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Sivashankari, Ramamoorthi M., Yuki Miyahara, and Takeharu Tsuge. "Poly(3-Hydroxybutyrate) Biosynthesis from [U-13C6]D-Glucose by Ralstonia eutropha NCIMB 11599 and Recombinant Escherichia coli." Microbiology Research 14, no. 4 (2023): 1894–906. http://dx.doi.org/10.3390/microbiolres14040129.
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The use of stable isotope-labeled polymers in in situ biodegradation tests provides detailed information on the degradation process. As isotope-labeled raw chemicals are generally expensive, it is desirable to prepare polymer samples with high production yields and high isotope-labeling ratios. The biodegradable plastic poly[(R)-3-hydroxybutyrate)] (P(3HB)) is produced by microorganisms. In this study, to produce carbon 13 (13C)-labeled P(3HB) from [U-13C6]D-glucose (13C-glucose), the culture conditions needed for high production yields and high 13C-labeling ratios were investigated using Ralstonia eutropha NCIMB 11599 and recombinant Escherichia coli JM109. We found that over 10 g/L of P(3HB) could be obtained when these microorganisms were cultured in Luria-Bertani (LB3) medium containing 3 g/L NaCl and 40 g/L 13C-glucose, while 1.4–4.7 g/L of P(3HB) was obtained when a mineral salt (MS) medium containing 20 g/L 13C-glucose was used. The 13C-labeling ratio of P(3HB) was determined by 1H nuclear magnetic resonance and gas chromatography-mass spectrometry (GC-MS), and both analytical methods yielded nearly identical results. High 13C-labeling ratios (97.6 atom% by GC-MS) were observed in the MS medium, whereas low 13C-labeling ratios (88.8–94.4 atom% by GC-MS) were observed in the LB3 medium. Isotope effects were observed for the P(3HB) content in cells cultured in the LB3 medium and the polydispersity of P(3HB).
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Ali, Rustam, Lindsay D. Clark, Jacob A. Zahm, et al. "Improved strategy for isoleucine 1H/13C methyl labeling in Pichia pastoris." Journal of Biomolecular NMR 73, no. 12 (2019): 687–97. http://dx.doi.org/10.1007/s10858-019-00281-1.
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Abstract Site specific methyl labeling combined with methyl TROSY offers a powerful NMR approach to study structure and dynamics of proteins and protein complexes of high molecular weight. Robust and cost-effective methods have been developed for site specific protein 1H/13C methyl labeling in an otherwise deuterated background in bacteria. However, bacterial systems are not suitable for expression and isotope labeling of many eukaryotic and membrane proteins. The yeast Pichia pastoris (P. pastoris) is a commonly used host for expression of eukaryotic proteins, and site-specific methyl labeling of perdeuterated eukaryotic proteins has recently been achieved with this system. However, the practical utility of methyl labeling and deuteration in P. pastoris is limited by high costs. Here, we describe an improved method for 1H/13C-labeling of the δ-methyl group of isoleucine residues in a perdeuterated background, which reduces the cost by ≥ 50% without compromising the efficiency of isotope enrichment. We have successfully implemented this method to label actin and a G-protein coupled receptor. Our approach will facilitate studies of the structure and dynamics of eukaryotic proteins by NMR spectroscopy.
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Miyatake, Tetsuro, Barbara J. MacGregor, and Henricus T. S. Boschker. "Linking Microbial Community Function to Phylogeny of Sulfate-Reducing Deltaproteobacteria in Marine Sediments by Combining Stable Isotope Probing with Magnetic-Bead Capture Hybridization of 16S rRNA." Applied and Environmental Microbiology 75, no. 15 (2009): 4927–35. http://dx.doi.org/10.1128/aem.00652-09.
