of anthropogenic land use change on Earth. Hence, understanding WF-ABL interactions and its effects is of significant scientific and societal importance.

Model-based analysis of coupled equilibrium-kinetic processes: indirect kinetic studies of thermodynamic parameters using the dynamic data.

PubMed

Emami, Fereshteh; Maeder, Marcel; Abdollahi, Hamid

2015-05-07

Thermodynamic studies of equilibrium chemical reactions linked with kinetic procedures are mostly impossible by traditional approaches. In this work, the new concept of generalized kinetic study of thermodynamic parameters is introduced for dynamic data. The examples of equilibria intertwined with kinetic chemical mechanisms include molecular charge transfer complex formation reactions, pH-dependent degradation of chemical compounds and tautomerization kinetics in micellar solutions. Model-based global analysis with the possibility of calculating and embedding the equilibrium and kinetic parameters into the fitting algorithm has allowed the complete analysis of the complex reaction mechanisms. After the fitting process, the optimal equilibrium and kinetic parameters together with an estimate of their standard deviations have been obtained. This work opens up a promising new avenue for obtaining equilibrium constants through the kinetic data analysis for the kinetic reactions that involve equilibrium processes.

DNA Replication Control During Drosophila Development: Insights into the Onset of S Phase, Replication Initiation, and Fork Progression

PubMed Central

Hua, Brian L.; Orr-Weaver, Terry L.

2017-01-01

Proper control of DNA replication is critical to ensure genomic integrity during cell proliferation. In addition, differential regulation of the DNA replication program during development can change gene copy number to influence cell size and gene expression. Drosophila melanogaster serves as a powerful organism to study the developmental control of DNA replication in various cell cycle contexts in a variety of differentiated cell and tissue types. Additionally, Drosophila has provided several developmentally regulated replication models to dissect the molecular mechanisms that underlie replication-based copy number changes in the genome, which include differential underreplication and gene amplification. Here, we review key findings and our current understanding of the developmental control of DNA replication in the contexts of the archetypal replication program as well as of underreplication and differential gene amplification. We focus on the use of these latter two replication systems to delineate many of the molecular mechanisms that underlie the developmental control of replication initiation and fork elongation. PMID:28874453

Structural Model for Antisocial Behavior: Generalization to Single-Mother Families.

ERIC Educational Resources Information Center

Baldwin, David V.; Skinner, Martie L.

1989-01-01

The purpose of this study was to replicate the "basic training" structural model in Patterson's (1982) coercion theory, extending its scope to single-mother families with younger (six- to eight-year-old) boys. Significance of the successful replication was seen to lie in implications for the generalizability of the model across family…

Manipulating 3D-Printed and Paper Models Enhances Student Understanding of Viral Replication

ERIC Educational Resources Information Center

Couper, Lisa; Johannes, Kristen; Powers, Jackie; Silberglitt, Matt; Davenport, Jodi

2016-01-01

Understanding key concepts in molecular biology requires reasoning about molecular processes that are not directly observable and, as such, presents a challenge to students and teachers. We ask whether novel interactive physical models and activities can help students understand key processes in viral replication. Our 3D tangible models are…

Development of Kinetic Mechanisms for Next-Generation Fuels and CFD Simulation of Advanced Combustion Engines

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

Pitz, William J.; McNenly, Matt J.; Whitesides, Russell

Predictive chemical kinetic models are needed to represent next-generation fuel components and their mixtures with conventional gasoline and diesel fuels. These kinetic models will allow the prediction of the effect of alternative fuel blends in CFD simulations of advanced spark-ignition and compression-ignition engines. Enabled by kinetic models, CFD simulations can be used to optimize fuel formulations for advanced combustion engines so that maximum engine efficiency, fossil fuel displacement goals, and low pollutant emission goals can be achieved.

DigOut: viewing differential expression genes as outliers.

PubMed

Yu, Hui; Tu, Kang; Xie, Lu; Li, Yuan-Yuan

2010-12-01

With regards to well-replicated two-conditional microarray datasets, the selection of differentially expressed (DE) genes is a well-studied computational topic, but for multi-conditional microarray datasets with limited or no replication, the same task is not properly addressed by previous studies. This paper adopts multivariate outlier analysis to analyze replication-lacking multi-conditional microarray datasets, finding that it performs significantly better than the widely used limit fold change (LFC) model in a simulated comparative experiment. Compared with the LFC model, the multivariate outlier analysis also demonstrates improved stability against sample variations in a series of manipulated real expression datasets. The reanalysis of a real non-replicated multi-conditional expression dataset series leads to satisfactory results. In conclusion, a multivariate outlier analysis algorithm, like DigOut, is particularly useful for selecting DE genes from non-replicated multi-conditional gene expression dataset.

Integrated and spectral energetics of the GLAS general circulation model

NASA Technical Reports Server (NTRS)

Tenenbaum, J.

1981-01-01

Integrated and spectral error energetics of the Goddard Laboratory for Atmospheric Sciences (GLAS) general circulation model are compared with observations for periods in January 1975, 1976, and 1977. For two cases the model shows significant skill in predicting integrated energetics quantities out to two weeks, and for all three cases, the integrated monthly mean energetics show qualitative improvements over previous versions of the model in eddy kinetic energy and barotropic conversions. Fundamental difficulties remain with leakage of energy to the stratospheric level. General circulation model spectral energetics predictions are compared with the corresponding observational spectra on a day by day basis. Eddy kinetic energy can be correct while significant errors occur in the kinetic energy of wavenumber three. Single wavenumber dominance in eddy kinetic energy and the correlation of spectral kinetic and potential energy are demonstrated.

Quantitative estimation of Nipah virus replication kinetics in vitro

PubMed Central

Chang, Li-Yen; Ali, AR Mohd; Hassan, Sharifah Syed; AbuBakar, Sazaly

2006-01-01

Background Nipah virus is a zoonotic virus isolated from an outbreak in Malaysia in 1998. The virus causes infections in humans, pigs, and several other domestic animals. It has also been isolated from fruit bats. The pathogenesis of Nipah virus infection is still not well described. In the present study, Nipah virus replication kinetics were estimated from infection of African green monkey kidney cells (Vero) using the one-step SYBR® Green I-based quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) assay. Results The qRT-PCR had a dynamic range of at least seven orders of magnitude and can detect Nipah virus from as low as one PFU/μL. Following initiation of infection, it was estimated that Nipah virus RNA doubles at every ~40 minutes and attained peak intracellular virus RNA level of ~8.4 log PFU/μL at about 32 hours post-infection (PI). Significant extracellular Nipah virus RNA release occurred only after 8 hours PI and the level peaked at ~7.9 log PFU/μL at 64 hours PI. The estimated rate of Nipah virus RNA released into the cell culture medium was ~0.07 log PFU/μL per hour and less than 10% of the released Nipah virus RNA was infectious. Conclusion The SYBR® Green I-based qRT-PCR assay enabled quantitative assessment of Nipah virus RNA synthesis in Vero cells. A low rate of Nipah virus extracellular RNA release and low infectious virus yield together with extensive syncytial formation during the infection support a cell-to-cell spread mechanism for Nipah virus infection. PMID:16784519

Pre-Steady State Kinetic Investigation of the Incorporation of Anti-Hepatitis B Nucleotide Analogs Catalyzed by Non-Canonical Human DNA Polymerases

PubMed Central

Brown, Jessica A.; Pack, Lindsey R.; Fowler, Jason D.; Suo, Zucai

2011-01-01

Antiviral nucleoside analogs have been developed to inhibit the enzymatic activities of the hepatitis B virus (HBV) polymerase, thereby preventing the replication and production of HBV. However, the usage of these analogs can be limited by drug toxicity because the 5′-triphosphates of these nucleoside analogs (nucleotide analogs) are potential substrates for human DNA polymerases to incorporate into host DNA. Although they are poor substrates for human replicative DNA polymerases, it remains to be established whether these nucleotide analogs are substrates for the recently discovered human X- and Y-family DNA polymerases. Using pre-steady state kinetic techniques, we have measured the substrate specificity values for human DNA polymerases β, λ, η, ι, κ, and Rev1 incorporating the active forms of the following anti-HBV nucleoside analogs approved for clinical use: adefovir, tenofovir, lamivudine, telbivudine, and entecavir. Compared to the incorporation of a natural nucleotide, most of the nucleotide analogs were incorporated less efficiently (2 to >122,000) by the six human DNA polymerases. In addition, the potential for entecavir and telbivudine, two drugs which possess a 3′-hydroxyl, to become embedded into human DNA was examined by primer extension and DNA ligation assays. These results suggested that telbivudine functions as a chain terminator while entecavir was efficiently extended by the six enzymes and was a substrate for human DNA ligase I. Our findings suggested that incorporation of anti-HBV nucleotide analogs catalyzed by human X- and Y-family polymerases may contribute to clinical toxicity. PMID:22132702

The logic of kinetic regulation in the thioredoxin system

PubMed Central

2011-01-01

Background The thioredoxin system consisting of NADP(H), thioredoxin reductase and thioredoxin provides reducing equivalents to a large and diverse array of cellular processes. Despite a great deal of information on the kinetics of individual thioredoxin-dependent reactions, the kinetic regulation of this system as an integrated whole is not known. We address this by using kinetic modeling to identify and describe kinetic behavioral motifs found within the system. Results Analysis of a realistic computational model of the Escherichia coli thioredoxin system revealed several modes of kinetic regulation in the system. In keeping with published findings, the model showed that thioredoxin-dependent reactions were adaptable (i.e. changes to the thioredoxin system affected the kinetic profiles of these reactions). Further and in contrast to other systems-level descriptions, analysis of the model showed that apparently unrelated thioredoxin oxidation reactions can affect each other via their combined effects on the thioredoxin redox cycle. However, the scale of these effects depended on the kinetics of the individual thioredoxin oxidation reactions with some reactions more sensitive to changes in the thioredoxin cycle and others, such as the Tpx-dependent reduction of hydrogen peroxide, less sensitive to these changes. The coupling of the thioredoxin and Tpx redox cycles also allowed for ultrasensitive changes in the thioredoxin concentration in response to changes in the thioredoxin reductase concentration. We were able to describe the kinetic mechanisms underlying these behaviors precisely with analytical solutions and core models. Conclusions Using kinetic modeling we have revealed the logic that underlies the functional organization and kinetic behavior of the thioredoxin system. The thioredoxin redox cycle and associated reactions allows for a system that is adaptable, interconnected and able to display differential sensitivities to changes in this redox cycle. This work provides a theoretical, systems-biological basis for an experimental analysis of the thioredoxin system and its associated reactions. PMID:21266044

Computational modeling approaches to quantitative structure-binding kinetics relationships in drug discovery.

PubMed

De Benedetti, Pier G; Fanelli, Francesca

2018-03-21

Simple comparative correlation analyses and quantitative structure-kinetics relationship (QSKR) models highlight the interplay of kinetic rates and binding affinity as an essential feature in drug design and discovery. The choice of the molecular series, and their structural variations, used in QSKR modeling is fundamental to understanding the mechanistic implications of ligand and/or drug-target binding and/or unbinding processes. Here, we discuss the implications of linear correlations between kinetic rates and binding affinity constants and the relevance of the computational approaches to QSKR modeling. Copyright © 2018 Elsevier Ltd. All rights reserved.

Autologous neutralizing antibody to human immunodeficiency virus-1 and replication-competent virus recovered from CD4+ T-cell reservoirs in pediatric HIV-1-infected patients on HAART.

PubMed

Ching, Natascha; Nielsen-Saines, Karin A; Deville, Jaime G; Wei, Lian S; Garratty, Eileen; Bryson, Yvonne J

2010-05-01

A patient's ability to produce autologous neutralizing antibody (ANAB) to current and past HIV isolates correlates with reduced disease progression and protects against maternal-fetal transmission. Little is known about the effects of prolonged viral suppression on the ANAB response in pediatric HIV-infected patients receiving HAART because the virus is hard to isolate, except by special methods. We therefore assessed ANAB to pre-HAART PBMC virus isolates and post-HAART replication-competent virus (RCV) isolates recovered from latent CD4(+) T-cell reservoirs in perinatally HIV-infected children by using a PBMC-based assay and 90% neutralization titers. We studied two infants and three children before and after HAART. At the time of RCV isolation (n = 4), plasma HIV RNA was <50 copies/ml. At baseline, four of five children had detectable ANAB titers to concurrent pre-HAART virus isolates. Although ANAB was detected in all subjects at several time points despite prolonged HAART and undetectable viremia, the response was variable. ANAB titers to concurrent post-HAART RCV and earlier pre-HAART plasma were present in 3 children suggesting prior exposure to this virus. Post-HAART RCV isolates had reduced replication kinetics in vitro compared to pre-HAART viruses. The presence of ANAB over time suggests that low levels of viral replication may still be ongoing despite HAART. The observation of baseline ANAB activity with earlier plasma against a later RCV suggests that the "latent" reservoir may be established early in life before HAART.

Inclusion Bodies Are a Site of Ebolavirus Replication

PubMed Central

Hoenen, Thomas; Shabman, Reed S.; Groseth, Allison; Herwig, Astrid; Weber, Michaela; Schudt, Gordian; Dolnik, Olga; Basler, Christopher F.; Becker, Stephan

2012-01-01

Inclusion bodies are a characteristic feature of ebolavirus infections in cells. They contain large numbers of preformed nucleocapsids, but their biological significance has been debated, and they have been suggested to be aggregates of viral proteins without any further biological function. However, recent data for other viruses that produce similar structures have suggested that inclusion bodies might be involved in genome replication and transcription. In order to study filovirus inclusion bodies, we fused mCherry to the ebolavirus polymerase L, which is found in inclusion bodies. The resulting L-mCherry fusion protein was functional in minigenome assays and incorporated into virus-like particles. Importantly, L-mCherry fluorescence in transfected cells was readily detectable and distributed in a punctate pattern characteristic for inclusion bodies. A recombinant ebolavirus encoding L-mCherry instead of L was rescued and showed virtually identical growth kinetics and endpoint titers to those for wild-type virus. Using this virus, we showed that the onset of inclusion body formation corresponds to the onset of viral genome replication, but that viral transcription occurs prior to inclusion body formation. Live-cell imaging further showed that inclusion bodies are highly dynamic structures and that they can undergo dramatic reorganization during cell division. Finally, by labeling nascent RNAs using click technology we showed that inclusion bodies are indeed the site of viral RNA synthesis. Based on these data we conclude that, rather than being inert aggregates of nucleocapsids, ebolavirus inclusion bodies are in fact complex and dynamic structures and an important site at which viral RNA replication takes place. PMID:22915810

Inclusion bodies are a site of ebolavirus replication.

PubMed

Hoenen, Thomas; Shabman, Reed S; Groseth, Allison; Herwig, Astrid; Weber, Michaela; Schudt, Gordian; Dolnik, Olga; Basler, Christopher F; Becker, Stephan; Feldmann, Heinz

2012-11-01

Inclusion bodies are a characteristic feature of ebolavirus infections in cells. They contain large numbers of preformed nucleocapsids, but their biological significance has been debated, and they have been suggested to be aggregates of viral proteins without any further biological function. However, recent data for other viruses that produce similar structures have suggested that inclusion bodies might be involved in genome replication and transcription. In order to study filovirus inclusion bodies, we fused mCherry to the ebolavirus polymerase L, which is found in inclusion bodies. The resulting L-mCherry fusion protein was functional in minigenome assays and incorporated into virus-like particles. Importantly, L-mCherry fluorescence in transfected cells was readily detectable and distributed in a punctate pattern characteristic for inclusion bodies. A recombinant ebolavirus encoding L-mCherry instead of L was rescued and showed virtually identical growth kinetics and endpoint titers to those for wild-type virus. Using this virus, we showed that the onset of inclusion body formation corresponds to the onset of viral genome replication, but that viral transcription occurs prior to inclusion body formation. Live-cell imaging further showed that inclusion bodies are highly dynamic structures and that they can undergo dramatic reorganization during cell division. Finally, by labeling nascent RNAs using click technology we showed that inclusion bodies are indeed the site of viral RNA synthesis. Based on these data we conclude that, rather than being inert aggregates of nucleocapsids, ebolavirus inclusion bodies are in fact complex and dynamic structures and an important site at which viral RNA replication takes place.

A resource facility for kinetic analysis: modeling using the SAAM computer programs.

PubMed

Foster, D M; Boston, R C; Jacquez, J A; Zech, L

1989-01-01

Kinetic analysis and integrated system modeling have contributed significantly to understanding the physiology and pathophysiology of metabolic systems in humans and animals. Many experimental biologists are aware of the usefulness of these techniques and recognize that kinetic modeling requires special expertise. The Resource Facility for Kinetic Analysis (RFKA) provides this expertise through: (1) development and application of modeling technology for biomedical problems, and (2) development of computer-based kinetic modeling methodologies concentrating on the computer program Simulation, Analysis, and Modeling (SAAM) and its conversational version, CONversational SAAM (CONSAM). The RFKA offers consultation to the biomedical community in the use of modeling to analyze kinetic data and trains individuals in using this technology for biomedical research. Early versions of SAAM were widely applied in solving dosimetry problems; many users, however, are not familiar with recent improvements to the software. The purpose of this paper is to acquaint biomedical researchers in the dosimetry field with RFKA, which, together with the joint National Cancer Institute-National Heart, Lung and Blood Institute project, is overseeing SAAM development and applications. In addition, RFKA provides many service activities to the SAAM user community that are relevant to solving dosimetry problems.

