Biomedical subjects

John Yin

Publications and source records attributed to John Yin.

At least 19 recordsLinked to original sources

PubMed2007

Rapid responses of ribosomal RNA synthesis to nutrient shifts.

A major challenge in systems biology is to integrate our mechanistic understanding of gene regulation to predict quantitatively how cells will respond to environmental changes. Living cells respond rapidly to the availability of nutrients in part by altering production of ribosomal RNA (rRNA), a limiting component in the biosynthesis of ribosomes. Studies of rRNA transcription by the RNA polymerase of Escherichia coli have identified regulatory roles for guanosine tetraphosphate (ppGpp), the initiating nucleotide, and the protein DksA. To what extent findings from in vitro studies can be used to quantitatively predict in vivo responses to changing nutrient environments is unknown. We developed a mechanistic mathematical model for rRNA transcriptional responses to such changes. Our model accounts for binding of RNAP to its rRNA promoter to form a closed complex, isomerization from a closed complex to an open complex, reversible incorporation of the initiating NTP (iNTP), transcript elongation, and clearance of the promoter. Further, the model incorporates interactions between ppGpp and DksA with transcription intermediates, and it includes an empirical correction to account for salt effects. The model biophysical parameters were determined using 33 single- and multi-round transcription experiments spanning 487 in vitro measurements. By incorporating in vivo measurements of ppGpp and ATP, the model correctly predicted rRNA production rates for cellular responses to nutrient upshifts, downshifts, and outgrowth into fresh medium. Inclusion of DksA was essential in all three cases. Our work provides a foundation for using data-driven computational models to predict the kinetics of in vivo transcriptional responses.

Adaptation, Physiological↗

PubMed2007

Thermostabilizing mutations in reovirus outer-capsid protein mu1 selected by heat inactivation of infectious subvirion particles.

The 76-kDa mu1 protein of nonfusogenic mammalian reovirus is a major component of the virion outer capsid, which contains 200 mu1 trimers arranged in an incomplete T=13 lattice. In virions, mu1 is largely covered by a second major outer-capsid protein, sigma3, which limits mu1 conformational mobility. In infectious subvirion particles, from which sigma3 has been removed, mu1 is broadly exposed on the surface and can be promoted to rearrange into a protease-sensitive and hydrophobic conformer, leading to membrane perforation or penetration. In this study, mutants that resisted loss of infectivity upon heat inactivation (heat-resistant mutants) were selected from infectious subvirion particles of reovirus strains Type 1 Lang and Type 3 Dearing. All of the mutants were found to have mutations in mu1, and the heat-resistance phenotype was mapped to mu1 by both recoating and reassortant genetics. Heat-resistant mutants were also resistant to rearrangement to the protease-sensitive conformer of mu1, suggesting that heat inactivation is associated with mu1 rearrangement, consistent with published results. Rate constants of heat inactivation were determined, and the dependence of inactivation rate on temperature was consistent with the Arrhenius relationship. The Gibbs free energy of activation was calculated with reference to transition-state theory and was found to be correlated with the degree of heat resistance in each of the analyzed mutants. The mutations are located in upper portions of the mu1 trimer, near intersubunit contacts either within or between trimers in the viral outer capsid. We propose that the mutants stabilize the outer capsid by interfering with unwinding of the mu1 trimer.

Animals↗

PubMed2006

A quantitative comet assay: imaging and analysis of virus plaques formed with a liquid overlay.

Although the plaque assay defines a "gold-standard" for measuring virus infectivity, its reliance on plaque counting limits its sensitivity. When the assay is performed with a liquid overlay, instead of agar overlay, spontaneous flows can promote a uni-directional spread of infection, creating elongated regions of cytopathology that resemble comets. As a model system comet and plaque cultures of vesicular stomatitis virus (VSV) on baby hamster kidney (BHK-21) cells were compared. Host-cell monolayers were infected with VSV particles, incubated 15 h in the presence of liquid or agar overlays and stained. VSV formed significantly larger comets than plaques, consistent with a mechanism of flow-enhanced spread. When antiviral drug (5-fluorouracil) was incorporated into the liquid overlay, comet sizes were reduced in a dose-dependent manner. Images of infected monolayers, acquired using a simple digital scanner, enabled a quantification of the inhibitory effect of the drug on infectivity. The resulting measure of drug susceptibility was found to be 18-fold more sensitive than the IC(50) measure attained by the traditional plaque-reduction assay. This quantitative comet assay has the potential to similarly enhance the sensitivity of infection measures for other plaque-forming viruses.

