Lectin-like oxidized low-density lipoprotein (LDL) receptor-1 (LOX-1), a receptor for oxidized-LDL, is up-regulated in activated endothelial cells, and it plays a role in atherothrombosis. However, its role in platelet aggregation is unclear. Both aspirin and HMG CoA reductase inhibitors (statins) reduce LOX-1 expression in endothelial cells. In this study, we investigated the effect of aspirin and pravastatin on LOX-1 expression on platelets. After ADP stimulation, mean fluorescence intensity of LOX-1 expression on platelets increased 1.5-to 2.0-fold. Blocking LOX-1 inhibited ADP-induced platelet aggregation in a concentration-and time-dependent manner. We also established that LOX-1 is important for ADP-stimulated inside-out activation of platelet ␣ IIb ␤ 3 and ␣ 2 ␤ 1 integrins (fibrinogen receptors). The specificity of this interaction was determined by arginine-glycine-aspartate-peptide inhibition. Furthermore, we found that LOX-1 inhibition of integrin activation is mediated by inhibition of protein kinase C activity. In other experiments, treatment with aspirin (1-10 mM) and pravastatin (1-5 M) reduced platelet LOX-1 expression, with a synergistic effect of the combination of aspirin and pravastatin. Aspirin and pravastatin both reduced reactive oxygen species (ROS) released by activated platelets measured as malonyldialdehyde (MDA) release and nitrate/nitrite ratio. Aspirin and pravastatin also enhanced nitric oxide (NO) release measured as nitrite/nitrite ϩ nitrate (NOx) ratio in platelet supernates. Small concentrations of aspirin and pravastatin had a synergistic effect on the inhibition of MDA release and enhancement of nitrite/NOx. Thus, LOX-1 is important for ADP-mediated platelet integrin activation, possibly through protein kinase C activation. Furthermore, aspirin and pravastatin inhibit LOX-1 expression on platelets in part by favorably affecting ROS and NO release from activated platelets.

Safety assessment of genetically modified organisms (GMOs) is a contentious topic. Proponents of GMOs assert that GMOs are safe since the FDA's policy of substantial equivalence considers GMOs "equivalent" to their non-GMO counterparts, and argue that genetic modification (GM) is simply an extension of a "natural" process of plant breeding, a form of "genetic modification", though done over longer time scales. Anti-GMO activists counter that GMOs are unsafe since substantial equivalence is unscientific and outdated since it originates in the 1970s to assess safety of medical devices, which are not comparable to the complexity of biological systems, and contend that targeted GM is not plant breeding. The heart of the debate appears to be on the methodology used to determine criteria for substantial equivalence. Systems biology, which aims to understand complexity of the whole organism, as a system, rather than just studying its parts in a reductionist manner, may provide a framework to determine appropriate criteria, as it recognizes that GM, small or large, may affect emergent properties of the whole system. Herein, a promising computational systems biology method couples known perturbations on five biomolecules caused by the CP4 EPSPS GM of Glycine max L. (soybean), with an integrative model of C1 metabolism and oxidative stress (two molecular systems critical to plant function). The results predict significant accumulation of formaldehyde and concomitant depletion of glutathione in the GMO, suggesting how a "small" and single GM creates "large" and systemic perturbations to molecular systems equilibria. Regulatory agencies, currently reviewing rules for GMO safety, may wish to adopt a systems biology approach using a combination of in silico, computational methods used herein, and subsequent targeted experimental in vitro and in vivo designs, to develop a systems understanding of "equivalence" using biomarkers, such as formaldehyde and glutathione, which predict metabolic disruptions, towards modernizing the safety assessment of GMOs.

Computational systems biology approaches provide insights to understand complex molecular phenomena in living systems. Such understanding demands the need to systematically interrogate and review existing literature to refine and distil key molecular pathways. This paper explores a methodological process to identify key molecular pathways from systematic bioinformatics literature review. This process is used to identify molecular pathways for a ubiquitous molecular process in all plant biological systems: C1 metabolism and formaldehyde detoxification, specific to maize. The C1 metabolism is essential for all organisms to provide one-carbon units for methylation and other types of modifications, as well as for nucleic acid, amino acid, and other biomolecule syntheses. Formaldehyde is a toxic one-carbon molecule which is produced endogenously and found in the environment, and whose detoxification is an important part of C1 metabolism. This systematic review involves a five-part process: 1) framing of the research question; 2) literature collection based on a parallel search strategy; 3) relevant study selection based on search refinement; 4) molecular pathway identification; and 5) integration of key molecular pathway mechanisms to yield a well-defined set molecular systems associated with a particular biochemical function. Findings from this systematic review produced three main molecular systems: a) methionine biosynthesis; b) the methylation cycle; and c) formaldehyde detoxification. Specific insights from the resulting molecular pathways indicate that normal C1 metabolism involves the transfer of a * Corresponding author. P. Deonikar et al.572 carbon group from serine through a folate-mediated pathway to methionine, and eventually the methylation of a biomolecule. In photosynthetic tissues, C1 metabolism often proceeds in reverse towards serine biosynthesis and formate oxidation. C1 metabolism, in maize, appears to be present in the developing embryo and endosperm indicating that these cells are vulnerable to perturbations in formaldehyde detoxification. These insights demonstrate the value of a systematic bioinformatics literature review process from a broad spectrum of domain literature to specific and relevant molecular pathways.