Thermodynamically Feasible Kinetic Models of Reaction Networks

PubMed Central

Ederer, Michael; Gilles, Ernst Dieter

2007-01-01

The dynamics of biological reaction networks are strongly constrained by thermodynamics. An holistic understanding of their behavior and regulation requires mathematical models that observe these constraints. However, kinetic models may easily violate the constraints imposed by the principle of detailed balance, if no special care is taken. Detailed balance demands that in thermodynamic equilibrium all fluxes vanish. We introduce a thermodynamic-kinetic modeling (TKM) formalism that adapts the concepts of potentials and forces from irreversible thermodynamics to kinetic modeling. In the proposed formalism, the thermokinetic potential of a compound is proportional to its concentration. The proportionality factor is a compound-specific parameter called capacity. The thermokinetic force of a reaction is a function of the potentials. Every reaction has a resistance that is the ratio of thermokinetic force and reaction rate. For mass-action type kinetics, the resistances are constant. Since it relies on the thermodynamic concept of potentials and forces, the TKM formalism structurally observes detailed balance for all values of capacities and resistances. Thus, it provides an easy way to formulate physically feasible, kinetic models of biological reaction networks. The TKM formalism is useful for modeling large biological networks that are subject to many detailed balance relations. PMID:17208985

Sabin-to-Mahoney Transition Model of Quasispecies Replication

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

2009-05-31

Qspp is an agent-based stochastic simulation model of the Poliovirus Sabin-to-Mahoney transition. This code simulates a cell-to-cell model of Poliovirus replication. The model tracks genotypes (virus genomes) as they are replicated in cells, and as the cells burst and release particles into the medium of a culture dish. An inoculum is then taken from the pool of virions and is used to inoculate cells on a new dish. This process repeats. The Sabin genotype comprises the initial inoculum. Nucleotide positions that match the Sabin1 (vaccine strain) and Mahoney (wild type) genotypes, as well as the neurovirulent phenotype (from the literature)more » are enumerated as constants.« less

Extraction kinetic modelling of total polyphenols and total anthocyanins from saffron floral bio-residues: Comparison of extraction methods.

PubMed

Da Porto, Carla; Natolino, Andrea

2018-08-30

Analysis of the extraction kinetic modelling for natural compounds is essential for industrial application. The second order rate model was applied to estimate the extraction kinetics of conventional solid-liquid extraction (CSLE), ultrasound-assisted extraction (UAE) and microwave-assisted extraction (MAE) of total polyphenols (TPC) from saffron floral bio-residues at different solid-to-liquid ratios (R S/L )(1:10, 1:20, 1:30, 1:50 g ml -1 ), ethanol 59% as solvent and 66 °C temperature. The optimum solid-to-liquid ratios for TPC kinetics were 1:20 for CLSE, 1:30 for UAE and 1:50 for MAE. The kinetics of total anthocyanins (TA) and antioxidant activity (AA) were investigated for the optimum R S/L for each method. The results showed a good prediction of the model for extraction kinetics in all experiments (R 2  > 0.99; NRMS 0.65-3.35%). The kinetic parameters were calculated and discussed. UAE, compared with the other methods, had the greater efficiency for TPC, TA and AA. Copyright © 2018 Elsevier Ltd. All rights reserved.

Optimized Replicating Renilla Luciferase Reporter HIV-1 Utilizing Novel Internal Ribosome Entry Site Elements for Native Nef Expression and Function.

PubMed

Alberti, Michael O; Jones, Jennifer J; Miglietta, Riccardo; Ding, Haitao; Bakshi, Rakesh K; Edmonds, Tara G; Kappes, John C; Ochsenbauer, Christina

2015-12-01

We previously developed replication-competent reporter HIV-1 (referred to herein as LucR.T2A reporter viruses), utilizing a "ribosome skipping" T2A peptide strategy to link Renilla luciferase (LucR) with Nef expression. The demonstrated utility for HIV-1 vaccine and transmission study applications included measurement of neutralizing antibody (NAb) activity in vaccine sera, improved cell-mediated virus inhibition assays, such as T cell-mediated virus inhibition and antibody-dependent cell-mediated cytotoxicity (ADCC) assays, and humanized mouse models. Herein, we extend our prior work and introduce reporter virus technology for applications that require fully functional Nef. We demonstrate that in CD4(+) T cells productively infected with LucR.T2A reporter viruses, T2A peptide-driven Nef expression and function, such as down-regulation of surface CD4 and MHC-I, were impaired. We overcame this limitation of LucR.T2A reporter viruses and achieved physiological Nef expression and function by engineering novel LucR reporter HIV-1 comprising 11 different internal ribosome entry site (IRES) elements chosen for size and relative activity. A range of Nef expression was observed in 293T cells transfected with the different LucR.IRES reporter virus constructs. Iteratively, we identified IRES reporter genomes that expressed Nef closest to physiological levels and produced virus with infectivity, titers, and replication kinetics similar to nonreporter viruses. Our results demonstrated that LucR reporter activity was stable over multiple replication cycles in peripheral blood mononuclear cells (PBMCs). Furthermore, we analyzed Nef functionality, i.e., down-modulation of MHC-I and CD4, following infection of T cell lines and PBMCs. Unlike LucR.T2A reporter virus, one of the redesigned LucR.IRES reporter viruses [containing the modified encephalomyocarditis virus (EMCV) 6ATR IRES element, "6ATRi"] demonstrated Nef expression and function similar to parental "nonreporter" virus. In a previously validated (nef-independent) T cell-based NAb neutralization assay, LucR.6ATRi reporter virus performed indistinguishably from LucR.T2A reporter virus. In summary, reporter viruses comprising the "6ATRi" element promise to augment HIV-1 vaccine and transmission research approaches requiring a sensitive reporter readout combined with wild-type Nef function.

Optimized Replicating Renilla Luciferase Reporter HIV-1 Utilizing Novel Internal Ribosome Entry Site Elements for Native Nef Expression and Function

PubMed Central

Alberti, Michael O.; Jones, Jennifer J.; Miglietta, Riccardo; Ding, Haitao; Bakshi, Rakesh K.; Edmonds, Tara G.; Kappes, John C.

2015-01-01

Abstract We previously developed replication-competent reporter HIV-1 (referred to herein as LucR.T2A reporter viruses), utilizing a “ribosome skipping” T2A peptide strategy to link Renilla luciferase (LucR) with Nef expression. The demonstrated utility for HIV-1 vaccine and transmission study applications included measurement of neutralizing antibody (NAb) activity in vaccine sera, improved cell-mediated virus inhibition assays, such as T cell-mediated virus inhibition and antibody-dependent cell-mediated cytotoxicity (ADCC) assays, and humanized mouse models. Herein, we extend our prior work and introduce reporter virus technology for applications that require fully functional Nef. We demonstrate that in CD4+ T cells productively infected with LucR.T2A reporter viruses, T2A peptide-driven Nef expression and function, such as down-regulation of surface CD4 and MHC-I, were impaired. We overcame this limitation of LucR.T2A reporter viruses and achieved physiological Nef expression and function by engineering novel LucR reporter HIV-1 comprising 11 different internal ribosome entry site (IRES) elements chosen for size and relative activity. A range of Nef expression was observed in 293T cells transfected with the different LucR.IRES reporter virus constructs. Iteratively, we identified IRES reporter genomes that expressed Nef closest to physiological levels and produced virus with infectivity, titers, and replication kinetics similar to nonreporter viruses. Our results demonstrated that LucR reporter activity was stable over multiple replication cycles in peripheral blood mononuclear cells (PBMCs). Furthermore, we analyzed Nef functionality, i.e., down-modulation of MHC-I and CD4, following infection of T cell lines and PBMCs. Unlike LucR.T2A reporter virus, one of the redesigned LucR.IRES reporter viruses [containing the modified encephalomyocarditis virus (EMCV) 6ATR IRES element, “6ATRi”] demonstrated Nef expression and function similar to parental “nonreporter” virus. In a previously validated (nef-independent) T cell-based NAb neutralization assay, LucR.6ATRi reporter virus performed indistinguishably from LucR.T2A reporter virus. In summary, reporter viruses comprising the “6ATRi” element promise to augment HIV-1 vaccine and transmission research approaches requiring a sensitive reporter readout combined with wild-type Nef function. PMID:26101895

Kinetic Behaviour of Failure Waves in a Filled Glass

NASA Astrophysics Data System (ADS)

Resnyansky, A. D.; Bourne, N. K.

2007-12-01

Experimental stress and velocity profiles in a lead filled glass demonstrate a pronounced kinetic behaviour for failure waves in the material during shock loading. The present work summarises the experimental proofs of the kinetic behaviour obtained with stress and velocity gauges. The work describes a model for this behaviour employing a kinetic description used earlier for fracture waves in Pyrex glass. This model is part of a family of two-phase, strain-rate sensitive models describing the behaviour of damaged brittle materials. The modelling results describe well both the stress decay of the failure wave precursor in the stress profiles and main pulse attenuation in the velocity profiles. The influences of the kinetic mechanisms and wave interactions within the test assembly on the reduction of this behaviour are discussed.

Kinetic Analysis for Macrocyclizations Involving Anionic Template at the Transition State

PubMed Central

Martí-Centelles, Vicente; Burguete, M. Isabel; Luis, Santiago V.

2012-01-01

Several kinetic models for the macrocyclization of a C2 pseudopeptide with a dihalide through a SN2 reaction have been developed. These models not only focus on the kinetic analysis of the main macrocyclization reaction, but also consider the competitive oligomerization/polymerization processes yielding undesired oligomeric/polymeric byproducts. The effect of anions has also been included in the kinetic models, as they can act as catalytic templates in the transition state reducing and stabilizing the transition state. The corresponding differential equation systems for each kinetic model can be solved numerically. Through a comprehensive analysis of these results, it is possible to obtain a better understanding of the different parameters that are involved in the macrocyclization reaction mechanism and to develop strategies for the optimization of the desired processes. PMID:22666148

Mutations of amino acids in the DNA-recognition domain of Epstein-Barr virus ZEBRA protein alter its sub-nuclear localization and affect formation of replication compartments

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

Park, Richard; Heston, Lee; Shedd, Duane

ZEBRA, a transcription factor and DNA replication protein encoded by the Epstein-Barr virus (EBV) BZLF1 gene, plays indispensable roles in the EBV lytic cycle. We recently described the phenotypes of 46 single amino acid substitutions introduced into the DNA-recognition region of ZEBRA [Heston, L., El-Guindy, A., Countryman, J., Dela Cruz, C., Delecluse, H.J., and Miller, G. 2006]. The 27 DNA-binding-proficient mutants exhibited distinct defects in their ability to activate expression of the kinetic classes of viral genes. Four phenotypic variants could be discerned: wild-type, defective at activating Rta, defective at activating early genes, and defective at activating late genes. Heremore » we analyze the distribution of ZEBRA within the nucleus and the localization of EA-D (the viral DNA polymerase processivity factor), an indicator of the development of replication compartments, in representatives of each phenotypic group. Plasmids encoding wild-type (WT) and mutant ZEBRA were transfected into 293 cells containing EBV-bacmids. WT ZEBRA protein was diffusely and smoothly distributed throughout the nucleus, sparing nucleoli, and partially recruited to globular replication compartments. EA-D induced by WT ZEBRA was present diffusely in some cells and concentrated in globular replication compartments in other cells. The distribution of ZEBRA and EA-D proteins was identical to WT following transfection of K188R, a mutant with a conservative change. The distribution of S186A mutant ZEBRA protein, defective for activation of Rta and EA-D, was identical to WT, except that the mutant ZEBRA was never found in globular compartments. Co-expression of Rta with S186A mutant rescued diffuse EA-D but not globular replication compartments. The most striking observation was that several mutant ZEBRA proteins defective in activating EA-D (R179A, K181A and A185V) and defective in activating lytic viral DNA replication and late genes (Y180E and K188A) were localized to numerous punctate foci. The speckled appearance of R179A and Y180E was more regular and clearly defined in EBV-positive than in EBV-negative 293 cells. The Y180E late-mutant induced EA-D, but prevented EA-D from localizing to globular replication compartments. These results show that individual amino acids within the basic domain influence localization of the ZEBRA protein and its capacity to induce EA-D to become located in mature viral replication compartments. Furthermore, these mutant ZEBRA proteins delineate several stages in the processes of nuclear re-organization which accompany lytic EBV replication.« less

Fiber-based modeling of in situ ankle ligaments with consideration of progressive failure.

PubMed

Nie, Bingbing; Forman, Jason L; Panzer, Matthew B; Mait, Alexander R; Donlon, John-Paul; Kent, Richard W

2017-08-16

Ligament sprains account for a majority of injuries to the foot and ankle complex among athletic populations. The infeasibility of measuring the in situ response and load paths of individual ligaments has precluded a complete characterization of their mechanical behavior via experiment. In the present study a fiber-based modeling approach of in situ ankle ligaments was developed and validated for determining the heterogeneous force-elongation characteristics and the consequent injury patterns. Nine major ankle ligaments were modeled as bundles of discrete elements, corresponding functionally to the structure of collagen fibers. To incorporate the progressive nature of ligamentous injury, the limit strain at the occurrence of fiber failure was described by a distribution function ranging from 12% to 18% along the width of the insertion site. The model was validated by comparing the structural kinetic and kinematic response obtained experimentally and computationally under well-controlled foot rotations. The simulation results replicated the 6 degree-of-freedom bony motion and ligamentous injuries and, by implication, the in situ deformations of the ligaments. Gross stiffness of the whole ligament derived from the fibers was comparable to existing experimental data. The present modeling approach provides a biomechanically realistic, interpretable and computationally efficient way to characterize the in situ ligament slack, sequential and heterogeneous uncrimping of collagen fascicles and failure propagation as the external load is applied. Applications of this model include functional ankle joint mechanics, injury prevention and countermeasure design for athletes. Copyright © 2017 Elsevier Ltd. All rights reserved.

Comparative evaluation of adsorption kinetics of diclofenac and isoproturon by activated carbon.

PubMed

Torrellas, Silvia A; Rodriguez, Araceli R; Escudero, Gabriel O; Martín, José María G; Rodriguez, Juan G

2015-01-01

Adsorption mechanism of diclofenac and isoproturon onto activated carbon has been proposed using Langmuir and Freundlich isotherms. Adsorption capacity and optimum adsorption isotherms were predicted by nonlinear regression method. Different kinetic equations, pseudo-first-order, pseudo-second-order, intraparticle diffusion model and Bangham kinetic model, were applied to study the adsorption kinetics of emerging contaminants on activated carbon in two aqueous matrices.

Validation of a thin-layer chromatography/densitometry method for the characterization of invertase activity.

PubMed

Ferey, Justine; Da Silva, David; Bravo-Veyrat, Sophie; Lafite, Pierre; Daniellou, Richard; Maunit, Benoît

2016-12-16

This paper presents a kinetic study of invertase, a specific fructofuranosidase cloned from the Leishmania major genome. The kinetic parameters of the β-d-fructofuranosidase from Leishmania major (BfrA) were determined using Thin-Layer Chromatography (TLC) and UV-densitometry (TLC@UV) specifically developed for the separation and detection of three carbohydrates namely sucrose, glucose and fructose. Separation was performed on TLC silica gel 60 F254 plates impregnated with sodium bisulphate and citrate and heated prior to development. This fast and easy separation was performed with two successive developments using ACN/H 2 O 80/20 (v/v) as mobile phase. Sensitive and repeatable derivatization of sugars was achieved by dipping the plates in a solution of 4-aminobenzoic acid. Quantification was performed by UV-detection. The method was validated according to ICH guidelines Q2(R1) in terms of specificity, limits of detection and quantification, precision and robustness (with n=3 replicates and CV ≤10%). The characterization of BfrA reaction kinetic was performed by monitoring the accumulation of either glucose or fructose detected by TLC@UV. Hydrolysis of sucrose was described by the Michaelis-Menten kinetic parameters (K M ; V max ) respectively equal to 63.09±7.590mM; 0.037±0.00094mM/min using glucose production and 83.01±14.39mM; 0.031±0.0021mM/min monitoring fructose. Hydrolyses of three alternative substrates, raffinose, stachyose and inulin, were also compared and the regiospecificity of the reaction was characterized. This TLC@UV method is shown to be suitable for the refined kinetic analysis of different reactions related to the hydrolysis of sugars. Copyright © 2016. Published by Elsevier B.V.

SVD-aided pseudo principal-component analysis: A new method to speed up and improve determination of the optimum kinetic model from time-resolved data

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

Oang, Key Young; Yang, Cheolhee; Muniyappan, Srinivasan

Determination of the optimum kinetic model is an essential prerequisite for characterizing dynamics and mechanism of a reaction. Here, we propose a simple method, termed as singular value decomposition-aided pseudo principal-component analysis (SAPPA), to facilitate determination of the optimum kinetic model from time-resolved data by bypassing any need to examine candidate kinetic models. We demonstrate the wide applicability of SAPPA by examining three different sets of experimental time-resolved data and show that SAPPA can efficiently determine the optimum kinetic model. In addition, the results of SAPPA for both time-resolved X-ray solution scattering (TRXSS) and transient absorption (TA) data of themore » same protein reveal that global structural changes of protein, which is probed by TRXSS, may occur more slowly than local structural changes around the chromophore, which is probed by TA spectroscopy.« less

A Rate-Theory-Phase-Field Model of Irradiation-Induced Recrystallization in UMo Nuclear Fuels

NASA Astrophysics Data System (ADS)

Hu, Shenyang; Joshi, Vineet; Lavender, Curt A.

