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Modeling virus self-assembly pathways: avoiding dynamics using geometric constraint decomposition.

We develop a model for elucidating the assembly pathways by which an icosahedral viral shell forms from 60 identical constituent protein monomers. This poorly understood process a remarkable example of macromolecular self-assembly occuring in nature and possesses many features that are desirable while engineering self-assembly at the nanoscale. The model uses static geometric and tensegrity constraints to represent the driving (weak) forces that cause a viral shell to assemble and hold it together. The goal is to answer focused questions about the structural properties of a successful assembly pathway. Pathways and their properties are carefully defined and computed using computational algebra and geometry, specifically state-of-art concepts in geometric constraint decomposition. The model is analyzable and refinable and avoids expensive dynamics. We show that it has a provably tractable and accurate computational simulation and that its predictions are roughly consistent with known information about viral shell assembly. Justifications for mathematical and biochemical assumptions are provided, and comparisons are drawn with other virus assembly models. A method for more conclusive experimental validation involving specific viruses is sketched. Overall, the paper indicates a strong and direct, mutually beneficial interplay between (a) the concepts underlying macromolecular assembly; and (b) a wide variety of established as well as novel concepts from combinatorial and computational algebra, geometry and algebraic complexity.

Computer Simulation↗

Accurate reading of morphogen concentrations by nuclear receptors: a formal model of complex transduction pathways.

Signal transduction in development follows multiple, interactive, and overlapping pathways. How does this contribute to accuracy and stability? I show that a formal model of retinoic acid receptors, based on the details of their molecular biology, demonstrates striking precision and robustness while converting a graded morphogen distribution into gene transcription patterns. Thus, transcription can be reliably established in a single row of cells, despite the absence, in the model, of intercellular signalling mechanisms. The subtle interplay of two nuclear receptor types is fundamental for this achievement: one of them ubiquitous, the other controlled itself by morphogen, they act as homodimers or heterodimers, ensuring that many errors cancel out by affecting both activation and repression pathways; regulatory molecular "reservoirs" are also formed. In spite of this robustness, some shifts in gene regulation may well have interesting evolutionary consequences. These conclusions regarding precision in transduction will remain of interest whether retinoic acid turns out to be a morphogen or not, and generalize easily to other experimental situations.

Animals↗

Pre-adolescent chumship as a buffer against psychopathology in adolescents with weak family support and weak parental bonding.

This study examines the degree to which the existence of a pre-adolescent "chum" interacts with family and social environments to buffer mental distress in adolescents. 831 high school students participated in this study, (male: 355; female: 476; mean age 16.7 +/- 1.0). Subjects were administered questionnaires assessing psychopathology and support systems. A pathway analyses model was used to investigate pathways and their interrelationships from chum to psychopathology and from social and family support to psychopathology. Only when adolescents experience weak parental bonding does chumship have a role in buffering distress.

Adolescent↗

Mathematical modeling of the nitric oxide/cGMP pathway in the vascular smooth muscle cell.

The nitric oxide (NO)/cGMP pathway in the vascular smooth muscle cell (VSMC) is an important cellular signaling system for the regulation of VSMC relaxation. We present a mathematical model to investigate the underlying mechanisms of this pathway. The model describes the flow of NO-driven signal transduction: NO activation of soluble guanylate cyclase (sGC), sGC- and phosphodiesterase-catalyzed cGMP production and degradation, cGMP-mediated regulation of protein targets including the Ca2+-activated K+ (KCa) channel, and the myosin contractile system. Model simulations reproduce major NO/cGMP-induced VSMC relaxation effects, including intracellular Ca2+ concentration reduction and Ca2+ desensitization of myosin phosphorylation and force generation. Using the model, we examine several testable principles. 1) Rapid sGC desensitization is caused by end-product cGMP feedback inhibition; a large fraction of the steady-state sGC population is in an inactivated intermediate state, and cGMP production is limited well below maximum. 2) NO activates the K(Ca) channel with both cGMP-dependent and -independent mechanisms; moderate NO concentration affects the K(Ca) via the cGMP-dependent pathway, whereas higher NO concentration is accommodated by a cGMP-independent mechanism. 3) Chronic NO synthase inhibition may cause underexpressions of K+ channels including inward rectifier and K(Ca) channels. 4) Ca2+ desensitization of the contractile system is distinguished from Ca2+ sensitivity of myosin phosphorylation. The model integrates these interactions among the heterogeneous components of the NO signaling system and can serve as a general modeling framework for studying NO-mediated VSMC relaxation under various physiological and pathological conditions. New data can be readily incorporated into this framework for interpretation and possible modification and improvement of the model.