An integrative computational, in silico, model of C1 metabolism is developed from molecular pathway systems identified from a recent, comprehensive systematic bioinformatics review of C1 metabolism. C1 metabolism is essential for all organisms to provide one-carbon units for methylation and other types of modifications, as well as for nucleic acid, amino acid, and other biomolecule syntheses. C1 metabolism consists of three important molecular pathway systems: 1) methionine biosynthesis, 2) methylation cycle, and 3) formaldehyde detoxification. Each of the three molecular pathway systems is individually modeled using the CytoSolve ® Collaboratory™, a proven and scalable computational systems biology platform for in silico modeling of complex molecular pathway systems. The individual models predict the temporal behavior of formaldehyde, formate, sarcosine, glutathione (GSH), and many other key biomolecules involved in C1 metabolism, which may be hard to measure experimentally. The individual models are then coupled and integrated dynamically using CytoSolve to produce, to the authors' knowledge, the first comprehensive computational model of C1 metabolism. In silico modeling of the individual and integrated C1 metabolism models enables the identification of the most sensitive parameters involved in the detoxification of formaldehyde. This integrative model of C1 metabolism, giving its systems-based nature, can likely serve as a platform for: 1) generalized research and study of C1 metabolism, 2) hypothesis generation that motivates focused and specific in vitro and in vivo testing in perhaps a more efficient manner, 3) expanding a systems biology understanding of plant bio-molecular systems by integrating other known molecular pathway systems associated with C1 metabolism, and 4) exploring and testing the potential effects of exogenous inputs on the C1 metabolism system.

This research provides, to the authors' knowledge, the first integrative model of oxidative stress and C1 metabolism in plants. Increased oxidative stress can cause irreversible damage to photosynthetic components and is harmful to plants. Perturbations at the genetic level may increase oxidative stress and upregulate antioxidant systems in plants. One of the key mechanisms involved in oxidative stress regulation is the ascorbate-glutathione cycle which operates in chloroplasts as well as the mitochondria and is responsible for removal of reactive oxygen species (ROS) generated during photosynthetic operations and respiration. In this research, the complexity of molecular pathway systems of oxidative stress is modeled and then integrated with a previously developed in silico model of C1 metabolism system. This molecular systems integration provides two important results: 1) demonstration of the scalability of the CytoSolve ® Collaboratory™, a computational systems biology platform that allows for modular integration of molecular pathway models, by coupling the in silico model of oxidative stress with the in silico model of C1 metabolism, and 2) derivation of new insights on the effects of oxidative stress on C1 metabolism relative to formaldehyde (HCHO), a toxic molecule, and glutathione (GSH), an important indicator of oxidative homeostasis in living systems. Previous in silico modeling of C1 metabolism, without oxidative stress, observed complete removal of formaldehyde via formaldehyde detoxification pathway and no change in glutathione concentrations. The results from this research of integrative oxidative stress with C1 metabolism, however, demonstrate significant upregulation of formaldehyde concentrations, with concomitant downregulation and depletion of glutathione. Sensitivity analysis indicates that kGSH-HCHO, the rate constant of GSH-HCHO binding, VSHMT, the rate of formation of sarcosine from glycine, and