2017-12-01

In this work, we developed a recrystallization model to study the effect of microstructures and radiation conditions on recrystallization kinetics in UMo fuels. The model integrates the rate theory of intragranular gas bubble and interstitial loop evolutions and a phase-field model of recrystallization zone evolution. A first passage method is employed to describe one-dimensional diffusion of interstitials with a diffusivity value several orders of magnitude larger than that of fission gas xenons. With the model, the effect of grain sizes on recrystallization kinetics is simulated. The results show that (1) recrystallization in large grains starts earlier than that in small grains, (2) the recrystallization kinetics (recrystallization volume fraction) decrease as the grain size increases, (3) the predicted recrystallization kinetics are consistent with the experimental results, and (4) the recrystallization kinetics can be described by the modified Avrami equation, but the parameters of the Avrami equation strongly depend on the grain size.

Exploring the Convergence of the Liberal Arts Model and the Ecuadorian Culture in a Latin American University

ERIC Educational Resources Information Center

Santana Paredes, Matias

2013-01-01

The replication of the U.S. cultural models in business and social organizations is a common practice in Latin America. In Ecuador, a university operates under the liberal arts model, understanding it as a replication of an U.S. cultural model, in an environment where the Ecuadorian cultural beliefs and values constitute the national cultural…

North Dakota's Experience with the Academy Model: A Successful Replication

ERIC Educational Resources Information Center

Hall, Tom; Clapper, Ann

2016-01-01

In this article, professors share how the district/university partnership model thriving at Kansas State University was successfully replicated in North Dakota, and was adapted to match their own department goals. While teacher leadership has become a theme among Kansas State academies, their model was created out of principal preparation efforts.…

Mechanisms and regulation of DNA replication initiation in eukaryotes

PubMed Central

Parker, Matthew W.; Botchan, Michael R.; Berger, James M.

2017-01-01

Cellular DNA replication is initiated through the action of multiprotein complexes that recognize replication start sites in the chromosome (termed origins) and facilitate duplex DNA melting within these regions. In a given cell cycle, initiation occurs only once per origin and each round of replication is tightly coupled to cell division. To avoid aberrant origin firing and re-replication, eukaryotes tightly regulate two events in the initiation process: loading of the replicative helicase, MCM2-7, onto chromatin by the Origin Recognition Complex (ORC), and subsequent activation of the helicase by incorporation into a complex known as the CMG. Recent work has begun to reveal the details of an orchestrated and sequential exchange of initiation factors on DNA that give rise to a replication-competent complex, the replisome. Here we review the molecular mechanisms that underpin eukaryotic DNA replication initiation – from selecting replication start sites to replicative helicase loading and activation – and describe how these events are often distinctly regulated across different eukaryotic model organisms. PMID:28094588

Mechanisms and regulation of DNA replication initiation in eukaryotes.

PubMed

Parker, Matthew W; Botchan, Michael R; Berger, James M

2017-04-01

Cellular DNA replication is initiated through the action of multiprotein complexes that recognize replication start sites in the chromosome (termed origins) and facilitate duplex DNA melting within these regions. In a typical cell cycle, initiation occurs only once per origin and each round of replication is tightly coupled to cell division. To avoid aberrant origin firing and re-replication, eukaryotes tightly regulate two events in the initiation process: loading of the replicative helicase, MCM2-7, onto chromatin by the origin recognition complex (ORC), and subsequent activation of the helicase by its incorporation into a complex known as the CMG. Recent work has begun to reveal the details of an orchestrated and sequential exchange of initiation factors on DNA that give rise to a replication-competent complex, the replisome. Here, we review the molecular mechanisms that underpin eukaryotic DNA replication initiation - from selecting replication start sites to replicative helicase loading and activation - and describe how these events are often distinctly regulated across different eukaryotic model organisms.

Green Energy Options for Consumer-Owned Business

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

Co-opPlus of Western Massachusetts

2006-05-01

The goal of this project was to define, test, and prototype a replicable business model for consumer-owned cooperatives. The result is a replicable consumer-owned cooperative business model for the generation, interconnection, and distribution of renewable energy that incorporates energy conservation and efficiency improvements.

Modelling nifedipine photodegradation, photostability and actinometric properties.

PubMed

Maafi, Wassila; Maafi, Mounir

2013-11-01

The photodegradation of drugs obeying unimolecular mechanisms such as that of nifedipine (NIF) were usually characterised in the literature by zero-, first- and second-order kinetics. This approach has been met with varying success. This paper addresses this issue and proposes a novel approach for unimolecular photodegradation kinetics. The photodegradation of the cardiovascular drug nifedipine is investigated within this framework. Experimental kinetic data of nifedipine photodegradation were obtained by continuous monochromatic irradiation and DAD analysis. Fourth-order Runge-Kutta calculated kinetic data served for the validation of the new semi-empirical integrated rate-law model proposed in this study. A new model equation has been developed and proposed which faithfully describes the kinetic behaviour of NIF in solution for non-isosbestic irradiations at wavelengths where both NIF and its photoproduct absorb. NIF absolute quantum yield values were determined and found to increase with irradiation wavelength according to a defined sigmoid relationship. The effects of increasing NIF or excipients' concentrations on NIF kinetics were successfully modelled and found to improve NIF photostability. The potential of NIF for actinometry has been explored and evaluated. A new reaction order (the so-called Φ-order) has been identified and specifically proposed for unimolecular photodegradation reactions. The semi-empirical and integrated rate-law models facilitated reliable kinetic studies of NIF photodegradation as an example of AB(1Φ) unimolecular reactions. It allowed filling a gap in kinetic studies of drugs since, thus far, thermal first-order or a combination of first- and zero- order kinetic equations were generally applied for drug photoreactions in the literature. Also, a new reaction order, the "Φ-order", has been evidenced and proposed as a specific alternative for photodegradation kinetics. Copyright © 2013 Elsevier B.V. All rights reserved.

Evaluation of porcine reproductive and respiratory syndrome virus replication in laboratory rodents

PubMed Central

Rosenfeld, Paul; Turner, Patricia V.; MacInnes, Janet I.; Nagy, Éva; Yoo, Dongwan

2009-01-01

Porcine reproductive and respiratory syndrome virus (PRRSV) is a major cause of economic losses in the swine industry. The disease is widespread worldwide, and so PRRSV-negative pigs are often difficult to find for the study of PRRSV in vivo. To determine if a small animal model could be developed for PRRSV, 3 strains of laboratory rodent were examined for their susceptibility to the virus. No virus replication was detected in BALB/c or SCID (severe combined immunodeficiency) mice after intraperitoneal inoculation. Moderate replication of PRRSV was detected in primary cotton rat lung cell cultures, but no viral replication was detected following intranasal or intraperitoneal inoculation. Following intratracheal inoculation, viral transcripts were detected in the lungs of cotton rats, but only for 1 day. This study indicates that PRRSV replication in common laboratory rodent species is inefficient, and suggests that a rodent model for this virus is not appropriate. PMID:20046635

Validation of variants in SLC28A3 and UGT1A6 as genetic markers predictive of anthracycline-induced cardiotoxicity in children.

PubMed

Visscher, H; Ross, C J D; Rassekh, S R; Sandor, G S S; Caron, H N; van Dalen, E C; Kremer, L C; van der Pal, H J; Rogers, P C; Rieder, M J; Carleton, B C; Hayden, M R

2013-08-01

The use of anthracyclines as effective antineoplastic drugs is limited by the occurrence of cardiotoxicity. Multiple genetic variants predictive of anthracycline-induced cardiotoxicity (ACT) in children were recently identified. The current study was aimed to assess replication of these findings in an independent cohort of children. . Twenty-three variants were tested for association with ACT in an independent cohort of 218 patients. Predictive models including genetic and clinical risk factors were constructed in the original cohort and assessed in the current replication cohort. . We confirmed the association of rs17863783 in UGT1A6 and ACT in the replication cohort (P = 0.0062, odds ratio (OR) 7.98). Additional evidence for association of rs7853758 (P = 0.058, OR 0.46) and rs885004 (P = 0.058, OR 0.42) in SLC28A3 was found (combined P = 1.6 × 10(-5) and P = 3.0 × 10(-5), respectively). A previously constructed prediction model did not significantly improve risk prediction in the replication cohort over clinical factors alone. However, an improved prediction model constructed using replicated genetic variants as well as clinical factors discriminated significantly better between cases and controls than clinical factors alone in both original (AUC 0.77 vs. 0.68, P = 0.0031) and replication cohort (AUC 0.77 vs. 0.69, P = 0.060). . We validated genetic variants in two genes predictive of ACT in an independent cohort. A prediction model combining replicated genetic variants as well as clinical risk factors might be able to identify high- and low-risk patients who could benefit from alternative treatment options. Copyright © 2013 Wiley Periodicals, Inc.

A Multi Water Bag model of drift kinetic electron plasmaa

NASA Astrophysics Data System (ADS)

Morel, Pierre; Ghiro, Florent Dreydemy; Berionni, Vincent; Coulette, David; Besse, Nicolas; Gürcan, Özgür D.

2014-08-01

A Multi Water Bag model is proposed for describing drift kinetic plasmas in a magnetized cylindrical geometry, relevant for various experimental devices, solar wind modeling... The Multi Water Bag (MWB) model is adapted to the description of a plasma with kinetic electrons as well as an arbitrary number of kinetic ions. This allows to describe the kinetic dynamics of the electrons, making possible the study of electron temperature gradient (ETG) modes, in addition to the effects of non adiabatic electrons on the ion temperature gradient (ITG) modes, that are of prime importance in the magnetized plasmas micro-turbulence [X. Garbet, Y. Idomura, L. Villard, T.H. Watanabe, Nucl. Fusion 50, 043002 (2010); J.A. Krommes, Ann. Rev. Fluid Mech. 44, 175 (2012)]. The MWB model is shown to link kinetic and fluid descriptions, depending on the number of bags considered. Linear stability of the ETG modes is presented and compared to the existing results regarding cylindrical ITG modes [P. Morel, E. Gravier, N. Besse, R. Klein, A. Ghizzo, P. Bertrand, W. Garbet, Ph. Ghendrih, V. Grandgirard, Y. Sarazin, Phys. Plasmas 14, 112109 (2007)].

Optimal bioprocess design through a gene regulatory network - growth kinetic hybrid model: Towards Replacing Monod kinetics.

PubMed

Tsipa, Argyro; Koutinas, Michalis; Usaku, Chonlatep; Mantalaris, Athanasios

2018-05-02

Currently, design and optimisation of biotechnological bioprocesses is performed either through exhaustive experimentation and/or with the use of empirical, unstructured growth kinetics models. Whereas, elaborate systems biology approaches have been recently explored, mixed-substrate utilisation is predominantly ignored despite its significance in enhancing bioprocess performance. Herein, bioprocess optimisation for an industrially-relevant bioremediation process involving a mixture of highly toxic substrates, m-xylene and toluene, was achieved through application of a novel experimental-modelling gene regulatory network - growth kinetic (GRN-GK) hybrid framework. The GRN model described the TOL and ortho-cleavage pathways in Pseudomonas putida mt-2 and captured the transcriptional kinetics expression patterns of the promoters. The GRN model informed the formulation of the growth kinetics model replacing the empirical and unstructured Monod kinetics. The GRN-GK framework's predictive capability and potential as a systematic optimal bioprocess design tool, was demonstrated by effectively predicting bioprocess performance, which was in agreement with experimental values, when compared to four commonly used models that deviated significantly from the experimental values. Significantly, a fed-batch biodegradation process was designed and optimised through the model-based control of TOL Pr promoter expression resulting in 61% and 60% enhanced pollutant removal and biomass formation, respectively, compared to the batch process. This provides strong evidence of model-based bioprocess optimisation at the gene level, rendering the GRN-GK framework as a novel and applicable approach to optimal bioprocess design. Finally, model analysis using global sensitivity analysis (GSA) suggests an alternative, systematic approach for model-driven strain modification for synthetic biology and metabolic engineering applications. Copyright © 2018. Published by Elsevier Inc.

Challenges in Reproducibility, Replicability, and Comparability of Computational Models and Tools for Neuronal and Glial Networks, Cells, and Subcellular Structures.

PubMed

Manninen, Tiina; Aćimović, Jugoslava; Havela, Riikka; Teppola, Heidi; Linne, Marja-Leena

2018-01-01

The possibility to replicate and reproduce published research results is one of the biggest challenges in all areas of science. In computational neuroscience, there are thousands of models available. However, it is rarely possible to reimplement the models based on the information in the original publication, let alone rerun the models just because the model implementations have not been made publicly available. We evaluate and discuss the comparability of a versatile choice of simulation tools: tools for biochemical reactions and spiking neuronal networks, and relatively new tools for growth in cell cultures. The replicability and reproducibility issues are considered for computational models that are equally diverse, including the models for intracellular signal transduction of neurons and glial cells, in addition to single glial cells, neuron-glia interactions, and selected examples of spiking neuronal networks. We also address the comparability of the simulation results with one another to comprehend if the studied models can be used to answer similar research questions. In addition to presenting the challenges in reproducibility and replicability of published results in computational neuroscience, we highlight the need for developing recommendations and good practices for publishing simulation tools and computational models. Model validation and flexible model description must be an integral part of the tool used to simulate and develop computational models. Constant improvement on experimental techniques and recording protocols leads to increasing knowledge about the biophysical mechanisms in neural systems. This poses new challenges for computational neuroscience: extended or completely new computational methods and models may be required. Careful evaluation and categorization of the existing models and tools provide a foundation for these future needs, for constructing multiscale models or extending the models to incorporate additional or more detailed biophysical mechanisms. Improving the quality of publications in computational neuroscience, enabling progressive building of advanced computational models and tools, can be achieved only through adopting publishing standards which underline replicability and reproducibility of research results.

Challenges in Reproducibility, Replicability, and Comparability of Computational Models and Tools for Neuronal and Glial Networks, Cells, and Subcellular Structures

PubMed Central

Manninen, Tiina; Aćimović, Jugoslava; Havela, Riikka; Teppola, Heidi; Linne, Marja-Leena

2018-01-01

The possibility to replicate and reproduce published research results is one of the biggest challenges in all areas of science. In computational neuroscience, there are thousands of models available. However, it is rarely possible to reimplement the models based on the information in the original publication, let alone rerun the models just because the model implementations have not been made publicly available. We evaluate and discuss the comparability of a versatile choice of simulation tools: tools for biochemical reactions and spiking neuronal networks, and relatively new tools for growth in cell cultures. The replicability and reproducibility issues are considered for computational models that are equally diverse, including the models for intracellular signal transduction of neurons and glial cells, in addition to single glial cells, neuron-glia interactions, and selected examples of spiking neuronal networks. We also address the comparability of the simulation results with one another to comprehend if the studied models can be used to answer similar research questions. In addition to presenting the challenges in reproducibility and replicability of published results in computational neuroscience, we highlight the need for developing recommendations and good practices for publishing simulation tools and computational models. Model validation and flexible model description must be an integral part of the tool used to simulate and develop computational models. Constant improvement on experimental techniques and recording protocols leads to increasing knowledge about the biophysical mechanisms in neural systems. This poses new challenges for computational neuroscience: extended or completely new computational methods and models may be required. Careful evaluation and categorization of the existing models and tools provide a foundation for these future needs, for constructing multiscale models or extending the models to incorporate additional or more detailed biophysical mechanisms. Improving the quality of publications in computational neuroscience, enabling progressive building of advanced computational models and tools, can be achieved only through adopting publishing standards which underline replicability and reproducibility of research results. PMID:29765315

Probe-level linear model fitting and mixture modeling results in high accuracy detection of differential gene expression.

PubMed

Lemieux, Sébastien

2006-08-25

The identification of differentially expressed genes (DEGs) from Affymetrix GeneChips arrays is currently done by first computing expression levels from the low-level probe intensities, then deriving significance by comparing these expression levels between conditions. The proposed PL-LM (Probe-Level Linear Model) method implements a linear model applied on the probe-level data to directly estimate the treatment effect. A finite mixture of Gaussian components is then used to identify DEGs using the coefficients estimated by the linear model. This approach can readily be applied to experimental design with or without replication. On a wholly defined dataset, the PL-LM method was able to identify 75% of the differentially expressed genes within 10% of false positives. This accuracy was achieved both using the three replicates per conditions available in the dataset and using only one replicate per condition. The method achieves, on this dataset, a higher accuracy than the best set of tools identified by the authors of the dataset, and does so using only one replicate per condition.

Kinetic models in industrial biotechnology - Improving cell factory performance.