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ABSTRACT We further developed the stable isotope probing, magnetic-bead capture method to make it applicable for linking microbial community function to phylogeny at the class and family levels. The main improvements were a substantial decrease in the protocol blank and an approximately 10-fold increase in the detection limit by using a micro-elemental analyzer coupled to isotope ratio mass spectrometry to determine 13C labeling of isolated 16S rRNA. We demonstrated the method by studying substrate utilization by Desulfobacteraceae, a dominant group of complete oxidizing sulfate-reducing Deltaproteobacteria in marine sediments. Stable-isotope-labeled [13C]glucose, [13C]propionate, or [13C]acetate was fed into an anoxic intertidal sediment. We applied a nested set of three biotin-labeled oligonucleotide probes to capture Bacteria, Deltaproteobacteria, and finally Desulfobacteraceae rRNA by using hydrophobic streptavidin-coated paramagnetic beads. The target specificities of the probes were examined with pure cultures of target and nontarget species and by determining the phylogenetic composition of the captured sediment rRNA. The specificity of the final protocol was generally very good, as more than 90% of the captured 16S rRNA belonged to the target range of the probes. Our results indicated that Desulfobacteraceae were important consumers of propionate but not of glucose. However, the results for acetate utilization were less conclusive due to lower and more variable labeling levels in captured rRNA. The main advantage of the method in this study over other nucleic acid-based stable isotope probing methods is that 13C labeling can be much lower, to the extent that δ13C ratios can be studied even at their natural abundances.
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Fahey, Timothy J., Kayla R. Jacobs, and Ruth E. Sherman. "Fine root turnover in sugar maple estimated by 13C isotope labeling." Canadian Journal of Forest Research 42, no. 10 (2012): 1792–95. http://dx.doi.org/10.1139/x2012-128.
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We evaluated variation in root turnover across five root orders in sugar maple ( Acer saccharum Marsh.) saplings growing in a northern hardwood forest in central New York, USA. We used a stable isotope approach in which root systems were labeled with 13C and root structural C sequentially sampled for 13C enrichment. Turnover of first- and second-order roots was apparently rapid with only about 5% of the 13C retained in living roots after two growing seasons. Although third- to fifth-order roots appeared to persist longer, differences among root orders were not statistically significant, probably mostly because of highly nonuniform initial labeling. This nonuniform labeling constrains the precision of root turnover quantification using this approach.
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Bagga, Puneet, Kevin L. Behar, Graeme F. Mason, Henk M. De Feyter, Douglas L. Rothman, and Anant B. Patel. "Characterization of Cerebral Glutamine Uptake from Blood in the Mouse Brain: Implications for Metabolic Modeling of 13C NMR Data." Journal of Cerebral Blood Flow & Metabolism 34, no. 10 (2014): 1666–72. http://dx.doi.org/10.1038/jcbfm.2014.129.
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13C Nuclear Magnetic Resonance (NMR) studies of rodent and human brain using [1-13C]/[1,6-13C2]glucose as labeled substrate have consistently found a lower enrichment (~25% to 30%) of glutamine-C4 compared with glutamate-C4 at isotopic steady state. The source of this isotope dilution has not been established experimentally but may potentially arise either from blood/brain exchange of glutamine or from metabolism of unlabeled substrates in astrocytes, where glutamine synthesis occurs. In this study, the contribution of the former was evaluated ex vivo using 1H-[13C]-NMR spectroscopy together with intravenous infusion of [U-13C5]glutamine for 3, 15, 30, and 60 minutes in mice. 13C labeling of brain glutamine was found to be saturated at plasma glutamine levels > 1.0 mmol/L. Fitting a blood–astrocyte–neuron metabolic model to the 13C enrichment time courses of glutamate and glutamine yielded the value of glutamine influx, VGln(in), 0.036 ± 0.002 μmol/g per minute for plasma glutamine of 1.8 mmol/L. For physiologic plasma glutamine level (~0.6 mmol/L), VGln(in) would be ~0.010 μmol/g per minute, which corresponds to ~6% of the glutamine synthesis rate and rises to ~11% for saturating blood glutamine concentrations. Thus, glutamine influx from blood contributes at most ~20% to the dilution of astroglial glutamine-C4 consistently seen in metabolic studies using [1-13C]glucose.
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Jin, S. J., and K. Y. Tserng. "Biogenesis of dicarboxylic acids in rat liver homogenate studied by 13C labeling." American Journal of Physiology-Endocrinology and Metabolism 261, no. 6 (1991): E719—E724. http://dx.doi.org/10.1152/ajpendo.1991.261.6.e719.
