Kinetic complexities of triacylglycerol accumulation in developing embryos from Camelina sativa provide evidence for multiple biosynthetic systems.

Quantitative flux maps describing glycerolipid synthesis can be important tools for rational engineering of lipid content and composition in oilseeds. Lipid accumulation in cultured embryos of Camelina sativa is known to mimic that of seeds in terms of rate of lipid synthesis and composition. To assess the kinetic complexity of the glycerolipid flux network, cultured embryos were incubated with [14C/13C]glycerol, and initial and steady state rates of [14C/13Cglyceryl] lipid accumulation were measured. At steady state, the linear accumulations of labeled lipid classes matched those expected from mass compositions. The system showed an apparently simple kinetic precursor-product relationship between the intermediate pool, dominated by diacylglycerol (DAG) and phosphatidylcholine (PC), and the triacylglycerol (TAG) product. We also conducted isotopomer analyses on hydrogenated lipid class species. [13C3glyceryl] labeling of DAG and PC, together with estimates of endogenous [12C3glyceryl] dilution, showed that each biosynthetically active lipid pool is ∼30% of the total by moles. This validates the concept that lipid sub-pools can describe lipid biosynthetic networks. By tracking the kinetics of [13C3glyceryl] and [13C2acyl] labeling, we observed two distinct TAG synthesis components. The major TAG synthesis flux (∼75%) was associated with >95% of the DAG/PC intermediate pool, with little glycerol being metabolized to fatty acids, and with little dilution from endogenous glycerol; a smaller flux exhibited converse characteristics. This kinetic heterogeneity was further explored using postlabeling embryo dissection and differential lipid extractions. The minor flux was tentatively localized to surface cells across the whole embryo. Such heterogeneity must be recognized in order to construct accurate gene expression patterns and metabolic networks describing lipid biosynthesis in developing embryos.

Bonded cumomer analysis of tumor metabolism based on 13 C magnetic resonance spectroscopy.

Bonded cumomers are sets of isotopomers of 13 C-labeled metabolites containing a particular sequence of contiguously or singly labeled carbon atoms. Only these isotopomers contribute to multiplet structure in the 13 C NMR spectrum. We discuss the application of this technique to the study of quantitative tumor metabolism, bioenergetics, and the Warburg effect. The advantages and sensitivity of bonded cumomer analysis over positional enrichment analysis are discussed. When sensitivity requirements are met, bonded cumomer analysis enables the extraction of fluxes through specific metabolic pathways with higher precision. In conjunction with isotopomer control analysis, we evaluate the sensitivity of experimentally measurable metabolite multiplets to determine the robustness of flux analysis in 13 C spectra of tumors. This review examines the role of glycolytic and tricarboxylic acid cycle metabolism with special emphasis on flux through the pentose phosphate pathway (PPP). The impact of reversibility of the nonoxidative branch of the PPP with various 13 C glucose tracers on fine-structure multiplets is analyzed.

Isotopomer analyses with the tricarboxylic acid cycle intermediates and exchanging metabolites from the rat kidney.

Renal metabolism is essential for kidney functions and energy homeostasis in the body. The TCA cycle is the hub of metabolism, but the metabolic activities of the cycle in the kidney have rarely been investigated. This study is to assess metabolic processes at the level of the TCA cycle in the kidney based on isotopomer distributions in multiple metabolites. Isolated rat kidneys were perfused with media containing common substrates including lactate and alanine for an hour. One group of kidneys received [U-13 C3 ]lactate instead of natural abundance lactate while the other group received [U-13 C3 ]alanine instead of natural abundance alanine. Perfused kidneys and effluent were prepared for analysis using NMR spectroscopy. 13 C-labeling patterns in glutamate, fumarate, aspartate and succinate from the kidney extracts showed that pyruvate carboxylase and oxidative metabolism through the TCA cycle were comparably very active, but pyruvate cycling and pyruvate dehydrogenase were relatively less active. Isotopomer analyses with fumarate and malate from effluent, however, indicated that pyruvate carboxylase was much more active than the TCA cycle and other metabolic processes. The reverse equilibrium of oxaloacetate with four-carbon intermediates of the cycle was nearly complete (92%), based on the ratio of [2,3,4-13 C3 ]/[1,2,3-13 C3 ] in aspartate or malate. 13 C enrichment in glucose with 13 C-lactate supply was higher than that with 13 C-alanine. Isotopomer analyses with multiple metabolites (i.e., glutamate, fumarate, aspartate, succinate and malate) allowed us to assess relative metabolic processes in the TCA cycle in the kidney supplied with [U-13 C3 ]lactate. Data from the analytes were generally consistent, indicating highly active pyruvate carboxylase and oxidative metabolism through the TCA cycle. Different 13 C-labeling patterns in analytes from the kidney extracts versus effluent suggested metabolic compartmentalization.

