Collisional fragmentation of central carbon metabolites in LC-MS/MS increases precision of ¹³C metabolic flux analysis

Experimental determination of fluxes by (13)C-tracers relies on detection of (13)C-patterns in metabolites or by-products. In the field of (13)C metabolic flux analysis, the most recent developments point toward recording labeling patterns by liquid chromatography (LC)-mass spectrometry (MS)/MS directly in intermediates in central carbon metabolism (CCM) to increase temporal resolution. Surprisingly, the flux studies published so far with LC-MS measurements were based on intact metabolic intermediates-thus neglected the potential benefits of using positional information to improve flux estimation. For the first time, we exploit collisional fragmentation to obtain more fine-grained (13)C-data on intermediates of CCM and investigate their impact in (13)C metabolic flux analysis. For the case study of Bacillus subtilis grown in mineral medium with (13)C-labeled glucose, we compare the flux estimates obtained by iterative isotopologue balancing of (13)C-data obtained either by LC-MS/MS for solely intact intermediates or LC-MS/MS for intact and fragmented intermediates of CCM. We show that with LC-MS/MS data, fragment information leads to more precise estimates of fluxes in pentose phosphate pathway, glycolysis, and to the tricarboxylic acid cycle. Additionally, we present an efficient analytical strategy to rapidly acquire large sets of (13)C-patterns by tandem MS, and an in-depth analysis of the collisional fragmentation of primary intermediates. In the future, this catalogue will enable comprehensive in silico calculability analyses to identify the most sensitive measurements and direct experimental design.     

What generates flux of tubulin in kinetochore microtubules?

Summary. We discuss models for production of tubulin flux in kinetochore microtubules. Current models concentrate solely on microtubules and their associated motors and enzymes. For example, in some models the driving force for flux is enzymes at the poles and the kinetochores; in others the driving force is motor molecules that are associated with a stationary spindle matrix. We present a different viewpoint, that microtubules are propelled poleward by forces arising from the spindle matrix, that the forces on the microtubules “activate” polymerising and depolymerising enzymes at kinetochores and poles, that matrix forces utilise actin, myosin, and microtubule motors, and that the matrix itself may not necessarily be static. Correspondence: A. Forer, Biology Department, York University, 4700 Keele Street, Toronto, ON M3J 1P3, Canada.     

Overcoming the metabolic burden of protein secretion in Schizosaccharomyces pombe – A quantitative approach using 13C-based metabolic flux analysis

Protein secretion in yeast is generally associated with a burden to cellular metabolism. To investigate this metabolic burden in Schizosaccharomyces pombe, we constructed a set of strains secreting the model protein maltase in different amounts. We quantified the influence of protein secretion on the metabolism applying ¹³C-based metabolic flux analysis in chemostat cultures. Analysis of the macromolecular biomass composition revealed an increase in cellular lipid content at elevated levels of protein secretion and we observed altered metabolic fluxes in the pentose phosphate pathway, the TCA cycle, and around the pyruvate node including mitochondrial NADPH supply. Supplementing acetate to glucose or glycerol minimal media was found to improve protein secretion, accompanied by an increased cellular lipid content and carbon flux through the TCA cycle as well as increased mitochondrial NADPH production. Thus, systematic metabolic analyses can assist in identifying factors limiting protein secretion and in deriving strategies to overcome these limitations.     