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The aim of this investigation is to assess whether long-chain fatty acids can be a substrate for omega-oxidation and the subsequent beta-oxidation to produce medium-chain dicarboxylic acids normally found in urine. Isolated rat liver 10,000 g supernatant and pellet fractions were used as the source of enzymes. The metabolism of palmitate was studied using [1,2,3,4-13C4]hexadecanoic acid as tracer. Selected ion monitoring mass spectrometry was utilized for the determination of isotope enrichments in precursor and products. Palmitate was found to be a good substrate for omega-oxidation; the rate was only slightly slower than decanoate. The beta-oxidation of [1,2,3,4-13C4]hexadecanedioic acid yielded labeled adipic, suberic, and sebacic acids. Isotope distribution in these dicarboxylic acids consisted mostly of unlabeled molecules (M + 0) and molecules labeled with four 13C (M + 4), in agreement with a beta-oxidation initiated equally from both carboxyl ends of the precursor. Significant enrichments (1-8%) with only two 13C labels (M + 2) indicate a partial bidirectional beta-oxidation. The direct metabolic conversion of hexadecanedioate to succinate was documented by the significant enrichment (1.40-1.90%) in M + 4 of succinate. These data indicate that long-chain fatty acids can be a substrate for the production of medium-chain dicarboxylates and the eventual direct conversion to succinate.
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Kim, Hee-Youn, Eun-Mi Hong, and Weon-Tae Lee. "Cost-effective isotope labeling technique developed for15N/13C-labeled proteins." Journal of the Korean Magnetic Resonance Society 15, no. 2 (2011): 115–27. http://dx.doi.org/10.6564/jkmrs.2011.15.2.115.
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Robins, Richard, Katarzyna Romek, Mathilde Grand, et al. "Difficulties in Differentiating Natural from Synthetic Alkaloids by Isotope Ratio Monitoring using 13C Nuclear Magnetic Resonance Spectrometry." Planta Medica 84, no. 12/13 (2018): 935–40. http://dx.doi.org/10.1055/a-0601-7157.
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AbstractWithin the food and pharmaceutical industries, there is an increasing legislative requirement for the accurate labeling of the productʼs origin. A key feature of this is to indicate whether the product is of natural or synthetic origin. With reference to this context, we have investigated three alkaloids commonly exploited for human use: nicotine, atropine, and caffeine. We have measured by 13C nuclear magnetic resonance spectrometry the position-specific distribution of 13C at natural abundance within several samples of each of these target molecules. This technique is well suited to distinguishing between origins, as the distribution of the 13C isotope reflects the primary source of the carbon atoms and the process by which the molecule was (bio)synthesized. Our findings indicate that labeling can be misleading, especially in relation to a supplied compound being labeled as “synthetic” even though its 13C profile indicates a natural origin.
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Novotny, Jessie Lanterman, and Karen Goodell. "Utility of carbon and nitrogen stable isotopes for inferring wild bee (Hymenoptera: Apoidea) use of adjacent foraging habitats." PLOS ONE 17, no. 7 (2022): e0271095. http://dx.doi.org/10.1371/journal.pone.0271095.
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Isotope analysis has proven useful for understanding diets of animals that are difficult to track for extended periods. Bees are small yet highly mobile and often forage from multiple habitats. However, current methods of assessing diet are limited in scope. Efficient methods of tracking bee diets that integrate across life stages, distinguish habitat use, and are sensitive to taxonomic differences will inform conservation strategies. We evaluated the utility of stable isotope analysis for estimating contributions of adjacent habitats to bees’ diets. We also investigated taxonomic variation in bee and flower isotope composition. We measured natural abundance of carbon and nitrogen stable isotopes in two body regions from three wild bee genera, as well as in 25 species of flowers that likely comprised their diets. Bee ∂13C and ∂15N varied with habitat and taxonomic groups (conflated with month), but did not match spatial or seasonal trends in their food plants. Flower ∂13C was lowest in the forest and in April–June, as expected if driven by water availability. However, bee ∂13C was elevated in the spring, likely from overwintering nutritional stress or unpredictable food availability. Bumble bees (Bombus) were enriched in ∂15N compared to others, possibly reflecting differences in larval feeding. Bee diet mixing models had high variation and should be interpreted with caution. Models estimated similar habitat contributions to diets of spring Andrena and overwintered Bombus queens. Summer Bombus queens and workers were indistinguishable. Sweat bees (Halictus) were estimated to use comparatively more field flowers than others. Overall, taxon more strongly influenced isotope composition than either foraging habitat or month, likely because of associated differences in sociality and timing of annual activity. Future studies seeking to reveal bee diets by isotope analysis may gain better resolution in more isotopically distinct habitats, in conjunction with controlled feeding or isotope labeling experiments.