NMR-based isotope editing, chemoselection and isotopomer distribution analysis in stable isotope resolved metabolomics.

NMR is a very powerful tool for identifying and quantifying compounds within complex mixtures without the need for individual standards or chromatographic separation. Stable Isotope Resolved Metabolomics (or SIRM) is an approach to following the fate of individual atoms from precursors through metabolic transformation, producing an atom-resolved metabolic fate map. However, extracts of cells or tissue give rise to very complex NMR spectra. While multidimensional NMR experiments may partially overcome the spectral overlap problem, additional tools may be needed to determine site-specific isotopomer distributions. NMR is especially powerful by virtue of its isotope editing capabilities using NMR active nuclei such as 13C, 15N, 19F and 31P to select molecules containing just these atoms in a complex mixture, and provide direct information about which atoms are present in identified compounds and their relative abundances. The isotope-editing capability of NMR can also be employed to select for those compounds that have been selectively derivatized with an NMR-active stable isotope at particular functional groups, leading to considerable spectral simplification. Here we review isotope analysis by NMR, and methods of chemoselection both for spectral simplification, and for enhanced isotopomer analysis.

Improved 13C metabolic flux analysis in Escherichia coli metabolism: application of a high-resolution MS (GC-EI-QTOF) for comprehensive assessment of MS/MS fragments.

Gas chromatography-tandem mass spectrometry with electron ionization (GC-EI-MS/MS) provides rich information on stable-isotope labeling for 13C-metabolic flux analysis (13C-MFA). To pave the way for the routine application of tandem MS data for metabolic flux quantification, we aimed to compile a comprehensive library of GC-EI-MS/MS fragments of tert-butyldimethylsilyl (TBDMS) derivatized proteinogenic amino acids. First, we established an analytical workflow that combines high-resolution gas chromatography-quadrupole time-of-flight mass spectrometry and fully 13C-labeled biomass to identify and structurally elucidate tandem MS amino acid fragments. Application of the high-mass accuracy MS procedure resulted into the identification of 129 validated precursor-product ion pairs of 13 amino acids with 30 fragments being accepted for 13C-MFA. The practical benefit of the novel tandem MS data was demonstrated by a proof-of-concept study, which confirmed the importance of the compiled library for high-resolution 13C-MFA. ONE SENTENCE SUMMARY: An analytical workflow that combines high-resolution mass spectrometry (MS) and fully 13C-labeled biomass to identify and structurally elucidate tandem MS amino acid fragments, which provide positional information and therefore offering significant advantages over traditional MS to improve 13C-metabolic flux analysis.

Establishment of a GC-MS-based 13 C-positional isotopomer approach suitable for investigating metabolic fluxes in plant primary metabolism.

13 C-Metabolic flux analysis (13 C-MFA) has greatly contributed to our understanding of plant metabolic regulation. However, the generation of detailed in vivo flux maps remains a major challenge. Flux investigations based on nuclear magnetic resonance have resolved small networks with high accuracy. Mass spectrometry (MS) approaches have broader potential, but have hitherto been limited in their power to deduce flux information due to lack of atomic level position information. Herein we established a gas chromatography (GC) coupled to MS-based approach that provides 13 C-positional labelling information in glucose, malate and glutamate (Glu). A map of electron impact (EI)-mediated MS fragmentation was created and validated by 13 C-positionally labelled references via GC-EI-MS and GC-atmospheric pressure chemical ionization-MS technologies. The power of the approach was revealed by analysing previous 13 C-MFA data from leaves and guard cells, and 13 C-HCO3 labelling of guard cells harvested in the dark and after the dark-to-light transition. We demonstrated that the approach is applicable to established GC-EI-MS-based 13 C-MFA without the need for experimental adjustment, but will benefit in the future from paired analyses by the two GC-MS platforms. We identified specific glucose carbon atoms that are preferentially labelled by photosynthesis and gluconeogenesis, and provide an approach to investigate the phosphoenolpyruvate carboxylase (PEPc)-derived 13 C-incorporation into malate and Glu. Our results suggest that gluconeogenesis and the PEPc-mediated CO2 assimilation into malate are activated in a light-independent manner in guard cells. We further highlight that the fluxes from glycolysis and PEPc toward Glu are restricted by the mitochondrial thioredoxin system in illuminated leaves.

