Physiological and metabolic flux screening of Saccharomyces cerevisiae single knockout mutants on different carbon sources

A novel method for high-throughput stoichiometric and metabolic flux profiling was developed and a set of deletion mutants of S. cerevisiae, which are known to be involved in central carbon metabolism were selected and investigated on glucose, galactose and fructose. On glucose and fructose, the growth was predominantly fermentative and on galactose, respiration was more active. mae1D strain did not show any significant growth phenotype on glucose, however, it had highest PPP flux on galactose, which could be due to redirection of NADPH production to the PPP. On fructose, mae1D strain had highest oxygen uptake rate with very low ethanol yield, which could be due to reduced PPP flux and to maintain NADPH levels either via NADPH specific -isocitrate dehydrogenase or -aldehyde dehydro-genase. imp2\u27D strain had lowest PPP flux and very high respiratory activity on galactose; and pck1D strain had lowest PPP flux on glucose, which might also point to a possible activation of malic enzyme. On fructose, hxt17D strain had highest sugar consumption and ethanol production rates and imp2\u27D strain had highest ethanol yield. The functional prediction of hypothetical genes by utilising this quantitative data using computational analyses suggested a possible role in glycolysis or pyruvate metabolism for YBR184W and low affinity transporter role for YIL170W.Es wurde eine neue Hochdurchsatzmethode für die Charakterisierung der Stöchiometrie und der metabolischen Flüsse entwickelt und auf ausgewählte Deletionsmutanten des Zentralstoffwechsels von S. cerevisiae angewendet, wobei Glucose, Galactose und Fructose als Substrate eingesetzt wurden. Während auf Glucose und Fructose das Wachstum vorwiegend fermentativ war, war es auf Galactose mehr respirativ. Der mae1D Stamm zeigte keinen Phänotyp auf Glucose, hatte aber auf Galactose einen sehr hohen Fluss in den Pentosephosphatweg (PPP) mit entsprechend hoher Bereitstellung von NADPH und auf Fructose die höchste Sauerstoffaufnahmerate mit zugleich sehr niedriger Ethanolausbeute, was auf einen reduzierten Fluss in den PPP und verstärkte Bildung von NADPH über die Isocitratdehydrogenase oder die Aldehyddehydrogenase hindeutet. Der imp2\u27D Stamm hatte einen sehr niedrigen PPP-Fluss und starke Respiration auf Galactose. Der pck1D Stamm hatte die niedrigsten PPP Fluss auf Glucose, was auf eine Aktivierung des Malatenzyms hindeutet. Auf Fructose zeigte der hxt17D Stamm höchste Zuckerverbrauchs- und Ethanolproduktionsraten und imp2\u27D hatte die höchste Ethanolausbeute. Numerische Analysen erlaubten eine erste Vorhersage möglicher Funktionen zweier hypothetischer Gene, in der Glykolyse oder im Pyruvatmetabolismus für YBR184W und als niedrig affinen Transporter für YIL170W

Antimalarial drug artemether inhibits neuroinflammation in BV2 microglia through Nrf2-dependent mechanisms

Artemether, a lipid-soluble derivative of artemisinin has been reported to possess anti-inflammatory properties. In this study, we have investigated the molecular mechanisms involved in the inhibition of neuroinflammation by the drug. The effects of artemether on neuroinflammation-mediated HT22 neuronal toxicity were also investigated in a BV2 microglia/HT22 neuron co-culture. To investigate effects on neuroinflammation, we used LPS-stimulated BV2 microglia treated with artemether (5-40µM) for 24 hours. ELISAs and western blotting were used to detect pro inflammatory cytokines, nitric oxide, PGE2, iNOS, COX-2 and mPGES-1. BACE-1 activity and Aβ levels were measured with ELISA kits. Protein levels of targets in NF-kappaB and p38 MAPK signalling, as well as HO-1, NQO1 and Nrf2 were also measured with western blot. NF-kappaB binding to the DNA was investigated using EMSA. MTT, DNA fragmentation and ROS assays in BV2-HT22 neuronal co-culture were used to evaluate the effects of artemether on neuroinflammation-induced neuronal death. The role of Nrf2 in the anti-inflammatory activity of artemether was investigated in BV2 cells transfected with Nrf2 siRNA. Artemether significantly suppressed pro-inflammatory mediators (NO/iNOS, PGE2/COX-2/mPGES-1, TNFα, and IL-6), Aβ and BACE-1 in BV2 cells following LPS stimulation. These effects of artemether were shown to be mediated through inhibition of NF-kappaB and p38MAPK signalling. Artemether produced increased levels of HO-1, NQO1 and GSH in BV2 microglia. The drug activated Nrf2 activity by increasing nuclear translocation of Nrf2 and its binding to antioxidant response elements in BV2 cells. Transfection of BV2 microglia with Nrf2 siRNA resulted in the loss of both anti-inflammatory and neuroprotective activities of artemether. We conclude that artemether induces Nrf2 expression and suggest that Nrf2 mediates the anti-inflammatory effect of artemether in BV2 microglia. Our results suggest that this drug has a therapeutic potential in neurodegenerative disorders