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Takeda, Mitsuhiro, Yohei Miyanoiri, Tsutomu Terauchi, and Masatsune Kainosho. "Conformational features and ionization states of Lys side chains in a protein studied using the stereo-array isotope labeling (SAIL) method." Magnetic Resonance 2, no. 1 (2021): 223–37. http://dx.doi.org/10.5194/mr-2-223-2021.
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Abstract. Although both the hydrophobic aliphatic chain and hydrophilic ζ-amino group of the Lys side chain presumably contribute to the structures and functions of proteins, the dual nature of the Lys residue has not been fully investigated using NMR spectroscopy, due to the lack of appropriate methods to acquire comprehensive information on its long consecutive methylene chain. We describe herein a robust strategy to address the current situation, using various isotope-aided NMR technologies. The feasibility of our approach is demonstrated for the Δ+PHS/V66K variant of staphylococcal nuclease (SNase), which contains 21 Lys residues, including the engineered Lys-66 with an unusually low pKa of ∼ 5.6. All of the NMR signals for the 21 Lys residues were sequentially and stereospecifically assigned using the stereo-array isotope-labeled Lys (SAIL-Lys), [U-13C,15N; β2,γ2,δ2,ε3-D4]-Lys. The complete set of assigned 1H, 13C, and 15N NMR signals for the Lys side-chain moieties affords useful structural information. For example, the set includes the characteristic chemical shifts for the 13Cδ, 13Cε, and 15Nζ signals for Lys-66, which has the deprotonated ζ-amino group, and the large upfield shifts for the 1H and 13C signals for the Lys-9, Lys-28, Lys-84, Lys-110, and Lys-133 side chains, which are indicative of nearby aromatic rings. The 13Cε and 15Nζ chemical shifts of the SNase variant selectively labeled with either [ε-13C;ε,ε-D2]-Lys or SAIL-Lys, dissolved in H2O and D2O, showed that the deuterium-induced shifts for Lys-66 were substantially different from those of the other 20 Lys residues. Namely, the deuterium-induced shifts of the 13Cε and 15Nζ signals depend on the ionization states of the ζ-amino group, i.e., −0.32 ppm for Δδ13Cε [NζD3+-NζH3+] vs. −0.21 ppm for Δδ13Cε [NζD2-NζH2] and −1.1 ppm for Δδ15Nζ[NζD3+-NζH3+] vs. −1.8 ppm for Δδ15Nζ[NζD2-NζH2]. Since the 1D 13C NMR spectrum of a protein selectively labeled with [ε-13C;ε,ε-D2]-Lys shows narrow (> 2 Hz) and well-dispersed 13C signals, the deuterium-induced shift difference of 0.11 ppm for the protonated and deprotonated ζ-amino groups, which corresponds to 16.5 Hz at a field strength of 14 T (150 MHz for 13C), could be accurately measured. Although the isotope shift difference itself may not be absolutely decisive to distinguish the ionization state of the ζ-amino group, the 13Cδ, 13Cε, and 15Nζ signals for a Lys residue with a deprotonated ζ-amino group are likely to exhibit distinctive chemical shifts as compared to the normal residues with protonated ζ-amino groups. Therefore, the isotope shifts would provide a useful auxiliary index for identifying Lys residues with deprotonated ζ-amino groups at physiological pH levels.
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Del Vecchio, Antonio, Alex Talbot, Fabien Caillé, et al. "Carbon isotope labeling of carbamates by late-stage [11C], [13C] and [14C]carbon dioxide incorporation." Chemical Communications 56, no. 78 (2020): 11677–80. http://dx.doi.org/10.1039/d0cc05031h.
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A procedure which allows labelling cyclic carbamates with all carbon isotopes has been developed. This protocol valorizes carbon dioxide, the universal building block for radiolabeling. A series of pharmaceuticals were obtained and a disconnection/reconnection strategy was implemented.
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Liu, Liang, and Shoushan Fan. "Isotope Labeling of Carbon Nanotubes and Formation of12C−13C Nanotube Junctions." Journal of the American Chemical Society 123, no. 46 (2001): 11502–3. http://dx.doi.org/10.1021/ja0167304.
