Glucose Induces Sensitivity to Oxygen Deprivation and Modulates Insulin/IGF-1 Signaling and Lipid Biosynthesis in Caenorhabditis elegans

Diet is a central environmental factor that contributes to the phenotype and physiology of individuals. At the root of many human health issues is the excess of calorie intake relative to calorie expenditure. For example, the increasing amount of dietary sugars in the human diet is contributing to the rise of obesity and type 2 diabetes. Individuals with obesity and type 2 diabetes have compromised oxygen delivery, and thus it is of interest to investigate the impact a high-sugar diet has on oxygen deprivation responses. By utilizing the Caenorhabditis elegans genetic model system, which is anoxia tolerant, we determined that a glucose-supplemented diet negatively impacts responses to anoxia and that the insulin-like signaling pathway, through fatty acid and ceramide synthesis, modulates anoxia survival. Additionally, a glucose-supplemented diet alters lipid localization and initiates a positive chemotaxis response. Use of RNA-sequencing analysis to compare gene expression responses in animals fed either a standard or glucose-supplemented diet revealed that glucose impacts the expression of genes involved with multiple cellular processes including lipid and carbohydrate metabolism, stress responses, cell division, and extracellular functions. Several of the genes we identified show homology to human genes that are differentially regulated in response to obesity or type 2 diabetes, suggesting that there may be conserved gene expression responses between C. elegans fed a glucose-supplemented diet and a diabetic and/or obesity state observed in humans. These findings support the utility of the C. elegans model for understanding the molecular mechanisms regulating dietary-induced metabolic diseases.     

Pressure gradient, not exposure duration, determines the extent of epithelial cell damage in a model of pulmonary airway reopening.

The reduction of tidal volume during mechanical ventilation has been shown to reduce mortality of patients with acute respiratory distress syndrome, but epithelial cell injury can still result from mechanical stresses imposed by the opening of occluded airways. To study these stresses, a fluid-filled parallel-plate flow chamber lined with epithelial cells was used as an idealized model of an occluded airway. Airway reopening was modeled by the progression of a semi-infinite bubble of air through the length of the channel, which cleared the fluid. In our laboratory's prior study, the magnitude of the pressure gradient near the bubble tip was directly correlated to the epithelial cell layer damage (Bilek AM, Dee KC, and Gaver DP III. J Appl Physiol 94: 770-783, 2003). However, in that study, it was not possible to discriminate the stress magnitude from the stimulus duration because the bubble propagation velocity varied between experiments. In the present study, the stress magnitude is modified by varying the viscosity of the occlusion fluid while fixing the reopening velocity across experiments. This approach causes the stimulus duration to be inversely related to the magnitude of the pressure gradient. Nevertheless, cell damage remains directly correlated with the pressure gradient, not the duration of stress exposure. The present study thus provides additional evidence that the magnitude of the pressure gradient induces cellular damage in this model of airway reopening. We explore the mechanism for acute damage and also demonstrate that repeated reopening and closure is shown to damage the epithelial cell layer, even under conditions that would not lead to extensive damage from a single reopening event.     

Quantitative Microbiological Analysis of Bacterial Community Shifts in a High-Rate Anaerobic Bioreactor Treating Sulfite Evaporator Condensate

The bacterial population of a high-rate, anaerobic, fixed-bed loop reactor treating sulfite evaporator condensate from the pulp industry was studied over a 14-month period. This period was divided into seven cycles that included a startup at the beginning of each cycle. Some 82% of the total biomass was immobilized on and between the porous glass rings filling the reactor. The range of the total number of microorganisms in these biofilms was 2 × 10⁹ to 7 × 10⁹ cells per ml. Enumeration and characterization by microbiological methods and by phase-contrast, epifluorescence, and electron microscopy showed that the samples consisted mainly of the following methanogens: a Methanobacterium sp., a Methanosarcina sp., a Methanobrevibacter sp., and a Methanothrix sp., as well as furfural-degrading sulfate-reducing bacteria resembling Desulfovibrio furfuralis. Viable counts of hydrogenotrophic methanogens were relatively stable (mostly within the range of 3.2 × 10⁸ to 7.5 × 10⁸ cells per ml), but Methanobrevibacter cells increased from <5 to 30% of the total hydrogenotrophic count after transfer of the fixed bed into a second reactor vessel. Acetotrophic methanogens reached their highest numbers of 1.3 × 10⁸ to 2.6 × 10⁸ cells per ml in the last fermentation cycles. They showed a morphological shift from sarcinalike packets in early samples to single coccoid forms in later phases of the fermentation. Furfural-degrading sulfate reducers reached counts of 1 × 10⁷ to 5.8 × 10⁷ cells per ml. The distribution of the chief metabolic groups between free fluid and biofilms was analyzed in the fifth fermentation cycle: 4.5 times more furfural degraders were found in the free fluid than in the biofilms. In contrast, 5.8 times more acetotrophic and 16.6 times more hydrogenotrophic methanogens were found in the biofilms than in the free liquid. The data concerning time shifts of morphotypes among the trophic groups of methanogens corroborated the trends observed by using immunological assays on the same samples.     

Effect of medium composition and sludge removal on the production, composition, and architecture of thermophilic (55 degrees C) acetate-utilizing granules from an upflow anaerobic sludge blanket reactor.