Inhibition of neuroinflammation in BV2 microglia by the biflavonoid kolaviron is dependent on the Nrf2/ARE antioxidant protective mechanism

Kolaviron is a mixture of bioflavonoids found in the nut of the West African edible seed Garcinia kola, and it has been reported to exhibit a wide range of pharmacological activities. In this study, we investigated the effects of kolaviron in neuroinflammation. The effects of kolaviron on the expression of nitric oxide/inducible nitric oxide synthase (iNOS), prostaglandin E2 (PGE2)/cyclooxygenase-2, cellular reactive oxygen species (ROS) and the pro-inflammatory cytokines were examined in lipopolysaccharide (LPS)-stimulated BV2 microglial cells. Molecular mechanisms of the effects of kolaviron on NF-B and Nrf2/ARE signalling pathways were analysed by immunoblotting, binding assay, and reporter assay. RNA interference was used to investigate the role of Nrf2 in the anti-inflammatory effect of kolaviron. Neuroprotective effect of kolaviron was assessed in a BV2 microglia/HT22 hippocampal neuron co-culture. Kolaviron inhibited the protein levels of NO/iNOS, PGE2/COX-2, cellular ROS and the proinflammatory cytokines (TNFα and IL-6) in LPS-stimulated microglia. Further mechanistic studies showed that kolaviron inhibited neuroinflammation by inhibiting IB/NF-B signalling pathway in LPS-activated BV2 microglia. Kolaviron produced antioxidant effect in BV2 microglia by increasing HO-1 via the Nrf2/ antioxidant response element (ARE) pathway. RNAi experiments revealed that Nrf2 is need for the anti-inflammatory effect of kolaviron. Kolaviron protected HT22 neurons from neuroinflammation-induced toxicity. Kolaviron inhibits neuroinflammation through Nrf2-dependent mechanisms. This compound may therefore be beneficial in neuroinflammation-related neurodegenerative disorders

Automated Analysis of Cryptococcal Macrophage Parasitism Using GFP-Tagged Cryptococci

The human fungal pathogens Cryptococcus neoformans and C. gattii cause life-threatening infections of the central nervous system. One of the major characteristics of cryptococcal disease is the ability of the pathogen to parasitise upon phagocytic immune effector cells, a phenomenon that correlates strongly with virulence in rodent models of infection. Despite the importance of phagocyte/Cryptococcus interactions to disease progression, current methods for assaying virulence in the acrophage system are both time consuming and low throughput. Here, we introduce the first stable and fully characterised GFP–expressing derivatives of two widely used cryptococcal strains: C. neoformans serotype A type strain H99 and C. gattii serotype B type strain R265. Both strains show unaltered responses to environmental and host stress conditions and no deficiency in virulence in the macrophage model system. In addition, we report the development of a method to effectively and rapidly investigate macrophage parasitism by flow cytometry, a technique that preserves the accuracy of current approaches but offers a four-fold improvement in speed