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Auclair, Julie, François Lépine, and Richard Villemur. "A liquid chromatography – mass spectrometry method to measure 13C-isotope enrichment for DNA stable-isotope probing." Canadian Journal of Microbiology 58, no. 3 (2012): 287–92. http://dx.doi.org/10.1139/w11-133.
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DNA stable-isotope probing (DNA-SIP) is a cultivation-independent technique that makes it possible to associate metabolic function and taxonomic identity in a wide range of terrestrial and aquatic environments. In DNA-SIP, DNA is labeled via the assimilation of a labeled growth substrate that is subsequently used to identify microorganisms involved in assimilation of the substrate. However, the labeling time has to be sufficient to obtain labeled DNA but not so long such that cross-feeding of 13C-labeled metabolites from the primary consumers to nontarget species can occur. Confirmation that the DNA is isotopically labeled in DNA-SIP assays can be achieved using an isotope ratio mass spectrometer. In this study, we describe the development of a method using liquid chromatography (HPLC) coupled to a quadrupole mass spectrometer (QMS) to measure the 13C enrichment of thymine incorporated into DNA in Escherichia coli cultures fed with [13C]acetate. The method involved the hydrolysis of DNA extracted from the cultures that released the nucleotides, followed by the separation of the thymine by HPLC on a reverse-phase C8 column in isocratic elution mode and the detection and quantification of 13C-labeled thymine by QMS. To mimic a DNA-SIP assay, a DNA mixture was made using 13C-labeled E. coli DNA with DNA extracted from five bacterial species. The HPLC–MS method was able to measure the correct proportion of 13C-DNA in the mix. This method can then be used as an alternative to the use of isotope ratio mass spectrometry in DNA-SIP assays.
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Gebbing, Thomas, and Hans Schnyder. "13C Labeling kinetics of sucrose in glumes indicates significant refixation of respiratory CO2 in the wheat ear." Functional Plant Biology 28, no. 10 (2001): 1047. http://dx.doi.org/10.1071/pp01072.
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Photosynthesis by the vegetative structures of the ear (i.e. glumes) of wheat (Triticum aestivum L.) may draw on two sources of CO2: atmospheric CO2 and CO2 originating from respiration within the ear. We exposed wheat plants to a changed C isotope composition (δ) of one of the two sources, atmospheric CO2, to assess the contributions of atmospheric and respiratory CO2 to ear photosynthesis by following the labeling kinetics of water-soluble carbohydrates (WSC; fructose, glucose, sucrose, fructan) in glumes. Experiments included sampling during diurnal cycles and after extended exposures (7 and 14 d). The labeling kinetics of sucrose in the flag leaf and grains was also determined. Significant diurnal changes in sucrose content (depletion during dark and accumulation during light periods) were observed in flag leaves and in glumes. In flag leaves (but not in glumes) the sucrose accumulating during the light period had a d that was close to the value expected if atmospheric CO2 (with changed δ) was the sole source of CO2 for photosynthesis. The δ of sucrose in glumes did not saturate after extended exposure to the labeling CO2 indicating the utilization of unlabeled (respiratory) CO2 for photosynthesis (i.e. refixation). Short-term labeling indicated that 73% of the sucrose accumulating in glumes after the start of labeling came from fixation of respiratory CO2. Stable C isotope discrimination during glume photosynthesis is discussed.
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Malloy, C. R., A. D. Sherry, and F. M. Jeffrey. "Analysis of tricarboxylic acid cycle of the heart using 13C isotope isomers." American Journal of Physiology-Heart and Circulatory Physiology 259, no. 3 (1990): H987—H995. http://dx.doi.org/10.1152/ajpheart.1990.259.3.h987.