PubMed

Almquist, Joachim; Cvijovic, Marija; Hatzimanikatis, Vassily; Nielsen, Jens; Jirstrand, Mats

2014-07-01

An increasing number of industrial bioprocesses capitalize on living cells by using them as cell factories that convert sugars into chemicals. These processes range from the production of bulk chemicals in yeasts and bacteria to the synthesis of therapeutic proteins in mammalian cell lines. One of the tools in the continuous search for improved performance of such production systems is the development and application of mathematical models. To be of value for industrial biotechnology, mathematical models should be able to assist in the rational design of cell factory properties or in the production processes in which they are utilized. Kinetic models are particularly suitable towards this end because they are capable of representing the complex biochemistry of cells in a more complete way compared to most other types of models. They can, at least in principle, be used to in detail understand, predict, and evaluate the effects of adding, removing, or modifying molecular components of a cell factory and for supporting the design of the bioreactor or fermentation process. However, several challenges still remain before kinetic modeling will reach the degree of maturity required for routine application in industry. Here we review the current status of kinetic cell factory modeling. Emphasis is on modeling methodology concepts, including model network structure, kinetic rate expressions, parameter estimation, optimization methods, identifiability analysis, model reduction, and model validation, but several applications of kinetic models for the improvement of cell factories are also discussed. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Combining experimental techniques with non-linear numerical models to assess the sorption of pesticides on soils

NASA Astrophysics Data System (ADS)

Magga, Zoi; Tzovolou, Dimitra N.; Theodoropoulou, Maria A.; Tsakiroglou, Christos D.

2012-03-01

The risk assessment of groundwater pollution by pesticides may be based on pesticide sorption and biodegradation kinetic parameters estimated with inverse modeling of datasets from either batch or continuous flow soil column experiments. In the present work, a chemical non-equilibrium and non-linear 2-site sorption model is incorporated into solute transport models to invert the datasets of batch and soil column experiments, and estimate the kinetic sorption parameters for two pesticides: N-phosphonomethyl glycine (glyphosate) and 2,4-dichlorophenoxy-acetic acid (2,4-D). When coupling the 2-site sorption model with the 2-region transport model, except of the kinetic sorption parameters, the soil column datasets enable us to estimate the mass-transfer coefficients associated with solute diffusion between mobile and immobile regions. In order to improve the reliability of models and kinetic parameter values, a stepwise strategy that combines batch and continuous flow tests with adequate true-to-the mechanism analytical of numerical models, and decouples the kinetics of purely reactive steps of sorption from physical mass-transfer processes is required.

Influence of mass transfer resistance on overall nitrate removal rate in upflow sludge bed reactors.

PubMed

Ting, Wen-Huei; Huang, Ju-Sheng

2006-09-01

A kinetic model with intrinsic reaction kinetics and a simplified model with apparent reaction kinetics for denitrification in upflow sludge bed (USB) reactors were proposed. USB-reactor performance data with and without sludge wasting were also obtained for model verification. An independent batch study showed that the apparent kinetic constants k' did not differ from the intrinsic k but the apparent Ks' was significantly larger than the intrinsic Ks suggesting that the intra-granule mass transfer resistance can be modeled by changes in Ks. Calculations of the overall effectiveness factor, Thiele modulus, and Biot number combined with parametric sensitivity analysis showed that the influence of internal mass transfer resistance on the overall nitrate removal rate in USB reactors is more significant than the external mass transfer resistance. The simulated residual nitrate concentrations using the simplified model were in good agreement with the experimental data; the simulated results using the simplified model were also close to those using the kinetic model. Accordingly, the simplified model adequately described the overall nitrate removal rate and can be used for process design.

Modeling of Non-isothermal Austenite Formation in Spring Steel

NASA Astrophysics Data System (ADS)

Huang, He; Wang, Baoyu; Tang, Xuefeng; Li, Junling

2017-12-01

The austenitization kinetics description of spring steel 60Si2CrA plays an important role in providing guidelines for industrial production. The dilatometric curves of 60Si2CrA steel were measured using a dilatometer DIL805A at heating rates of 0.3 K to 50 K/s (0.3 °C/s to 50 °C/s). Based on the dilatometric curves, a unified kinetics model using the internal state variable (ISV) method was derived to describe the non-isothermal austenitization kinetics of 60Si2CrA, and the abovementioned model models the incubation and transition periods. The material constants in the model were determined using a genetic algorithm-based optimization technique. Additionally, good agreement between predicted and experimental volume fractions of transformed austenite was obtained, indicating that the model is effective for describing the austenitization kinetics of 60Si2CrA steel. Compared with other modeling methods of austenitization kinetics, this model, which uses the ISV method, has some advantages, such as a simple formula and explicit physics meaning, and can be probably used in engineering practice.

Kinetic modeling of antimony(III) oxidation and sorption in soils.

PubMed

Cai, Yongbing; Mi, Yuting; Zhang, Hua

2016-10-05

Kinetic batch and saturated column experiments were performed to study the oxidation, adsorption and transport of Sb(III) in two soils with contrasting properties. Kinetic and column experiment results clearly demonstrated the extensive oxidation of Sb(III) in soils, and this can in return influence the adsorption and transport of Sb. Both sorption capacity and kinetic oxidation rate were much higher in calcareous Huanjiang soil than in acid red Yingtan soil. The results indicate that soil serve as a catalyst in promoting oxidation of Sb(III) even under anaerobic conditions. A PHREEQC model with kinetic formulations was developed to simulate the oxidation, sorption and transport of Sb(III) in soils. The model successfully described Sb(III) oxidation and sorption data in kinetic batch experiment. It was less successful in simulating the reactive transport of Sb(III) in soil columns. Additional processes such as colloid facilitated transport need to be quantified and considered in the model. Copyright © 2016 Elsevier B.V. All rights reserved.

Epigenetically-inherited centromere and neocentromere DNA replicates earliest in S-phase.

PubMed

Koren, Amnon; Tsai, Hung-Ji; Tirosh, Itay; Burrack, Laura S; Barkai, Naama; Berman, Judith

2010-08-19

Eukaryotic centromeres are maintained at specific chromosomal sites over many generations. In the budding yeast Saccharomyces cerevisiae, centromeres are genetic elements defined by a DNA sequence that is both necessary and sufficient for function; whereas, in most other eukaryotes, centromeres are maintained by poorly characterized epigenetic mechanisms in which DNA has a less definitive role. Here we use the pathogenic yeast Candida albicans as a model organism to study the DNA replication properties of centromeric DNA. By determining the genome-wide replication timing program of the C. albicans genome, we discovered that each centromere is associated with a replication origin that is the first to fire on its respective chromosome. Importantly, epigenetic formation of new ectopic centromeres (neocentromeres) was accompanied by shifts in replication timing, such that a neocentromere became the first to replicate and became associated with origin recognition complex (ORC) components. Furthermore, changing the level of the centromere-specific histone H3 isoform led to a concomitant change in levels of ORC association with centromere regions, further supporting the idea that centromere proteins determine origin activity. Finally, analysis of centromere-associated DNA revealed a replication-dependent sequence pattern characteristic of constitutively active replication origins. This strand-biased pattern is conserved, together with centromere position, among related strains and species, in a manner independent of primary DNA sequence. Thus, inheritance of centromere position is correlated with a constitutively active origin of replication that fires at a distinct early time. We suggest a model in which the distinct timing of DNA replication serves as an epigenetic mechanism for the inheritance of centromere position.

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

Seapan, M.; Crynes, B.L.; Dale, S.

The objectives of this study were to analyze alternate crudes kinetic hydrotreatment data in the literature, develop a mathematical model for interpretation of these data, develop an experimental procedure and apparatus to collect accurate kinetic data, and finally, to combine the model and experimental data to develop a general model which, with a few experimental parameters, could be used in design of future hydrotreatment processes. These objectives were to cover a four year program (1980 to 1984) and were subjective to sufficient funding. Only partial funding has been available thus far to cover activities for two years. A hydrotreatment datamore » base is developed which contains over 2000 citations, stored in a microcomputer. About 50% of these are reviewed, classified and can be identified by feedstock, catalyst, reactor type and other process characteristics. Tests of published hydrodesulfurization data indicate the problems with simple n-th order, global kinetic models, and point to the value of developing intrinsic reaction kinetic models to describe the reaction processes. A Langmuir-Hinshelwood kinetic model coupled with a plug flow reactor design equation has been developed and used for published data evaluation. An experimental system and procedure have been designed and constructed, which can be used for kinetic studies. 30 references, 4 tables.« less

Development of high performance particle in cell code for the exascale age

NASA Astrophysics Data System (ADS)

Lapenta, Giovanni; Amaya, Jorge; Gonzalez, Diego; Deep-Est H2020 Consortium Collaboration

2017-10-01

Magnetized plasmas are most effectively described by magneto-hydrodynamics, MHD, a fluid theory based on describing some fields defined in space: electromagnetic fields, density, velocity and temperature of the plasma. However, microphysics processes need kinetic theory, where statistical distributions of particles are governed by the Boltzmann equation. While fluid models are based on the ordinary space and time, kinetic models require a six dimensional space, called phase space, besides time. The two methods are not separated but rather interact to determine the system evolution. Arriving at a single self-consistent model is the goal of our research. We present a new approach developed with the goal of extending the reach of kinetic models to the fluid scales. Kinetic models are a higher order description and all fluid effects are included in them. However, the cost in terms of computing power is much higher and it has been so far prohibitively expensive to treat space weather events fully kinetically. We have now designed a new method capable of reducing that cost by several orders of magnitude making it possible for kinetic models to study macroscopic systems. H2020 Deep-EST consortium (European Commission).

Effects of replicative fitness on competing HIV strains.

PubMed

Chirove, Faraimunashe; Lungu, Edward M

2013-07-01

We develop an n-strain model to show the effects of replicative fitness of competing viral strains exerting selective density-dependant infective pressure on each other. A two strain model is used to illustrate the results. A perturbation technique and numerical simulations were used to establish the existence and stability of steady states. More than one infected steady states governed by the replicative fitness resulted from the model exhibiting either strain replacement or co-infection. We found that the presence of two or more HIV strains could result in a disease-free state that, in general, is not globally stable. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Release kinetics of volatile organic compounds from roasted and ground coffee: online measurements by PTR-MS and mathematical modeling.

PubMed

Mateus, Maria-L; Lindinger, Christian; Gumy, Jean-C; Liardon, Remy

2007-12-12

The present work shows the possibilities and limitations in modeling release kinetics of volatile organic compounds (VOCs) from roasted and ground coffee by applying physical and empirical models such as the diffusion and Weibull models. The release kinetics of VOCs were measured online by proton transfer reaction-mass spectrometry (PTR-MS). Compounds were identified by GC-MS, and the contribution of the individual compounds to different mass fragments was elucidated by GC/PTR-MS. Coffee samples roasted to different roasting degrees and ground to different particle sizes were studied under dry and wet stripping conditions. To investigate the accuracy of modeling the VOC release kinetics recorded using PTR-MS, online kinetics were compared with kinetics reconstituted from purge and trap samplings. Results showed that uncertainties in ion intensities due to the presence of isobaric species may prevent the development of a robust mathematical model. Of the 20 identified compounds, 5 were affected to a lower extent as their contribution to specific m/z intensity varied by <15% over the stripping time. The kinetics of these compounds were fitted using physical and statistical models, respectively, the diffusion and Weibull models, which helped to identify the underlying release mechanisms. For dry stripping, the diffusion model allowed a good representation of the release kinetics, whereas for wet stripping conditions, release patterns were very complex and almost specific for each compound analyzed. In the case of prewetted coffee, varying particle size (approximately 400-1200 microm) had no significant effect on the VOC release rate, whereas for dry coffee, the release was faster for smaller particles. The absence of particle size effect in wet coffee was attributed to the increase of opened porosity and compound diffusivity by solubilization and matrix relaxation. To conclude, the accurate modeling of VOC release kinetics from coffee allowed small variations in compound release to be discriminated. Furthermore, it evidenced the different aroma compositions that may be obtained depending on the time when VOCs are recovered.

The Influence of Hydroxylation on Maintaining CpG Methylation Patterns: A Hidden Markov Model Approach.

PubMed

Giehr, Pascal; Kyriakopoulos, Charalampos; Ficz, Gabriella; Wolf, Verena; Walter, Jörn

2016-05-01

DNA methylation and demethylation are opposing processes that when in balance create stable patterns of epigenetic memory. The control of DNA methylation pattern formation by replication dependent and independent demethylation processes has been suggested to be influenced by Tet mediated oxidation of 5mC. Several alternative mechanisms have been proposed suggesting that 5hmC influences either replication dependent maintenance of DNA methylation or replication independent processes of active demethylation. Using high resolution hairpin oxidative bisulfite sequencing data, we precisely determine the amount of 5mC and 5hmC and model the contribution of 5hmC to processes of demethylation in mouse ESCs. We develop an extended hidden Markov model capable of accurately describing the regional contribution of 5hmC to demethylation dynamics. Our analysis shows that 5hmC has a strong impact on replication dependent demethylation, mainly by impairing methylation maintenance.

Replicates in high dimensions, with applications to latent variable graphical models.

PubMed

Tan, Kean Ming; Ning, Yang; Witten, Daniela M; Liu, Han

2016-12-01

In classical statistics, much thought has been put into experimental design and data collection. In the high-dimensional setting, however, experimental design has been less of a focus. In this paper, we stress the importance of collecting multiple replicates for each subject in this setting. We consider learning the structure of a graphical model with latent variables, under the assumption that these variables take a constant value across replicates within each subject. By collecting multiple replicates for each subject, we are able to estimate the conditional dependence relationships among the observed variables given the latent variables. To test the null hypothesis of conditional independence between two observed variables, we propose a pairwise decorrelated score test. Theoretical guarantees are established for parameter estimation and for this test. We show that our proposal is able to estimate latent variable graphical models more accurately than some existing proposals, and apply the proposed method to a brain imaging dataset.

Integrated and spectral energetics of the GLAS general circulation model

NASA Technical Reports Server (NTRS)

Tenenbaum, J.

1982-01-01

Integrated and spectral error energetics of the GLAS General circulation model are compared with observations for periods in January 1975, 1976, and 1977. For two cases the model shows significant skill in predicting integrated energetics quantities out to two weeks, and for all three cases, the integrated monthly mean energetics show qualitative improvements over previous versions of the model in eddy kinetic energy and barotropic conversions. Fundamental difficulties remain with leakage of energy to the stratospheric level, particularly above strong initial jet streams associated in part with regions of steep terrain. The spectral error growth study represents the first comparison of general circulation model spectral energetics predictions with the corresponding observational spectra on a day by day basis. The major conclusion is that eddy kinetics energy can be correct while significant errors occur in the kinetic energy of wavenumber 3. Both the model and observations show evidence of single wavenumber dominance in eddy kinetic energy and the correlation of spectral kinetics and potential energy.

Energy Landscape and Pathways for Transitions between Watson-Crick and Hoogsteen Base Pairing in DNA.

PubMed

Chakraborty, Debayan; Wales, David J

2018-01-04

The recent discovery that Hoogsteen (HG) base pairs are widespread in DNA across diverse sequences and positional contexts could have important implications for understanding DNA replication and DNA-protein recognition. While evidence is emerging that the Hoogsteen conformation could be a thermodynamically accessible conformation of the DNA duplex and provide a means to expand its functionality, relatively little is known about the molecular mechanism underlying the Watson-Crick (WC) to HG transition. In this Perspective, we describe pathways and kinetics for this transition at an atomic level of detail, using the energy landscape perspective. We show that competition between the duplex conformations results in a double funnel landscape, which explains some recent experimental observations. The interconversion pathways feature a number of intermediates, with a variable number of WC and HG base pairs. The relatively slow kinetics, with possible deviations from two-state behavior, suggest that this conformational switch is likely to be a challenging target for both simulation and experiment.

Kinetics of the association of dengue virus capsid protein with the granular component of nucleolus.

PubMed

Tiwary, Ashish Kumar; Cecilia, D

2017-02-01

Dengue virus (DENV) replicates in the cytoplasm but translocation of the capsid protein (C) to the nucleoli of infected cells has been shown to facilitate virus multiplication for DENV-2. This study demonstrates that the nucleolar localization of C occurs with all four serotypes of DENV. The interaction of C with the nucleolus was found to be dynamic with a mobile fraction of 66% by FRAP. That the C shuttled between the nucleus and cytoplasm was suggested by FLIP and translation inhibition experiments. Colocalization with B23 indicated that DENV C targeted the granular component (GC) of the nucleolus. Presence of DENV C in the nucleolus affected the recovery kinetics of B23 in infected and transfected cells. Sub-nucleolar localization of DENV C of all serotypes to the GC, its mobility in and out of the nucleolus and its affect on the dynamics of B23 is being shown for the first time. Copyright © 2016 Elsevier Inc. All rights reserved.

Effect of electrolyte nature on kinetics of remazol yellow G removal by electrocoagulation

NASA Astrophysics Data System (ADS)

Rajabi, M.; Bagheri-Roochi, M.; Asghari, A.

2011-10-01

The present study describes an electrocoagulation process for the removal of remazol yellow G from dye solutions using Iron as the anode and Steel as the cathode. Pseudo-first-order, pseudo-second-order and intraparticle diffusion models were used to analyze the kinetic data obtained at different concentrations in different conditions. The adsorption kinetics was well described by the pseudo-second-order kinetic model.