Static v. Expandable TLIF Cage Outcomes

Static cages were introduced in the 1990s as a solution to degenerative spondylolisthesis, recurrent disc herniation and spinal stenosis. As this procedure was popularized, a new class of expandable Transforaminal Lumbar Interbody Fusion devices was introduced to further improve outcomes that will be studied in this project. It will be explored how expandable cages compare to static cages in TLIF procedures in patient-reported outcomes, complications and restoration of appropriate lumbar lordosis. We conducted a retrospective cohort review comparing those who received expandable and static cages. Eligible patients received TLIF procedure at the Rothman Institute, were ≥18 years of age and had radiographic follow-up at 3 months and 1 year postoperatively. Outcomes were measured in lumbar lordosis via calculating angles via radiographic images preoperatively and 3 month and 1 year postoperatively as well as pre- and post-operative SF-12 surveys. At this time, data acquisition is ongoing and no preliminary data has been generated. However, we anticipate better patient reported outcomes and greater and sustained restoration of Lumbar Lordosis in patients who received expandable cages. Data collection is scheduled to be completed shortly. Once completed, this will be a study of greater magnitude and will address the shortage of investigations into the surgical outcomes of static and expandable cages and clarify the theorized benefits of expandable cages. Recent emphasis has been placed on restoring appropriate lumbar lordosis in fusion surgeries and this project was designed to investigate lordosis at different time posts as compared to patient-reported outcomes

Insight into the prebiotic concept: lessons from an exploratory, double blind intervention study with inulin-type fructans in obese women

Objective To highlight the contribution of the gut microbiota to the modulation of host metabolism by dietary inulin-type fructans (ITF prebiotics) in obese women. Methods A double blind, placebo controlled, intervention study was performed with 30 obese women treated with ITF prebiotics (inulin/oligofructose 50/50 mix; n=15) or placebo (maltodextrin; n=15) for 3 months (16 g/day). Blood, faeces and urine sampling, oral glucose tolerance test, homeostasis model assessment and impedancemetry were performed before and after treatment. The gut microbial composition in faeces was analysed by phylogenetic microarray and qPCR analysis of 16S rDNA. Plasma and urine metabolic profiles were analysed by 1H-NMR spectroscopy. Results Treatment with ITF prebiotics, but not the placebo, led to an increase in Bifidobacterium and Faecalibacterium prausnitzii; both bacteria negatively correlated with serum lipopolysaccharide levels. ITF prebiotics also decreased Bacteroides intestinalis, Bacteroides vulgatus and Propionibacterium, an effect associated with a slight decrease in fat mass and with plasma lactate and phosphatidylcholine levels. No clear treatment clustering could be detected for gut microbial analysis or plasma and urine metabolomic profile analyses. However, ITF prebiotics led to subtle changes in the gut microbiota that may importantly impact on several key metabolites implicated in obesity and/or diabetes. Conclusions ITF prebiotics selectively changed the gut microbiota composition in obese women, leading to modest changes in host metabolism, as suggested by the correlation between some bacterial species and metabolic endotoxaemia or metabolomic signatures

Salmonella Typhimurium impairs glycolysismediated acidification of phagosomes to evade macrophage defense

Regulation of cellular metabolism is now recognized as a crucial mechanism for the activation of innate and adaptive immune cells upon diverse extracellular stimuli. Macrophages, for instance, increase glycolysis upon stimulation with pathogen-associated molecular patterns (PAMPs). Conceivably, pathogens also counteract these metabolic changes for their own survival in the host. Despite this dynamic interplay in host-pathogen interactions, the role of immunometabolism in the context of intracellular bacterial infections is still unclear. Here, employing unbiased metabolomic and transcriptomic approaches, we investigated the role of metabolic adaptations of macrophages upon Salmonella enterica serovar Typhimurium (S. Typhimurium) infections. Importantly, our results suggest that S. Typhimurium abrogates glycolysis and its modulators such as insulin-signaling to impair macrophage defense. Mechanistically, glycolysis facilitates glycolytic enzyme aldolase A mediated v- ATPase assembly and the acidification of phagosomes which is critical for lysosomal degradation. Thus, impairment in the glycolytic machinery eventually leads to decreased bacterial clearance and antigen presentation in murine macrophages (BMDM). Collectively, our results highlight a vital molecular link between metabolic adaptation and phagosome maturation in macrophages, which is targeted by S. Typhimurium to evade cell-autonomous defense. Copyright

Spatial and temporal expression of laccase in Coprinopsis cinerea using galectin promoters

