Impact of intra- and interspecies variation of occludin on its function as coreceptor for authentic hepatitis C virus particles

Hepatitis C virus (HCV) is characterized by a narrow host range and high interindividual variability in the clinical course of infection. Both of these traits are thought to be largely due to genetic variation between species and between individual hosts. The tight junction component occludin (OCLN) is essential for HCV entry into host cells, and the differences between human and murine OCLN are thought to account in part for the inability of HCV to infect mice and hence preclude their use as a convenient small-animal model. This study assesses the impact of genetic variation in OCLN on cell culture-grown HCV (HCVcc) using a newly generated and characterized OCLN(low) subclone of the Huh-7.5 cell line (Huh-7.5 subclone in which endogenous OCLN expression has been downregulated by a short hairpin RNA). We report the frequency of coding nonsynonymous single nucleotide polymorphisms, i.e., polymorphisms resulting in amino acid exchanges, present in the human population and determine their ability to function as HCV (co)receptors. Moreover, we show that murine OCLN can sustain HCVcc entry, albeit with about 5-fold reduced efficiency compared to that of human OCLN. This reduction in efficiency is due solely to two amino acid residues previously identified by others using an HCV pseudoparticle approach. Finally, we use the Huh-7.5/OCLN(low) cell line to show that HCV spread between neighboring cells is strictly dependent on OCLN.     

The mechanism of high Mr thioredoxin reductase from Drosophila melanogaster

Drosophila melanogaster thioredoxin reductase-1 (DmTrxR-1) is a key flavoenzyme in dipteran insects, where it substitutes for glutathione reductase. DmTrxR-1 belongs to the family of dimeric, high Mr thioredoxin reductases, which catalyze reduction of thioredoxin by NADPH. Thioredoxin reductase has an N-terminal redox-active disulfide (Cys57-Cys62) adjacent to the flavin and a redox-active C-terminal cysteine pair (Cys489'-Cys490' in the other subunit) that transfer electrons from Cys57-Cys62 to the substrate thioredoxin. Cys489'-Cys490' functions similarly to Cys495-Sec496 (Sec = selenocysteine) and Cys535-XXXX-Cys540 in human and parasite Plasmodium falciparum enzymes, but a catalytic redox center formed by adjacent Cys residues, as observed in DmTrxR-1, is unprecedented. Our data show, for the first time in a high Mr TrxR, that DmTrxR-1 oscillates between the 2-electron reduced state, EH2, and the 4-electron state, EH4, in catalysis, after the initial priming reduction of the oxidized enzyme (Eox) to EH2. The reductive half-reaction consumes 2 eq of NADPH in two observable steps to produce EH4. The first equivalent yields a FADH--NADP+ charge-transfer complex that reduces the adjacent disulfide to form a thiolate-flavin charge-transfer complex. EH4 reacts with thioredoxin rapidly to produce EH2. In contrast, Eox formation is slow and incomplete; thus, EH2 of wild-type cannot reduce thioredoxin at catalytically competent rates. Mutants lacking the C-terminal redox center, C489S, C490S, and C489S/C490S, are incapable of reducing thioredoxin and can only be reduced to EH2 forms. Additional data suggest that Cys57 attacks Cys490' in the interchange reaction between the N-terminal dithiol and the C-terminal disulfide.     

A structured approach for the engineering of biochemical network models, illustrated for signalling pathways

Quantitative models of biochemical networks (signal transduction cascades, metabolic pathways, gene regulatory circuits) are a central component of modern systems biology. Building and managing these complex models is a major challenge that can benefit from the application of formal methods adopted from theoretical computing science. Here we provide a general introduction to the field of formal modelling, which emphasizes the intuitive biochemical basis of the modelling process, but is also accessible for an audience with a background in computing science and/or model engineering. We show how signal transduction cascades can be modelled in a modular fashion, using both a qualitative approach--qualitative Petri nets, and quantitative approaches--continuous Petri nets and ordinary differential equations (ODEs). We review the major elementary building blocks of a cellular signalling model, discuss which critical design decisions have to be made during model building, and present a number of novel computational tools that can help to explore alternative modular models in an easy and intuitive manner. These tools, which are based on Petri net theory, offer convenient ways of composing hierarchical ODE models, and permit a qualitative analysis of their behaviour. We illustrate the central concepts using signal transduction as our main example. The ultimate aim is to introduce a general approach that provides the foundations for a structured formal engineering of large-scale models of biochemical networks.     