A simple method for quantifying de novo lipogenesis rate and substrate selection in cell cultures by 13 C NMR isotopomer analysis of the crude lipid fraction.

PURPOSE: De novo lipogenesis (DNL) is critical for cell growth and maintenance, and acetyl-CoA precursors can be derived from different substrates. We developed a 13 C NMR analysis of lipid extracts from cultured microglia cells administered with [U-13 C]glucose that informs overall lipogenic activity as well as the contribution of glucose to lipogenic acetyl-CoA. METHODS: BV-2 microglial cell line cultured with glucose and glutamine was provided with [U-13 C]glucose and unlabeled glutamine for 24 h and studied in either the presence or absence of lipopolysaccharide (LPS). Cells were then extracted for lipids and the crude lipid fraction was analyzed by 13 C NMR. 13 C-isotopomer signals in the fatty acid ω - 1 and ω - 2 signals representing consecutive or non-consecutive enrichment of the fatty acid chain by [1,2-13 C2 ]acetyl-CoA were quantified and applied to a probabilistic model of acetyl-CoA precursor and fatty acid enrichment. RESULTS: Glucose contributed 72 ± 2% of lipogenic acetyl-CoA while DNL from all sources accounted for 16 ± 2% of lipid turnover. With LPS, there was a significant decrease in glucose contribution (59 ± 4%, p < 0.05) while DNL was unchanged (11 ± 3%). CONCLUSIONS: A simple 13 C NMR analysis of the crude lipid fractions of BV-2 cells administered with [U-13 C]glucose informs DNL activity and the contribution of glucose to the acetyl-CoA precursors. While DNL was preserved in the presence of LPS, there was redirection of lipogenic acetyl-CoA sources from glucose to other substrates. Thus, in the present article, we describe a novel and simple 13 C NMR analysis approach to disclose the overall lipogenic activity and substrate contribution to DNL, suitable for evaluating DNL rates in cell cultures.

Organic fertility inputs synergistically increase denitrification-derived nitrous oxide emissions in agroecosystems.

Soil fertility in organic agriculture relies on microbial cycling of nutrient inputs from legume cover crops and animal manure. However, large quantities of labile carbon (C) and nitrogen (N) in these amendments may promote the production and emission of nitrous oxide (N2 O) from soils. Better ecological understanding of the N2 O emission controls may lead to new management strategies to reduce these emissions. We measured soil N2 O emission for two growing seasons in four corn-soybean-winter grain rotations with tillage, cover crop, and manure management variations typical of organic agriculture in temperate and humid North America. To identify N2 O production pathways and mitigation opportunities, we supplemented N2 O flux measurements with determinations of N2 O isotopomer composition and microbiological genomic DNA abundances in microplots where we manipulated cover crop and manure additions. The N input from legume-rich cover crops and manure prior to corn planting made the corn phase the main source of N2 O emissions, averaging 9.8 kg/ha of N2 O-N and representing 80% of the 3-yr rotations' total emissions. Nitrous oxide emissions increased sharply when legume cover crop and manure inputs exceeded 1.8 and 4 Mg/ha (dry matter), respectively. Removing the legume aboveground biomass before corn planting to prevent co-location of fresh biomass and manure decreased N2 O emissions by 60% during the corn phase. The co-occurrence of peak N2 O emission and high carbon dioxide emission suggests that oxygen (O2 ) consumption likely caused hypoxia and bacterial denitrification. This interpretation is supported by the N2 O site preference values trending towards denitrification during peak emissions with limited N2 O reduction, as revealed by the N2 O δ15 N and δ18 O and the decrease in clade I nosZ gene abundance following incorporation of cover crops and manure. Thus, accelerated microbial O2 consumption seems to be a critical control of N2 O emissions in systems with large additions of decomposable C and N substrates. Because many agricultural systems rely on combined fertility inputs from legumes and manures, our research suggests that controlling the rate and timing of organic input additions, as well as preventing the co-location of legume cover crops and manure, could mitigate N2 O emissions.