Quantitation of “autophagic flux” in mature skeletal muscle

Reliable and quantitative assays to measure in vivo autophagy are essential. Currently, there are varied methods for monitoring autophagy; however, it is a challenge to measure “autophagic flux” in an in vivo model system. Conversion and subsequent degradation of the microtubule-associated protein 1 light chain 3 (MAP1-LC3/LC3) to the autophagosome associated LC3-II isoform can be evaluated by immunoblot. However, static levels of endogenous LC3-II protein may render possible misinterpretations since LC3-II levels can increase, decrease or remain unchanged in the setting of autophagic induction. Therefore, it is necessary to measure LC3-II protein levels in the presence and absence of lysomotropic agents that block the degradation of LC3-II, a technique aptly named the “autophagometer.” In order to measure autophagic flux in mouse skeletal muscle, we treated animals with the microtubule depolarizing agent colchicine. Two days of 0.4 mg/kg/day intraperitoneal colchicine blocked autophagosome maturation to autolysosomes and increased LC3-II protein levels in mouse skeletal muscle by >100%. the addition of an autophagic stimulus such as dietary restriction or rapamycin led to an additional increase in LC3-II above that seen with colchicine alone. Moreover, this increase was not apparent in the absence of a “colchicine block.” Using this assay, we evaluated the autophagic response in skeletal muscle upon denervation induced atrophy. Our studies highlight the feasibility of performing an “in vivo autophagometer” study using colchicine in skeletal muscle.Key words: autophagy, rapamycin, skeletal muscle     

Airway smooth muscle electrophysiology in a state of flux?

Activation of chloride currents and release of internally sequestered Ca(2+) in airway smooth muscle have long been associated with excitation and contraction. Surprisingly, however, two recent publications (Deshpande DA, Wang WC, McIlmoyle EL, Robinett KS, Schillinger RM, An SS, Sham JS, Liggett SB. Nat Med 16: 1299-1304, 2010; Gallos G, Yim P, Chang S, Zhang Y, Xu D, Cook JM, Gerthoffer WT, Emala CW Sr. Am J Physiol Lung Cell Mol Physiol 302: L248-L256, 2012) have linked both events to relaxation. This begs a closer look at our understanding of airway smooth muscle electrophysiology and its contribution to excitation-contraction coupling. This Editorial Focus highlights those two aforementioned studies and several other equally paradoxical findings and proposes some possible reinterpretations of the data and/or new directions of research in which the answers might be found.     

¹³C metabolic flux analysis identifies an unusual route for pyruvate dissimilation in mycobacteria which requires isocitrate lyase and carbon dioxide fixation

Mycobacterium tuberculosis requires the enzyme isocitrate lyase (ICL) for growth and virulence in vivo. The demonstration that M. tuberculosis also requires ICL for survival during nutrient starvation and has a role during steady state growth in a glycerol limited chemostat indicates a function for this enzyme which extends beyond fat metabolism. As isocitrate lyase is a potential drug target elucidating the role of this enzyme is of importance; however, the role of isocitrate lyase has never been investigated at the level of in vivo fluxes. Here we show that deletion of one of the two icl genes impairs the replication of Mycobacterium bovis BCG at slow growth rate in a carbon limited chemostat. In order to further understand the role of isocitrate lyase in the central metabolism of mycobacteria the effect of growth rate on the in vivo fluxes was studied for the first time using 13C-metabolic flux analysis (MFA). Tracer experiments were performed with steady state chemostat cultures of BCG or M. tuberculosis supplied with 13C labeled glycerol or sodium bicarbonate. Through measurements of the 13C isotopomer labeling patterns in protein-derived amino acids and enzymatic activity assays we have identified the activity of a novel pathway for pyruvate dissimilation. We named this the GAS pathway because it utilizes the Glyoxylate shunt and Anapleurotic reactions for oxidation of pyruvate, and Succinyl CoA synthetase for the generation of succinyl CoA combined with a very low flux through the succinate--oxaloacetate segment of the tricarboxylic acid cycle. We confirm that M. tuberculosis can fix carbon from CO? into biomass. As the human host is abundant in CO? this finding requires further investigation in vivo as CO? fixation may provide a point of vulnerability that could be targeted with novel drugs. This study also provides a platform for further studies into the metabolism of M. tuberculosis using 13C-MFA.     

Nitrogen transformations and nitrous oxide flux in a tropical deciduous forest in México

Summary Emissions of nitrous oxide and soil nitrogen pools and transformations were measured over an annual cycle in two forests and one pasture in tropical deciduous forest near Chamela, México. Nitrous oxide flux was moderately high (0.5–2.5 ng cm^–2 h^–1) during the wet season and low (<0.3 ng cm^–2 h^–1) during the dry season. Annual emissions of nitrogen as nitrous oxide were calculated to be 0.5–0.7 kg ha^–1 y^–1, with no substantial difference between the forests and pasture. Wetting of dry soil caused a large but short-lived pulse of N₂O flux that accounted for <2% of annual flux. Variation in soil water through the season was the primary controlling factor for pool sizes of ammonium and nitrate, nitrogen transformations, and N₂O flux.     