A thermophilic upflow anaerobic sludge blanket (UASB) reactor degrading acetate was started by applying published methods (W. M. Wiegant and A. W. A. de Man, Biotechnol. Bioeng. 28:718-77, 1986) for production of granules dominated by Methanothrix spp. The reactor was inoculated with thermophilic digested sludge. No granules were observed during the first 7 months of start-up of the UASB reactor. However, after the concentrations of potassium, phosphate, ammonium, and magnesium in the medium were gradually increased, granules developed, indicating that there was a critical concentration of one or more of the ions required for production of granules from the starting material. After several years of stable operation, the effect of removing 60% of the granular sludge was investigated. Immunologic qualitative and quantitative studies showed that removal of the granular sludge resulted in an increase in the number of the predominant methanogens, antigenically related to Methanosarcina thermophila TM-1 and Methanosarcina mazeii S-6, and Methanobacterium thermoautotrophicum delta H and GC1. These changes were accompanied by modifications of the microanatomy of the granules, as demonstrated histochemically and immunohistochemically. The results indicated that different catabolic pathways dominated in different regions of the granules, i.e., acetate oxidation in the middle of the granules, where there is a low acetate concentration, and an aceticlastic reaction in the outer surfaces, with a high acetate concentration. The results also showed that removal of granules from a UASB reactor which has been under steady-state operation for a long period can improve the reactor's performance via formation of denser and larger granules with improved microbial activities.     

Antibody analysis of relationships among methanogenic bacteria

A bank of antisera to the majority of methanogenic bacteria is now available. Three antibody probes, R, S, and T, were derived from each antiserum in the bank and used for analysis of antigenic relatedness among methanogens by immunofluorescence. The T probe reacted only with the immunizing (or homologous) strain, the S probe gave strong cross-reactions with strains of the same species, and the R probe revealed some interspecies relationships. The results were confirmed and extended by enzyme immunoassays and standard serological methods involving serial dilution analysis, cross-adsorptions, and the use of reference strains. The immunological methods and standardized antibody probes are useful for rapid identification of methanogens and measurements of antigenic relationships which aid in the classification of these bacteria.     

Effect of magnesium on methanogenic subpopulations in a thermophilic acetate-degrading granular consortium.

The effects of Mg2+ on thermophilic (55 degrees C) granules grown on acetate in 0.2-liter upflow anaerobic sludge blanket reactors were studied. The methanogens in the granules were identified and counted by using antibody probes and the antigenic fingerprinting method. Packets of large coccoidal cells antigenically related to Methanosarcina thermophila TM-1 were scarce in the absence of Mg2+ but increased with increasing Mg2+ concentrations up to 30 mM; Methanosarcina packets immunologically related to Methanosarcina barkeri R1M3 showed a similar trend, and their numbers increased up to 100 mM Mg2+. The number of single cells antigenically related to TM-1, R1M3, and Methanosarcina mazei S-6 were scarce at low Mg2+ concentrations but increased drastically at 30 and 100 mM Mg2+. The number of rod-shaped bacteria antigenically related to Methanobacterium thermoautotrophicum GC1 and delta H was highest with no Mg2+ present, and their numbers decreased with increasing concentrations of the cation. These quantitative data, obtained by counting cells in suspensions made from disrupted granules, were confirmed by microscopic observation of the methanogenic subpopulations in thin histologic sections of the granules.     

Immunochemical differences among Methanosarcina mazei S-6 morphologic forms.

Methanosarcinae are the only archaeobacteria known to undergo major morphologic changes during growth involving unicellular and multicellular forms, and Methanosarcina mazei S-6 is the only strain for which three distinct forms, packets, single cells, and lamina, have so far been observed. It is reported that two pairs of these forms, either packets and single cells or single cells and lamina, grew and interconverted in medium with the same composition, Ca2+ and Mg2+ concentrations, and growth substrate, and that the two forms in each pair displayed distinctive differences revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting, the same growth medium-substrate notwithstanding.     

Isolation and characterization of Methanocorpusculum parvum,gen. nov., spec. nov., a new tungsten requiring,coccoid methanogen

A new mesophilic, monotrichously flagellated methane-producing coccus of 1m in diameter was isolated from an anaerobic sour whey digester, originally inoculated with sewage sludge. Growth and methane production were observed with H₂/CO₂, formate and — less effectively — with 2-propanol/CO₂. The isolate grew at temperatures between 15° C and 45° C with the optimum at around 37° C. Acetate, yeast extract and tungstate were required in the medium. Clarified rumen fluid stimulated growth.The DNA of the new methanogen has a G+C content of 48.5 mol%. Comparative 16 S rRNA oligonucleotide cataloguing allows to define the new isolate as a member of a new genus of the order Methanomicrobiales. Further evidence for this is provided by the antigenic crossreactivity with anti-S probes and by metabolic features.Because of its small size the new methanogen is named Methanocorpusculum parvum.This work was supported by a grant of the Deutsche Forschungsgemeinschaft DFG to J. W. and E. S. Immunologic studies were supported in part by grants No. DE-FGO2-84 R 13197 from the U.S. Department of Energy, and No. 261.81/82 from the North Atlantic Treaty Organization (NATO)