Animals↗

PubMed2006

Model-based design of growth-attenuated viruses.

Live-virus vaccines activate both humoral and cell-mediated immunity, require only a single boosting, and generally provide longer immune protection than killed or subunit vaccines. However, growth of live-virus vaccines must be attenuated to minimize their potential pathogenic effects, and mechanisms of attenuation by conventional serial-transfer viral adaptation are not well-understood. New methods of attenuation based on rational engineering of viral genomes may offer a potentially greater control if one can link defined genetic modifications to changes in virus growth. To begin to establish such links between genotype and growth phenotype, we developed a computer model for the intracellular growth of vesicular stomatitis virus (VSV), a well-studied, nonsegmented, negative-stranded RNA virus. Our model incorporated established regulatory mechanisms of VSV while integrating key wild-type infection steps: hijacking of host resources, transcription, translation, and replication, followed by assembly and release of progeny VSV particles. Generalization of the wild-type model to allow for genome rearrangements matched the experimentally observed attenuation ranking for recombinant VSV strains that altered the genome position of their nucleocapsid gene. Finally, our simulations captured previously reported experimental results showing how altering the positions of other VSV genes has the potential to attenuate the VSV growth while overexpressing the immunogenic VSV surface glycoprotein. Such models will facilitate the engineering of new live-virus vaccines by linking genomic manipulations to controlled changes in virus gene-expression and growth.

Animals↗

PubMed2006

Spatial patterns of protein expression in focal infections of human cytomegalovirus.

Human cytomegalovirus (HCMV) is a medically significant human pathogen that infects a wide range of cell and tissue types. During infection, HCMV activates a variety of signal transduction pathways that induce profound changes in cellular processes and dramatically affect cellular gene expression patterns. To better define how these virus-host interactions affect the local microenvironment and influence the spatial and temporal spread of HCMV, we initiated HCMV focal infections on normal human dermal fibroblast monolayers and monitored viral gene expression patterns and infection spread over 45 days. To establish baseline temporal measurements of HCMV infection and spread in cell monolayers, we characterized the influence of three experimental variables on viral gene expression: cell plating density, the presence of serum, and neutralization of cellular antiviral responses with an antibody against interferon-beta. We found that high cell plating density or the inclusion of serum correlated with enhanced HCMV infection spread. Dramatic differences in the expression pattern of the viral immediate early 2 (IE2) gene were observed under these conditions as compared to low plating density or the absence of serum. In the latter case round, uniform foci were observed with a clear wave of IE2 expression visible in advance of a late stage viral protein, envelope glycoprotein B. By contrast, larger irregular foci with arms of IE2 expression were observed in the presence of serum. Addition of the antibody had little effect on the rate of spread, which is consistent with the knowledge that HCMV represses antiviral responses during infection. This experimental system provides a useful means to visualize and quantify complex virus-host interactions.

Antibodies↗

PubMed2006

Dynamic tradeoffs in the raft-mediated entry of human immunodeficiency virus type 1 into cells.

To initiate an infection human immunodeficiency virus type 1 (HIV-1) particles must first bind to receptors on the surface of their host cells, a process that eventually leads to fusion of viral and cellular membranes and release of the viral genome into the cytoplasm. Understanding the molecular mechanisms of these processes may enable the development of new anti-HIV strategies. Disagreement currently prevails on the role in virus entry of microdomains within the cellular plasma membrane known as lipid rafts. Experiments have suggested that lipid rafts, in their interactions with cellular receptors and viral particles, either promote or have minimal effect on viral entry. Here we develop a dynamic model for HIV-1 entry that enables us to identify and quantitatively assess tradeoffs that can arise from the clustering of receptors in rafts. Specifically, receptor clustering can be detrimental to the initiation of viral infection by reducing the probability that a virus particle finds its primary receptor, CD4. However, receptor clustering can also enable a virus particle, once bound, to rapidly form multivalent interactions with receptors and co-receptors that are required for virus-cell membrane fusion. We show how the resolution of such tradeoffs hinges on the level and spatial distribution of receptors and co-receptors on the cell surface, and we discuss implications of these effects for the design of therapeutics that inhibit HIV-1 entry.

CD4 Antigens↗

PubMed2005

In silico mutagenesis of RNA splicing in HIV-1.

