Human cytomegalovirus (HCMV) infection causes dramatic alterations of intermediary metabolism, similar to those found in tumor cells. In infected cells, glucose carbon is not completely broken down by the tricarboxylic acid (TCA) cycle for energy; instead it is used biosynthetically. This process requires increased glucose uptake, increased glycolysis and the diversion of glucose carbon, in the form of citrate, from the TCA cycle for use in HCMV-induced fatty acid biosynthesis. The diversion of citrate from the TCA cycle (cataplerosis) requires induction of enzymes to promote glutaminolysis, the conversion of glutamine to -ketoglutarate in order to maintain the TCA cycle (anaplerosis) and ATP production. Such changes could result in heretofore uncharacterized pathogenesis, potentially implicating HCMV as a subtle co-factor in many maladies, including oncogenesis. Recognition of the effects of HCMV, and other viruses, on host cell metabolism will provide new understanding of viral pathogenesis and novel avenues for antiviral therapy.

The hepatitis B virus (HBV) X protein (HBx) is a multifunctional protein that regulates numerous cellular signal transduction pathways, including those that modulate apoptosis. However, different HBx-dependent effects on apoptosis have been reported; these differences are likely the consequence of the exact conditions and cell types used in a study. Many of the previously reported studies that analyzed HBx regulation of apoptosis were conducted in immortalized or transformed cells, and the alterations that have transformed or immortalized these cells likely impact apoptotic pathways. We examined the effects of HBx on apoptotic pathways in cultured primary rat hepatocytes, a biologically relevant system that mimics normal hepatocytes in the liver. We analyzed the effects of HBx on apoptosis both when HBx was expressed in the absence of other HBV proteins and in the context of HBV replication. HBx stimulation of NF-B inhibited the activation of apoptotic pathways in cultured primary rat hepatocytes. However, when HBx-induced activation of NF-B was blocked, HBx stimulated apoptosis; blocking the activity of the mitochondrial permeability transition pore inhibited HBx activation of apoptosis. These results suggest that HBx can be either proapoptotic or antiapoptotic in hepatocytes, depending on the status of NF-B, and confirm previous studies that link some HBx activities to modulation of the mitochondrial permeability transition pore. Overall, our studies define apoptotic pathways that are regulated by HBx in cultured primary hepatocytes and provide potential mechanisms for the development of HBV-associated liver cancer.

Over 350 million people are chronically infected with hepatitis B virus (HBV), and a significant number of chronically infected individuals develop primary liver cancer. HBV encodes seven viral proteins, including the nonstructural X (HBx) protein. The results of studies with immortalized or transformed cells and withHBx-transgenic mice demonstrated that HBx can interact with mitochondria. However, no studies with normal hepatocytes have characterized the precise mitochondrial localization of HBx or the effect of HBx on mitochondrial physiology. We have used cultured primary rat hepatocytes as a model system to characterize the mitochondrial localization of HBx and the effect of HBx expression on mitochondrial physiology. We now show that a fraction of HBx colocalizes with density-gradient-purified mitochondria and associates with the outer mitochondrial membrane. We also demonstrate that HBx regulates mitochondrial membrane potential in hepatocytes and that this function of HBx varies depending on the status of NF-B activity. In primary rat hepatocytes, HBx activation of NF-B prevented mitochondrial membrane depolarization; however, when NF-B activity was inhibited, HBx induced membrane depolarization through modulation of the mitochondrial permeability transition pore. Collectively, these results define potential pathways through which HBx may act in order to modulate mitochondrial physiology, thereby altering many cellular activities and ultimately contributing to the development of HBV-associated liver cancer.Worldwide, an estimated 350 to 400 million people are chronically infected with human hepatitis B virus (HBV) (82,97). HBV is the prototype member of the Hepadnaviridae, a family of hepatotropic viruses that can infect both birds and mammals (82). HBV has a partially double-stranded, circular DNA genome containing four overlapping, open reading frames that encode the viral envelope, capsid, polymerase, and X (HBx) proteins (82). Chronic infections with HBV are associated with the development of primary liver cancer, hepatocellular carcinoma (HCC) (3). The underlying molecular mechanisms that link chronic HBV infections to the development of HCC are incompletely understood, and both immunemediated destruction of HBV-infected hepatocytes and concomitant liver regeneration, as well as activities of the HBx protein, are thought to be involved (82).Numerous functions have been attributed to HBx, and how HBx influences HBV replication and HCC development is the subject of considerable debate. HBx expression facilitates development of HCC in HBx-transgenic mice, although whether HBx directly induces tumor formation or only sensitizes mice to carcinogens varies depending on the genetic background of the HBx-transgenic mice (43,44,56,85,101). Chronic infections with mammalian hepadnaviruses, all of which encode a X protein, cause HCC, while chronic infections with avian hepadnaviruses, which either do not encode a X protein or encode a highly divergent form of the protein, are not associated with the development...

For nanotechnology to meet its potential as a game-changing and sustainable technology, it is important to ensure that the engineered nanomaterials and nanoenabled products that gain entry to the marketplace are safe and effective. Tools and methods are needed for regulatory purposes to allow rapid material categorization according to human health and environmental risk potential, so that materials of high concern can be targeted for additional scrutiny, while material categories that pose the least risk can receive expedited review. Using carbon nanotubes as an example, we discuss how data from alternative testing strategies can be used to facilitate engineered nanomaterial categorization according to risk potential and how such an approach could facilitate regulatory decision-making in the future.

