Verónica S. Martínez

INTRODUCTION: The human genome-scale metabolic reconstruction details all known metabolic reactions occurring in humans, and thereby holds substantial promise for studying complex diseases and phenotypes. Capturing the whole human metabolic reconstruction is an on-going task and since the last community effort generated a consensus reconstruction, several updates have been developed. OBJECTIVES: We report a new consensus version, Recon 2.2, which integrates various alternative versions with significant additional updates. In addition to re-establishing a consensus reconstruction, further key objectives included providing more comprehensive annotation of metabolites and genes, ensuring full mass and charge balance in all reactions, and developing a model that correctly predicts ATP production on a range of carbon sources. METHODS: Recon 2.2 has been developed through a combination of manual curation and automated error checking. Specific and significant manual updates include a respecification of fatty acid metabolism, oxidative phosphorylation and a coupling of the electron transport chain to ATP synthase activity. All metabolites have definitive chemical formulae and charges specified, and these are used to ensure full mass and charge reaction balancing through an automated linear programming approach. Additionally, improved integration with transcriptomics and proteomics data has been facilitated with the updated curation of relationships between genes, proteins and reactions. RESULTS: Recon 2.2 now represents the most predictive model of human metabolism to date as demonstrated here. Extensive manual curation has increased the reconstruction size to 5324 metabolites, 7785 reactions and 1675 associated genes, which now are mapped to a single standard. The focus upon mass and charge balancing of all reactions, along with better representation of energy generation, has produced a flux model that correctly predicts ATP yield on different carbon sources. CONCLUSION: Through these updates we have achieved the most complete and best annotated consensus human metabolic reconstruction available, thereby increasing the ability of this resource to provide novel insights into normal and disease states in human. The model is freely available from the Biomodels database (http://identifiers.org/biomodels.db/MODEL1603150001).

A transgenic mouse strain that expresses the hepatitis B virus (HBV) large envelope protein in the liver was used to determine the extent of oxidative DNA damage that occurs during chronic HBV infection. This mouse strain develops a chronic necroinflammatory liver disease that mimics the inflammation, cellular hyperplasia, and increased risk for cancer that is evident in human chronic active hepatitis. When perfused in situ with nitroblue tetrazolium, an indicator for superoxide formation, the liver of transgenic mice displayed intense formazan deposition in Kupffer cells, indicating oxygen radical production, and S-phase hepatocytes were commonly seen adjacent to the stained Kupffer cells. Similar changes were not observed in nontransgenic control livers. To determine whether these events were associated with oxidative DNA damage, genomic DNA from the livers of transgenic mice and nontransgenic controls was isolated and examined for 8-oxo-2'-deoxyguanosine, an oxidatively modified adduct of deoxyguanosine. Results showed a significant, sustained accumulation in steady-state 8-oxo-2'-deoxyguanosine that started early in life exclusively in the transgenic mice and increased progressively with advancing disease. The most pronounced increase occurred in livers exhibiting microscopic nodular hyperplasia, adenomas, and hepatocellular carcinoma. Thus, HBV transgenic mice with chronic active hepatitis display greatly increased hepatic oxidative DNA damage. Moreover, the DNA damage occurs in the presence of heightened hepatocellular proliferation, increasing the probability of fixation of the attendant genetic and chromosomal abnormalities and the development of hepatocellular carcinoma.

Mammalian cell cultures typically exhibit an energy inefficient phenotype characterized by the consumption of large quantities of glucose and the concomitant production of large quantities of lactate. Under certain conditions, mammalian cells can switch to a more energy efficient state during which lactate is consumed. Using a metabolic model derived from a mouse genome scale model we performed flux balance analysis of Chinese hamster ovary cells before and after a metabolic switch from lactate production (in the presence of glucose) to lactate consumption (after glucose depletion). Despite a residual degree of freedom after accounting for measurements, the calculated flux ranges and associated errors were narrow enough to enable investigation of metabolic changes across the metabolic switch. Surprisingly, the fluxes through the lower part of the TCA cycle from oxoglutarate to malate were very similar (around 60 µmol/gDW/h) for both phases. A detailed analysis of the energy metabolism showed that cells consuming lactate have an energy efficiency (total ATP produced per total C-mol substrate consumed) six times greater than lactate producing cells.

Transgenic mice that express the hepatitis B virus core protein were used to examine factors that influence the intracellular localization of nucleocapsid particles in the primary hepatocyte in vivo. In this model, viral nucleocapsid particles are strictly localized to the nucleus of the hepatocyte except when the nuclear membrane dissolves during cell division, at which time they enter the cytoplasm. The cytoplasmic nucleocapsid particles do not reenter the nucleus, however, when the nuclear membrane re-forms after cell division. The data support the notion that nucleocapsid particles can form de novo within the nucleus, and they suggest that performed nucleocapsid particles cannot be transported across the intact nuclear membrane in either direction. The results imply that nucleocapsid disassembly is probably required for entry of the hepadnaviral genome into the nucleus, and they question the role of the intranuclear viral nucleocapsid particle during the viral life cycle.