Effects of eCG and FSH on ovarian response, recovery rate and number and quality of oocytes obtained by ovum pick-up in Holstein cows

The goal of the present study was to compare the ovarian response, oocyte yields per animal, and the morphological quality of oocytes collected by ultrasound guided follicular aspiration from Holstein cows treated either with FSH or eCG. Twenty four normal cyclic, German Holstein cows were randomly divided into two groups. Fourteen cows received 3000 IU eCG on day-4 prior to ovum pick-up (OPU) (day 0), 2 days later (day-2), 625 microg cloprostenol was administered. On day-1 GnRH was administered i.m. and 24h later OPU (day 0) was performed. In ten cows a total dose of 500 IU follicle stimulating hormone (Pluset) was administered intramuscularly in a constant dosage for 4 days with intervals of 12h, starting on day-5. Luteolysis was induced by application of 625 microg cloprostenol on day-2. On day-1 (24h after the last FSH treatment) GnRH was administered i.m. and 24h later OPU (day 0) was performed. Ovarian follicles were visualized on the ultrasound monitor, counted and recorded. All visible antral follicles were punctured. Recovered oocytes were graded morphologically based on the cumulus investment. Average follicle number in ovaries was higher in FSH group than eCG group (p<0.05). Oocyte yields per animal did not differ between FSH and eCG groups. The proportion of grade A oocytes was higher in the FSH group in the than eCG group (p<0.05). Likewise, rate of grade C oocytes in FSH group were lower than eCG group (p<0.05). In conclusion, these results suggest that ovarian response, follicle number in ovaries and oocyte quality are affected by the type of gonadotropin and FSH is better alternative than eCG for OPU treatment.     

Selective permeability of different connexin channels to the second messenger cyclic AMP

Gap junctions are intercellular conduits that are formed in vertebrates by connexin proteins and allow diffusion exchange of intracellular ions and small molecules. At least 20 different connexin genes in the human and mouse genome are cell-type specifically expressed with overlapping expression patterns. A possible explanation for this diversity could be different permeability of biologically important molecules, such as second messenger molecules. We have recently demonstrated that cyclic nucleotide-gated channels can be used to quantify gap junction-mediated diffusion of cyclic AMP. Using this method we have compared the relative permeability of gap junction channels composed of connexin 26, 32, 36, 43, 45, or 47 proteins toward the second messenger cAMP. Here we show that cAMP permeates through the investigated connexin channels with up to 30-fold different efficacy. Our results suggest that intercellular cAMP signaling in different cell types can be affected by the connexin expression pattern.     

A unique transformation from ordinary differential equations to reaction networks

Many models in Systems Biology are described as a system of Ordinary Differential Equations, which allows for transient, steady-state or bifurcation analysis when kinetic information is available. Complementary structure-related qualitative analysis techniques have become increasingly popular in recent years, like qualitative model checking or pathway analysis (elementary modes, invariants, flux balance analysis, graph-based analyses, chemical organization theory, etc.). They do not rely on kinetic information but require a well-defined structure as stochastic analysis techniques equally do. In this article, we look into the structure inference problem for a model described by a system of Ordinary Differential Equations and provide conditions for the uniqueness of its solution. We describe a method to extract a structured reaction network model, represented as a bipartite multigraph, for example, a continuous Petri net (CPN), from a system of Ordinary Differential Equations (ODEs). A CPN uniquely defines an ODE, and each ODE can be transformed into a CPN. However, it is not obvious under which conditions the transformation of an ODE into a CPN is unique, that is, when a given ODE defines exactly one CPN. We provide biochemically relevant sufficient conditions under which the derived structure is unique and counterexamples showing the necessity of each condition. Our method is implemented and available; we illustrate it on some signal transduction models from the BioModels database. A prototype implementation of the method is made available to modellers at http://contraintes.inria.fr/~soliman/ode2pn.html, and the data mentioned in the "Results" section at http://contraintes.inria.fr/~soliman/ode2pn_data/. Our results yield a new recommendation for the import/export feature of tools supporting the SBML exchange format.     

Cation-exchange chromatography of monoclonal antibodies: Characterization of a novel stationary phase designed for production-scale purification