Animals↗

A model for the role of the proline-linked pentose-phosphate pathway in phenolic phytochemical bio-synthesis and mechanism of action for human health and environmental applications.

The combination of immunodeficiency, inflammatory process and nutritional status that is characteristic of infective and food-borne illness is more evident in chronic diet- and environment-influenced chronic diseases such as diabetes, obesity, cardiovascular disease, cancer, arthritis and neuro-degeneration diseases. These chronic diseases tend to be oxidation-linked and may manifest in communities around the world, irrespective of income. In addressing the challenges of the above diseases, a significant role for dietary phytochemicals is emerging. Phytochemicals are required from a spectrum of food for at least their antioxidant role, if not for other properties, to protect tissues from activities that manifest themselves into what we call chronic disease. Among the diverse groups of phytochemicals, phenolic antioxidants and antimicrobials from food plants are being targeted for designed dietary intervention to manage major oxidation-linked diseases such as diabetes, cardiovascular diseases, arthritis, cognition diseases and cancer. Foods containing phenolic phytochemicals are also being targeted to manage bacterial infections associated with chronic diseases such as peptic ulcer, urinary tract infections, dental caries and food-borne bacterial infections. Plants produce phenolic metabolites as a part of growth, developmental and stress adaptation response. These stress and developmental responses are being harnessed to design consistent phytochemical profiles for safety and clinical relevancy using novel tissue culture and bioprocessing technologies. The biochemical strategy for harnessing phenolic phytochemicals for human health and wellness is based on the hypothesis that phenolic metabolites in plants are efficiently produced through an alternative mode of metabolism linking proline synthesis with pentose-phosphate pathway. In this model, stress-induced proline biosynthesis is coupled to pentose-phosphate pathway, driving the synthesis of NADPH(2) and sugar phosphates for anabolic pathways, including phenolic and antioxidant response pathways, while simultaneously providing reducing equivalents needed for mitochondrial oxidative phosphorylation in the form of proline as an alternative to NADH from Krebs/TCA cycle. Based on this model, tissue culture techniques and elicitation concepts have been used to stimulate phenolic metabolites with an antioxidant response in germinating seeds, sprouts and clonal lines of dietary plants. From our initial investigations, a model has been proposed in which the proline-linked pentose-phosphate pathway is suggested to be critical for modulating protective antioxidant response pathways in diverse biological systems, including biochemical and cellular pathways important for human health. The proposed proline-linked pentose-phosphate pathway model provides a mechanism for understanding the mode of action of phenolic phytochemicals in modulating antioxidant pathways and provides avenues by which dietary approaches may manage oxidation-linked chronic and infectious diseases. The model also has implications for the development of antimicrobial phenolic phytochemicals against bacterial pathogens in an era of increasing antibiotic resistance. Further, this model also has relevance for improving fungal and yeast-based food bioprocessing for designing functional foods and for environmental bioremediation using plant and microbial systems, as well as for improving agricultural and food systems in harsh environments.

Antioxidants↗

Metabolic control analysis of monoclonal antibody synthesis.

A general route for protein synthesis in eukaryotic cells has been proposed and applied to monoclonal antibody (MAb) synthesis. It takes into account transcription of the gene, binding of ribosomes to mRNA, and polypeptide elongation including binding to SRP (signal recognition particles) and SRP-receptor, competing translocation, folding and glycosylation, assembly of the heavy and light chains in a tetrameric protein and Golgi processing and secretion. A comprehensive model was built on the basis of the proposed pathway. The model takes into account the mechanism of each step. Metabolic control analysis (MCA) principles were applied to the general pathway using the proposed model, and control coefficients were calculated. The results show a shared flux control (of both pathway flux and flux ratio at the branch) among different steps, i.e., transcription, folding, glycosylation, translocation and building blocks synthesis. The steps sharing the control depend on the concentration of building blocks, pathway flux and levels of OST (oligosacharyl transferase), BiP (heavy chain binding protein) and PDI (protein disulfide isomerase). Model predictions compare well with experimental data for MAb synthesis, explaining the control structure of the route and the heterogeneity of the product and also addressing future targets for improvement of the production rate of MAbs.