The nonstructural hepatitis B virus (HBV) protein HBx has an important role in HBV replication and in HBV-associated liver disease. Many activities have been linked to HBx expression; however, the molecular mechanisms underlying many of these activities are unknown. One proposed HBx function is the regulation of cytosolic calcium. We analyzed calcium levels in HepG2 cells that expressed HBx or replicating HBV, and we demonstrated that HBx, expressed in the absence of other HBV proteins or in the context of HBV replication, elevates cytosolic calcium. We linked this elevation of cytosolic calcium to the association of HBx with the mitochondrial permeability transition pore.

Engineered metal/mineral, lipid and biochemical macromolecule nanomaterials (NMs) have potential applications in food. Methodologies for the assessment of NM digestion and bioavailability in the gastrointestinal tract are nascent and require refinement. A working group was tasked by the International Life Sciences Institute NanoRelease Food Additive project to review existing models of the gastrointestinal tract in health and disease, and the utility of these models for the assessment of the uptake of NMs intended for food. Gastrointestinal digestion and absorption could be addressed in a tiered approach using in silico computational models, in vitro non-cellular fluid systems and in vitro cell culture models, after which the necessity of ex vivo organ culture and in vivo animal studies can be considered. Examples of NM quantification in gastrointestinal tract fluids and tissues are emerging; however, few standardized analytical techniques are available. Coupling of these techniques to gastrointestinal models, along with further standardization, will further strengthen methodologies for risk assessment.

The mammalian target of rapamycin (mTOR) kinase is present in 2 functionally distinct complexes, mTOR complex 1 (mTORC1) and complex 2 (mTORC2). Active mTORC1 mediates phosphorylation of eIF4E-binding protein (4E-BP) and p70 S6 kinase (S6K), which is important for maintaining translation. During human cytomegalovirus (HCMV) infection, cellular stress responses are activated that normally inhibit mTORC1; however, previous data show that HCMV infection circumvents stress responses and maintains mTOR kinase activity. Amino acid deprivation is a stress response that normally inhibits mTORC1 activity. Amino acids can signal to mTORC1 through the Rag proteins, which promote the colocalization of mTORC1 with its activator Rheb-GTP in a perinuclear region, thereby inducing 4E-BP and S6K phosphorylation. As expected, our results show that amino acid depletion in mock-infected cells caused loss of mTORC1 activity and loss of the perinuclear localization; however, there was no loss of activity or perinuclear localization in HCMV-infected cells where the perinuclear localization of Rheb-GTP and mTOR coincided with the perinuclear assembly compartment (AC). This suggested that HCMV infection bypasses normal Rag-dependent amino acid signaling. This was demonstrated by short hairpin RNA (shRNA) depletion of Rag proteins, which had little effect on mTORC1 activity in infected cells but inhibited activity in mock-infected cells. Our data show that HCMV maintains mTORC1 activity in an amino acid-and Rag-independent manner through the colocalization of mTOR and Rheb-GTP, which occurs in association with the formation of the AC, thus bypassing inhibition that may result from lowered amino acid levels.

The mammalian target of rapamycin (mTOR) kinase occurs in mTOR complex 1 (mTORC1) and complex 2 (mTORC2), primarily differing by the substrate specificity factors raptor (in mTORC1) and rictor (in mTORC2). Both complexes are activated during human cytomegalovirus (HCMV) infection. mTORC1 phosphorylates eukaryotic initiation factor 4E (eIF4E)-binding protein (4E-BP1) and p70S6 kinase (S6K) in uninfected cells, and this activity is lost upon raptor depletion. In infected cells, 4E-BP1 and S6K phosphorylation is maintained when raptor or rictor is depleted, suggesting that either mTOR complex can phosphorylate 4E-BP1 and S6K. Studies using the mTOR inhibitor Torin1 show that phosphorylation of 4E-BP1 and S6K in infected cells depends on mTOR kinase. The total levels of 4E-BP1 and viral proteins representative of all temporal classes were lowered by Torin1 treatment and by raptor, but not rictor, depletion, suggesting that mTORC1 is involved in the production of all classes of HCMV proteins. We also show that Torin1 inhibition of mTOR kinase is rapid and most deleterious at early times of infection. While Torin1 treatment from the beginning of infection significantly inhibited translation of viral proteins, its addition at later time points had far less effect. Thus, with respect to mTOR's role in translational control, HCMV depends on it early in infection but can bypass it at later times of infection. Depletion of 4E-BP1 by use of short hairpin RNAs (shRNAs) did not rescue HCMV growth in Torin1-treated human fibroblasts as it has been shown to in murine cytomegalovirus (MCMV)-infected 4E-BP1 ؊/؊ mouse embryo fibroblasts (MEFs), suggesting that during HCMV infection mTOR kinase has additional roles other than phosphorylating and inactivating 4E-BP1. Overall, our data suggest a dynamic relationship between HCMV and mTOR kinase which changes during the course of infection.