KiMoSys: a web-based repository of experimental data for KInetic MOdels of biological SYStems

PubMed Central

2014-01-01

Background The kinetic modeling of biological systems is mainly composed of three steps that proceed iteratively: model building, simulation and analysis. In the first step, it is usually required to set initial metabolite concentrations, and to assign kinetic rate laws, along with estimating parameter values using kinetic data through optimization when these are not known. Although the rapid development of high-throughput methods has generated much omics data, experimentalists present only a summary of obtained results for publication, the experimental data files are not usually submitted to any public repository, or simply not available at all. In order to automatize as much as possible the steps of building kinetic models, there is a growing requirement in the systems biology community for easily exchanging data in combination with models, which represents the main motivation of KiMoSys development. Description KiMoSys is a user-friendly platform that includes a public data repository of published experimental data, containing concentration data of metabolites and enzymes and flux data. It was designed to ensure data management, storage and sharing for a wider systems biology community. This community repository offers a web-based interface and upload facility to turn available data into publicly accessible, centralized and structured-format data files. Moreover, it compiles and integrates available kinetic models associated with the data. KiMoSys also integrates some tools to facilitate the kinetic model construction process of large-scale metabolic networks, especially when the systems biologists perform computational research. Conclusions KiMoSys is a web-based system that integrates a public data and associated model(s) repository with computational tools, providing the systems biology community with a novel application facilitating data storage and sharing, thus supporting construction of ODE-based kinetic models and collaborative research projects. The web application implemented using Ruby on Rails framework is freely available for web access at http://kimosys.org, along with its full documentation. PMID:25115331

Comparison of the performance of tracer kinetic model-driven registration for dynamic contrast enhanced MRI using different models of contrast enhancement.

PubMed

Buonaccorsi, Giovanni A; Roberts, Caleb; Cheung, Sue; Watson, Yvonne; O'Connor, James P B; Davies, Karen; Jackson, Alan; Jayson, Gordon C; Parker, Geoff J M

2006-09-01

The quantitative analysis of dynamic contrast-enhanced (DCE) magnetic resonance imaging (MRI) data is subject to model fitting errors caused by motion during the time-series data acquisition. However, the time-varying features that occur as a result of contrast enhancement can confound motion correction techniques based on conventional registration similarity measures. We have therefore developed a heuristic, locally controlled tracer kinetic model-driven registration procedure, in which the model accounts for contrast enhancement, and applied it to the registration of abdominal DCE-MRI data at high temporal resolution. Using severely motion-corrupted data sets that had been excluded from analysis in a clinical trial of an antiangiogenic agent, we compared the results obtained when using different models to drive the tracer kinetic model-driven registration with those obtained when using a conventional registration against the time series mean image volume. Using tracer kinetic model-driven registration, it was possible to improve model fitting by reducing the sum of squared errors but the improvement was only realized when using a model that adequately described the features of the time series data. The registration against the time series mean significantly distorted the time series data, as did tracer kinetic model-driven registration using a simpler model of contrast enhancement. When an appropriate model is used, tracer kinetic model-driven registration influences motion-corrupted model fit parameter estimates and provides significant improvements in localization in three-dimensional parameter maps. This has positive implications for the use of quantitative DCE-MRI for example in clinical trials of antiangiogenic or antivascular agents.

The Artemisinin Derivative Artemisone Is a Potent Inhibitor of Human Cytomegalovirus Replication.

PubMed

Oiknine-Djian, E; Weisblum, Y; Panet, A; Wong, H N; Haynes, R K; Wolf, D G

2018-04-30

Human cytomegalovirus (HCMV) is a major cause of disease in immunocompromised individuals and the most common cause of congenital infection and neuro-sensorial disease. The expanding target populations for HCMV antiviral treatment along with the limitations of the currently available HCMV DNA polymerase inhibitors underscore the need for new antiviral agents with alternative modes of action. The anti-malarial artemisinin derivative artesunate was shown to inhibit HCMV in vitro , yet has demonstrated limited antiviral efficacy in vivo , prompting our search for more potent anti-HCMV artemisinin derivatives. Here we show that the innovative artemisinin derivative artemisone, which has been screened against malaria in human clinical studies, is a potent and non-cytotoxic inhibitor of HCMV. Artemisone exhibited an antiviral efficacy comparable to ganciclovir (EC 50 1.20 ± 0.46 μM) in human foreskin fibroblasts, with enhanced relative potency in lung fibroblasts and epithelial cells. Significantly, the antiviral efficacy of artemisone was consistently ≥10-fold superior to that of artesunate in all cells. Artemisone effectively inhibited both laboratory-adapted and low-passage clinical strains, as well as drug-resistant HCMV strains. By using quantitative viral kinetics and gene expression studies, we showed that artemisone is a reversible inhibitor, targeting an earlier phase of the viral replication cycle than ganciclovir. Importantly, artemisone most effectively inhibited HCMV infection ex vivo in a clinically-relevant multicellular model of integral human placental tissues maintained in organ culture. Our promising findings encourage preclinical and clinical studies of artemisone as a new inhibitor against HCMV. Copyright © 2018 American Society for Microbiology.

Rapid spectrofluorometric screening of poly-hydroxyalkanoate-producing bacteria from microbial mats.

PubMed

Berlanga, Mercedes; Montero, M T; Fernández-Borrell, Jordi; Guerrero, Ricardo

2006-06-01

Microbial mat ecosystems are characterized by both seasonal and diel fluctuations in several physicochemical variables, so that resident microorganisms must frequently adapt to the changing conditions of their environment. It has been pointed out that, under stress conditions, bacterial cells with higher contents of poly-hydroxyalkanoates (PHA) survive longer than those with lower PHA content. In the present study, PHA-producing strains from Ebro Delta microbial mats were selected using the Nile red dying technique and the relative accumulation of PHA was monitored during further laboratory cultivation. The number of heterotrophic isolates in trypticase soy agar (TSA) was ca. 107 colony-forming units/g microbial mat. Of these, 100 randomly chosen colonies were replicated on mineral salt agar limited in nitrogen, and Nile red was added to the medium to detect PHA. Orange fluorescence, produced upon binding of the dye to polymer granules in the cell, was detected in approximately 10% of the replicated heterotrophic isolates. The kinetics of PHA accumulation in Pseudomonas putida, and P. oleovorans were compared with those of several of the environmental isolates spectrofluorometry. PHA accumulation, measured as relative fluorescence intensity, resulted in a steady-state concentration after 48 h of incubation in all strains assayed. At 72 h, the maximum fluorescence intensity of each strain incubated with glucose and fructose was usually similar. MAT-28 strain accumulated more PHA than the other isolates. The results show that data obtained from environmental isolates can highly improve studies based on modeling-simulation programs, and that microbial mats constitute an excellent source for the isolation of PHA-producing strains with industrial applications.

Diversifying the STEM Pipeline: The Model Replication Institutions Program

ERIC Educational Resources Information Center

Cullinane, Jenna

2009-01-01

In 2006, the National Science Foundation (NSF) began funding the Model Replication Institutions (MRI) program, which sought to improve the quality, availability, and diversity of science, technology, engineering, and mathematics (STEM) education. Faced with pressing national priorities in the STEM fields and chronic gaps in postsecondary…

Polarization and Compressibility of Oblique Kinetic Alfven Waves

NASA Technical Reports Server (NTRS)

Hunana, Peter; Goldstein, M. L.; Passot, T.; Sulem, P. L.; Laveder, D.; Zank, G. P.

2012-01-01

Even though solar wind, as a collisionless plasma, is properly described by the kineticMaxwell-Vlasov description, it can be argued that much of our understanding of solar wind observational data comes from an interpretation and numerical modeling which is based on a fluid description of magnetohydrodynamics. In recent years, there has been a significant interest in better understanding the importance of kinetic effects, i.e. the differences between the kinetic and usual fluid descriptions. Here we concentrate on physical properties of oblique kinetic Alfvn waves (KAWs), which are often recognized as one of the key ingredients in the solar wind turbulence cascade. We use three different fluid models with various degrees of complexity and calculate polarization and magnetic compressibility of oblique KAWs (propagation angle q = 88), which we compare to solutions derived from linear kinetic theory. We explore a wide range of possible proton plasma b = [0.1,10.0] and a wide range of length scales krL = [0.001,10.0]. It is shown that the classical isotropic two-fluid model is very compressible in comparison with kinetic theory and that the largest discrepancy occurs at scales larger than the proton gyroscale. We also show that the two-fluid model contains a large error in the polarization of electric field, even at scales krL 1. Furthermore, to understand these discrepancies between the two-fluid model and the kinetic theory, we employ two versions of the Landau fluid model that incorporate linear low-frequency kinetic effects such as Landau damping and finite Larmor radius (FLR) corrections into the fluid description. It is shown that Landau damping significantly reduces the magnetic compressibility and that FLR corrections (i.e. nongyrotropic contributions) are required to correctly capture the polarization.We also show that, in addition to Landau damping, FLR corrections are necessary to accurately describe the damping rate of KAWs. We conclude that kinetic effects are important even at scales which are significantly larger than the proton gyroscale krL 1.

Evaluation of γ-Induced Apoptosis in Human Peripheral Blood Lymphocytes

NASA Astrophysics Data System (ADS)

Baranova, Elena; Boreyko, Alla; Ravnachka, Ivanka; Saveleva, Maria

2010-01-01

Several experiments have been performed to study regularities in the induction of apoptotic cells in human lymphocytes by 60Co γ-rays at different times after irradiation. Apoptosis induction by 60Co γ-rays in human lymphocytes in different cell cycle phases (G0, S, G1, and G2) has been studied. The maximal apoptosis output in lymphocyte cells was observed in the S phase. Modifying effect of replicative and reparative DNA synthesis inhibitors—1- β -D-arabinofuranosylcytosine (Ara-C) and hydroxyurea (Hu)—on the kinetics of 60Co γ-rays induced apoptosis in human lymphocytes has been studied.

Direct Estimation of Kinetic Parametric Images for Dynamic PET

PubMed Central

Wang, Guobao; Qi, Jinyi

2013-01-01

Dynamic positron emission tomography (PET) can monitor spatiotemporal distribution of radiotracer in vivo. The spatiotemporal information can be used to estimate parametric images of radiotracer kinetics that are of physiological and biochemical interests. Direct estimation of parametric images from raw projection data allows accurate noise modeling and has been shown to offer better image quality than conventional indirect methods, which reconstruct a sequence of PET images first and then perform tracer kinetic modeling pixel-by-pixel. Direct reconstruction of parametric images has gained increasing interests with the advances in computing hardware. Many direct reconstruction algorithms have been developed for different kinetic models. In this paper we review the recent progress in the development of direct reconstruction algorithms for parametric image estimation. Algorithms for linear and nonlinear kinetic models are described and their properties are discussed. PMID:24396500

A study of the kinetics of isothermal nicotine desorption from silicon dioxide

NASA Astrophysics Data System (ADS)

Adnadjevic, Borivoj; Lazarevic, Natasa; Jovanovic, Jelena

2010-12-01

The isothermal kinetics of nicotine desorption from silicon dioxide (SiO 2) was investigated. The isothermal thermogravimetric curves of nicotine at temperatures of 115 °C, 130 °C and 152 °C were recorded. The kinetic parameters ( Ea, ln A) of desorption of nicotine were calculated using various methods (stationary point, model constants and differential isoconversion method). By applying the "model-fitting" method, it was found that the kinetic model of nicotine desorption from silicon dioxide was a phase boundary controlled reaction (contracting volume). The values of the kinetic parameters, Ea,α and ln Aα, complexly change with changing degree of desorption and a compensation effect exists. A new mechanism of activation for the desorption of the absorbed molecules of nicotine was suggested in agreement with model of selective energy transfer.

Oxygen Diffusion and Reaction Kinetics in Continuous Fiber Ceramic Matrix Composites

NASA Technical Reports Server (NTRS)

Halbig, Michael C.; Eckel, Andrew J.; Cawley, James D.

1999-01-01

Previous stressed oxidation tests of C/SiC composites at elevated temperatures (350 C to 1500 C) and sustained stresses (69 MPa and 172 MPa) have led to the development of a finite difference cracked matrix model. The times to failure in the samples suggest oxidation occurred in two kinetic regimes defined by the rate controlling mechanisms (i.e. diffusion controlled and reaction controlled kinetics). Microstructural analysis revealed preferential oxidation along as-fabricated, matrix microcracks and also suggested two regimes of oxidation kinetics dependent on the oxidation temperature. Based on experimental results, observation, and theory, a finite difference model was developed. The model simulates the diffusion of oxygen into a matrix crack bridged by carbon fibers. The model facilitates the study of the relative importance of temperature, the reaction rate constant, and the diffusion coefficient on the overall oxidation kinetics.

Establishing a coherent and replicable measurement model of the Edinburgh Postnatal Depression Scale.

PubMed

Martin, Colin R; Redshaw, Maggie

2018-06-01

The 10-item Edinburgh Postnatal Depression Scale (EPDS) is an established screening tool for postnatal depression. Inconsistent findings in factor structure and replication difficulties have limited the scope of development of the measure as a multi-dimensional tool. The current investigation sought to robustly determine the underlying factor structure of the EPDS and the replicability and stability of the most plausible model identified. A between-subjects design was used. EPDS data were collected postpartum from two independent cohorts using identical data capture methods. Datasets were examined with confirmatory factor analysis, model invariance testing and systematic evaluation of relational and internal aspects of the measure. Participants were two samples of postpartum women in England assessed at three months (n = 245) and six months (n = 217). The findings showed a three-factor seven-item model of the EPDS offered an excellent fit to the data, and was observed to be replicable in both datasets and invariant as a function of time point of assessment. Some EPDS sub-scale scores were significantly higher at six months. The EPDS is multi-dimensional and a robust measurement model comprises three factors that are replicable. The potential utility of the sub-scale components identified requires further research to identify a role in contemporary screening practice. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Computational Evaluation of the Strict Master and Random Template Models of Endogenous Retrovirus Evolution

PubMed Central

Nascimento, Fabrícia F.; Rodrigo, Allen G.

2016-01-01

Transposable elements (TEs) are DNA sequences that are able to replicate and move within and between host genomes. Their mechanism of replication is also shared with endogenous retroviruses (ERVs), which are also a type of TE that represent an ancient retroviral infection within animal genomes. Two models have been proposed to explain TE proliferation in host genomes: the strict master model (SMM), and the random template (or transposon) model (TM). In SMM only a single copy of a given TE lineage is able to replicate, and all other genomic copies of TEs are derived from that master copy. In TM, any element of a given family is able to replicate in the host genome. In this paper, we simulated ERV phylogenetic trees under variations of SMM and TM. To test whether current phylogenetic programs can recover the simulated ERV phylogenies, DNA sequence alignments were simulated and maximum likelihood trees were reconstructed and compared to the simulated phylogenies. Results indicate that visual inspection of phylogenetic trees alone can be misleading. However, if a set of statistical summaries is calculated, we are able to distinguish between models with high accuracy by using a data mining algorithm that we introduce here. We also demonstrate the use of our data mining algorithm with empirical data for the porcine endogenous retrovirus (PERV), an ERV that is able to replicate in human and pig cells in vitro. PMID:27649303

Examination and evaluation of the use of screen heaters for the measurement of the high temperature pyrolysis kinetics of polyethene and polypropene

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

Westerhout, R.W.J.; Balk, R.H.P.; Meijer, R.

1997-08-01

A screen heater with a gas sweep was developed and applied to study the pyrolysis kinetics of low density polyethene (LDPE) and polypropene (PP) at temperatures ranging from 450 to 530 C. The aim of this study was to examine the applicability of screen heaters to measure these kinetics. On-line measurement of the rate of volatiles formation using a hydrocarbon analyzer was applied to enable the determination of the conversion rate over the entire conversion range on the basis of a single experiment. Another important feature of the screen heater used in this study is the possibility to measure pyrolysismore » kinetics under nearly isothermal conditions. The kinetic constants for LDPE and PP pyrolysis were determined, using a first order model to describe the conversion rate in the 70--90% conversion range and the random chain dissociation model for the entire conversion range. In addition to the experimental work two single particle models have been developed which both incorporate a mass and a (coupled) enthalpy balance, which were used to assess the influence of internal and external heat transfer processes on the pyrolysis process. The first model assumes a variable density and constant volume during the pyrolysis process, whereas the second model assumes a constant density and a variable volume. An important feature of these models is that they can accommodate kinetic models for which no analytical representation of the pyrolysis kinetics is available.« less

Metabolically Coupled Replicator Systems: Overview of an RNA-world model concept of prebiotic evolution on mineral surfaces.

PubMed

Czárán, Tamás; Könnyű, Balázs; Szathmáry, Eörs

2015-09-21

Metabolically Coupled Replicator Systems (MCRS) are a family of models implementing a simple, physico-chemically and ecologically feasible scenario for the first steps of chemical evolution towards life. Evolution in an abiotically produced RNA-population sets in as soon as any one of the RNA molecules become autocatalytic by engaging in template directed self-replication from activated monomers, and starts increasing exponentially. Competition for the finite external supply of monomers ignites selection favouring RNA molecules with catalytic activity helping self-replication by any possible means. One way of providing such autocatalytic help is to become a replicase ribozyme. An additional way is through increasing monomer supply by contributing to monomer synthesis from external resources, i.e., by evolving metabolic enzyme activity. Retroevolution may build up an increasingly autotrophic, cooperating community of metabolic ribozymes running an increasingly complicated and ever more efficient metabolism. Maintaining such a cooperating community of metabolic replicators raises two serious ecological problems: one is keeping the system coexistent in spite of the different replicabilities of the cooperating replicators; the other is constraining parasitism, i.e., keeping "cheaters" in check. Surface-bound MCRS provide an automatic solution to both problems: coexistence and parasite resistance are the consequences of assuming the local nature of metabolic interactions. In this review we present an overview of results published in previous articles, showing that these effects are, indeed, robust in different MCRS implementations, by considering different environmental setups and realistic chemical details in a few different models. We argue that the MCRS model framework naturally offers a suitable starting point for the future modelling of membrane evolution and extending the theory to cover the emergence of the first protocell in a self-consistent manner. The coevolution of metabolic, genetic and membrane functions is hypothesized to follow the progressive sequestration scenario, the conceptual blueprint for the earliest steps of protocell evolution. Copyright © 2015 Elsevier Ltd. All rights reserved.