Antibodies, Monoclonal↗

Bacterial lactoferrin receptors.

Lactoferrin is thought to play a pivotal role in prevention of infection in the host and its ability to sequester iron from potential pathogens has been considered an important component of its antimicrobial function. A number of bacterial species in the Neisseriaceae have developed a mechanism for acquiring iron directly from this host glycoprotein which involves surface receptors capable of specifically binding lactoferrin. Initial attempts at identifying the receptor proteins in Neisseria and Moraxella species using affinity isolation with immobilized lactoferrin under high stringency conditions presumptively identified a single 100 kDa receptor protein, LbpA (formerly Lbp1). Under modified affinity isolation conditions a second 84 kDa lactoferrin binding protein was isolated and had been presumptively identified as LbpB. This protein was not isolated from a CopB-ve isogenic mutant of Moraxella catarrhalis, indicating that it was in fact CopB. However, another lactoferrin binding protein isolated under high stringency conditions, that comigrated with LbpA in most, but not all, M. catarrhalis strains, was identified by convalescent antisera. Its biochemical properties suggested that it indeed was LbpB. The identity of these proteins was confirmed by preparing isogenic mutants with the lbpA and lbpB genes. Growth studies with isogenic mutants deficient in LbpB, LbpA, CopB or FbpA were performed to evaluate their role in iron acquisition from lactoferrin. LbpA and FbpA were essential for this process, supporting prior models of the iron acquisition pathway. LbpB was not essential which is remniscent of studies with the bacterial transferrin receptors. The isogenic CopB-ve isogenic mutants were deficient in iron acquisition from both transferrin and lactoferrin, suggesting that it is a key component in both pathways. A model providing an alternate explanation of the data is presented. The role and surface accessibility of the lactoferrin receptor proteins suggests that they might be useful vaccine antigens and the preferentially reactivity of convalescent antisera with LbpB suggests that it may be the prime candidate.

Bacteria↗

Electric fields in the human body resulting from 60-Hz contact currents.

Contact currents occur when a person touches conductive surfaces at different potentials and completes a path for current flow through the body. Such currents provide an additional coupling mechanism to that, due to the direct field effect between the human body and low-frequency external fields. The scalar potential finite difference method, with minor modifications, is applied to assess current density and electric field within excitable tissue and bone marrow due to contact current. An anatomically correct adult model is used, as well as a proportionally downsized child model. Three pathways of contact current are modeled: hand to opposite hand and both feet, hand to hand only, and hand to both feet. Because of its larger size relative to the child, the adult model has lower electric field and current-density values in tissues/unit of contact current. For a contact current of 1 mA [the occupational reference level set by the International Commission on Non-ionizing Protection (ICNIRP)], the current density in brain does not exceed the basic restriction of 10 mA/m2. The restriction is exceeded slightly in the spine, and by a factor of more than 2 in the heart. For a contact current of 0.5 mA (ICNIRP general public reference level), the basic restriction of 2 mA/m2 is exceeded several-fold in the spine and heart. Several microamperes of contact current produces tens of mV/m within the child's lower arm bone marrow.

Adult↗

BioMiner--modeling, analyzing, and visualizing biochemical pathways and networks.

MOTIVATION: Understanding the biochemistry of a newly sequenced organism is an essential task for post-genomic analysis. Since, however, genome and array data grow much faster than biochemical information, it is necessary to infer reactions by comparative analysis. No integrated and easy to use software tool for this purpose exists as yet. RESULTS: We present a new software system--BioMiner--for analyzing and visualizing biochemical pathways and networks. BioMiner is based on a new comprehensive, extensible and reusable data model--BioCore--which can be used to model biochemical pathways and networks. As a first application we present PathFinder, a new tool predicting biochemical pathways by comparing groups of related organisms based on sequence similarity. We successfully tested PathFinder with a number of experiments, e.g. the well studied glycolysis in bacteria. Additionally, an application called PathViewer for the visualization of metabolic networks is presented. PathViewer is the first application we are aware of which supports the graphical comparison of metabolic networks of different organisms. AVAILABILITY: http://www.zbi.uni-saarland.de/chair/projects/BioMiner SUPPLEMENTARY INFORMATION: Additional information on experimental results can be found on our web site.