Calling Behavior and Sex Pheromone Release and Storage in the Moth Chloridea virescens.

Female moths release sex pheromone to attract mates. In most species, sex pheromone is produced in, and released from, a specific gland. In a previous study, we used empirical data and compartmental modeling to account for the major pheromone gland processes of female Chloridea virescens: synthesis, storage, catabolism and release; we found that females released little (20-30%) of their pheromone, with most catabolized. The recent publication of a new pheromone collection method led us to reinvestigate pheromone release and catabolism in C. virescens on the basis that our original study might have underestimated release rate (thereby overestimating catabolism) due to methodology and females not calling (releasing) continuously. Further we wished to compare pheromone storage/catabolism between calling and non-calling females. First, we observed calling intermittency of females. Then, using decapitated females, we used the new collection method, along with compartmental modeling, gland sampling and stable isotope labeling, to determine differences in pheromone release, catabolism and storage between (forced) simulated calling and non-calling females. We found, (i) intact 1 d females call intermittently; (ii) pheromone is released at a higher rate than previously determined, with simulations estimating that continuously calling females release ca. 70% of their pheromone (only 30% catabolized); (iii) extension (calling)/retraction of the ovipositor is a highly effective "on/off' mechanism for release; (iv) both calling and non-calling females store most pheromone on or near the gland surface, but calling females catabolize less pheromone; (v) females are capable of producing and releasing pheromone very rapidly. Thus, not only is the moth pheromone gland efficient, in terms of the proportion of pheromone released Vs. catabolized, but it is highly effective at shutting on/off a high flux of pheromone for release.

Novel Insights into Dietary Phytosterol Utilization and Its Fate in Honey Bees (Apis mellifera L.).

Poor nutrition is an important factor in global bee population declines. A significant gap in knowledge persists regarding the role of various nutrients (especially micronutrients) in honey bees. Sterols are essential micronutrients in insect diets and play a physiologically vital role as precursors of important molting hormones and building blocks of cellular membranes. Sterol requirements and metabolism in honey bees are poorly understood. Among all pollen sterols, 24-methylenecholesterol is considered the key phytosterol required by honey bees. Nurse bees assimilate this sterol from dietary sources and store it in their tissues as endogenous sterol, to be transferred to the growing larvae through brood food. This study examined the duration of replacement of such endogenous sterols in honey bees. The dietary 13C-labeled isotopomer of 24-methylenecholesterol added to artificial bee diet showed differential, progressive in vivo assimilation across various honey bee tissues. Significantly higher survival, diet consumption, head protein content and abdominal lipid content were observed in the dietary sterol-supplemented group than in the control group. These findings provide novel insights into phytosterol utilization and temporal pattern of endogenous 24-methylenecholesterol replacement in honey bees.

A model for determining cardiac mitochondrial substrate utilisation using stable 13C-labelled metabolites.

INTRODUCTION: Relative oxidation of different metabolic substrates in the heart varies both physiologically and pathologically, in order to meet metabolic demands under different circumstances. 13C labelled substrates have become a key tool for studying substrate use-yet an accurate model is required to analyse the complex data produced as these substrates become incorporated into the Krebs cycle. OBJECTIVES: We aimed to generate a network model for the quantitative analysis of Krebs cycle intermediate isotopologue distributions measured by mass spectrometry, to determine the 13C labelled proportion of acetyl-CoA entering the Krebs cycle. METHODS: A model was generated, and validated ex vivo using isotopic distributions measured from isolated hearts perfused with buffer containing 11 mM glucose in total, with varying fractions of universally labelled with 13C. The model was then employed to determine the relative oxidation of glucose and triacylglycerol by hearts perfused with 11 mM glucose and 0.4 mM equivalent Intralipid (a triacylglycerol mixture). RESULTS: The contribution of glucose to Krebs cycle oxidation was measured to be 79.1 ± 0.9%, independent of the fraction of buffer glucose which was U-13C labelled, or of which Krebs cycle intermediate was assessed. In the presence of Intralipid, glucose and triglyceride were determined to contribute 58 ± 3.6% and 35.6 ± 0.8% of acetyl-CoA entering the Krebs cycle, respectively. CONCLUSION: These results demonstrate the accuracy of a functional model of Krebs cycle metabolism, which can allow quantitative determination of the effects of therapeutics and pathology on cardiac substrate metabolism.