A novel cation-exchange resin, Eshmuno™ S, was compared to Fractogel^® SO₃⁻ (M) and Toyopearl GigaCap S-650M. The stationary phases have different base matrices and carry specific types of polymeric surface modifications. Three monoclonal antibodies (mAbs) were used as model proteins to characterize these chromatographic resins. Results from gradient elutions, stirred batch adsorptions and confocal laser scanning microscopic investigations were used to elucidate binding behavior of mAbs onto Eshmuno™ S and Fractogel^® SO₃⁻ and the corresponding transport mechanisms on these two resins. The number of charges involved in mAb binding for Eshmuno™ S is lower than for Fractogel^® SO₃⁻, indicating a slightly weaker electrostatic interaction. Kinetics from batch uptake experiments are compared to kinetic data obtained from confocal laser scanning microscopy images. Both experimental approaches show an accelerated protein adsorption for the novel stationary phase. The influence of pH, salt concentrations and residence times on dynamic binding capacities was determined. A higher dynamic binding capacity for Eshmuno™ S over a wider range of pH values and residence times was found compared to Fractogel^® SO₃⁻ and Toyopearl GigaCap S-650M. The capture of antibodies from cell culture supernatant, as well as post-protein A eluates, were analyzed with respect to their host cell protein (hcp) removal capabilities. Comparable or even better hcp clearance was observed at much higher protein loading for Eshmuno™ S than Fractogel^® SO₃⁻ or Toyopearl GigaCap S-650M.Key words: ion-exchange chromatography, dynamic binding capacity, tentacle surface modification, linear gradient elution, hcp removal     

Dietary Determinants of Changes in Waist Circumference Adjusted for Body Mass Index – a Proxy Measure of Visceral Adiposity

Background

Given the recognized health effects of visceral fat, the understanding of how diet can modulate changes in the phenotype “waist circumference for a given body mass index (WC_BMI)”, a proxy measure of visceral adiposity, is deemed necessary. Hence, the objective of the present study was to assess the association between dietary factors and prospective changes in visceral adiposity as measured by changes in the phenotype WC_BMI.

Methods and Findings

We analyzed data from 48,631 men and women from 5 countries participating in the European Prospective Investigation into Cancer and Nutrition (EPIC) study. Anthropometric measurements were obtained at baseline and after a median follow-up time of 5.5 years. WC_BMI was defined as the residuals of waist circumference regressed on body mass index, and annual change in WC_BMI (ΔWC_BMI, cm/y) was defined as the difference between residuals at follow-up and baseline, divided by follow-up time. The association between energy, energy density (ED), macronutrients, alcohol, glycemic index (GI), glycemic load (GL), fibre and ΔWC_BMI was modelled using centre-specific adjusted linear regression, and random-effects meta-analyses to obtain pooled estimates. Men and women with higher ED and GI diets showed significant increases in their WC_BMI, compared to those with lower ED and GI [1 kcal/g greater ED predicted a ΔWC_BMI of 0.09 cm (95% CI 0.05 to 0.13) in men and 0.15 cm (95% CI 0.09 to 0.21) in women; 10 units greater GI predicted a ΔWC_BMI of 0.07 cm (95% CI 0.03 to 0.12) in men and 0.06 cm (95% CI 0.03 to 0.10) in women]. Among women, lower fibre intake, higher GL, and higher alcohol consumption also predicted a higher ΔWC_BMI.

Conclusions

Results of this study suggest that a diet with low GI and ED may prevent visceral adiposity, defined as the prospective changes in WC_BMI. Additional effects may be obtained among women of low alcohol, low GL, and high fibre intake.     

Food composition of the diet in relation to changes in waist circumference adjusted for body mass index

Background

Dietary factors such as low energy density and low glycemic index were associated with a lower gain in abdominal adiposity. A better understanding of which food groups/items contribute to these associations is necessary.

Objective

To ascertain the association of food groups/items consumption on prospective annual changes in “waist circumference for a given BMI” (WC_BMI), a proxy for abdominal adiposity.

Design

We analyzed data from 48,631 men and women from 5 countries participating in the European Prospective Investigation into Cancer and Nutrition (EPIC) study. Anthropometric measurements were obtained at baseline and after a median follow-up time of 5.5 years. WC_BMI was defined as the residuals of waist circumference regressed on BMI, and annual change in WC_BMI (ΔWC_BMI, cm/y) was defined as the difference between residuals at follow-up and baseline, divided by follow-up time. The association between food groups/items and ΔWC_BMI was modelled using centre-specific adjusted linear regression, and random-effects meta-analyses to obtain pooled estimates.

Results

Higher fruit and dairy products consumption was associated with a lower gain in WC_BMI whereas the consumption of white bread, processed meat, margarine, and soft drinks was positively associated with ΔWC_BMI. When these six food groups/items were analyzed in combination using a summary score, those in the highest quartile of the score – indicating a more favourable dietary pattern –showed a ΔWC_BMI of −0.11 (95% CI −0.09 to −0.14) cm/y compared to those in the lowest quartile.

Conclusion

A dietary pattern high in fruit and dairy and low in white bread, processed meat, margarine, and soft drinks may help to prevent abdominal fat accumulation.