Biochemistry↗

Nitrogen-bisphosphonates block retinoblastoma phosphorylation and cell growth by inhibiting the cholesterol biosynthetic pathway in a keratinocyte model for esophageal irritation.

The surprising discovery that nitrogen-containing bisphosphonates (N-BPs) act via inhibition of the mevalonate-to-cholesterol pathway raised the possibility that esophageal irritation by N-BPs is mechanism-based. We used normal human epidermal keratinocytes (NHEKs) to model N-BP effects on stratified squamous epithelium of the esophagus. The N-BPs alendronate and risedronate inhibited NHEK growth in a dose-dependent manner without inducing apoptosis. N-BPs (30 microM) caused accumulation of cells in S phase and increased binucleation (inhibited cytokinesis). Consistent with N-BP inhibition of isoprenylation, geranylgeraniol or farnesol prevented accumulation in S phase. Binucleation was also induced by the 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor lovastatin and by the squalene synthase inhibitor zaragozic acid A and was prevented by adding low-density lipoprotein. At 300 microM, N-BPs reduced expression of cyclin-dependent kinase (cdk) 2 and cdk4 and enhanced expression of p21(waf1) and p27(kip1) and their binding to cdks with corollary hypophosphorylation of retinoblastoma. Lovastatin and zaragozic acid A produced similar effects, except that p21(waf1) expression and binding to cdks was not induced. Growth inhibition, but not binucleation, was also caused by the geranylgeranyl transferase I inhibitor, GGTI-298, which also enhanced cdk2 and cdk4 association with p27(kip1). These findings are consistent with suppression of epithelial cell growth by N-BPs via inhibition of the mevalonate pathway and the consequent reduction in cholesterol synthesis, which blocks cytokinesis, and in geranylgeranylation, which interferes with progression through the cell cycle.

Cell Cycle↗

Suicide: a four-pathway clinical-biochemical model.

This chapter, based on a review of recent research as well as data presented in this report, proposes four hypothetical pathways leading to suicide in clinical depression: (1) an acute pathway involving severe anxiety/agitation associated with high brain corticotrophin-releasing factor (CRF or CRH) levels, (2) trait baseline and reactivity hopelessness, (3) severe anhedonia, and (4) trait impulsiveness associated with low brain serotonin turnover and low total cholesterol as a possible peripheral correlate. Clinical research showing evidence for acute versus chronic high-risk suicide factors and other studies linking severe anxiety/agitation to high CRF levels will be presented as associated with acute suicidal risk, which is potentially reversible with recognition and treatment. Evidence for anhedonia severity as a risk factor and trait, as well as evidence that baseline hopelessness and sensitivity are traits related to chronic suicide risk, will be presented. Finally, evidence relating low serum cholesterol to suicide in depressed inpatients will be presented in the context of literature suggesting a relationship between low serum cholesterol and violent death and suicide in population studies. Data suggesting a relationship between low serum cholesterol and decreased CSF 5-HIAA, suggesting reduced serotonin turnover, will be presented, in light of prior studies relating low CSF 5-HIAA and violent suicide. These data taken together suggest four pathways to suicide that are worth investigating in order to better understand the mechanisms leading to this behavior. Future possibilities and applications of these findings are discussed.

Cholesterol↗

Organ slice viability extended for pathway characterization: an in vitro model to investigate fibrosis.