Human immunodeficiency virus type-1 (HIV-1) relies on both partial and complete splicing of its full-length RNA transcripts to generate a distribution of essential spliced mRNA products. The complexity of the splicing process, which can employ multiple alternative splice sites, challenges our ability to understand how mutations in splice sites may influence the composition of the resulting mRNA pool and, more broadly, the development of viral progeny. Here, we begin to systematically address these issues by developing a mechanistic mathematical model for the splicing process. We identify as key parameters the probabilities that the cellular splice machinery selects specific splice acceptors, and we show how the splicing process depends on these probabilities. Further, by incorporating this splicing model into a detailed kinetic model for HIV-1 intracellular development we find that an increase in the fraction of either rev or tat mRNA in the HIV-1 mRNA pool is generally beneficial for HIV-1 growth. However, a splice site mutation that excessively increases the fraction of either mRNA can be detrimental due to the corresponding reduction in the other mRNA, suggesting that a balance of Rev and Tat is needed in order for HIV-1 to optimize its growth. Although our model is based on still very limited quantitative data on RNA splicing, Rev-mediated splicing regulation and nuclear export, and the effects of associated mutations, it serves as a starting point for better understanding how variations in essential post-transcriptional functions can impact the intracellular development of HIV-1.

Computational Biology↗

PubMed2005

Formation of peptides in the dry state.

Although many potential pathways exist for the prebiotic condensation of amino acids to form simple peptides, minimal conditions for such a reaction in the dry state have yet to be defined. In this work, water was evaporated from a solution of alanine and copper chloride (CuCl2), creating a dry residue. Incubation of this residue at moderate temperatures over 25 days produced even greater amounts of di-alanine, as determined by high performance liquid chromatographic characterization of the re-dissolved residue. Copper(II) and chloride were required for the reaction and di-peptide yields were highest for 1:2 molar ratios of copper:alanine. These results define minimal conditions for a dry-state pathway that plausibly played a role in the prebiotic formation of simple peptides.

Alanine↗

PubMed2005

Robust growth of human immunodeficiency virus type 1 (HIV-1).

The persistence of human immunodeficiency virus type-1 (HIV-1) has long been attributed to its high mutation rate and the capacity of its resulting heterogeneous virus populations to evade host immune responses and antiviral drugs. However, this view is incomplete because it does not explain how the virus persists in light of the adverse effects mutations in the viral genome and variations in host functions can potentially have on viral functions and growth. Here we show that the resilience of HIV-1 can be credited, at least in part, to a robust response to perturbations that emerges as an intrinsic property of its intracellular development. Specifically, robustness in HIV-1 arises through the coupling of two feedback loops: a Rev-mediated negative feedback and a Tat-mediated positive feedback. By employing a mechanistic kinetic model for its growth we found that HIV-1 buffers the effects of many potentially detrimental variations in essential viral and cellular functions, including the binding of Rev to mRNA; the level of rev mRNA in the pool of fully spliced mRNA; the splicing of mRNA; the Rev-mediated nuclear export of incompletely-spliced mRNAs; and the nuclear import of Tat and Rev. The virus did not, however, perform robustly to perturbations in all functions. Notably, HIV-1 tended to amplify rather than buffer adverse effects of variations in the interaction of Tat with viral mRNA. This result shows how targeting therapeutics against molecular components of the viral positive-feedback loop open new possibilities and potential in the effective treatment of HIV-1.

Cell Proliferation↗

PubMed2005

Fidelity of micropatterned cell cultures.

Methods that enable the culture of micropatterned cells may help advance our fundamental understanding of cell-cell and cell-surface interactions, while facilitating the development and implementation of cell-based biological assays. However, the long-term stability of the cell patterns can limit the time scales over which such methods can be informative. Here we used self-assembling monolayers (SAMs) to localize the adsorption of baby hamster kidney (BHK-21) cells as well as cells from a murine astrocytoma-derived cell line (delayed brain tumor) in linear arrays. We tested the effects of surface chemistries, fibronectin pre-treatments, array dimensions, and cell types on pattern fidelity. Changes in patterns were monitored by phase-contrast microscopy up to 96 h post-plating, followed by digital imaging, and these changes were quantified by measuring an "intrusion distance" or the average distance cells extend beyond the initial adhesive/non-adhesive boundary. Loss of pattern boundaries involved different mechanisms for different cells. Treatment of patterned surfaces with fibronectin prior to plating of cells tended to promote earlier loss of pattern fidelity, and the extent of pattern loss was further augmented for SAMs formed using hydrophobic monolayers. Finally, reduction of gap spacing between adjacent cell arrays promoted pattern loss.

