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

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

Differential Lipid Partitioning Between Adipocytes and Tissue Macrophages Modulates Macrophage Lipotoxicity and M2/M1 Polarization in Obese Mice

Objective: obesity-associated insulin resistance is characterized by a state of chronic, low-grade inflammation that is associated with the accumulation of M1 proinflammatory macrophages in adipose tissue. Although different evidence explains the mechanisms linking the expansion of adipose tissue and adipose tissue macrophage (ATM) polarization, in the current study we investigated the concept of lipid-induced toxicity as the pathogenic link that could explain the trigger of this response. Research design and methods: we addressed this question using isolated ATMs and adipocytes from genetic and diet-induced murine models of obesity. Through transcriptomic and lipidomic analysis, we created a model integrating transcript and lipid species networks simultaneously occurring in adipocytes and ATMs and their reversibility by thiazolidinedione treatment. Results: we show that polarization of ATMs is associated with lipid accumulation and the consequent formation of foam cell-like cells in adipose tissue. Our study reveals that early stages of adipose tissue expansion are characterized by M2-polarized ATMs and that progressive lipid accumulation within ATMs heralds the M1 polarization, a macrophage phenotype associated with severe obesity and insulin resistance. Furthermore, rosiglitazone treatment, which promotes redistribution of lipids toward adipocytes and extends the M2 ATM polarization state, prevents the lipid alterations associated with M1 ATM polarization. Conclusions:our data indicate that the M1 ATM polarization in obesity might be a macrophage-specific manifestation of a more general lipotoxic pathogenic mechanism. This indicates that strategies to optimize fat deposition and repartitioning toward adipocytes might improve insulin sensitivity by preventing ATM lipotoxicity and M1 polarization.</p

The gut microbiota modulates host energy and lipid metabolism in mice

Peer reviewe

Association of Lipidome Remodeling in the Adipocyte Membrane with Acquired Obesity in Humans

The authors describe a new approach to studying cellular lipid profiles and propose a compensatory mechanism that may help maintain the normal membrane function of adipocytes in the context of obesity

Elective cancer surgery in COVID-19-free surgical pathways during the SARS-CoV-2 pandemic: An international, multicenter, comparative cohort study

PURPOSE As cancer surgery restarts after the first COVID-19 wave, health care providers urgently require data to determine where elective surgery is best performed. This study aimed to determine whether COVID-19–free surgical pathways were associated with lower postoperative pulmonary complication rates compared with hospitals with no defined pathway. PATIENTS AND METHODS This international, multicenter cohort study included patients who underwent elective surgery for 10 solid cancer types without preoperative suspicion of SARS-CoV-2. Participating hospitals included patients from local emergence of SARS-CoV-2 until April 19, 2020. At the time of surgery, hospitals were defined as having a COVID-19–free surgical pathway (complete segregation of the operating theater, critical care, and inpatient ward areas) or no defined pathway (incomplete or no segregation, areas shared with patients with COVID-19). The primary outcome was 30-day postoperative pulmonary complications (pneumonia, acute respiratory distress syndrome, unexpected ventilation). RESULTS Of 9,171 patients from 447 hospitals in 55 countries, 2,481 were operated on in COVID-19–free surgical pathways. Patients who underwent surgery within COVID-19–free surgical pathways were younger with fewer comorbidities than those in hospitals with no defined pathway but with similar proportions of major surgery. After adjustment, pulmonary complication rates were lower with COVID-19–free surgical pathways (2.2% v 4.9%; adjusted odds ratio [aOR], 0.62; 95% CI, 0.44 to 0.86). This was consistent in sensitivity analyses for low-risk patients (American Society of Anesthesiologists grade 1/2), propensity score–matched models, and patients with negative SARS-CoV-2 preoperative tests. The postoperative SARS-CoV-2 infection rate was also lower in COVID-19–free surgical pathways (2.1% v 3.6%; aOR, 0.53; 95% CI, 0.36 to 0.76). CONCLUSION Within available resources, dedicated COVID-19–free surgical pathways should be established to provide safe elective cancer surgery during current and before future SARS-CoV-2 outbreaks

Elective Cancer Surgery in COVID-19-Free Surgical Pathways During the SARS-CoV-2 Pandemic: An International, Multicenter, Comparative Cohort Study.

PURPOSE: As cancer surgery restarts after the first COVID-19 wave, health care providers urgently require data to determine where elective surgery is best performed. This study aimed to determine whether COVID-19-free surgical pathways were associated with lower postoperative pulmonary complication rates compared with hospitals with no defined pathway. PATIENTS AND METHODS: This international, multicenter cohort study included patients who underwent elective surgery for 10 solid cancer types without preoperative suspicion of SARS-CoV-2. Participating hospitals included patients from local emergence of SARS-CoV-2 until April 19, 2020. At the time of surgery, hospitals were defined as having a COVID-19-free surgical pathway (complete segregation of the operating theater, critical care, and inpatient ward areas) or no defined pathway (incomplete or no segregation, areas shared with patients with COVID-19). The primary outcome was 30-day postoperative pulmonary complications (pneumonia, acute respiratory distress syndrome, unexpected ventilation). RESULTS: Of 9,171 patients from 447 hospitals in 55 countries, 2,481 were operated on in COVID-19-free surgical pathways. Patients who underwent surgery within COVID-19-free surgical pathways were younger with fewer comorbidities than those in hospitals with no defined pathway but with similar proportions of major surgery. After adjustment, pulmonary complication rates were lower with COVID-19-free surgical pathways (2.2% v 4.9%; adjusted odds ratio [aOR], 0.62; 95% CI, 0.44 to 0.86). This was consistent in sensitivity analyses for low-risk patients (American Society of Anesthesiologists grade 1/2), propensity score-matched models, and patients with negative SARS-CoV-2 preoperative tests. The postoperative SARS-CoV-2 infection rate was also lower in COVID-19-free surgical pathways (2.1% v 3.6%; aOR, 0.53; 95% CI, 0.36 to 0.76). CONCLUSION: Within available resources, dedicated COVID-19-free surgical pathways should be established to provide safe elective cancer surgery during current and before future SARS-CoV-2 outbreaks

Physiological and metabolic flux screening of <i>Saccharomyces cerevisiae</i> single knockout mutants on different carbon sources:Dissertation

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'D 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'D 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'D 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'D 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