Nitrous oxide production by lithotrophic ammonia-oxidizing bacteria and implications for engineered nitrogen-removal systems

Chemolithoautotrophic AOB (ammonia-oxidizing bacteria) form a crucial component in microbial nitrogen cycling in both natural and engineered systems. Under specific conditions, including transitions from anoxic to oxic conditions and/or excessive ammonia loading, and the presence of high nitrite (NO??) concentrations, these bacteria are also documented to produce nitric oxide (NO) and nitrous oxide (N?O) gases. Essentially, ammonia oxidation in the presence of non-limiting substrate concentrations (ammonia and O?) is associated with N?O production. An exceptional scenario that leads to such conditions is the periodical switch between anoxic and oxic conditions, which is rather common in engineered nitrogen-removal systems. In particular, the recovery from, rather than imposition of, anoxic conditions has been demonstrated to result in N?O production. However, applied engineering perspectives, so far, have largely ignored the contribution of nitrification to N?O emissions in greenhouse gas inventories from wastewater-treatment plants. Recent field-scale measurements have revealed that nitrification-related N?O emissions are generally far higher than emissions assigned to heterotrophic denitrification. In the present paper, the metabolic pathways, which could potentially contribute to NO and N?O production by AOB have been conceptually reconstructed under conditions especially relevant to engineered nitrogen-removal systems. Taken together, the reconstructed pathways, field- and laboratory-scale results suggest that engineering designs that achieve low effluent aqueous nitrogen concentrations also minimize gaseous nitrogen emissions.     

Structural Characterization of the Glycoprotein GP2 Core Domain from the CAS Virus, a Novel Arenavirus-Like Species

Fusion of the viral and host cell membranes is a necessary first step for infection by enveloped viruses and is mediated by the envelope glycoprotein. The transmembrane subunits from the structurally defined “class I” glycoproteins adopt an α-helical “trimer-of-hairpins” conformation during the fusion pathway. Here, we present our studies on the envelope glycoprotein transmembrane subunit, GP2, of the CAS virus (CASV). CASV was recently identified from annulated tree boas (Corallus annulatus) with inclusion body disease and is implicated in the disease etiology. We have generated and characterized two protein constructs consisting of the predicted CASV GP2 core domain. The crystal structure of the CASV GP2 post-fusion conformation indicates a trimeric α-helical bundle that is highly similar to those of Ebola virus and Marburg virus GP2 despite CASV genome homology to arenaviruses. Denaturation studies demonstrate that the stability of CASV GP2 is pH dependent with higher stability at lower pH; we propose that this behavior is due to a network of interactions among acidic residues that would destabilize the α-helical bundle under conditions where the side chains are deprotonated. The pH-dependent stability of the post-fusion structure has been observed in Ebola virus and Marburg virus GP2, as well as other viruses that enter via the endosome. Infection experiments with CASV and the related Golden Gate virus support a mechanism of entry that requires endosomal acidification. Our results suggest that, despite being primarily arenavirus like, the transmembrane subunit of CASV is extremely similar to the filoviruses.     

Analysis of tumor metabolism reveals mitochondrial glucose oxidation in genetically diverse human glioblastomas in the mouse brain in vivo

Dysregulated metabolism is a hallmark of cancer cell lines, but little is known about the fate of glucose and other nutrients in tumors growing in their native microenvironment. To study tumor metabolism in vivo, we used an orthotopic mouse model of primary human glioblastoma (GBM). We infused (13)C-labeled nutrients into mice bearing three independent GBM lines, each with a distinct set of mutations. All three lines displayed glycolysis, as expected for aggressive tumors. They also displayed unexpected metabolic complexity, oxidizing glucose via pyruvate dehydrogenase and the citric acid cycle, and using glucose to supply anaplerosis and other biosynthetic activities. Comparing the tumors to surrounding brain revealed obvious metabolic differences, notably the accumulation of a large glutamine pool within the tumors. Many of these same activities were conserved in cells cultured ex vivo from the tumors. Thus GBM cells utilize mitochondrial glucose oxidation during aggressive tumor growth in vivo.     

