Central metabolism in Mycobacterium smegmatis during the transition from O2-rich to O2-poor conditions as studied by isotopomer-assisted metabolite analysis

Isotopomer-assisted metabolite analysis was used to investigate the central metabolism of Mycobacterium smegmatis and its transition from normal growth to a non-replicating state under a hypoxic environment. Tween 80 significantly promoted aerobic growth by improving O2 transfer, while only small amount was degraded and metabolized via the TCA cycle for biomass synthesis. As the bacillus encountered hypoxic stress, isotopomer analysis suggested: (1) isocitrate lyase activity increased, which further induced glyoxylate pathway and glycine dehydrogenase for replenishing NAD+; (2) the relative amount of acetyl-CoA entering the TCA cycle was doubled, whereas little entered the glycolytic and pentose phosphate pathways

Novel composite implant in craniofacial bone reconstruction

Bioactive glass (BAG) and polymethyl methacrylate (PMMA) have been used in clinical applications. Antimicrobial BAG has the ability to attach chemically to surrounding bone, but it is not possible to bend, drill or shape BAG during the operation. PMMA has advantages in terms of shaping during the operation, but it does not attach chemically to the bone and is an exothermic material. To increase the usefulness of BAG and PMMA in skull bone defect reconstructions, a new composite implant containing BAG and PMMA in craniofacial reconstructions is presented. Three patients had pre-existing large defects in the calvarial and one in the midface area. An additive manufacturing (AM) model was used preoperatively for treatment planning and custom-made implant production. The trunk of the PMMA implant was coated with BAG granules. Clinical and radiological follow-up was performed postoperatively at 1 week, and 3, 6 and 12 months, and thereafter annually up to 5 years. Computer tomography (CT) and positron emission tomography (PET-CT) were performed at 12 and 24 months postoperatively. Uneventful clinical recovery with good esthetic and functional outcome was seen. CT and PET-CT findings supported good clinical outcome. The BAG–PMMA implant seems to be a promising craniofacial reconstruction alternative. However, more clinical experience is needed

Planktonic events may cause polymictic-dimictic regime shifts in temperate lakes

Water transparency affects the thermal structure of lakes, and within certain lake depth ranges, it can determine whether a lake mixes regularly (polymictic regime) or stratifies continuously (dimictic regime) from spring through summer. Phytoplankton biomass can influence transparency but the effect of its seasonal pattern on stratification is unknown. Therefore we analysed long term field data from two lakes of similar depth, transparency and climate but one polymictic and one dimictic, and simulated a conceptual lake with a hydrodynamic model. Transparency in the study lakes was typically low during spring and summer blooms and high in between during the clear water phase (CWP), caused when zooplankton graze the spring bloom. The effect of variability of transparency on thermal structure was stronger at intermediate transparency and stronger during a critical window in spring when the rate of lake warming is highest. Whereas the spring bloom strengthened stratification in spring, the CWP weakened it in summer. The presence or absence of the CWP influenced stratification duration and under some conditions determined the mixing regime. Therefore seasonal plankton dynamics, including biotic interactions that suppress the CWP, can influence lake temperatures, stratification duration, and potentially also the mixing regime

Chlorogenic Acid Stimulates Glucose Transport in Skeletal Muscle via AMPK Activation: A Contributor to the Beneficial Effects of Coffee on Diabetes

Chlorogenic acid (CGA) has been shown to delay intestinal glucose absorption and inhibit gluconeogenesis. Our aim was to investigate the role of CGA in the regulation of glucose transport in skeletal muscle isolated from db/db mice and L6 skeletal muscle cells. Oral glucose tolerance test was performed on db/db mice treated with CGA and soleus muscle was isolated for 2-deoxyglucose transport study. 2DG transport was also examined in L6 myotubes with or without inhibitors such as wortmannin or compound c. AMPK was knocked down with AMPKα1/2 siRNA to study its effect on CGA-stimulated glucose transport. GLUT 4 translocation, phosphorylation of AMPK and Akt, AMPK activity, and association of IRS-1 and PI3K were investigated in the presence of CGA. In db/db mice, a significant decrease in fasting blood sugar was observed 10 minutes after the intraperitoneal administration of 250 mg/kg CGA and the effect persisted for another 30 minutes after the glucose challenge. Besides, CGA stimulated and enhanced both basal and insulin-mediated 2DG transports in soleus muscle. In L6 myotubes, CGA caused a dose- and time-dependent increase in glucose transport. Compound c and AMPKα1/2 siRNA abrogated the CGA-stimulated glucose transport. Consistent with these results, CGA was found to phosphorylate AMPK and ACC, consistent with the result of increased AMPK activities. CGA did not appear to enhance association of IRS-1 with p85. However, we observed activation of Akt by CGA. These parallel activations in turn increased translocation of GLUT 4 to plasma membrane. At 2 mmol/l, CGA did not cause any significant changes in viability or proliferation of L6 myotubes. Our data demonstrated for the first time that CGA stimulates glucose transport in skeletal muscle via the activation of AMPK. It appears that CGA may contribute to the beneficial effects of coffee on Type 2 diabetes mellitus