Liver slice viability is extended to 96 h for rat, expanding the use of this in vitro model for studying mechanisms of injury and repair, including pathways of fibrosis. The contributing factors to increased organ slice survival consist of the use of a preservation solution for liver perfusion and slice preparation, obtaining rats that are within the weight range of 250-325 g, placing a cellulose filter atop the titanium mesh roller-insert to support the slice, and maintaining the slices in an optimized culture medium which is replaced daily. The liver slices remain metabolically active, synthesizing adenosine triphosphate (ATP), glutathione, and glycogen, and exhibit preserved organelle integrity and slice morphology. Slice preparation results in 2-cut surfaces which likely triggers a repair and regenerative response. The fibrogenic pathways are evident by the activation of stellate cells, the proliferation of myofibroblast-like cells, and an increased collagen deposition by 48 h. Markers indicative of activated stellate cells, alpha-smooth muscle actin, collagen 1a1, desmin, and HSP47 are substantiated by real time-PCR. Increased staining of alpha-smooth muscle actin initially around the vessels and by 72-96 h in the tissue is accompanied by increased collagen staining. Microarray gene expression revealed extracellular matrix changes with the up-regulation of cytoskeleton, filaments, collagens, and actin genes; and the down-regulation of genes linked with lipid metabolism. The improvements in extending liver slice survival, in conjunction with its three-dimensional multi-cellular complexity, increases the application of this in vitro model for investigating pathways of injury and repair, and fibrosis.

Adenosine Triphosphate↗

Modelling the dynamics of the yeast pheromone pathway.

We present a mathematical model of the dynamics of the pheromone pathways in haploid yeast cells of mating type MATa after stimulation with pheromone alpha-factor. The model consists of a set of differential equations and describes the dynamics of signal transduction from the receptor via several steps, including a G protein and a scaffold MAP kinase cascade, up to changes in the gene expression after pheromone stimulation in terms of biochemical changes (complex formations, phosphorylations, etc.). The parameters entering the models have been taken from the literature or adapted to observed time courses or behaviour. Using this model we can follow the time course of the various complex formation processes and of the phosphorylation states of the proteins involved. Furthermore, we can explain the phenotype of more than a dozen well-characterized mutants and also the graded response of yeast cells to varying concentrations of the stimulating pheromone.

Cell Cycle Proteins↗

From the lab to the clinic: activation of an alpha-2 agonist pathway is neuroprotective in models of retinal and optic nerve injury.

PURPOSE: The selective alpha-2 agonist brimonidine was used as a pharmacological probe to activate alpha 2 receptor-mediated neuroprotective signaling pathways and quantitate the enhancement of retinal ganglion cell survival and function in animals with ischemic retinal and optic nerve injury. METHODS: Two animal models were used to achieve different methods of neuronal insult. The first model involved mechanical injury of the rat optic nerve after treatment with a single intraperitoneal (i.p.) dose of brimonidine or a control vehicle. The second model involving acute retinal ischemic/reperfusion injury was used in a variety of experiments in which rats were treated with either intraperitoneal brimonidine or single dose topical brimonidine at various strengths. In all cases retinal ischemia was induced and maintained followed by reperfusion. In some cases, TUNEL staining was performed on histologic sections of the retinas of rats that had been sacrificed after 24 hours. To examine the activation of neuronal survival pathways at the molecular level, rats were injected with i.p. brimonidine followed by the isolation of mRNAs from whole retinas 24 hours after ischemic injury. RESULTS: Intraperitoneal brimonidine enhanced rat RGC survival and function in the partial crush injury model, and neuroprotection was dose-dependent. Topical application of brimonidine 1 hour before injury was effective in decreasing ischemic retinal injury. Ischemic retinas treated with brimonidine resulted with a large decrease in TUNEL staining. CONCLUSIONS: Treatment with the alpha 2 adrenoreceptor agonist brimonidine was found to confer neuroprotection to retinal ganglion cells in two distinct models of neuronal injury resulting from acute retinal ischemia/reperfusion and calibrated optic nerve compression.

Adrenergic alpha-Agonists↗

Overview of the actions of glucocorticoids on the immune response: a good model to characterize new pathways of immunosuppression for new treatment strategies.

Glucocorticoids have been used for over 50 years in the treatment of inflammatory and autoimmune diseases and in preventing graft rejection. Today, knowledge of their molecular, cellular, and pharmacological properties allows a better understanding of glucocorticoid-mediated immunosuppression. Glucocorticoids exert both negative and positive effects with a dynamic and bi-directional spectrum of activities on various limbs and components of the immune response. They modulate genes involved in the priming of the innate immune response, while their actions on the adaptive immune response are to suppress cellular (Th1) immunity and promote humoral (Th2) immunity. Interestingly, glucocorticoids can also induce tolerance to specific antigens by influencing dendritic cell maturation and function and promoting the development of regulatory high IL-10-producing T cells. The ex vivo therapeutic use of glucocorticoids could therefore represent an adjuvant treatment to cell therapy in autoimmune diseases, avoiding the long-term deleterious adverse effects of glucocorticoids. Thus, the panoramic view of glucocorticoid actions on the immune system provides an interesting model for characterizing important biological pathways of immunosuppression.

