Determining Host Metabolic Limitations on Viral Replication via Integrated Modeling and Experimental Perturbation

Viral replication relies on host metabolic machinery and precursors to produce large numbers of progeny - often very rapidly. A fundamental example is the infection of Escherichia coli by bacteriophage T7. The resource draw imposed by viral replication represents a significant and complex perturbation to the extensive and interconnected network of host metabolic pathways. To better understand this system, we have integrated a set of structured ordinary differential equations quantifying T7 replication and an E. coli flux balance analysis metabolic model. Further, we present here an integrated simulation algorithm enforcing mutual constraint by the models across the entire duration of phage replication. This method enables quantitative dynamic prediction of virion production given only specification of host nutritional environment, and predictions compare favorably to experimental measurements of phage replication in multiple environments. The level of detail of our computational predictions facilitates exploration of the dynamic changes in host metabolic fluxes that result from viral resource consumption, as well as analysis of the limiting processes dictating maximum viral progeny production. For example, although it is commonly assumed that viral infection dynamics are predominantly limited by the amount of protein synthesis machinery in the host, our results suggest that in many cases metabolic limitation is at least as strict. Taken together, these results emphasize the importance of considering viral infections in the context of host metabolism.     

The perspective of GABA replenishment therapy in the epilepsies: A critical evaluation of hopes and concerns

Impaired GABA-mediated inhibition is probably one of the cellular abnormalities leading to Focal Epilepsy. The role of GABA in generalized seizures, particularly of Petit Mal type, is unknown. Various approaches are available to potentiate GABA function. Merits and flaws of each one of them are critically evaluated. In some forms of epilepsy, GABA agonists may replenish depleted pools, and in some others may nonspecifically raise the general excitability threshold of the brain, yet in other forms they may exert a glutamate/aspartate antagonistic effect. The available experimental evidence suggests that in bilaterally synchronous spike and wave epilepsies, GABA agonists are either ineffective or pejorative.     

Identification of a direct hemolytic effect dependent on the catalytic activity induced by phospholipase‐D (dermonecrotic toxin) from brown spider venom

Brown spiders have world‐wide distribution and are the cause of health problems known as loxoscelism. Necrotic cutaneous lesions surrounding the bites and less intense systemic signs like renal failure, DIC, and hemolysis were observed. We studied molecular mechanism by which recombinant toxin, biochemically characterized as phospholipase‐D , causes direct hemolysis (complement independent). Human erythrocytes treated with toxin showed direct hemolysis in a dose‐dependent and time‐dependent manner, as well as morphological changes in cell size and shape. Erythrocytes from human, rabbit, and sheep were more susceptible than those from horse. Hemolysis was not dependent on ABO group or Rhesus system. Confocal and FACS analyses using antibodies or GFP‐phospholipase‐D protein showed direct toxin binding to erythrocytes membrane. Moreover, toxin‐treated erythrocytes reacted with annexin‐V and showed alterations in their lipid raft profile. Divalent ion chelators significantly inhibited hemolysis evoked by phospholipase‐D , which has magnesium at the catalytic domain. Chelators were more effective than PMSF (serine‐protease inhibitor) that had no effect on hemolysis. By site‐directed mutation at catalytic domain (histidine 12 by alanine), hemolysis and morphologic changes of erythrocytes (but not the toxin's ability of membrane binding) were inhibited, supporting that catalytic activity is involved in hemolysis and cellular alterations but not toxin cell binding. The results provide evidence that L. intermedia venom phospholipase‐D triggers direct human blood cell hemolysis in a catalytic‐dependent manner. J. Cell. Biochem. 107: 655–666, 2009. © 2009 Wiley‐Liss, Inc.     

Discovery of a novel series of CXCR3 antagonists

The discovery of a novel series of CXCR3 antagonists is described. Starting from an HTS positive, iterative optimization gave potent compounds (IC₅₀ 15 nM in a chemotaxis assay). The strategy employed to improve the metabolic stability of these derivatives is described.     