Origin-Dependent Inverted-Repeat Amplification: Tests of a Model for Inverted DNA Amplification.

PubMed

Brewer, Bonita J; Payen, Celia; Di Rienzi, Sara C; Higgins, Megan M; Ong, Giang; Dunham, Maitreya J; Raghuraman, M K

2015-12-01

DNA replication errors are a major driver of evolution--from single nucleotide polymorphisms to large-scale copy number variations (CNVs). Here we test a specific replication-based model to explain the generation of interstitial, inverted triplications. While no genetic information is lost, the novel inversion junctions and increased copy number of the included sequences create the potential for adaptive phenotypes. The model--Origin-Dependent Inverted-Repeat Amplification (ODIRA)-proposes that a replication error at pre-existing short, interrupted, inverted repeats in genomic sequences generates an extrachromosomal, inverted dimeric, autonomously replicating intermediate; subsequent genomic integration of the dimer yields this class of CNV without loss of distal chromosomal sequences. We used a combination of in vitro and in vivo approaches to test the feasibility of the proposed replication error and its downstream consequences on chromosome structure in the yeast Saccharomyces cerevisiae. We show that the proposed replication error-the ligation of leading and lagging nascent strands to create "closed" forks-can occur in vitro at short, interrupted inverted repeats. The removal of molecules with two closed forks results in a hairpin-capped linear duplex that we show replicates in vivo to create an inverted, dimeric plasmid that subsequently integrates into the genome by homologous recombination, creating an inverted triplication. While other models have been proposed to explain inverted triplications and their derivatives, our model can also explain the generation of human, de novo, inverted amplicons that have a 2:1 mixture of sequences from both homologues of a single parent--a feature readily explained by a plasmid intermediate that arises from one homologue and integrates into the other homologue prior to meiosis. Our tests of key features of ODIRA lend support to this mechanism and suggest further avenues of enquiry to unravel the origins of interstitial, inverted CNVs pivotal in human health and evolution.

A Transient Kinetic Approach to Investigate Nucleoside Inhibitors of Mitochondrial DNA polymerase γ

PubMed Central

Anderson, Karen S.

2010-01-01

Nucleoside analogs play an essential role in treating human immunodeficiency virus (HIV) infection since the beginning of the AIDS epidemic and work by inhibition of HIV-1 reverse transcriptase (RT), a viral polymerase essential for DNA replication. Today, over 90% of all regimens for HIV treatment contain at least one nucleoside. Long-term use of nucleoside analogs has been associated with adverse effects including mitochondrial toxicity due to inhibition of the mitochondrial polymerase, DNA polymerase gamma (mtDNA pol ©). In this review, we describe our efforts to delineate the molecular mechanism of nucleoside inhibition of HIV-1 RT and mtDNA pol © based upon a transient kinetic approach using rapid chemical quench methodology. Using transient kinetic methods, the maximum rate of polymerization (kpol), the dissociation constant for the ground state binding (Kd), and the incorporation efficiency (kpol/Kd) can be determined for the nucleoside analogs and their natural substrates. This analysis allowed us to develop an understanding of the structure activity relationships that allow correlation between the structural and stereochemical features of the nucleoside analog drugs with their mechanistic behavior toward the viral polymerase, RT, and the host cell polymerase, mtDNA pol γ. An in-depth understanding of the mechanisms of inhibition of these enzymes is imperative in overcoming problems associated with toxicity. PMID:20573564

Comparison of skating kinetics and kinematics on ice and on a synthetic surface.

PubMed

Stidwill, T J; Pearsall, David; Turcotte, Rene

2010-03-01

The recent popularization and technological improvements of synthetic or artificial ice surfaces provide an attractive alternative to real ice in venues where the latter is impractical to install. Potentially, synthetic ice (SI) may be installed in controlled laboratory settings to permit detailed biomechanical analysis of skating manoeuvres. Unknown, however, is the extent to which skating on SI replicates skating on traditional ice (ICE). Hence, the purpose of this study was to compare kinetic and kinematic forward skating parameters between SI and ICE surfaces. With 11 male hockey players, a portable strain gauge system adhered to the outside of the skate blade holder was used to measure skate propulsive force synchronized with electrogoniometers for tracking dynamic knee and ankle movements during forward skating acceleration. In general, the kinetic and kinematic variables investigated in this study showed minimal differences between the two surfaces (P > 0.06), and no individual variable differences were identified between the two surfaces (P > or = 0.1) with the exception of greater knee extension on SI than ICE (15.2 degrees to 11.0 degrees; P < or = 0.05). Overall, SI surfaces permit comparable mechanics for on-ice forward skating, and thus offer the potential for valid analogous conditions for in-lab testing and training.

A KINETIC MODEL FOR CELL DENSITY DEPENDENT BACTERIAL TRANSPORT IN POROUS MEDIA

EPA Science Inventory

A kinetic transport model with the ability to account for variations in cell density of the aqueous and solid phases was developed for bacteria in porous media. Sorption kinetics in the advective-dispersive-sorptive equation was described by assuming that adsorption was proportio...

Engineering 3D Models of Tumors and Bone to Understand Tumor-Induced Bone Disease and Improve Treatments.

PubMed

Kwakwa, Kristin A; Vanderburgh, Joseph P; Guelcher, Scott A; Sterling, Julie A

2017-08-01

Bone is a structurally unique microenvironment that presents many challenges for the development of 3D models for studying bone physiology and diseases, including cancer. As researchers continue to investigate the interactions within the bone microenvironment, the development of 3D models of bone has become critical. 3D models have been developed that replicate some properties of bone, but have not fully reproduced the complex structural and cellular composition of the bone microenvironment. This review will discuss 3D models including polyurethane, silk, and collagen scaffolds that have been developed to study tumor-induced bone disease. In addition, we discuss 3D printing techniques used to better replicate the structure of bone. 3D models that better replicate the bone microenvironment will help researchers better understand the dynamic interactions between tumors and the bone microenvironment, ultimately leading to better models for testing therapeutics and predicting patient outcomes.

Nuclear DNA Replication in Trypanosomatids: There Are No Easy Methods for Solving Difficult Problems.

PubMed

da Silva, Marcelo S; Pavani, Raphael S; Damasceno, Jeziel D; Marques, Catarina A; McCulloch, Richard; Tosi, Luiz Ricardo Orsini; Elias, Maria Carolina

2017-11-01

In trypanosomatids, etiological agents of devastating diseases, replication is robust and finely controlled to maintain genome stability and function in stressful environments. However, these parasites encode several replication protein components and complexes that show potentially variant composition compared with model eukaryotes. This review focuses on the advances made in recent years regarding the differences and peculiarities of the replication machinery in trypanosomatids, including how such divergence might affect DNA replication dynamics and the replication stress response. Comparing the DNA replication machinery and processes of parasites and their hosts may provide a foundation for the identification of targets that can be used in the development of chemotherapies to assist in the eradication of diseases caused by these pathogens. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Experimentally Guided Models Reveal Replication Principles that Shape the Mutation Distribution of RNA Viruses

DTIC Science & Technology

2015-01-30

intracel- lular replication. Two classic replication modes have been described for single-stranded RNA viruses: the ‘stamping machine’ mode ( Stent ...Journal of Theoretical Biology 218:309–321. doi: 10.1006/jtbi.2002.3078. Stent GS. 1963. Molecular Biology of Bacterial Viruses. San Francisco, Calif: W H

Charter School Replication. Policy Guide

ERIC Educational Resources Information Center

Rhim, Lauren Morando

2009-01-01

"Replication" is the practice of a single charter school board or management organization opening several more schools that are each based on the same school model. The most rapid strategy to increase the number of new high-quality charter schools available to children is to encourage the replication of existing quality schools. This policy guide…

Mechanistic Kinetic Modeling of Thiol-Michael Addition Photopolymerizations via Photocaged "Superbase" Generators: An Analytical Approach.

PubMed

Claudino, Mauro; Zhang, Xinpeng; Alim, Marvin D; Podgórski, Maciej; Bowman, Christopher N

2016-11-08

A kinetic mechanism and the accompanying mathematical framework are presented for base-mediated thiol-Michael photopolymerization kinetics involving a photobase generator. Here, model kinetic predictions demonstrate excellent agreement with a representative experimental system composed of 2-(2-nitrophenyl)propyloxycarbonyl-1,1,3,3-tetramethylguanidine (NPPOC-TMG) as a photobase generator that is used to initiate thiol-vinyl sulfone Michael addition reactions and polymerizations. Modeling equations derived from a basic mechanistic scheme indicate overall polymerization rates that follow a pseudo-first-order kinetic process in the base and coreactant concentrations, controlled by the ratio of the propagation to chain-transfer kinetic parameters ( k p / k CT ) which is dictated by the rate-limiting step and controls the time necessary to reach gelation. Gelation occurs earlier as the k p / k CT ratio reaches a critical value, wherefrom gel times become nearly independent of k p / k CT . The theoretical approach allowed determining the effect of induction time on the reaction kinetics due to initial acid-base neutralization for the photogenerated base caused by the presence of protic contaminants. Such inhibition kinetics may be challenging for reaction systems that require high curing rates but are relevant for chemical systems that need to remain kinetically dormant until activated although at the ultimate cost of lower polymerization rates. The pure step-growth character of this living polymerization and the exhibited kinetics provide unique potential for extended dark-cure reactions and uniform material properties. The general kinetic model is applicable to photobase initiators where photolysis follows a unimolecular cleavage process releasing a strong base catalyst without cogeneration of intermediate radical species.

Computer-Aided Construction of Chemical Kinetic Models

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

Green, William H.

2014-12-31

The combustion chemistry of even simple fuels can be extremely complex, involving hundreds or thousands of kinetically significant species. The most reasonable way to deal with this complexity is to use a computer not only to numerically solve the kinetic model, but also to construct the kinetic model in the first place. Because these large models contain so many numerical parameters (e.g. rate coefficients, thermochemistry) one never has sufficient data to uniquely determine them all experimentally. Instead one must work in “predictive” mode, using theoretical rather than experimental values for many of the numbers in the model, and as appropriatemore » refining the most sensitive numbers through experiments. Predictive chemical kinetics is exactly what is needed for computer-aided design of combustion systems based on proposed alternative fuels, particularly for early assessment of the value and viability of proposed new fuels before those fuels are commercially available. This project was aimed at making accurate predictive chemical kinetics practical; this is a challenging goal which requires a range of science advances. The project spanned a wide range from quantum chemical calculations on individual molecules and elementary-step reactions, through the development of improved rate/thermo calculation procedures, the creation of algorithms and software for constructing and solving kinetic simulations, the invention of methods for model-reduction while maintaining error control, and finally comparisons with experiment. Many of the parameters in the models were derived from quantum chemistry calculations, and the models were compared with experimental data measured in our lab or in collaboration with others.« less

The Human Cytomegalovirus UL51 Protein Is Essential for Viral Genome Cleavage-Packaging and Interacts with the Terminase Subunits pUL56 and pUL89

PubMed Central

Borst, Eva Maria; Kleine-Albers, Jennifer; Gabaev, Ildar; Babić, Marina; Wagner, Karen; Binz, Anne; Degenhardt, Inga; Kalesse, Markus; Jonjić, Stipan; Bauerfeind, Rudolf

2013-01-01

Cleavage of human cytomegalovirus (HCMV) genomes as well as their packaging into capsids is an enzymatic process mediated by viral proteins and therefore a promising target for antiviral therapy. The HCMV proteins pUL56 and pUL89 form the terminase and play a central role in cleavage-packaging, but several additional viral proteins, including pUL51, had been suggested to contribute to this process, although they remain largely uncharacterized. To study the function of pUL51 in infected cells, we constructed HCMV mutants encoding epitope-tagged versions of pUL51 and used a conditionally replicating virus (HCMV-UL51-ddFKBP), in which pUL51 levels could be regulated by a synthetic ligand. In cells infected with HCMV-UL51-ddFKBP, viral DNA replication was not affected when pUL51 was knocked down. However, no unit-length genomes and no DNA-filled C capsids were found, indicating that cleavage of concatemeric HCMV DNA and genome packaging into capsids did not occur in the absence of pUL51. pUL51 was expressed mainly with late kinetics and was targeted to nuclear replication compartments, where it colocalized with pUL56 and pUL89. Upon pUL51 knockdown, pUL56 and pUL89 were no longer detectable in replication compartments, suggesting that pUL51 is needed for their correct subnuclear localization. Moreover, pUL51 was found in a complex with the terminase subunits pUL56 and pUL89. Our data provide evidence that pUL51 is crucial for HCMV genome cleavage-packaging and may represent a third component of the viral terminase complex. Interference with the interactions between the terminase subunits by antiviral drugs could be a strategy to disrupt the HCMV replication cycle. PMID:23175377

Attenuated and Replication-Competent Vaccinia Virus Strains M65 and M101 with Distinct Biology and Immunogenicity as Potential Vaccine Candidates against Pathogens

PubMed Central

Sánchez-Sampedro, Lucas; Gómez, Carmen Elena; Mejías-Pérez, Ernesto; Pérez-Jiménez, Eva; Oliveros, Juan Carlos

2013-01-01

Replication-competent poxvirus vectors with an attenuation phenotype and with a high immunogenic capacity of the foreign expressed antigen are being pursued as novel vaccine vectors against different pathogens. In this investigation, we have examined the replication and immunogenic characteristics of two vaccinia virus (VACV) mutants, M65 and M101. These mutants were generated after 65 and 101 serial passages of persistently infected Friend erythroleukemia (FEL) cells. In cultured cells of different origins, the mutants are replication competent and have growth kinetics similar to or slightly reduced in comparison with those of the parental Western Reserve (WR) virus strain. In normal and immune-suppressed infected mice, the mutants showed different levels of attenuation and pathogenicity in comparison with WR and modified vaccinia Ankara (MVA) strains. Wide genome analysis after deep sequencing revealed selected genomic deletions and mutations in a number of viral open reading frames (ORFs). Mice immunized in a DNA prime/mutant boost regimen with viral vectors expressing the LACK (Leishmania homologue for receptors of activated C kinase) antigen of Leishmania infantum showed protection or a delay in the onset of cutaneous leishmaniasis. Protection was similar to that triggered by MVA-LACK. In immunized mice, both polyfunctional CD4+ and CD8+ T cells with an effector memory phenotype were activated by the two mutants, but the DNA-LACK/M65-LACK protocol preferentially induced CD4+ whereas DNA-LACK/M101-LACK preferentially induced CD8+ T cell responses. Altogether, our findings showed the adaptive changes of the WR genome during long-term virus-host cell interaction and how the replication competency of M65 and M101 mutants confers distinct biological properties and immunogenicity in mice compared to those of the MVA strain. These mutants could have applicability for understanding VACV biology and as potential vaccine vectors against pathogens and tumors. PMID:23596295

Diversifying the STEM Pipeline: Recommendations from the Model Replication Institutions Program

ERIC Educational Resources Information Center

Institute for Higher Education Policy, 2010

2010-01-01

Launched in 2006 to address issues of national competitiveness and equity in science, technology, engineering, and mathematics (STEM) fields, the National Science Foundation-funded Model Replication Institutions (MRI) program sought to improve the quality, availability, and diversity of STEM education. The project offered technical assistance to…

Modeling Ebola Virus Genome Replication and Transcription with Minigenome Systems.

PubMed

Cressey, Tessa; Brauburger, Kristina; Mühlberger, Elke

2017-01-01

In this chapter, we describe the minigenome system for Ebola virus (EBOV), which reconstitutes EBOV polymerase activity in cells and can be used to model viral genome replication and transcription. This protocol comprises all steps including cell culture, plasmid preparation, transfection, and luciferase reporter assay readout.

Model Development and Replication: Introducing and Sustaining Change

ERIC Educational Resources Information Center

Garland, Corinne W.

2005-01-01

This article describes the three-decade experience in model development and replication and in introducing and sustaining organization change of Child Development Resources (CDR), of Williamsburg, Virginia. CDR provides an integrated system of services for children with disabilities and developmental delays as well as for children who are at risk.…

Breakdown parameter for kinetic modeling of multiscale gas flows.

PubMed

Meng, Jianping; Dongari, Nishanth; Reese, Jason M; Zhang, Yonghao

2014-06-01

Multiscale methods built purely on the kinetic theory of gases provide information about the molecular velocity distribution function. It is therefore both important and feasible to establish new breakdown parameters for assessing the appropriateness of a fluid description at the continuum level by utilizing kinetic information rather than macroscopic flow quantities alone. We propose a new kinetic criterion to indirectly assess the errors introduced by a continuum-level description of the gas flow. The analysis, which includes numerical demonstrations, focuses on the validity of the Navier-Stokes-Fourier equations and corresponding kinetic models and reveals that the new criterion can consistently indicate the validity of continuum-level modeling in both low-speed and high-speed flows at different Knudsen numbers.

Hybrid simulations of magnetic reconnection with kinetic ions and fluid electron pressure anisotropy

DOE PAGES

Le, A.; Daughton, W.; Karimabadi, H.; ...