Has Arabidopsis SWEETIE protein a role in sugar flux and utilization?

In plants, sugars affect growth and development and play an important role in the intricate machinery of signal transduction. Understanding the mechanisms behind the flux of sugar in the plant is of central interest. We recently characterized an Arabidopsis mutant: sweetie, which is defective in the control of growth and development, sterile, shows premature senescence and affects sugar metabolism. Our microarray analysis showed that 15 genes annotated as sugar transporter related proteins were found to be upregulated in sweetie while one sugar transporter gene was found to be downregulated. Most of them are unspecified sugar transporters but four genes have been annotated as monosaccharide transporters and one has been annotated as a disaccharide transporter. Moreover, as computer analyses predicted that SWEETIE might be a membrane protein and might have a function of glycosyl transferase, our data suggest that SWEETIE could be involved in the general control of sugar flux and modulates many important processes such as morphogenesis, flowering, stress responses and senescence.Key words: Arabidopsis thaliana, sweetie mutant, microarray, sugar flux, sugar transport     

Phosphorus in sediments — speciation and analysis

Characterization of sediment phosphorus is commonly based on sequential chemical extractions, in which phosphorus is supposed to be selectively removed from different compounds in the sediments. The first extraction schemes were designed to quantify discrete chemical or mineralogical compounds. As extraction schemes have been tested on different sediments, several systematic errors have been detected and the schemes have been modified and simplified accordingly. Other chemical extractions or treatments have attempted to determine phosphorus bound to particles with a certain strength or binding energy, the purpose being to determine the labile, loosely bound, exchangeable, mobile or algal-available fraction of sediment phosphorus. All extraction procedures yield operationally defined fractions and cannot be used for identification of discrete phosphorus compounds. The many methodological modifications make it necessary to be cautious when comparing results from the literature in this field.     

Determination of the gas exchange phenology in an evergreen coniferous forest from 7 years of eddy covariance flux data using an extended big-leaf analysis

We defined gas exchange phenology as the seasonality of the gas exchange characteristics of a forest canopy, and investigated how the gas exchange phenology could be directly detected from an eddy covariance (EC) dataset and its influence on the canopy fluxes within an evergreen Japanese cypress forest. For the detection of gas exchange phenology, we derived three bulk parameters of the extended big-leaf model (Kosugi et al. 2005) inversely from EC flux data over a 7-year period: surface conductance (g _c), maximum rate of carboxylation of the “big leaf” (V _CMAX), and intercellular CO₂ concentration of the “big leaf” (C _I). The relationship between g _c and the vapor pressure deficit declined in winter and spring. The relationship between the daily ecosystem respiration and air temperature was greater in the spring than in the other seasons. The temperature dependence curve of V _CMAX decreased substantially in the winter and was different from that of an evergreen broadleaved forest. A decrease in C _I was occasionally coupled with the decrease in canopy gross primary production during April and August, indicating that stomatal closure was responsible for a decline in canopy photosynthesis. Gas exchange phenology should be quantified when understanding the determining factors of the seasonality of canopy fluxes at evergreen coniferous forests.     

Multiple glucose 6-phosphate pools or channelling of flux in diverse pathways?

Glucose 6-phosphate is an intermediate of pathways of glucose utilization and production as well as a regulator of enzyme activity and gene expression. Studies on the latter functions are in part based on measurement of the glucose 6-phosphate content in a whole-cell extract. Several studies have suggested that there are multiple subcellular pools of glucose 6-phosphate. It is proposed that this data can be interpreted in terms of channelling of metabolic intermediates through multiple pathways of glucose metabolism with leakage of glucose 6-phosphate from the channels into a single free pool. It is also proposed that measurement of total tissue content of glucose 6-phosphate approximates the free pool.     