Adsorption↗

PubMed2005

Arrested spread of vesicular stomatitis virus infections in vitro depends on interferon-mediated antiviral activity.

A quantitative understanding of the innate immune response will enable its recruitment against emerging, poorly characterized, or weaponized viral pathogens. To gain insights into how the innate responses can limit viral spread, we used quantitative focal infections to study how the spread of recombinant vesicular stomatitis viruses (VSV) on baby hamster kidney (BHK) and delayed brain tumor (DBT) cell monolayers is affected by innate cellular antiviral responses. We observed that rates of infection spread correlated with one-step growth rankings for four ectopic VSV strains: N1, N2, N3, and N4. However, this correlation was lost for M51R, a recombinant VSV mutant that lacks the ability to shut-off host gene expression. In BHK cells, M51R spread at two-thirds the rate of the recombinant control virus, XK3.1, even though their one-step growth was comparable. In DBT cells, M51R infections failed to spread beyond the site of inoculation. Addition of anti-interferon antibody restored M51R spread and one-step growth to wild-type levels. Interestingly, the antibody enhanced the spread of wild-type virus but not its growth. These results suggest that while the rate of viral spread generally correlates with the rate of viral growth, the induction of cellular antiviral activities can be in some cases, the overriding factor in both spread and growth. In summary, focal infections enabled us to visualize and quantify how viral spread was inhibited by cellular antiviral activities. This study demonstrates a mechanism for quantifying how innate cellular responses can mitigate infection spread in vitro.

Animals↗

PubMed2005

Localization of receptors in lipid rafts can inhibit signal transduction.

Processes of cell survival, division, differentiation, and death are guided by the binding of signal molecules to receptors, which activates intracellular signaling networks and ultimately elicits genetic, biochemical, or biomechanical responses within the cell. While intracellular mechanisms for these processes have been well studied, little attention has been given to the role extracellular ligand transport and binding may play in signal initiation. Recent studies have found that the localization of receptors in lipid rafts is critical for the functions of many signaling pathways. By concentrating membrane components, rafts may promote essential interactions for signaling. Lipid rafts can also have negative effects on signaling, but mechanisms remain elusive. We propose that raft-mediated receptor clustering can reduce signaling by prolonging the diffusion of ligands to their receptors. We quantify this effect using a simple diffusion-limited binding model that accounts for the spatial distribution of lipid rafts and receptors on the cell surface. We find that receptor clustering can reduce the apparent rate of receptor binding by up to 80%, consistent with observed increases in epidermal growth factor (EGF) binding by up to 100% following disruption of lipid rafts (Pike and Casey 2002 Biochemistry 41:10315-10322; Roepstorff et al. 2002 J Biol Chem 277:18954-18960). Failure to account for the effects of receptor clustering on rates of ligand binding can skew the interpretation of current methods of cancer diagnosis and treatment. Finally, we discuss how the activation of particular signaling pathways can change over time, depending, in part, on the overall level and spatial distribution of the receptors.

Absorption↗

PubMed2004

Energy-efficient growth of phage Q Beta in Escherichia coli.

The role of natural selection in the optimal design of organisms is controversial. Optimal forms, functions, or behaviors of organisms have long been claimed without knowledge of how genotype contributes to phenotype, delineation of design constraints, or reference to alternative designs. Moreover, arguments for optimal designs have been often based on models that were difficult, if not impossible, to test. Here, we begin to address these issues by developing and probing a kinetic model for the intracellular growth of bacteriophage Q beta in Escherichia coli. The model accounts for the energetic costs of all template-dependent polymerization reactions, in ATP equivalents, including RNA-dependent RNA elongation by the phage replicase and synthesis of all phage proteins by the translation machinery of the E. coli host cell. We found that translation dominated phage growth, requiring 85% of the total energy expenditure. Only 10% of the total energy was applied to activities other than the direct synthesis of progeny phage components, reflecting primarily the cost of making the negative-strand RNA template that is needed for replication of phage genomic RNA. Further, we defined an energy efficiency of phage growth and showed its direct relationship to the yield of phage progeny. Finally, we performed a sensitivity analysis and found that the growth of wild-type phage was optimized for progeny yield or energy efficiency, suggesting that phage Q beta has evolved to optimally utilize the finite resources of its host cells.

Coliphages↗

PubMed2004

Effects of lipid rafts on dynamics of retroviral entry and trafficking: Quantitative analysis.