Animals↗

Coronary artery spasm does not depend on the intracellular calcium store but is substantially mediated by the protein kinase C-mediated pathway in a swine model with interleukin-1 beta in vivo.

BACKGROUND: The intracellular mechanism for coronary artery spasm is still unknown. Since the protein kinase C (PKC)-mediated pathway and Ca2+ release from sarcoplasmic reticulum (SR) are important intracellular mechanisms of vascular smooth muscle contraction, we examined the possible role of these two mechanisms in the pathogenesis of coronary spasm in our swine model in vivo. METHODS AND RESULTS: In 25 pigs, interleukin-1 beta (IL-1 beta) was applied chronically to the coronary arteries from the adventitia to induce an inflammatory/proliferative lesion. Two weeks after the operation, either intracoronary serotonin or histamine repeatedly induced coronary spasm at the IL-1 beta-treated site. At those spastic sites, phorbol-12, 13-dibutyrate, a PKC-activating phorbol ester, also induced coronary spasm, which was blocked by pretreatment with the PKC inhibitors staurosporine and sphingosine. Serotonin- and histamine-induced coronary spasm was also significantly inhibited by pretreatment with staurosporine, sphingosine, or nifedipine (an L-type Ca2+ channel antagonist) but not by ryanodine (an inhibitor of Ca(2+)-induced Ca2+ release from SR) or thapsigargin (an inhibitor of Ca(2+)-ATPase of SR). Bay K 8644 (an L-type Ca2+ channel agonist) also induced coronary spasm at the IL-1 beta-treated site, which was significantly inhibited by pretreatment with staurosporine, sphingosine, and nifedipine. In contrast, coronary vasoconstriction induced by prostaglandin F2 alpha was not affected by pretreatment with staurosporine or sphingosine but was significantly inhibited by pretreatment with ryanodine, thapsigargin, or nifedipine. CONCLUSIONS: These results suggest that (1) PKC activation largely accounts for the serotonin- and histamine-induced coronary spasm; (2) at the spastic site, the calcium influx through L-type Ca2+ channels may be augmented via the PKC-mediated pathway; and (3) the Ca2+ release from the SR into the cytosol may not play a primary role in coronary spasm.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

Parent and child expressed affect and children's social competence: modeling direct and indirect pathways.

This study examined links between parents' and children's expressed affect during parent-child play and children's social functioning with peers. A total of 116 kindergarten-age children and their parents (114 mothers, 102 fathers) were observed during physical play interactions and were coded on global measures of expressed positive and negative affect. Kindergarten and 1st-grade teachers and peers provided measures of social competence. Latent variable path analysis with partial least squares was used to examine models that included "direct" and "indirect" pathways. Relations between parental positive affect and children's social competence were mediated by children's expressed positive affect. Parental negative affect was associated with negative social outcomes in children; however, these relations were not mediated by children's negative expressions. The strongest support for the hypothesized models was found in same-sex dyads.

Affect↗

South African adolescents: pathways to risky sexual behavior.

This study tested a developmental model of pathways to risky sexual behavior among South African adolescents. Participants comprised 633 adolescents, 12-17 years old, recruited from households in Durban, South Africa. Data were collected using in-person interviews. Topics included adolescents' sexual behaviors, household poverty levels, vulnerable personality and behavioral attributes, parent-child relations, and deviant peers. Structural equation modeling was used to assess the pathways to risky sexual behavior among the adolescents. The goodness-of-fit index (GFI) was .93. One major pathway indicated that family poverty was associated with difficulty in the parent-child relationship. This was related to vulnerable personality and behavioral attributes and to association with deviant peers, which, in turn, were related to risky sexual behavior. Findings suggest that poverty, parent-child relations, personality and behavioral vulnerabilities, and peer influences should be among factors addressed by prevention and intervention programs to reduce sexual risk behaviors by South African adolescents.

Adolescent↗