Energy expenditure and water flux of Rüppell's foxes in Saudi Arabia

Scattered populations of Rüppell's foxes (Vulpes rueppelli) occur across the deserts of northern Africa and Arabia. Little is known about the biology of these canids, especially the physiological mechanisms that contribute to their ability to live in such harsh environments. For individuals from Saudi Arabia, we tested the hypotheses that Rüppell's foxes have a reduced basal metabolic rate and total evaporative water loss (TEWL), parameters measured in the laboratory, and a reduced field metabolic rate (FMR) and water flux when free-living. Under basal conditions in the laboratory, males, which averaged 1,858 g in body mass, had an oxygen consumption of 914.9 mL O(2)/h, whereas females, which weighed on average 1,233 g, consumed 682.9 mL O(2)/h; rates of oxygen consumption translated to 441.4 kJ/d and 329.4 kJ/d, respectively. TEWL averaged 52.6 g H(2)O/d for males and 47.5 g H(2)O/d for females. We found no evidence that basal metabolism is reduced in Rüppell's foxes, but their TEWL was remarkably low: 50.9% of allometric prediction for males and 64.5% for females. In the wild during winter, males expended energy at a rate of 1,306.5 kJ/d, whereas females had an expenditure of 722.8 kJ/d. Analysis of covariance with FMR as the dependent variable, sex as a fixed factor, and body mass as a covariate showed no statistical difference in FMR between sexes. Water flux did not differ significantly between sexes and averaged 123 mL H(2)O/d, a value 30% lower than the kit fox from the deserts of southwestern North America. FMR was positively related to nocturnal activity levels as FMR (kJ/d) = -2,900.1+55.5 (% of time moving). The water content of prey items varied between 1.9 and 4.1 g H(2)O/g dry matter consumed. Based on these values and knowledge of their diet, we calculated that foxes captured about one rodent and a variety of anthropods per night of foraging.     

N-Acyl taurines trigger insulin secretion by increasing calcium flux in pancreatic β-cells

Pancreatic β-cells secrete insulin in response to various stimuli to control blood glucose levels. This insulin release is the result of a complex interplay between signaling, membrane potential and intracellular calcium levels. Various nutritional and hormonal factors are involved in regulating this process. N-Acyl taurines are a group of fatty acids which are amidated (or conjugated) to taurine and little is known about their physiological functions. In this study, treatment of pancreatic β-cell lines (HIT-T15) and rat islet cell lines (INS-1) with N-acyl taurines (N-arachidonoyl taurine and N-oleoyl taurine), induced a high frequency of calcium oscillations in these cells. Treatment with N-arachidonoyl taurine and N-oleoyl taurine also resulted in a significant increase in insulin secretion from pancreatic β-cell lines as determined by insulin release assay and immunofluorescence (p < 0.05). Our data also show that the transient receptor potential vanilloid 1 (TRPV1) channel is involved in insulin secretion in response to N-arachidonoyl taurine and N-oleoyl taurine treatment. However our data also suggest that receptors other than TRPV1 are involved in the insulin secretion response to treatment with N-oleoyl taurine.     

Functional redundancy of the two 5-hydroxylases in monolignol biosynthesis of Populus trichocarpa: LC–MS/MS based protein quantification and metabolic flux analysis

Flowering plants have syringyl and guaiacyl subunits in lignin in contrast to the guaiacyl lignin in gymnosperms. The biosynthesis of syringyl subunits is initiated by coniferaldehyde 5-hydroxylase (CAld5H). In Populus trichocarpa there are two closely related CAld5H enzymes (PtrCAld5H1 and PtrCAld5H2) associated with lignin biosynthesis during wood formation. We used yeast recombinant PtrCAld5H1 and PtrCAld5H2 proteins to carry out Michaelis-Menten and inhibition kinetics with LC-MS/MS based absolute protein quantification. CAld5H, a monooxygenase, requires a cytochrome P450 reductase (CPR) as an electron donor. We cloned and expressed three P. trichocarpa CPRs in yeast and show that all are active with both CAld5Hs. The kinetic analysis shows both CAld5Hs have essentially the same biochemical functions. When both CAld5Hs are coexpressed in the same yeast membranes, the resulting enzyme activities are additive, suggesting functional redundancy and independence of these two enzymes. Simulated reaction flux based on Michaelis-Menten kinetics and inhibition kinetics confirmed the redundancy and independence. Subcellular localization of both CAld5Hs as sGFP fusion proteins expressed in P. trichocarpa differentiating xylem protoplasts indicate that they are endoplasmic reticulum resident proteins. These results imply that during wood formation, 5-hydroxylation in monolignol biosynthesis of P. trichocarpa requires the combined metabolic flux of these two CAld5Hs to maintain adequate biosynthesis of syringyl lignin. The combination of genetic analysis, absolute protein quantitation-based enzyme kinetics, homologous CPR specificity, SNP characterization, and ER localization provides a more rigorous basis for a comprehensive systems understanding of 5-hydroxylation in lignin biosynthesis.     