2016-03-16

We present the first hybrid simulations with kinetic ions and recently developed equations of state for the electron fluid appropriate for reconnection with a guide field. The equations of state account for the main anisotropy of the electron pressure tensor.Magnetic reconnection is studied in two systems, an initially force-free current sheet and a Harris sheet. The hybrid model with the equations of state is compared to two other models, hybrid simulations with isothermal electrons and fully kinetic simulations. Including the anisotropicequations of state in the hybrid model provides a better match to the fully kinetic model. In agreement with fullymore » kinetic results, the main feature captured is the formation of an electron current sheet that extends several ion inertial lengths. This electron current sheet modifies the Hall magnetic field structure near the X-line, and it is not observed in the standard hybrid model with isotropic electrons. The saturated reconnection rate in this regime nevertheless remains similar in all three models. Here, implications for global modeling are discussed.« less

Model of Inclusion Evolution During Calcium Treatment in the Ladle Furnace

NASA Astrophysics Data System (ADS)

Tabatabaei, Yousef; Coley, Kenneth S.; Irons, Gordon A.; Sun, Stanley

2018-04-01

Calcium treatment of steel is typically employed to modify alumina inclusions to liquid calcium aluminates. However, injected calcium also reacts with the dissolved sulfur to form calcium sulfide. The current work aims to develop a kinetic model for the evolution of oxide and sulfide inclusions in Al-killed alloyed steel during Ca treatment in the ladle refining process. The model considers dissolution of the calcium from the calcium bubbles into the steel and reduction of calcium oxide in the slag to dissolved calcium. A steel-inclusion kinetic model is used for mass transfer to the inclusion interface and diffusion within the calcium aluminate phases formed on the inclusion. The inclusion-steel kinetic model is then coupled with a previously developed steel-slag kinetic model. The coupled inclusion-steel-slag kinetic model is applied to the chemical composition changes in molten steel, slag, and evolution of inclusions in the ladle. The result of calculations is found to agree well with an industrial heat for species in the steel as well as inclusions during Ca treatment.

Nanobacteria: Fact or Fiction? Characteristics, Detection and Medical Importance of Novel Self-Replicating, Calcifying Nanoparticles

NASA Technical Reports Server (NTRS)

Ciftcioglu, Neva; Mckay, David S.; Mathew, Grace; Kajander, E. Olavi

2006-01-01

There is some debate in microbiology as to whether Nanobacteria (NB) are alive. This paper reviews some aspects of NB. In summary, Nanobacteria is a perfect model for studying biogenic mineralization/calcification because NB a) are self-replicating particles and have less complicated metabolic pathways b) accumulate calcium and phosphate under physiological conditions, c)produce a calcium phosphate mineral similar to bone, d) exist in physical conditions (pH, gravity, temperature, etc) that are easy to manipulate, and which can be replicated for the physiological model.

Eukaryotic Replicative Helicase Subunit Interaction with DNA and Its Role in DNA Replication

PubMed Central

Martinez, Matthew P.; Wacker, Amanda L.; Bruck, Irina; Kaplan, Daniel L.

2017-01-01

The replicative helicase unwinds parental double-stranded DNA at a replication fork to provide single-stranded DNA templates for the replicative polymerases. In eukaryotes, the replicative helicase is composed of the Cdc45 protein, the heterohexameric ring-shaped Mcm2-7 complex, and the tetrameric GINS complex (CMG). The CMG proteins bind directly to DNA, as demonstrated by experiments with purified proteins. The mechanism and function of these DNA-protein interactions are presently being investigated, and a number of important discoveries relating to how the helicase proteins interact with DNA have been reported recently. While some of the protein-DNA interactions directly relate to the unwinding function of the enzyme complex, other protein-DNA interactions may be important for minichromosome maintenance (MCM) loading, origin melting or replication stress. This review describes our current understanding of how the eukaryotic replicative helicase subunits interact with DNA structures in vitro, and proposed models for the in vivo functions of replicative helicase-DNA interactions are also described. PMID:28383499

Analysis of a kinetic multi-segment foot model part II: kinetics and clinical implications.

PubMed

Bruening, Dustin A; Cooney, Kevin M; Buczek, Frank L

2012-04-01

Kinematic multi-segment foot models have seen increased use in clinical and research settings, but the addition of kinetics has been limited and hampered by measurement limitations and modeling assumptions. In this second of two companion papers, we complete the presentation and analysis of a three segment kinetic foot model by incorporating kinetic parameters and calculating joint moments and powers. The model was tested on 17 pediatric subjects (ages 7-18 years) during normal gait. Ground reaction forces were measured using two adjacent force platforms, requiring targeted walking and the creation of two sub-models to analyze ankle, midtarsal, and 1st metatarsophalangeal joints. Targeted walking resulted in only minimal kinematic and kinetic differences compared with walking at self selected speeds. Joint moments and powers were calculated and ensemble averages are presented as a normative database for comparison purposes. Ankle joint powers are shown to be overestimated when using a traditional single-segment foot model, as substantial angular velocities are attributed to the mid-tarsal joint. Power transfer is apparent between the 1st metatarsophalangeal and mid-tarsal joints in terminal stance/pre-swing. While the measurement approach presented here is limited to clinical populations with only minimal impairments, some elements of the model can also be incorporated into routine clinical gait analysis. Copyright © 2011 Elsevier B.V. All rights reserved.

NS5A inhibitors unmask differences in functional replicase complex half-life between different hepatitis C virus strains

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

Benzine, Tiffany; Brandt, Ryan; Lovell, William C.

We synthesized the Hepatitis C virus (HCV) RNA by the replicase complex (RC), a macromolecular assembly composed of viral non-structural proteins and cellular co-factors. Inhibitors of the HCV NS5A protein block formation of new RCs but do not affect RNA synthesis by preformed RCs. Without new RC formation, existing RCs turn over and are eventually lost from the cell. We aimed to use NS5A inhibitors to estimate the half-life of the functional RC of HCV. We compared different cell culture-infectious strains of HCV that may be grouped based on their sensitivity to lipid peroxidation: robustly replicating, lipid peroxidation resistant (LPOmore » R) viruses (e.g. JFH-1 or H77D) and more slowly replicating, lipid peroxidation sensitive (LPO S) viruses (e.g. H77S.3 and N.2). Furthermore, in luciferase assays, LPO S HCV strains declined under NS5A inhibitor therapy with much slower kinetics compared to LPO R HCV strains. This difference in rate of decline was not observed for inhibitors of the NS5B RNAdependent RNA polymerase suggesting that the difference was not simply a consequence of differences in RNA stability. In further analyses, we compared two isoclonal HCV variants: the LPO S H77S.3 and the LPO R H77D that differ only by 12 amino acids. Differences in rate of decline between H77S.3 and H77D following NS5A inhibitor addition were not due to amino acid sequences in NS5A but rather due to a combination of amino acid differences in the non-structural proteins that make up the HCV RC. The mathematical modeling of intracellular HCV RNA dynamics suggested that differences in RC stability (half-lives of 3.5 and 9.9 hours, for H77D and H77S.3, respectively) are responsible for the different kinetics of antiviral suppression between LPO S and LPO R viruses. In nascent RNA capture assays, the rate of RNA synthesis decline following NS5A inhibitor addition was significantly faster for H77D compared to H77S.3 indicating different half-lives of functional RCs.« less

NS5A inhibitors unmask differences in functional replicase complex half-life between different hepatitis C virus strains

DOE PAGES

Benzine, Tiffany; Brandt, Ryan; Lovell, William C.; ...

2017-06-08

We synthesized the Hepatitis C virus (HCV) RNA by the replicase complex (RC), a macromolecular assembly composed of viral non-structural proteins and cellular co-factors. Inhibitors of the HCV NS5A protein block formation of new RCs but do not affect RNA synthesis by preformed RCs. Without new RC formation, existing RCs turn over and are eventually lost from the cell. We aimed to use NS5A inhibitors to estimate the half-life of the functional RC of HCV. We compared different cell culture-infectious strains of HCV that may be grouped based on their sensitivity to lipid peroxidation: robustly replicating, lipid peroxidation resistant (LPOmore » R) viruses (e.g. JFH-1 or H77D) and more slowly replicating, lipid peroxidation sensitive (LPO S) viruses (e.g. H77S.3 and N.2). Furthermore, in luciferase assays, LPO S HCV strains declined under NS5A inhibitor therapy with much slower kinetics compared to LPO R HCV strains. This difference in rate of decline was not observed for inhibitors of the NS5B RNAdependent RNA polymerase suggesting that the difference was not simply a consequence of differences in RNA stability. In further analyses, we compared two isoclonal HCV variants: the LPO S H77S.3 and the LPO R H77D that differ only by 12 amino acids. Differences in rate of decline between H77S.3 and H77D following NS5A inhibitor addition were not due to amino acid sequences in NS5A but rather due to a combination of amino acid differences in the non-structural proteins that make up the HCV RC. The mathematical modeling of intracellular HCV RNA dynamics suggested that differences in RC stability (half-lives of 3.5 and 9.9 hours, for H77D and H77S.3, respectively) are responsible for the different kinetics of antiviral suppression between LPO S and LPO R viruses. In nascent RNA capture assays, the rate of RNA synthesis decline following NS5A inhibitor addition was significantly faster for H77D compared to H77S.3 indicating different half-lives of functional RCs.« less

Integrating Kinetic Model of E. coli with Genome Scale Metabolic Fluxes Overcomes Its Open System Problem and Reveals Bistability in Central Metabolism

PubMed Central

Mannan, Ahmad A.; Toya, Yoshihiro; Shimizu, Kazuyuki; McFadden, Johnjoe; Kierzek, Andrzej M.; Rocco, Andrea

2015-01-01

An understanding of the dynamics of the metabolic profile of a bacterial cell is sought from a dynamical systems analysis of kinetic models. This modelling formalism relies on a deterministic mathematical description of enzyme kinetics and their metabolite regulation. However, it is severely impeded by the lack of available kinetic information, limiting the size of the system that can be modelled. Furthermore, the subsystem of the metabolic network whose dynamics can be modelled is faced with three problems: how to parameterize the model with mostly incomplete steady state data, how to close what is now an inherently open system, and how to account for the impact on growth. In this study we address these challenges of kinetic modelling by capitalizing on multi-‘omics’ steady state data and a genome-scale metabolic network model. We use these to generate parameters that integrate knowledge embedded in the genome-scale metabolic network model, into the most comprehensive kinetic model of the central carbon metabolism of E. coli realized to date. As an application, we performed a dynamical systems analysis of the resulting enriched model. This revealed bistability of the central carbon metabolism and thus its potential to express two distinct metabolic states. Furthermore, since our model-informing technique ensures both stable states are constrained by the same thermodynamically feasible steady state growth rate, the ensuing bistability represents a temporal coexistence of the two states, and by extension, reveals the emergence of a phenotypically heterogeneous population. PMID:26469081

The cellular Mre11 protein interferes with adenovirus E4 mutant DNA replication.

PubMed

Mathew, Shomita S; Bridge, Eileen

2007-09-01

Adenovirus type 5 (Ad5) relocalizes and degrades the host DNA repair protein Mre11, and efficiently initiates viral DNA replication. Mre11 associates with Ad E4 mutant DNA replication centers and is important for concatenating viral genomes. We have investigated the role of Mre11 in the E4 mutant DNA replication defect. RNAi-mediated knockdown of Mre11 dramatically rescues E4 mutant DNA replication in cells that do or do not concatenate viral genomes, suggesting that Mre11 inhibits DNA replication independent of genome concatenation. The mediator of DNA damage checkpoint 1 (Mdc1) protein is involved in recruiting and sustaining Mre11 at sites of DNA damage following ionizing radiation. We observe foci formation by Mdc1 in response to viral infection, indicating that this damage response protein is activated. However, knockdown of Mdc1 does not prevent Mre11 from localizing at viral DNA replication foci or rescue E4 mutant DNA replication. Our results are consistent with a model in which Mre11 interferes with DNA replication when it is localized at viral DNA replication foci.

Estimation of foot joint kinetics in three and four segment foot models using an existing proportionality scheme: Application in paediatric barefoot walking.

PubMed

Deschamps, Kevin; Eerdekens, Maarten; Desmet, Dirk; Matricali, Giovanni Arnoldo; Wuite, Sander; Staes, Filip

2017-08-16

Recent studies which estimated foot segment kinetic patterns were found to have inconclusive data on one hand, and did not dissociate the kinetics of the chopart and lisfranc joint. The current study aimed therefore at reproducing independent, recently published three-segment foot kinetic data (Study 1) and in a second stage expand the estimation towards a four-segment model (Study 2). Concerning the reproducibility study, two recently published three segment foot models (Bruening et al., 2014; Saraswat et al., 2014) were reproduced and kinetic parameters were incorporated in order to calculate joint moments and powers of paediatric cohorts during gait. Ground reaction forces were measured with an integrated force/pressure plate measurement set-up and a recently published proportionality scheme was applied to determine subarea total ground reaction forces. Regarding Study 2, moments and powers were estimated with respect to the Instituto Ortopedico Rizzoli four-segment model. The proportionality scheme was expanded in this study and the impact of joint centre location on kinetic data was evaluated. Findings related to Study 1 showed in general good agreement with the kinetic data published by Bruening et al. (2014). Contrarily, the peak ankle, midfoot and hallux powers published by Saraswat et al. (2014) are disputed. Findings of Study 2 revealed that the chopart joint encompasses both power absorption and generation, whereas the Lisfranc joint mainly contributes to power generation. The results highlights the necessity for further studies in the field of foot kinetic models and provides a first estimation of the kinetic behaviour of the Lisfranc joint. Copyright © 2017 Elsevier Ltd. All rights reserved.

A general theory of kinetics and thermodynamics of steady-state copolymerization.

PubMed

Shu, Yao-Gen; Song, Yong-Shun; Ou-Yang, Zhong-Can; Li, Ming

2015-06-17

Kinetics of steady-state copolymerization has been investigated since the 1940s. Irreversible terminal and penultimate models were successfully applied to a number of comonomer systems, but failed for systems where depropagation is significant. Although a general mathematical treatment of the terminal model with depropagation was established in the 1980s, a penultimate model and higher-order terminal models with depropagation have not been systematically studied, since depropagation leads to hierarchically-coupled and unclosed kinetic equations which are hard to solve analytically. In this work, we propose a truncation method to solve the steady-state kinetic equations of any-order terminal models with depropagation in a unified way, by reducing them into closed steady-state equations which give the exact solution of the original kinetic equations. Based on the steady-state equations, we also derive a general thermodynamic equality in which the Shannon entropy of the copolymer sequence is explicitly introduced as part of the free energy dissipation of the whole copolymerization system.

Pseudo-second order models for the adsorption of safranin onto activated carbon: comparison of linear and non-linear regression methods.

PubMed

Kumar, K Vasanth

2007-04-02

Kinetic experiments were carried out for the sorption of safranin onto activated carbon particles. The kinetic data were fitted to pseudo-second order model of Ho, Sobkowsk and Czerwinski, Blanchard et al. and Ritchie by linear and non-linear regression methods. Non-linear method was found to be a better way of obtaining the parameters involved in the second order rate kinetic expressions. Both linear and non-linear regression showed that the Sobkowsk and Czerwinski and Ritchie's pseudo-second order models were the same. Non-linear regression analysis showed that both Blanchard et al. and Ho have similar ideas on the pseudo-second order model but with different assumptions. The best fit of experimental data in Ho's pseudo-second order expression by linear and non-linear regression method showed that Ho pseudo-second order model was a better kinetic expression when compared to other pseudo-second order kinetic expressions.

Pecan nutshell as biosorbent to remove Cu(II), Mn(II) and Pb(II) from aqueous solutions.

PubMed

Vaghetti, Julio C P; Lima, Eder C; Royer, Betina; da Cunha, Bruna M; Cardoso, Natali F; Brasil, Jorge L; Dias, Silvio L P

2009-02-15

In the present study we reported for the first time the feasibility of pecan nutshell (PNS, Carya illinoensis) as an alternative biosorbent to remove Cu(II), Mn(II) and Pb(II) metallic ions from aqueous solutions. The ability of PNS to remove the metallic ions was investigated by using batch biosorption procedure. The effects such as, pH, biosorbent dosage on the adsorption capacities of PNS were studied. Four kinetic models were tested, being the adsorption kinetics better fitted to fractionary-order kinetic model. Besides that, the kinetic data were also fitted to intra-particle diffusion model, presenting three linear regions, indicating that the kinetics of adsorption should follow multiple sorption rates. The equilibrium data were fitted to Langmuir, Freundlich, Sips and Redlich-Peterson isotherm models. Taking into account a statistical error function, the data were best fitted to Sips isotherm model. The maximum biosorption capacities of PNS were 1.35, 1.78 and 0.946mmolg(-1) for Cu(II), Mn(II) and Pb(II), respectively.

Kinetic modeling of kraft delignification of Eucalyptus globulus

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

Santos, A.; Rodriguez, F.; Gilarranz, M.A.

1997-10-01

A kinetic model for the kraft pulping delignification of Eucalyptus globulus is proposed. This model is discriminated among some kinetic expressions often used in the literature, and the kinetic parameters are determined by fitting of experimental results. A total of 25 isothermal experiments at liquor-to-wood ratios of 50 and 5 L/kg have been carried out. Initial, bulk, and residual delignification stages have been observed during the lignin removal, the transitions being, referring to the lignin initial content, about 82 and 3%. Carbohydrate removal and effective alkali-metal and hydrosulfide consumption have been related with the lignin removal by means of effectivemore » stoichiometric coefficients for each stage, coefficients also being calculated by fitting of the experimental data. The kinetic model chosen has been used to simulate typical kraft pulping experiments carried out at nonisothermal conditions, using a temperature ramp. The model yields simulated values close to those obtained experimentally for the wood studied and also ably reproduces the trends of the literature data.« less

Kinetic model for binary homogeneous nucleation in the H2O-H2SO4 system: comparison with experiments and classical theory of nucleation.

