Systems-level organization of yeast methylotrophic lifestyle

BACKGROUND: Some yeasts have evolved a methylotrophic lifestyle enabling them to utilize the single carbon compound methanol as a carbon and energy source. Among them, Pichia pastoris (syn. Komagataella sp.) is frequently used for the production of heterologous proteins and also serves as a model organism for organelle research. Our current knowledge of methylotrophic lifestyle mainly derives from sophisticated biochemical studies which identified many key methanol utilization enzymes such as alcohol oxidase and dihydroxyacetone synthase and their localization to the peroxisomes. C1 assimilation is supposed to involve the pentose phosphate pathway, but details of these reactions are not known to date. RESULTS: In this work we analyzed the regulation patterns of 5,354 genes, 575 proteins, 141 metabolites, and fluxes through 39 reactions of P. pastoris comparing growth on glucose and on a methanol/glycerol mixed medium, respectively. Contrary to previous assumptions, we found that the entire methanol assimilation pathway is localized to peroxisomes rather than employing part of the cytosolic pentose phosphate pathway for xylulose-5-phosphate regeneration. For this purpose, P. pastoris (and presumably also other methylotrophic yeasts) have evolved a duplicated methanol inducible enzyme set targeted to peroxisomes. This compartmentalized cyclic C1 assimilation process termed xylose-monophosphate cycle resembles the principle of the Calvin cycle and uses sedoheptulose-1,7-bisphosphate as intermediate. The strong induction of alcohol oxidase, dihydroxyacetone synthase, formaldehyde and formate dehydrogenase, and catalase leads to high demand of their cofactors riboflavin, thiamine, nicotinamide, and heme, respectively, which is reflected in strong up-regulation of the respective synthesis pathways on methanol. Methanol-grown cells have a higher protein but lower free amino acid content, which can be attributed to the high drain towards methanol metabolic enzymes and their cofactors. In context with up-regulation of many amino acid biosynthesis genes or proteins, this visualizes an increased flux towards amino acid and protein synthesis which is reflected also in increased levels of transcripts and/or proteins related to ribosome biogenesis and translation. CONCLUSIONS: Taken together, our work illustrates how concerted interpretation of multiple levels of systems biology data can contribute to elucidation of yet unknown cellular pathways and revolutionize our understanding of cellular biology. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12915-015-0186-5) contains supplementary material, which is available to authorized users

Energy Estimation of Cosmic Rays with the Engineering Radio Array of the Pierre Auger Observatory

The Auger Engineering Radio Array (AERA) is part of the Pierre Auger Observatory and is used to detect the radio emission of cosmic-ray air showers. These observations are compared to the data of the surface detector stations of the Observatory, which provide well-calibrated information on the cosmic-ray energies and arrival directions. The response of the radio stations in the 30 to 80 MHz regime has been thoroughly calibrated to enable the reconstruction of the incoming electric field. For the latter, the energy deposit per area is determined from the radio pulses at each observer position and is interpolated using a two-dimensional function that takes into account signal asymmetries due to interference between the geomagnetic and charge-excess emission components. The spatial integral over the signal distribution gives a direct measurement of the energy transferred from the primary cosmic ray into radio emission in the AERA frequency range. We measure 15.8 MeV of radiation energy for a 1 EeV air shower arriving perpendicularly to the geomagnetic field. This radiation energy -- corrected for geometrical effects -- is used as a cosmic-ray energy estimator. Performing an absolute energy calibration against the surface-detector information, we observe that this radio-energy estimator scales quadratically with the cosmic-ray energy as expected for coherent emission. We find an energy resolution of the radio reconstruction of 22% for the data set and 17% for a high-quality subset containing only events with at least five radio stations with signal.Comment: Replaced with published version. Added journal reference and DO

Measurement of the Radiation Energy in the Radio Signal of Extensive Air Showers as a Universal Estimator of Cosmic-Ray Energy

We measure the energy emitted by extensive air showers in the form of radio emission in the frequency range from 30 to 80 MHz. Exploiting the accurate energy scale of the Pierre Auger Observatory, we obtain a radiation energy of 15.8 \pm 0.7 (stat) \pm 6.7 (sys) MeV for cosmic rays with an energy of 1 EeV arriving perpendicularly to a geomagnetic field of 0.24 G, scaling quadratically with the cosmic-ray energy. A comparison with predictions from state-of-the-art first-principle calculations shows agreement with our measurement. The radiation energy provides direct access to the calorimetric energy in the electromagnetic cascade of extensive air showers. Comparison with our result thus allows the direct calibration of any cosmic-ray radio detector against the well-established energy scale of the Pierre Auger Observatory.Comment: Replaced with published version. Added journal reference and DOI. Supplemental material in the ancillary file

Oxidative stress in hypobaric hypoxia and influence on vessel-tone modifying mediators