Pseudomonas putida KT2440 metabolism undergoes sequential modifications during exponential growth in a complete medium as compounds are gradually consumed

Pseudomonas putida is a soil bacterium with a versatile and robust metabolism. When confronted with mixtures of carbon sources, it prioritizes the utilization of the preferred compounds, optimizing metabolism and growth. This response is particularly strong when growing in a complex medium such as LB. This work examines the changes occurring in P. putida KT2440 metabolic fluxes, while it grows exponentially in LB medium and sequentially consumes the compounds available. Integrating the uptake rates for each compound at three different moments during the exponential growth with the changes observed in the proteome, and with the metabolic fluxes predicted by the iJN1411 metabolic model for this strain, allowed the metabolic rearrangements that occurred to be determined. The results indicate that the bacterium changes significantly the configuration of its metabolism during the early, mid and late exponential phases of growth. Sugars served as an energy source during the early phase and later as energy and carbon source. The configuration of the tricarboxylic acids cycle varied during growth, providing no energy in the early phase, and turning to a reductive mode in the mid phase and to an oxidative mode later on. This work highlights the dynamism and flexibility of P. putida metabolism.     

Active-Site Inhibitors of mTOR Target Rapamycin-Resistant Outputs of mTORC1 and mTORC2

The mammalian target of rapamycin (mTOR) regulates cell growth and survival by integrating nutrient and hormonal signals. These signaling functions are distributed between at least two distinct mTOR protein complexes: mTORC1 and mTORC2. mTORC1 is sensitive to the selective inhibitor rapamycin and activated by growth factor stimulation via the canonical phosphoinositide 3-kinase (PI3K)→Akt→mTOR pathway. Activated mTORC1 kinase up-regulates protein synthesis by phosphorylating key regulators of mRNA translation. By contrast, mTORC2 is resistant to rapamycin. Genetic studies have suggested that mTORC2 may phosphorylate Akt at S473, one of two phosphorylation sites required for Akt activation; this has been controversial, in part because RNA interference and gene knockouts produce distinct Akt phospho-isoforms. The central role of mTOR in controlling key cellular growth and survival pathways has sparked interest in discovering mTOR inhibitors that bind to the ATP site and therefore target both mTORC2 and mTORC1. We investigated mTOR signaling in cells and animals with two novel and specific mTOR kinase domain inhibitors (TORKinibs). Unlike rapamycin, these TORKinibs (PP242 and PP30) inhibit mTORC2, and we use them to show that pharmacological inhibition of mTOR blocks the phosphorylation of Akt at S473 and prevents its full activation. Furthermore, we show that TORKinibs inhibit proliferation of primary cells more completely than rapamycin. Surprisingly, we find that mTORC2 is not the basis for this enhanced activity, and we show that the TORKinib PP242 is a more effective mTORC1 inhibitor than rapamycin. Importantly, at the molecular level, PP242 inhibits cap-dependent translation under conditions in which rapamycin has no effect. Our findings identify new functional features of mTORC1 that are resistant to rapamycin but are effectively targeted by TORKinibs. These potent new pharmacological agents complement rapamycin in the study of mTOR and its role in normal physiology and human disease.     

Transferrin Receptor 2 Is Frequently and Highly Expressed in Glioblastomas

Under physiological conditions, transferrin receptor 2 (TfR2) is expressed in the liver and its balance is related to the cell cycle rather than to intracellular iron levels. We recently showed that TfR2 is highly expressed in glioblastoma cell lines. Here, we demonstrate that, in these cells, TfR2 appears to localize in lipid rafts, induces extracellular signal-regulated kinase 1/2 phosphorylation after transferrin binding, and contributes to cell proliferation, as shown by RNA silencing experiments. In vitro hypoxic conditions induce a significant TfR2 up-regulation, suggesting a role in tumor angiogenesis. As assessed by immunohistochemistry, the level of TfR2 expression in astrocytic tumors is related to histologic grade, with the highest expression observed in glioblastomas. The level of TfR2 expression represents a favorable prognostic factor, which is associated with the higher sensitivity to temozolomide of TfR2-positive tumor cells in vitro. The endothelial cells of glioblastoma vasculature also stain for TfR2, whereas those of the normal brain vessels do not. Importantly, TfR2 is expressed by the subpopulation of glioblastoma cells with properties of cancer-initiating cells. TfR2-positive glioblastoma cells retain their TfR2 expression on xenografting in immunodeficient mice. In conclusion, our observations demonstrate that TfR2 is a neoantigen for astrocytomas that seems attractive for developing target therapies.