Marburg virus glycoprotein GP2: pH-dependent stability of the ectodomain α-helical bundle

Marburg virus (MARV) and Ebola virus (EBOV) constitute the family Filoviridae of enveloped viruses (filoviruses) that cause severe hemorrhagic fever. Infection by MARV requires fusion between the host cell and viral membranes, a process that is mediated by the two subunits of the envelope glycoprotein, GP1 (surface subunit) and GP2 (transmembrane subunit). Upon viral attachment and uptake, it is believed that the MARV viral fusion machinery is triggered by host factors and environmental conditions found in the endosome. Next, conformational rearrangements in the GP2 ectodomain result in the formation of a highly stable six-helix bundle; this refolding event provides the energetic driving force for membrane fusion. Both GP1 and GP2 from EBOV have been extensively studied, but there is little information available for the MARV glycoproteins. Here we have expressed two variants of the MARV GP2 ectodomain in Escherichia coli and analyzed their biophysical properties. Circular dichroism indicates that the MARV GP2 ectodomain adopts an α-helical conformation, and one variant sediments as a trimer by equilibrium analytical ultracentrifugation. Denaturation studies indicate the α-helical structure is highly stable at pH 5.3 (unfolding energy, ΔG(unf,H(2)O), of 33.4 ± 2.5 kcal/mol and melting temperature, T(m), of 75.3 ± 2.1 °C for one variant). Furthermore, we found the α-helical stability to be strongly dependent on pH, with higher stability under lower-pH conditions (T(m) values ranging from ~92 °C at pH 4.0 to ~38 °C at pH 8.0). Mutational analysis suggests two glutamic acid residues (E579 and E580) are partially responsible for this pH-dependent behavior. On the basis of these results, we hypothesize that the pH-dependent folding stability of the MARV GP2 ectodomain provides a mechanism for controlling conformational preferences such that the six-helix bundle "postfusion" state is preferred under conditions of appropriately matured endosomes.     

Maternal obesity at conception programs obesity in the offspring

Risk of obesity in adult life is subject to programming during gestation. To examine whether in utero exposure to maternal obesity increases the risk of obesity in offspring, we developed an overfeeding-based model of maternal obesity in rats utilizing intragastric feeding of diets via total enteral nutrition. Feeding liquid diets to adult female rats at 220 kcal/kg(3/4) per day (15% excess calories/day) compared with 187 kcal/kg(3/4) per day for 3 wk caused substantial increase in body weight gain, adiposity, serum insulin, leptin, and insulin resistance. Lean or obese female rats were mated with ad libitum AIN-93G-fed male rats. Exposure to obesity was ensured to be limited only to the maternal in utero environment by cross-fostering pups to lean dams having ad libitum access to AIN-93G diets throughout lactation. Numbers of pups, birth weight, and size were not affected by maternal obesity. Male offspring from each group were weaned at postnatal day (PND)21 to either AIN-93G diets or high-fat diets (45% fat calories). Body weights of offspring from obese dams did not differ from offspring of lean dams when fed AIN-93G diets through PND130. However, offspring from obese dams gained remarkably greater (P < 0.005) body weight and higher % body fat when fed a high-fat diet. Body composition was assessed by NMR, X-ray computerized tomography, and weights of adipose tissues. Adipose histomorphometry, insulin sensitivity, and food intake were also assessed in the offspring. Our data suggest that maternal obesity at conception leads to fetal programming of offspring, which could result in obesity in later life.     

Cytokine and chemokine expression associated with steatohepatitis and hepatocyte proliferation in rats fed ethanol via total enteral nutrition

To determine the temporal relationship between alcohol-induced changes in cytokines and chemokines, development of liver pathology and stimulation of hepatocyte proliferation, male Sprague-Dawley rats were intragastrically fed low carbohydrate-containing ethanol (EtOH) diets via total enteral nutrition (TEN) for up to 49 d. Induction of EtOH metabolism and appearance of steatosis preceded development of oxidative stress, inflammation, and cell death. A transitory peak of tumor necrosis factor (TNFalpha) and interferon gamma (IFN gamma) was observed at 14 d followed by reduced expression of TNFalpha, IFN gamma and another Th1 cytokine IL-12 accompanied by reduced expression of the Th1 regulators T-bet and STAT4. After 35-49 d of EtOH, at a time when hepatocyte proliferation was stimulated, IL-12 returned to control values and a second peak of TNFalpha occurred. The Th2 cytokine IL-4 remained suppressed throughout the study and was accompanied by reductions in the Th2 regulator GATA3. There was no temporal effect of EtOH on expression of IL-6 or TGFbeta. IL-5 and IL-13 mRNA were undetectable. Chemokine CXCL-2 expression increased progressively up to 35 d and preceded the appearance of inflammatory infiltrates. These data suggest that steatosis, increased ethanol metabolism, a transient induction of the innate immune response and suppression of Th2 responses were acute consequences of ethanol treatment and were followed by suppression of Th1 responses. However, the majority of necrosis, apoptosis and a late peak of TNFalpha only occurred after 6-7 weeks of ethanol, coincided with the appearance of inflammatory infiltrates and were associated with stimulation of hepatocyte proliferation.