Increased pulmonary artery pressure is a well-known phenomenon of hypoxia and is seen in patients with chronic pulmonary diseases, and also in mountaineers on high altitude expedition. Different mediators are known to regulate pulmonary artery vessel tone. However, exact mechanisms are not fully understood and a multimodal process consisting of a whole panel of mediators is supposed to cause pulmonary artery vasoconstriction. We hypothesized that increased hypoxemia is associated with an increase in vasoconstrictive mediators and decrease of vasodilatators leading to a vasoconstrictive net effect. Furthermore, we suggested oxidative stress being partly involved in changement of these parameters. Oxygen saturation (Sao2) and clinical parameters were assessed in 34 volunteers before and during a Swiss research expedition to Mount Muztagh Ata (7549 m) in Western China. Blood samples were taken at four different sites up to an altitude of 6865 m. A mass spectrometry-based targeted metabolomic platform was used to detect multiple parameters, and revealed functional impairment of enzymes that require oxidation-sensitive cofactors. Specifically, the tetrahydrobiopterin (BH4)-dependent enzyme nitric oxide synthase (NOS) showed significantly lower activities (citrulline-to-arginine ratio decreased from baseline median 0.21 to 0.14 at 6265 m), indicating lower NO availability resulting in less vasodilatative activity. Correspondingly, an increase in systemic oxidative stress was found with a significant increase of the percentage of methionine sulfoxide from a median 6% under normoxic condition to a median level of 30% (p<0.001) in camp 1 at 5533 m. Furthermore, significant increase in vasoconstrictive mediators (e.g., tryptophan, serotonin, and peroxidation-sensitive lipids) were found. During ascent up to 6865 m, significant altitude-dependent changes in multiple vessel-tone modifying mediators with excess in vasoconstrictive metabolites could be demonstrated. These changes, as well as highly significant increase in systemic oxidative stress, may be predictive for increase in acute mountain sickness score and changes in Sao2

Stability of human plasma “metabotypes” in response to dietary challenges

Introduction and Objective – Metabolic phenotyping in humans is usually done in fasting state not taking into account that metabolism is highly dynamic with major changes in space (organs) and time. To assess this flexibility of the human metabolic system we performed standardized challenge tests combined with metabolite profiling of plasma and tissues as part of the NutriTech project.Methods – 72 volunteers (men and women, age = 60±3 yrs., BMI = 30±2 kg/m²) were recruited, submitted to whole body MRI and underwent an oral glucose tolerance test (OGTT) and a mixed meal tolerance test (MMTT). The two tests were repeated after 13 weeks with 40 volunteers on a diet providing 20 % less calories, while 32 followed a standardized diet without energy restriction. Metabolite profiling was performed in plasma samples using LC-MS/MS, GC-MS and NMR platforms with around 260 entities covered. Results – Despite a mean weight loss of 4.5±0.2 kg, caloric restriction did not induce major changes in plasma metabolite concentration profiles during the challenge tests. Assessed by ANOVA, the overall contribution of the caloric restriction to the observed variations of metabolite concentrations was negligible, ranging from 0.25% for lyso PC C18:2, to about 2.3% for Alanine. This indicates the individual responses to the challenges were highly reproducible on the two occasions, identifying “robust metabotypes”. Despite little effect of the intervention, inter-individual variability is extremely high. Taking all metabolites analyzed into consideration, on average inter-individual differences account for more than 50% of the observed variability; ranging from 10% for non-esterified fatty acids to more than 80% for cholesterol in VLDL particles. Conclusion – The results demonstrate that on a time axis of three months, metabolic phenotypes derived from challenge experiments such as OGTT or MMTT are highly reproducible despite large inter-individual differences

Vertical atlantoaxial distraction injuries: radiological criteria and clinical implications.

OBJECT: The authors sought to establish radiological criteria for the diagnosis of C1-2 vertical distraction injuries. METHODS: Conventional radiography, computerized tomography (CT), and magnetic resonance (MR) imaging findings in five patients with a C1-2 vertical distraction injury were correlated with their clinical history, operative findings, and autopsy findings. The basion-dens interval (BDI) and the C-1 and C-2 lateral mass interval (LMI) were measured in 93 control patients who underwent CT angiography; these measurements were used to define the normal BDI and LMI. The MR imaging results obtained in 30 healthy individuals were used to characterize the normal signal intensity of the C1-2 joint. The MR imaging results were compared with MR images obtained in five patients with distraction injuries. In the 93 patients, the BDI averaged 4.7 mm (standard deviation [SD] 1.7 mm, range 0.6-9 mm) and the LMI averaged 1.7 mm (SD 0.48 mm, range 0.7-3.3 mm). Based on CT scanning in the five patients with distraction injuries, the BDIs (mean 11.9 mm, SD 3.2 mm; p \u3c 0.001) and LMIs (mean 5.5 mm, SD 2 mm; p \u3c 0.0001) were significantly greater than in the control group. Fast-spin echo inversion-recovery MR images obtained in these five patients revealed markedly increased signal distributed throughout the C1-2 lateral mass articulations bilaterally. CONCLUSIONS: In 95% of healthy individuals, the LMI ranged between 0.7 and 2.6 mm. An LMI greater than 2.6 mm indicates the possibility of a distraction injury, which can be confirmed using MR imaging. Patients with a suspected C1-2 distraction injury may be candidates for surgical fusion of C1-2