The association of cell surface receptors with sterol-sphingolipid-enriched microdomains of the plasma membrane, so-called lipid rafts, may affect the receptor-mediated entry and trafficking dynamics of viruses. A model retrovirus, subgroup A avian sarcoma and leukosis virus (ASLV-A), can initiate infection by binding to either of two forms of the tumor virus subgroup A (TVA) receptor, a lipid-raft-associated glycosylphosphatidylinositol (GPI)-anchored receptor (TVA800) or a transmembrane receptor (TVA950). Narayan et al. previously found that virus particles bound to TVA950 were more rapidly internalized than virions bound to TVA800, and the internalization via TVA950 exhibited biphasic kinetics. To explore potential molecular mechanisms for these results we developed a mathematical model that accounts for internalization of viruses through cellular pits, trafficking to an endosomal compartment where fusion occurs, and viral DNA synthesis. By fitting the model to experimental data we found that viruses bound to TVA950 were internalized up to 2.6-fold more rapidly than viruses bound to TVA800. Two- to threefold greater lateral diffusivities of transmembrane proteins, relative to GPI-anchored proteins, observed in other systems, suggest that the internalization rate of ASLV-A is diffusion-limited. Furthermore, by allowing for recycling of internalized TVA950-bound viruses back to the cell surface, we can account for the observed biphasic internalization kinetics. This mechanism is also consistent with the observed slower rate of DNA synthesis for viruses that enter via TVA950. Overall, the model provides a means to generate new experimentally testable hypotheses and sets a foundation for building a quantitative and integrated understanding of viral entry, trafficking, and intracellular dynamics.

Animals↗

PubMed2004

Genome function--a virus-world view.

By studying viruses one may begin to understand how static genomes can define dynamic processes of development. This talk will describe some of the approaches we are taking, using computer simulations and laboratory experiments, to account for the many molecular-level processes and interactions that occur when a common bacterium, E. coli, is infected by one of its viruses, phage T7. We accounted for processes of phage genome entry, transcription, translation, and DNA replication, including protein-DNA and protein-protein regulatory interactions, and we predicted the dynamics of phage progeny formation. The simulations have enabled us to identify limiting host-cell resources in phage growth, discover novel anti-viral strategies, and suggest frameworks for mining data from global mRNA and protein studies.

Bacteriophage T7↗

PubMed2004

Quantitative analysis of a parasitic antiviral strategy.

We extended a computer simulation of viral intracellular growth to study a parasitic antiviral strategy that diverts the viral replicase toward parasite growth. This strategy inhibited virus growth over a wide range of conditions, while minimizing host cell perturbations. Such parasitic strategies may inhibit the development of drug-resistant virus strains.

Allolevivirus↗

PubMed2003

Propagation of viruses on micropatterned host cells.

We have developed a technique to characterize the in vitro propagation of viruses. Microcontact printing was used to generate linear arrays of alkanethiols on gold surfaces, which served as substrates for the patterned culture of baby hamster kidney (BHK-21) cells. Vesicular stomatitis virus (VSV) was added to unpatterned cell reservoirs adjacent to the patterned cells and incubated, setting in motion a continuously advancing viral infection into the patterned cells. At different incubation times, multiple arrays were chemically fixed to stop the viral propagation. Viral propagation distances into the patterned cells were determined by indirect immunofluorescent labeling and visualization of the VSV surface glycoprotein (G). The infection spread at approximately 50 microm/h in the 140-microm lines. Moreover, different temporal stages of the infection process were simultaneously visualized along individual lines. These stages included initiation of infection, based on G protein expression; cell-cell fusion, based on virus-induced clustering of cell nuclei; and cytoskeletal degradation, based on localized release of cells from the surface. This work sets a foundation for parallel, high-throughput characterization of viral and cellular processes.

Animals↗

PubMed2003

Modeling biological systems using Dynetica--a simulator of dynamic networks.

UNLABELLED: We present Dynetica, a user-friendly simulator of dynamic networks for constructing, visualizing, and analyzing kinetic models of biological systems. In addition to generic reaction networks, Dynetica facilitates construction of models of genetic networks, where many reactions are gene expression and interactions among gene products. Further, it integrates the capability of conducting both deterministic and stochastic simulations. AVAILABILITY AND SUPPLEMENTARY INFORMATION: Dynetica 1.0, example models, and the user's guide are available at http://www.its.caltech.edu/~you/Dynetica/Dynetica_page.htm

Algorithms↗