Novel genetic analysis in Beh?et's disease

Behçet''s disease (BD) is characterized by oral and genital ulceration and is complicated by eye, skin, joint and central nervous system lesions. It has long been understood that BD has a strong genetic component, but to date the identified genes account for only around 30% of the risk for developing the disease, and the work has mostly been based on candidate gene analysis. In a recent report, Fei and coworkers presented the results of the first genome-wide analysis of patients with BD. These findings suggest new pathways for investigation in this complex disease.Error, like straws, upon the surface flowHe who would search for pearls must dive belowJohn Dryden, 1678Fei and coworkers [1] identified novel single nucleotide polymorphisms (SNPs) in five genes (KIAA1529, CPVL, LOC100129342, UBASH3B and UBAC2) that encode proteins with both known and unknown functions. Moreover, subset analysis showed links between some of the SNPs and particular manifestations of Behçet''s disease (BD).KIAA1529 and LOC100129342 have no known function, but the latter SNP on chromosome 1p34 confirms a locus previously identified in a multiplex family study [2]. UBASH3B and UBAC2 encode ubiquitin-associated proteins. The ubiquitin system is best described in targeting misfolded or damaged proteins for proteosomal degradation, but it is also involved in several other cellular processes, including regulation of nuclear factor-κB function and autophagy. Such processes are involved in cells of both innate and adaptive immune responses, which is of interest with respect to the ongoing debate on whether BD is an autoinflammatory or an autoimmune disorder [3]. Dysfunction of the ubiquitin pathway has been implicated in cancer, neurodegenerative diseases and type 2 diabetes [4,5].Carboxypeptidase vitellogenic-like protein is upregulated in monocytes on conversion to macrophages, in which it co-localizes to the secreted proteins tumour necrosis factor and the chemokine CCL3 (C-C chemokine ligand 3). It has been implicated in the processing/transport of peptides for loading onto major histocompatibility complex (MHC) class I molecules [6]. The strongest genetic association with BD is human leucocyte antigen (HLA)-B*5101 and HLA-B*5108, an MHC class I molecule. Microsatellite analysis confirms HLA-{"type":"entrez-nucleotide","attrs":{"text":"B85101","term_id":"2926233","term_text":"B85101"}}B85101/5108 as the most likely causative gene in BD, but SNPs in other genes in close proximity on chromosome 6 – MICA, MICB and TNF – have also been reported [7]. Moreover, the mechanism of action of HLA-B*5101/5108 in BD has not been elucidated. Alteration in the process of peptide production by the CVLP SNP could have important implications in the expression or maintenance of MHC class I molecules on the cell surface and be linked to the pathogenesis of the disease.In subset analysis the UBASH3B SNP was more common in patients with ocular and vascular manifestations, whereas the KIAA1529 SNP was more common in patients without such involvement. The authors correctly stated that the numbers with each manifestation make such an analysis very preliminary, but several other SNPs have been linked to ocular disease specifically, so these findings are not unexpected [8]. It should be noted, however, that eye and vascular disease can take many forms in BD, and more detailed analysis will be required when greater numbers are tested for these SNPs.There are certain caveats to the findings. Genome-wide analysis (GWAS) is normally performed on a large number of samples, which is not easy for a rare condition such as BD. The authors addressed this point by performing the initial analysis on pooled samples and then, having identified potential SNPs, validating the findings in each sample individually. However, it is important for these results to be validated in other cohorts of BD patients. There are extensive data describing ethnic differences in SNPs studied by candidate analysis, and the association of these newly identified SNPs in BD patients from different geographical areas will be important. Similarly, in several studies males have been shown to have a worse prognosis, and the association of these SNPs with sex should be examined. Other GWAS are ongoing or planned in Japanese, European and Turkish patients with BD, and comparison with the current study will be of great interest. Finally, and most importantly, the functional relevance of these SNPs will need to be investigated and – if found – tested in different cell types such as lymphoid, myeloid, epithelial and endothelial cells that are involved in BD.BD is a complex disease. Different patients will experience different symptoms, and there is a clear geographical distribution of the disease. The candidate gene approach has been useful in identifying susceptibility and severity genes in BD, but the ability to undertake GWAS has led to the identification of several new SNPs in many human diseases, increasing our understanding of the pathogenic mechanisms involved. Fei and coworkers [1] are to be commended for such a study in BD.     