Production and Distribution of Aldehyde and Alcohol Sex Pheromone Components in the Pheromone Gland of Females of the Moth Chloridea virescens.

Aldehydes are components of many moth sex pheromones, and are thought to be produced from analogous alcohols by oxidase(s) in the cell membrane or the gland cuticle. This implies that the two types of components are produced and/or stored in different parts of the gland: alcohols in cells and aldehydes in cuticle. Few studies have investigated the distribution of components in moth pheromone glands. Using rinse/extract sampling, stable isotope tracer/tracee methods, and decapitation/ pheromone biosynthesis activating neuropeptide stimulation, we studied production and distribution of (Z)-11-hexadecenal (Z11-16:Ald) and (Z)-hexadecenol (Z11-16:OH) in the gland of Chloridea virescens (formerly Heliothis virescens). The rinse, which likely sampled the surface and outer cuticle, contained large amounts of aldehyde and small amounts of alcohol. By contrast, the residual extract, which likely sampled cells and less solvent-accessible (inner) cuticle, had large amounts of alcohol and small amounts of aldehyde. When a tracer (U-13C-glucose) was fed to females, the aldehyde had higher isotopic enrichment than the alcohol in the rinse, but not in the residual extract, showing that in the rinse pool, Z11-16:Ald was, on average, synthesized before Z11-16:OH. This is consistent with greater aldehyde than alcohol flux through the cuticle. While our results are consistent with cell/cuticle synthesis sites for alcohol/aldehyde components, we cannot rule out both being synthesized in gland cells. We propose two alternative conceptual models for how site of production, cuticular transport and catabolism/metabolism might explain the relative masses of Z11-16:Ald and Z11-16:OH translocated to the pheromone gland surface in female C. virescens.

Acetaminophen cytotoxicity in HepG2 cells is associated with a decoupling of glycolysis from the TCA cycle, loss of NADPH production, and suppression of anabolism.

Acetaminophen (APAP) is one of the most commonly used analgesics worldwide, and overdoses are associated with lactic acidosis, hepatocyte toxicity, and acute liver failure due to oxidative stress and mitochondrial dysfunction. Hepatoma cell lines typically lack the CYP450 activity to generate the reactive metabolite of APAP observed in vivo, but are still subject to APAP cytotoxicity. In this study, we employed metabolic profiling and isotope labelling approaches to investigate the metabolic impact of acute exposure to cytotoxic doses of APAP on the widely used HepG2 cell model. We found that APAP exposure leads to limited cellular death and substantial growth inhibition. Metabolically, we observed an up-regulation of glycolysis and lactate production with a concomitant reduction in carbon from glucose entering the pentose-phosphate pathway and the TCA cycle. This was accompanied by a depletion of cellular NADPH and a reduction in the de novo synthesis of fatty acids and the amino acids serine and glycine. These events were not associated with lower reduced glutathione levels and no glutathione conjugates were seen in cell extracts. Co-treatment with a specific inhibitor of the lactate/H+ transporter MCT1, AZD3965, led to increased apoptosis in APAP-treated cells, suggesting that lactate accumulation could be a cause of cell death in this model. In conclusion, we show that APAP toxicity in HepG2 cells is largely independent of oxidative stress, and is linked instead to a decoupling of glycolysis from the TCA cycle, lactic acidosis, reduced NADPH production, and subsequent suppression of the anabolic pathways required for rapid growth.

Deuterated active pharmaceutical ingredients: A science-based proposal for synthesis, analysis, and control. Part 1: Framing the problem.

The International Consortium for Innovation & Quality (IQ) in Pharmaceutical Development recently established a working group focused on the development of a guidance to address Deuterated Active Pharmaceutical Ingredients. Deuteration of an Active Pharmaceutical Ingredient (API) in some cases can retard and/or alter API metabolism by exploiting the primary kinetic isotope effect. Several deuterated APIs have entered into the clinic, and one has recently been approved. In most cases, it is very difficult to nearly impossible to synthesize a 100% isotopically pure compound. This raises synthetic, analytical, and regulatory questions that warrant a science-based assessment and recommendations for synthetic methods, analytical methods, and specifications. A cross functional team of scientists with expertise in isotope chemistry, process chemistry, analytical chemistry, and drug metabolism and pharmacokinetics have been meeting under the auspices of IQ to define and address these questions. This paper strives to frame chemistry, manufacturing, and controls challenges.

d2ome, Software for in Vivo Protein Turnover Analysis Using Heavy Water Labeling and LC-MS, Reveals Alterations of Hepatic Proteome Dynamics in a Mouse Model of NAFLD.