Altered Metabolism and Persistent Starvation Behaviors Caused by Reduced AMPK Function in Drosophila

Organisms must utilize multiple mechanisms to maintain energetic homeostasis in the face of limited nutrient availability. One mechanism involves activation of the heterotrimeric AMP-activated protein kinase (AMPK), a cell-autonomous sensor to energetic changes regulated by ATP to AMP ratios. We examined the phenotypic consequences of reduced AMPK function, both through RNAi knockdown of the gamma subunit (AMPKγ) and through expression of a dominant negative alpha (AMPKα) variant in Drosophila melanogaster. Reduced AMPK signaling leads to hypersensitivity to starvation conditions as measured by lifespan and locomotor activity. Locomotor levels in flies with reduced AMPK function were lower during unstressed conditions, but starvation-induced hyperactivity, an adaptive response to encourage foraging, was significantly higher than in wild type. Unexpectedly, total dietary intake was greater in animals with reduced AMPK function yet total triglyceride levels were lower. AMPK mutant animals displayed starvation-like lipid accumulation patterns in metabolically key liver-like cells, oenocytes, even under fed conditions, consistent with a persistent starved state. Measurements of O2 consumption reveal that metabolic rates are greater in animals with reduced AMPK function. Lastly, rapamycin treatment tempers the starvation sensitivity and lethality associated with reduced AMPK function. Collectively, these results are consistent with models that AMPK shifts energy usage away from expenditures into a conservation mode during nutrient-limited conditions at a cellular level. The highly conserved AMPK subunits throughout the Metazoa, suggest such findings may provide significant insight for pharmaceutical strategies to manipulate AMPK function in humans

LC–MS-based absolute metabolite quantification:Application to metabolic flux measurement in trypanosomes

Human African trypanosomiasis is a neglected tropical disease caused by the protozoan parasite, Trypanosoma brucei. In the mammalian bloodstream, the trypanosome’s metabolism differs significantly from that of its host. For example, the parasite relies exclusively on glycolysis for energy source. Recently, computational and mathematical models of trypanosome metabolism have been generated to assist in understanding the parasite metabolism with the aim of facilitating drug development. Optimisation of these models requires quantitative information, including metabolite concentrations and/or metabolic fluxes that have been hitherto unavailable on a large scale. Here, we have implemented an LC–MS-based method that allows large scale quantification of metabolite levels by using U-13C-labelled E. coli extracts as internal standards. Known amounts of labelled E. coli extract were added into the parasite samples, as well as calibration standards, and used to obtain calibration curves enabling us to convert intensities into concentrations. This method allowed us to reliably quantify the changes of 43 intracellular metabolites and 32 extracellular metabolites in the medium over time. Based on the absolute quantification, we were able to compute consumption and production fluxes. These quantitative data can now be used to optimise computational models of parasite metabolism

Inhibition of Melanogenesis by the Pyridinyl Imidazole Class of Compounds: Possible Involvement of the Wnt/β-Catenin Signaling Pathway

While investigating the role of p38 MAPK in regulating melanogenesis, we found that pyridinyl imidazole inhibitors class compounds as well as the analog compound SB202474, which does not inhibit p38 MAPK, suppressed both α-MSH-induced melanogenesis and spontaneous melanin synthesis. In this study, we demonstrated that the inhibitory activity of the pyridinyl imidazoles correlates with inhibition of the canonical Wnt/β-catenin pathway activity. Imidazole-treated cells showed a reduction in the level of Tcf/Lef target genes involved in the β-catenin signaling network, including ubiquitous genes such as Axin2, Lef1, and Wisp1 as well as cell lineage-restricted genes such as microphthalmia-associated transcription factor and dopachrome tautomerase. Although over-expression of the Wnt signaling pathway effector β-catenin slightly restored the melanogenic program, the lack of complete reversion suggested that the imidazoles interfered with β-catenin-dependent transcriptional activity rather than with β-catenin expression. Accordingly, we did not observe any significant change in β-catenin protein expression. The independence of p38 MAPK activity from the repression of Wnt/β-catenin signaling pathway was confirmed by small interfering RNA knockdown of p38 MAPK expression, which by contrast, stimulated β-catenin-driven gene expression. Our data demonstrate that the small molecule pyridinyl imidazoles possess two distinct and opposite mechanisms that modulate β-catenin dependent transcription: a p38 inhibition-dependent effect that stimulates the Wnt pathway by increasing β-catenin protein expression and an off-target mechanism that inhibits the pathway by repressing β-catenin protein functionality. The p38-independent effect seems to be dominant and, at least in B16-F0 cells, results in a strong block of the Wnt/β-catenin signaling pathway