Oscillatory glucose flux in INS 1 pancreatic β cells: a self-referencing microbiosensor study

Signaling and insulin secretion in β cells have been reported to demonstrate oscillatory modes, with abnormal oscillations associated with type 2 diabetes. We investigated cellular glucose influx in β cells with a self-referencing (SR) microbiosensor based on nanomaterials with enhanced performance. Dose–response analyses with glucose and metabolic inhibition studies were used to study oscillatory patterns and transporter kinetics. For the first time, we report a stable and regular oscillatory uptake of glucose (averaged period 2.9 ± 0.6 min), which corresponds well with an oscillator model. This oscillatory behavior is part of the feedback control pathway involving oxygen, cytosolic Ca²⁺/ATP, and insulin secretion (periodicity approximately 3 min). Glucose stimulation experiments show that the net Michaelis–Menten constant (6.1 ± 1.5 mM) is in between GLUT2 and GLUT9. Phloretin inhibition experiments show an EC₅₀ value of 28 ± 1.6 μM phloretin for class I GLUT proteins and a concentration of 40 ± 0.6 μM phloretin caused maximum inhibition with residual nonoscillating flux, suggesting that the transporters not inhibited by phloretin are likely responsible for the remaining nonoscillatory uptake, and that impaired uptake via GLUT2 may be the cause of the oscillation loss in type 2 diabetes. Transporter studies using the SR microbiosensor will contribute to diabetes research and therapy development by exploring the nature of oscillatory transport mechanisms.     

Leaps in cardiovascular diseases after a decrease of hard cosmic ray flux and atmospheric pressure in Vilnius city in 2004–2007

The prognostic relationship between a decrease of hard cosmic ray flux (HCRF) and subsequent leaps in cardiovascular disease (CVD) occurrence within 1–3 days is well known. The influence of meteorological situations on human health is also known. However, no correlation was found between a simultaneous decrease of HCRF and atmospheric pressure and the leaps of CVD on some days. For the analysis of a prognostic connection between these parameters, empirical criteria have been put forward. To achieve effective results from analysis, the criteria of a continuous decrease of HCRF to 200 impulses or more within 4 h and of atmospheric pressure by 2 mmHg or more were used. CVD leaps were considered real when their number increased by 10% or more in comparison with the average monthly value. The highest prognostic correlation between HCRF decrease and CVD leaps within 1–2 days was from 64 to 76%. The correlation within 2–3 days was lower. The correlation between atmospheric pressure decrease and leaps of CVD in 1–2 days and on the same day was 25–44%. It means that, while studying the meteorological effects on human organisms, other parameters such as temperature, humidity, wind velocity, etc. should also be taken into account. An essential role in reducing the prognostic accuracy of CVD leaps by a decrease of HCRF relates to the human factor.     

Signalling molecule transport analysis in lacunar–canalicular system

Biomechanics and Modeling in Mechanobiology - Mechanical loading-induced fluid flow in lacunar–canalicular space (LCS) of bone excites osteocyte cells to release signalling molecules which...