Metabolic labeling with heavy water followed by LC-MS is a high throughput approach to study proteostasis in vivo. Advances in mass spectrometry and sample processing have allowed consistent detection of thousands of proteins at multiple time points. However, freely available automated bioinformatics tools to analyze and extract protein decay rate constants are lacking. Here, we describe d2ome-a robust, automated software solution for in vivo protein turnover analysis. d2ome is highly scalable, uses innovative approaches to nonlinear fitting, implements Grubbs' outlier detection and removal, uses weighted-averaging of replicates, applies a data dependent elution time windowing, and uses mass accuracy in peak detection. Here, we discuss the application of d2ome in a comparative study of protein turnover in the livers of normal vs Western diet-fed LDLR-/- mice (mouse model of nonalcoholic fatty liver disease), which contained 256 LC-MS experiments. The study revealed reduced stability of 40S ribosomal protein subunits in the Western diet-fed mice.

Dose-dependent quantitative effects of acute fructose administration on hepatic de novo lipogenesis in healthy humans.

Fructose feeding increases hepatic de novo lipogenesis (DNL) and is associated with nonalcoholic fatty liver disease. Little is known, however, about individual variation in susceptibility to fructose stimulation of DNL. In this three-period crossover study, 17 healthy male subjects were enrolled to evaluate the within- and between-subject variability of acute fructose feeding on hepatic fractional DNL. During each assessment, [1-13C1]acetate was infused to measure DNL in the fasting state and during fructose feeding. Subjects randomly received a high dose of fructose (10 mg·kg fat-free mass-1·min-1) on two occasions and a low dose (5 mg·kg fat-free mass-1·min-1) on another. Fructose solutions were administered orally every 30 min for 9.5 h. Ten subjects completed all three study periods. DNL was assessed as the fractional contribution of newly synthesized palmitate into very-low-density lipoprotein triglycerides using mass isotopomer distribution analysis. Mean fasting DNL was 5.3 ± 2.8%, with significant within- and between-subject variability. DNL increased dose dependently during fructose feeding to 15 ± 2% for low- and 29 ± 2% for high-dose fructose. The DNL response to high-dose fructose was very reproducible within an individual ( r = 0.93, P < 0.001) and independent of fasting DNL. However, it was variable between individuals and significantly correlated to influx of unlabeled acetyl-CoA ( r = 0.7, P < 0.001). Unlike fasting DNL, fructose-stimulated DNL is a robust and reproducible measure of hepatic lipogenic activity for a given individual and may be a useful indicator of metabolic disease susceptibility and treatment response.

Funaria hygrometrica Hedw. elevated tolerance to D2O: its use for the production of highly deuterated metabolites.

MAIN CONCLUSION: The method introduced here to grow F. hygrometrica in high concentrations of D 2 O is an excellent alternative to produce highly deuterated metabolites with broad applications in metabolic studies. Our mass spectrometry experiments strongly indicate the successful incorporation of deuterium into organic compounds. Deuterated metabolites are useful tracers for metabolic studies, yet their wide utilization in research is limited by the multi-step total synthesis required to produce them in the laboratory. Alternatively, deuterated metabolites can be obtained from organisms grown in D2O or deuterated nutrients. This approach also has limitations as D2O in high concentrations negatively affects the survival of most organisms. Here we report the moss Funaria hygrometrica as an unusual high tolerant to D2O in liquid culture. We found that this moss is able to grow in up to 90% D2O, a condition lethal for many eukaryotes. Mass spectrometric analyses of F. hygrometrica extracts showed a strong deuteration pattern. The ability to tolerate high concentrations of D2O together with the development of a rich molecular toolbox makes F. hygrometrica an ideal system for the production of valuable deuterated metabolites.

Novel quantitative insights into carbon sources for synthesis of poly hydroxybutyrate in Synechocystis PCC 6803.