In blood vessels, nitric oxide homeostasis is maintained by its formation by endothelial nitric oxide synthase and its consumption in smooth muscle cells and in vascular lumen by red blood cell (RBC) encapsulated hemoglobin (Hb). Free hemoglobin has a very high reaction rate (k(Hb-NO) approximately 10(7) M(-1) s(-1)) with NO as compared to RBC-Hb. Mechanisms of reduced NO uptake by RBC-Hb has been extensively studied in recent years. A critical factor in the investigation of NO-RBC interactions is delivery of NO. Common NO delivery methods include use of NO donors and bolus saturated NO solutions, which delivers NO homogeneously and only in the vicinity of bolus, respectively. In this study, we developed a flow system that uses gaseous delivery of NO through a polymeric semi-permeable membrane to obtain precise and uniform NO concentrations for NO-RBC interactions. We conducted experiments using the flow system to study the effect of NO concentrations, hematocrit and RBC suspension flow rates on NO-RBC interactions. We developed a computational model to simulate NO transport and to estimate the reaction rate constant for NO-RBC interaction in the flow system. Our results showed that NO consumption of RBCs (i) increased linearly with an increase in available NO, and (ii) decreased with increase in RBCs suspension flow rate. We estimated the reaction rate constant for NO-RBC interactions to be 0.2 x 10(5) M(-1) s(-1) which is approximately 1250-fold lower than NO consumption by free hemoglobin and approximately 2.5-20 fold slower than reported NO-RBC reaction rate.

Nitric oxide (NO) is a potent vasodilator and its homeostasis depends on interaction with RBCs. A key factor in understanding NO-RBC interactions in vascular lumen is a comprehensive analysis of product identification and quantification. In this context, administration of NO during in vitro NO-RBC interactions becomes a crucial variable. In this study, we designed a bioreactor that maintains a precise NO concentration in the headspace that diffuses to RBCs suspension to study the quantitative effect of NO concentration and hematocrit (Hct) on NO-RBC interactions. The products of NO-RBC reaction (nitrite and total nitrogen species (total NOx)) were measured by chemiluminescence assay. A mathematical model simulating NO biotransport to a single RBC was developed to 1) estimate NO-RBC reaction rate constant; 2) predict the NO concentrations in the bulk RBC suspension and at the RBC membrane for RBC membrane NO permeability (P m ) values of 0.0415-40 cm/s. Measured nitrite and total NOx concentrations increased with increase in headspace NO concentration whereas nitrite concentrations decreased with hematocrit and total NOx concentrations increased with hematocrit. This indicates that the extracellular resistance is a controlling factor for RBC uptake of NO. Modeling results showed that the effective reaction rate constant (k eff ) for NO-RBC interactions was 2.32 × 10 4 -1.08 × 10 6 M −1 s −1 . Results also predict that the membrane permeability in the range of 0.0415 -0.4 cm/s is required to maintain physiologically relevant levels of NO at the smooth muscle cell layer. The effective reaction rate constant increased with increase in P m and magnitude of increase was higher at 45% Hct. For all P m values, the k hb /k eff ratios were lower for 45% Hct as compared to 5% Hct indicating extracellular resistance is important for RBC NO uptake. Our experimental and mathematical analyses of NO-RBC interactions indicate that both unstirred layer and RBC membrane have a significant effect on NO transport to RBCs. In addition, the membrane permeability in the range of 0.0415 -0.4 cm/s is required to maintain sufficient NO concentrations at the smooth muscle cell layer.

Bioavailability of vasoactive endothelium-derived nitric oxide (NO) in vasculature is a critical factor in regulation of many physiological processes. Consumption of NO by RBC plays a crucial role in maintaining NO bioavailability. Recently, Deonikar et al (Deonikar and Kavdia, 2009b) reported a effective NO-RBC reaction rate constant of 0.2×105 M−1s−1 that is ~7 times lower than the commonly used NO-RBC reaction rate constant of 1.4×105 M−1s−1. To study the effect of lower NO-RBC reaction rate constant and nitrite and nitrate formation (products of NO metabolism in blood), we developed a 2D mathematical model of NO biotransport in 50 and 200 μm ID arterioles to calculate NO concentration in radial and axial direction in the vascular lumen and vascular wall of the arterioles. We also simulated the effect of blood velocity on NO distribution in the arterioles to determine whether NO can be transported to downstream locations in the arteriolar lumen. The results indicate that lowering the NO-RBC reaction rate constant increased the NO concentration in the vascular lumen as well as the vascular wall. Increasing the velocity also led to increase in NO concentration. We predict increased NO concentration gradient along the axial direction with an increase in the velocity. The predicted NO concentration were 281–1163 nM in the smooth muscle cell layer for 50 μm arteriole over the blood velocity range of 0.5–4 cm/s for kNO-RBC of 0.2×105 M−1 s−1, which are much higher than the reported values from earlier mathematical modeling studies. The NO concentrations are similar to the experimentally measured vascular wall NO concentration range of 300–1000 nM in several different vascular beds. The results are significant from the perspective that the downstream transport of NO is possible under right circumstances.