Comunicación presentada al VI Meeting on Genetics and Cellular Biology of Basidiomycetes (GCBB-VI), organizado por y celebrado en la Universidad Pública de Navarra el 3-6 de junio de 2005.Galectins are ß-galactoside binding lectins defined by a conserved sequence. In mammals, there is a family of galectins that interact with glycoproteins in both extracellular and intracellular milieu and regulate various biological phenomena including cell growth, cell differentiation, cell adhesion, and apoptosis. Outside of the animal kingdom, genes for galectins are known in Arabidopsis and a few basidiomycetes. In fungi, galectins have been studied in detail in Coprinopsis cinerea. The C. cinerea galectins Cgl1 and Cgl2 are specific to the fruiting body. When nutrients are exhausted, Cgl2 is expressed under dark conditions in the mycelium at places of fruiting body initiation and primary hyphal knot formation. Cgl2 expression continues within the initial stages of fruiting body development till primordia formation is completed. In contrast, expression of Cgl1 starts later with the light-induced formation of secondary hyphal knots. Cgl1 expression also continues during primordia development to end at the stage of meiosis. In the genome, there is a gene for a third galectin (cgl3) but nothing is yet known about its expression. In this study, we establish the C. cinerea laccase gene lcc1 as a reporter gene to study spatial and temporal regulation of galectin gene promoters during fruiting body development.The author's laboratory is funded by the DBU (Deutsche Bundesstiftung Umwelt)

Metabolomic Profiling of Extracellular Vesicles and Alternative Normalization Methods Reveal Enriched Metabolites and Strategies to Study Prostate Cancer-Related Changes

Body fluids are a rich source of extracellular vesicles (EVs), which carry cargo derived from the secreting cells. So far, biomarkers for pathological conditions have been mainly searched from their protein, (mi) RNA, DNA and lipid cargo. Here, we explored the small molecule metabolites from urinary and platelet EVs relative to their matched source samples. As a proof-of-concept study of intra-EV metabolites, we compared alternative normalization methods to profile urinary EVs from prostate cancer patients before and after prostatectomy and from healthy controls. Methods: We employed targeted ultra-performance liquid chromatography-tandem mass spectrometry to profile over 100 metabolites in the isolated EVs, original urine samples and platelets. We determined the enrichment of the metabolites in the EVs and analyzed their subcellular origin, pathways and relevant enzymes or transporters through data base searches. EV-and urine-derived factors and ratios between metabolites were tested for normalization of the metabolomics data. Results: Approximately 1 x 10(10) EVs were sufficient for detection of metabolite profiles from EVs. The profiles of the urinary and platelet EVs overlapped with each other and with those of the source materials, but they also contained unique metabolites. The EVs enriched a selection of cytosolic metabolites including members from the nucleotide and spermidine pathways, which linked to a number of EV-resident enzymes or transporters. Analysis of the urinary EVs from the patients indicated that the levels of glucuronate, D-ribose 5-phosphate and isobutyryl-L-carnitine were 2-26-fold lower in all pre-prostatectomy samples compared to the healthy control and post-prostatectomy samples (p <0.05). These changes were only detected from EVs by normalization to EV-derived factors or with metabolite ratios, and not from the original urine samples. Conclusions: Our results suggest that metabolite analysis of EVs from different samples is feasible using a high-throughput platform and relatively small amount of sample material. With the knowledge about the specific enrichment of metabolites and normalization methods, EV metabolomics could be used to gain novel biomarker data not revealed by the analysis of the original EV source materials.Peer reviewe

Multiple hydrophobin genes in mushrooms

Comunicación presentada al VI Meeting on Genetics and Cellular Biology of Basidiomycetes (GCBB-VI), organizado por y celebrado en la Universidad Pública de Navarra el 3-6 de junio de 2005.Hydrophobins are small secreted fungal proteins that form amphipathic films on the hyphal surfaces. In the wood-rotting fungus Schizophyllum commune, four different hydrophobins are known with well established functions during vegetative growth and fruiting body development. Our study aims at elucidating the role of these proteins in wood penetration and lignocellulose degradation. Blast searches of the genome of the dung fungus Coprinopsis cinerea revealed a surprising number of 34 different hydrophobin genes in this species. Functional analysis of these genes is in progress.Part of this work was conducted within the NHN (Niedersächsisches Kompetenznetz für Nachhaltige Holznutzung). Financial support by the Ministry for Science and Culture of Lower Saxony and by EFRE (European Fund for Regional Development, grant 2001.085). The section Molecular Wood Biotechnology at the Institute for Forest Botany is funded by the DBU (Deutsche Bundesstiftung Umwelt)