Many freshwater cyanobacteria accumulate polyhydroxybutyrate (PHB) under nitrogen or phosphorus deprivation. While prior literature has shed lights on transcriptomic and metabolomic changes in the model cyanobacterium Synechocystis PCC 6803 cells, the quantitative contributions of the newly fixed carbon following nitrogen deprivation or the externally added acetate to PHB synthesis are not clear. Similarly, it is not clear how photomixotrophy affects precursor contributions. In this study, we show that (i) the pre-growth mode (photoautotrophic or photomixotrophic), while significantly impacting glycogen levels, does not have any significant effect on PHB levels, (ii) the carbon fixed following nitrogen deprivation contributes 26% of C for PHB synthesis in photoautotrophically pre-grown cells and its contribution to the PHB synthesis goes down with the addition of acetate at the resuspension phase or with photomixotrophic pre-growth, (iii) the acetate added at the start of nitrogen deprivation, doubles the intracellular PHB levels and contributes 44-48% to PHB synthesis and this value is not greatly affected by how the cells were pre-grown. Indirectly, the labeling studies also show that the intracellular C recycling is the most important source of precursors for PHB synthesis, contributing about 74-87% of the C for PHB synthesis in the absence of acetate. The addition of acetate significantly reduces its contribution. In photoautotrophic pre-growth followed by acetate addition under nitrogen starvation, the contribution of intracellular C reduces to about 34%. Thus, our study provides several novel quantitative insights on how prior nutritional status affects the precursor contributions for PHB synthesis.

Differential Pheromone Sampling of the Gland of Female Heliothis Virescens Moths Reveals Glandular Differences in Composition and Quantity.

By differentially sampling the pheromone gland of females of the moth Heliothis virescens, we explored differences in pheromone on the surface, or outer distal layer(s) of the gland, and that located more proximally. For this, we used two sampling approaches, (i) a solid phase microextraction fiber rub followed by solvent extraction of residual pheromone (SPME rub/extract), and (ii) rapid solvent rinsing followed by solvent extraction of residual pheromone (rinse/extract). The SPME rub showed differences in component ratio between the dorsal and ventral gland surfaces. The rinse sampled a greater amount of pheromone than the SPME rub, sampling the whole gland surface as well as likely deeper into the gland. Compared to the other samplings, pheromone in the rinse was depleted in the minor component; consequently, the corresponding residual extract was highly enriched in the minor component. Further rinses of the gland yielded only small amounts of pheromone, with a similar component ratio as the first rinse, suggesting that the residual pheromone was less accessible and required extraction in solvent to be liberated. Sampling over the photoperiod showed that the more volatile minor component was depleted (relative to the major component) on the surface/outer cuticle over the period when females called. Together, these data suggest that the pheromone is stored, at least in part, on and in the gland cuticle and that distinct pools may be transported to different topographic regions. Females fed with a stable isotope tracer, incorporated label into pheromone in the gland very rapidly, with the labeled pheromone appearing on the gland surface ca. 1 min later.

Detecting long-term metabolic shifts using isotopomers: CO2-driven suppression of photorespiration in C3 plants over the 20th century.

Terrestrial vegetation currently absorbs approximately a third of anthropogenic CO2 emissions, mitigating the rise of atmospheric CO2. However, terrestrial net primary production is highly sensitive to atmospheric CO2 levels and associated climatic changes. In C3 plants, which dominate terrestrial vegetation, net photosynthesis depends on the ratio between photorespiration and gross photosynthesis. This metabolic flux ratio depends strongly on CO2 levels, but changes in this ratio over the past CO2 rise have not been analyzed experimentally. Combining CO2 manipulation experiments and deuterium NMR, we first establish that the intramolecular deuterium distribution (deuterium isotopomers) of photosynthetic C3 glucose contains a signal of the photorespiration/photosynthesis ratio. By tracing this isotopomer signal in herbarium samples of natural C3 vascular plant species, crops, and a Sphagnum moss species, we detect a consistent reduction in the photorespiration/photosynthesis ratio in response to the ∼100-ppm CO2 increase between ∼1900 and 2013. No difference was detected in the isotopomer trends between beet sugar samples covering the 20th century and CO2 manipulation experiments, suggesting that photosynthetic metabolism in sugar beet has not acclimated to increasing CO2 over >100 y. This provides observational evidence that the reduction of the photorespiration/photosynthesis ratio was ca. 25%. The Sphagnum results are consistent with the observed positive correlations between peat accumulation rates and photosynthetic rates over the Northern Hemisphere. Our results establish that isotopomers of plant archives contain metabolic information covering centuries. Our data provide direct quantitative information on the "CO2 fertilization" effect over decades, thus addressing a major uncertainty in Earth system models.