Substrate cycling between de novo lipogenesis and lipid oxidation: a thermogenic mechanism against skeletal muscle lipotoxicity and glucolipotoxicity

Life is a combustion, but how the major fuel substrates that sustain human life compete and interact with each other for combustion has been at the epicenter of research into the pathogenesis of insulin resistance ever since Randle proposed a 'glucose-fatty acid cycle' in 1963. Since then, several features of a mutual interaction that is characterized by both reciprocality and dependency between glucose and lipid metabolism have been unravelled, namely: 1. the inhibitory effects of elevated concentrations of fatty acids on glucose oxidation (via inactivation of mitochondrial pyruvate dehydrogenase or via desensitization of insulin-mediated glucose transport), 2. the inhibitory effects of elevated concentrations of glucose on fatty acid oxidation (via malonyl-CoA regulation of fatty acid entry into the mitochondria), and more recently 3. the stimulatory effects of elevated concentrations of glucose on de novo lipogenesis, that is, synthesis of lipids from glucose (via SREBP1c regulation of glycolytic and lipogenic enzymes). This paper first revisits the physiological significance of these mutual interactions between glucose and lipids in skeletal muscle pertaining to both blood glucose and intramyocellular lipid homeostasis. It then concentrates upon emerging evidence, from calorimetric studies investigating the direct effect of leptin on thermogenesis in intact skeletal muscle, of yet another feature of the mutual interaction between glucose and lipid oxidation: that of substrate cycling between de novo lipogenesis and lipid oxidation. It is proposed that this energy-dissipating substrate cycling that links glucose and lipid metabolism to thermogenesis could function as a 'fine-tuning' mechanism that regulates intramyocellular lipid homeostasis, and hence contributes to the protection of skeletal muscle against lipotoxicity

Network analysis of the transcriptional pattern of young and old cells of Escherichia coli during lag phase

Background: The aging process of bacteria in stationary phase is halted if cells are subcultured and enter lag phase and it is then followed by cellular division. Network science has been applied to analyse the transcriptional response, during lag phase, of bacterial cells starved previously in stationary phase for 1 day (young cells) and 16 days (old cells). Results: A genome scale network was constructed for E. coli K-12 by connecting genes with operons, transcription and sigma factors, metabolic pathways and cell functional categories. Most of the transcriptional changes were detected immediately upon entering lag phase and were maintained throughout this period. The lag period was longer for older cells and the analysis of the transcriptome revealed different intracellular activity in young and old cells. The number of genes differentially expressed was smaller in old cells (186) than in young cells (467). Relatively, few genes (62) were up- or down-regulated in both cultures. Transcription of genes related to osmotolerance, acid resistance, oxidative stress and adaptation to other stresses was down-regulated in both young and old cells. Regarding carbohydrate metabolism, genes related to the citrate cycle were up-regulated in young cells while old cells up-regulated the Entner Doudoroff and gluconate pathways and down-regulated the pentose phosphate pathway. In both old and young cells, anaerobic respiration and fermentation pathways were down-regulated, but only young cells up-regulated aerobic respiration while there was no evidence of aerobic respiration in old cells.Numerous genes related to DNA maintenance and replication, translation, ribosomal biosynthesis and RNA processing as well as biosynthesis of the cell envelope and flagellum and several components of the chemotaxis signal transduction complex were up-regulated only in young cells. The genes for several transport proteins for iron compounds were up-regulated in both young and old cells. Numerous genes encoding transporters for carbohydrates and organic alcohols and acids were down-regulated in old cells only. Conclusion: Network analysis revealed very different transcriptional activities during the lag period in old and young cells. Rejuvenation seems to take place during exponential growth by replicative dilution of old cellular components