PubMed

Sorokin, A; Vancassel, X; Mirabel, P

2005-12-22

A kinetic model to predict nucleation rates in the sulfuric acid-water system is presented. It allows calculating steady-state nucleation rates and the corresponding time lag, using a direct solution of a system of kinetic equations that describe the populations of sub- and near-critical clusters. This kinetic model takes into account cluster-cluster collisions and decay of clusters into smaller clusters. The model results are compared with some predictions obtained with the classical nucleation theory (CNT) and also with available measurement data obtained in smog chambers or flow tubes. It is shown that in the case of slow nucleation processes, the kinetic model and the CNT as used by Shugard et al. [J. Chem. Phys. 75, 5298 (1974)] give the same results. However, in the case of intensive nucleation, a large part of the nucleation flux is due to cluster-cluster collisions and the CNT underestimates the nucleation rates.

The use of modified and non-natural nucleotides provide unique insights into pro-mutagenic replication catalyzed by polymerase eta

PubMed Central

Choi, Jung-Suk; Dasari, Anvesh; Hu, Peter; Benkovic, Stephen J.; Berdis, Anthony J.

2016-01-01

This report evaluates the pro-mutagenic behavior of 8-oxo-guanine (8-oxo-G) by quantifying the ability of high-fidelity and specialized DNA polymerases to incorporate natural and modified nucleotides opposite this lesion. Although high-fidelity DNA polymerases such as pol δ and the bacteriophage T4 DNA polymerase replicating 8-oxo-G in an error-prone manner, they display remarkably low efficiencies for TLS compared to normal DNA synthesis. In contrast, pol η shows a combination of high efficiency and low fidelity when replicating 8-oxo-G. These combined properties are consistent with a pro-mutagenic role for pol η when replicating this DNA lesion. Studies using modified nucleotide analogs show that pol η relies heavily on hydrogen-bonding interactions during translesion DNA synthesis. However, nucleobase modifications such as alkylation to the N2 position of guanine significantly increase error-prone synthesis catalyzed by pol η when replicating 8-oxo-G. Molecular modeling studies demonstrate the existence of a hydrophobic pocket in pol η that participates in the increased utilization of certain hydrophobic nucleotides. A model is proposed for enhanced pro-mutagenic replication catalyzed by pol η that couples efficient incorporation of damaged nucleotides opposite oxidized DNA lesions created by reactive oxygen species. The biological implications of this model toward increasing mutagenic events in lung cancer are discussed. PMID:26717984

Model-based variance-stabilizing transformation for Illumina microarray data.

PubMed

Lin, Simon M; Du, Pan; Huber, Wolfgang; Kibbe, Warren A

2008-02-01

Variance stabilization is a step in the preprocessing of microarray data that can greatly benefit the performance of subsequent statistical modeling and inference. Due to the often limited number of technical replicates for Affymetrix and cDNA arrays, achieving variance stabilization can be difficult. Although the Illumina microarray platform provides a larger number of technical replicates on each array (usually over 30 randomly distributed beads per probe), these replicates have not been leveraged in the current log2 data transformation process. We devised a variance-stabilizing transformation (VST) method that takes advantage of the technical replicates available on an Illumina microarray. We have compared VST with log2 and Variance-stabilizing normalization (VSN) by using the Kruglyak bead-level data (2006) and Barnes titration data (2005). The results of the Kruglyak data suggest that VST stabilizes variances of bead-replicates within an array. The results of the Barnes data show that VST can improve the detection of differentially expressed genes and reduce false-positive identifications. We conclude that although both VST and VSN are built upon the same model of measurement noise, VST stabilizes the variance better and more efficiently for the Illumina platform by leveraging the availability of a larger number of within-array replicates. The algorithms and Supplementary Data are included in the lumi package of Bioconductor, available at: www.bioconductor.org.

C. botulinum inactivation kinetics implemented in a computational model of a high-pressure sterilization process.

PubMed

Juliano, Pablo; Knoerzer, Kai; Fryer, Peter J; Versteeg, Cornelis

2009-01-01

High-pressure, high-temperature (HPHT) processing is effective for microbial spore inactivation using mild preheating, followed by rapid volumetric compression heating and cooling on pressure release, enabling much shorter processing times than conventional thermal processing for many food products. A computational thermal fluid dynamic (CTFD) model has been developed to model all processing steps, including the vertical pressure vessel, an internal polymeric carrier, and food packages in an axis-symmetric geometry. Heat transfer and fluid dynamic equations were coupled to four selected kinetic models for the inactivation of C. botulinum; the traditional first-order kinetic model, the Weibull model, an nth-order model, and a combined discrete log-linear nth-order model. The models were solved to compare the resulting microbial inactivation distributions. The initial temperature of the system was set to 90 degrees C and pressure was selected at 600 MPa, holding for 220 s, with a target temperature of 121 degrees C. A representation of the extent of microbial inactivation throughout all processing steps was obtained for each microbial model. Comparison of the models showed that the conventional thermal processing kinetics (not accounting for pressure) required shorter holding times to achieve a 12D reduction of C. botulinum spores than the other models. The temperature distribution inside the vessel resulted in a more uniform inactivation distribution when using a Weibull or an nth-order kinetics model than when using log-linear kinetics. The CTFD platform could illustrate the inactivation extent and uniformity provided by the microbial models. The platform is expected to be useful to evaluate models fitted into new C. botulinum inactivation data at varying conditions of pressure and temperature, as an aid for regulatory filing of the technology as well as in process and equipment design.

Sterol Binding by the Tombusviral Replication Proteins Is Essential for Replication in Yeast and Plants.

PubMed

Xu, Kai; Nagy, Peter D

2017-04-01

Membranous structures derived from various organelles are important for replication of plus-stranded RNA viruses. Although the important roles of co-opted host proteins in RNA virus replication have been appreciated for a decade, the equally important functions of cellular lipids in virus replication have been gaining full attention only recently. Previous work with Tomato bushy stunt tombusvirus (TBSV) in model host yeast has revealed essential roles for phosphatidylethanolamine and sterols in viral replication. To further our understanding of the role of sterols in tombusvirus replication, in this work we showed that the TBSV p33 and p92 replication proteins could bind to sterols in vitro The sterol binding by p33 is supported by cholesterol recognition/interaction amino acid consensus (CRAC) and CARC-like sequences within the two transmembrane domains of p33. Mutagenesis of the critical Y amino acids within the CRAC and CARC sequences blocked TBSV replication in yeast and plant cells. We also showed the enrichment of sterols in the detergent-resistant membrane (DRM) fractions obtained from yeast and plant cells replicating TBSV. The DRMs could support viral RNA synthesis on both the endogenous and exogenous templates. A lipidomic approach showed the lack of enhancement of sterol levels in yeast and plant cells replicating TBSV. The data support the notion that the TBSV replication proteins are associated with sterol-rich detergent-resistant membranes in yeast and plant cells. Together, the results obtained in this study and the previously published results support the local enrichment of sterols around the viral replication proteins that is critical for TBSV replication. IMPORTANCE One intriguing aspect of viral infections is their dependence on efficient subcellular assembly platforms serving replication, virion assembly, or virus egress via budding out of infected cells. These assembly platforms might involve sterol-rich membrane microdomains, which are heterogeneous and highly dynamic nanoscale structures usurped by various viruses. Here, we demonstrate that TBSV p33 and p92 replication proteins can bind to sterol in vitro Mutagenesis analysis of p33 within the CRAC and CARC sequences involved in sterol binding shows the important connection between the abilities of p33 to bind to sterol and to support TBSV replication in yeast and plant cells. Together, the results further strengthen the model that cellular sterols are essential as proviral lipids during viral replication. Copyright © 2017 American Society for Microbiology.

Identification of HIV-1 determinants for replication in vivo.

PubMed

Su, L; Kaneshima, H; Bonyhadi, M L; Lee, R; Auten, J; Wolf, A; Du, B; Rabin, L; Hahn, B H; Terwilliger, E; Mccune, J M

1997-01-06

Pathogenic organisms are frequently attenuated after long-term culture in vitro. The mechanisms of the attenuation process are not clear, but probably involve mutations of functions required for replication and pathogenicity in vivo. To identify these functions, a direct comparison must be made between attenuated genomes and those that remain pathogenic in vivo. In this study, we used the heterochimeric SCID-hu Thy/Liv mouse as an in vivo model to define human immunodeficiency virus type 1 (HIV-1) determinants which are uniquely required for replication in vivo. The Lai/IIIB isolate and its associated infectious molecular clones (e.g., HXB2) were found to infect T cell lines but failed to replicate in the SCID-hu Thy/Liv model. When a lab worker was accidentally infected by Lai/IIIB, however, HIV-1 was isolated only from infection of primary PBMC, and not from infection of T cell lines. We hypothesized that the lab worker was exposed to a heterogeneous viral stock which had been attenuated by passage in immortalized T cell lines. Either a rare family member from this stock was selected for in vivo replication or, alternatively, an attenuated genotype dominant in vitro may have reverted to become more infectious in vivo. To address this hypothesis, we have used the SCID-hu Thy/Liv model to study the replication of HXB2 and of HXB2 recombinant viruses with HIV-1 fragments isolated from the infected lab worker. HXB2 showed no or very low levels of replication in the Thy/Liv organ. Replacement of its subgenomic fragment encoding the envelope gene with a corresponding fragment from the lab worker isolate generated a recombinant virus (HXB2/LW) which replicated actively in SCID-hu mice. The NEF mutation in the HXB2 genome is still present in HXB2/LW. Thus, the LW sequences encode HIV-1 determinants which enhance HIV replication in vivo in a NEF-independent mechanism. The specific determinants have been mapped to the V1-V3 regions of the HIV-1 genome. Six unique mutations in the V3 loop region of HXB2/LW have been identified which contribute to the increased replication in vivo.

Human Papillomavirus E6E7-Mediated Adenovirus Cell Killing: Selectivity of Mutant Adenovirus Replication in Organotypic Cultures of Human Keratinocytes

PubMed Central

Balagué, Cristina; Noya, Francisco; Alemany, Ramon; Chow, Louise T.; Curiel, David T.

2001-01-01

Replication-competent adenoviruses are being investigated as potential anticancer agents. Exclusive virus replication in cancer cells has been proposed as a safety trait to be considered in the design of oncolytic adenoviruses. From this perspective, we have investigated several adenovirus mutants for their potential to conditionally replicate and promote the killing of cells expressing human papillomavirus (HPV) E6 and E7 oncoproteins, which are present in a high percentage of anogenital cancers. For this purpose, we have employed an organotypic model of human stratified squamous epithelium derived from primary keratinocytes that have been engineered to express HPV-18 oncoproteins stably. We show that, whereas wild-type adenovirus promotes a widespread cytopathic effect in all infected cells, E1A- and E1A/E1B-deleted adenoviruses cause no deleterious effect regardless of the coexpression of HPV18 E6E7. An adenovirus deleted in the CR2 domain of E1A, necessary for binding to the pRB family of pocket proteins, shows no selectivity of replication as it efficiently kills all normal and E6E7-expressing keratinocytes. Finally, an adenovirus mutant deleted in the CR1 and CR2 domains of E1A exhibits preferential replication and cell killing in HPV E6E7-expressing cultures. We conclude that the organotypic keratinocyte culture represents a distinct model to evaluate adenovirus selectivity and that, based on this model, further modifications of the adenovirus genome are required to restrict adenovirus replication to tumor cells. PMID:11462032

Management and control of self-replicating systems: A systems model

NASA Technical Reports Server (NTRS)

Vontiesenhausen, G.

1982-01-01

In 1980, a conceptual engineering approach to self-replicating systems was achieved. The design was based on von Newmann's kinematic version of self-replicating automata. The systems management and control and the organization of the control elements are reported. After developing the functional requirements of such a system, a hierarchy of three management and control levels is described. These are an autonomous, an external, and an intelligent management and control system. Systems recycling, systems specialization, and information replication are discussed.

Trajectory of Externalizing Child Behaviors in a KEEP Replication

ERIC Educational Resources Information Center

Uretsky, Mathew C.; Lee, Bethany R.; Greeno, Elizabeth J.; Barth, Richard P.

2017-01-01

Objective: The purpose of this study is to examine the correlates of child behavior change over time in a replication of the KEEP intervention. Method: The study sample was drawn from the treatment group of the Maryland replication of KEEP (n=65). Change over time was analyzed using multilevel linear mixed modeling. Results: Parents' use of…

qPIPSA: Relating enzymatic kinetic parameters and interaction fields

PubMed Central

Gabdoulline, Razif R; Stein, Matthias; Wade, Rebecca C

2007-01-01

Background The simulation of metabolic networks in quantitative systems biology requires the assignment of enzymatic kinetic parameters. Experimentally determined values are often not available and therefore computational methods to estimate these parameters are needed. It is possible to use the three-dimensional structure of an enzyme to perform simulations of a reaction and derive kinetic parameters. However, this is computationally demanding and requires detailed knowledge of the enzyme mechanism. We have therefore sought to develop a general, simple and computationally efficient procedure to relate protein structural information to enzymatic kinetic parameters that allows consistency between the kinetic and structural information to be checked and estimation of kinetic constants for structurally and mechanistically similar enzymes. Results We describe qPIPSA: quantitative Protein Interaction Property Similarity Analysis. In this analysis, molecular interaction fields, for example, electrostatic potentials, are computed from the enzyme structures. Differences in molecular interaction fields between enzymes are then related to the ratios of their kinetic parameters. This procedure can be used to estimate unknown kinetic parameters when enzyme structural information is available and kinetic parameters have been measured for related enzymes or were obtained under different conditions. The detailed interaction of the enzyme with substrate or cofactors is not modeled and is assumed to be similar for all the proteins compared. The protein structure modeling protocol employed ensures that differences between models reflect genuine differences between the protein sequences, rather than random fluctuations in protein structure. Conclusion Provided that the experimental conditions and the protein structural models refer to the same protein state or conformation, correlations between interaction fields and kinetic parameters can be established for sets of related enzymes. Outliers may arise due to variation in the importance of different contributions to the kinetic parameters, such as protein stability and conformational changes. The qPIPSA approach can assist in the validation as well as estimation of kinetic parameters, and provide insights into enzyme mechanism. PMID:17919319

Kinetic modelling of anaerobic hydrolysis of solid wastes, including disintegration processes

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

García-Gen, Santiago; Sousbie, Philippe; Rangaraj, Ganesh

2015-01-15

Highlights: • Fractionation of solid wastes into readily and slowly biodegradable fractions. • Kinetic coefficients estimation from mono-digestion batch assays. • Validation of kinetic coefficients with a co-digestion continuous experiment. • Simulation of batch and continuous experiments with an ADM1-based model. - Abstract: A methodology to estimate disintegration and hydrolysis kinetic parameters of solid wastes and validate an ADM1-based anaerobic co-digestion model is presented. Kinetic parameters of the model were calibrated from batch reactor experiments treating individually fruit and vegetable wastes (among other residues) following a new protocol for batch tests. In addition, decoupled disintegration kinetics for readily and slowlymore » biodegradable fractions of solid wastes was considered. Calibrated parameters from batch assays of individual substrates were used to validate the model for a semi-continuous co-digestion operation treating simultaneously 5 fruit and vegetable wastes. The semi-continuous experiment was carried out in a lab-scale CSTR reactor for 15 weeks at organic loading rate ranging between 2.0 and 4.7 g VS/L d. The model (built in Matlab/Simulink) fit to a large extent the experimental results in both batch and semi-continuous mode and served as a powerful tool to simulate the digestion or co-digestion of solid wastes.« less

On the biophysics and kinetics of toehold-mediated DNA strand displacement

PubMed Central

Srinivas, Niranjan; Ouldridge, Thomas E.; Šulc, Petr; Schaeffer, Joseph M.; Yurke, Bernard; Louis, Ard A.; Doye, Jonathan P. K.; Winfree, Erik

2013-01-01

Dynamic DNA nanotechnology often uses toehold-mediated strand displacement for controlling reaction kinetics. Although the dependence of strand displacement kinetics on toehold length has been experimentally characterized and phenomenologically modeled, detailed biophysical understanding has remained elusive. Here, we study strand displacement at multiple levels of detail, using an intuitive model of a random walk on a 1D energy landscape, a secondary structure kinetics model with single base-pair steps and a coarse-grained molecular model that incorporates 3D geometric and steric effects. Further, we experimentally investigate the thermodynamics of three-way branch migration. Two factors explain the dependence of strand displacement kinetics on toehold length: (i) the physical process by which a single step of branch migration occurs is significantly slower than the fraying of a single base pair and (ii) initiating branch migration incurs a thermodynamic penalty, not captured by state-of-the-art nearest neighbor models of DNA, due to the additional overhang it engenders at the junction. Our findings are consistent with previously measured or inferred rates for hybridization, fraying and branch migration, and they provide a biophysical explanation of strand displacement kinetics. Our work paves the way for accurate modeling of strand displacement cascades, which would facilitate the simulation and construction of more complex molecular systems. PMID:24019238