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13C-nuclear magnetic resonance (NMR) spectroscopy provides a new approach to the analysis of metabolic pathways, because it detects an interaction between adjacent 13C nuclei. Previous models of isotope distribution in the tricarboxylic acid cycle were designed for analysis of radioisotope data and did not consider the information provided by 13C-13C coupling. A mathematical model of the tricarboxylic acid cycle was developed that preserves all isotope isomer (isotopomer) information and yields simple relationships between 13C-NMR spectra of glutamate and metabolic parameters under steady-state conditions. With the use of relative peak areas measured from the spectra of tissues supplied with 13C-enriched substrate(s), the relative fluxes through both oxidative (acetyl-CoA utilization) and nonoxidative (anaplerotic) pathways of the tricarboxylic acid cycle can be determined. Furthermore, with the judicious selection of 13C-labeling patterns in a mixture of substrates, direct substrate competition experiments can be performed. The perchloric acid extracts of Langendorff and working rat hearts oxidizing 13C-enriched fatty acids or carbohydrates are analyzed to illustrate this approach, and the importance of measuring the fractional enrichment of the available substrate is demonstrated. The technique can of course be used with all tissues, not just heart, and is applicable to the analysis of in vivo 13C-NMR spectra.
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C�bron, Aur�lie, Levente Bodrossy, Nancy Stralis-Pavese, et al. "Nutrient Amendments in Soil DNA Stable Isotope Probing Experiments Reduce the Observed Methanotroph Diversity." Applied and Environmental Microbiology 73, no. 3 (2006): 798–807. http://dx.doi.org/10.1128/aem.01491-06.
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ABSTRACT Stable isotope probing (SIP) can be used to analyze the active bacterial populations involved in a process by incorporating 13C-labeled substrate into cellular components such as DNA. Relatively long incubation times are often used with laboratory microcosms in order to incorporate sufficient 13C into the DNA of the target organisms. Addition of nutrients can be used to accelerate the processes. However, unnatural concentrations of nutrients may artificially change bacterial diversity and activity. In this study, methanotroph activity and diversity in soil was examined during the consumption of 13CH4 with three DNA-SIP experiments, using microcosms with natural field soil water conditions, the addition of water, and the addition of mineral salts solution. Methanotroph population diversity was studied by targeting 16S rRNA and pmoA genes. Clone library analyses, denaturing gradient gel electrophoresis fingerprinting, and pmoA microarray hybridization analyses were carried out. Most methanotroph diversity (type I and type II methanotrophs) was observed in nonamended SIP microcosms. Although this treatment probably best reflected the in situ environmental conditions, one major disadvantage of this incubation was that the incorporation of 13CH4 was slow and some cross-feeding of 13C occurred, thereby leading to labeling of nonmethanotroph microorganisms. Conversely, microcosms supplemented with mineral salts medium exhibited rapid consumption of 13CH4, resulting in the labeling of a less diverse population of only type I methanotrophs. DNA-SIP incubations using water-amended microcosms yielded faster incorporation of 13C into active methanotrophs while avoiding the cross-feeding of 13C.
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Yagi, Hirokazu, Saeko Yanaka, Rina Yogo, et al. "Silkworm Pupae Function as Efficient Producers of Recombinant Glycoproteins with Stable-Isotope Labeling." Biomolecules 10, no. 11 (2020): 1482. http://dx.doi.org/10.3390/biom10111482.
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Baculovirus-infected silkworms are promising bioreactors for producing recombinant glycoproteins, including antibodies. Previously, we developed a method for isotope labeling of glycoproteins for nuclear magnetic resonance (NMR) studies using silkworm larvae reared on an artificial diet containing 15N-labeled yeast crude protein extract. Here, we further develop this method by introducing a technique for the expression of isotope-labeled glycoproteins by silkworm pupae, which has several potential advantages relative to larvae-based techniques in terms of production yield, ease of handling, and storage. Here, we fed fifth instar larvae an artificial diet with an optimized composition containing [methyl-13C]methionine, leading to pupation. Nine-day-old pupae were then injected with recombinant Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid for expression of recombinant human immunoglobulin G (IgG). From the whole-body homogenates of pupae, 0.35 mg/pupa of IgG was harvested, which is a yield that is five times higher than can be obtained from larvae. Recombinant IgG, thus prepared, exhibited mainly three kinds of pauci-mannose-type oligosaccharides and had a 13C-enrichment ratio of approximately 80%. This enabled selective observation of NMR signals originating from the methionyl methyl group of IgG, confirming its conformational integrity. These data demonstrate the utility of silkworm pupae as factories for producing recombinant glycoproteins with amino-acid-selective isotope labeling.
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Nogués, Salvador, Guillaume Tcherkez, Gabriel Cornic, and Jaleh Ghashghaie. "Respiratory Carbon Metabolism following Illumination in Intact French Bean Leaves Using 13C/12C Isotope Labeling." Plant Physiology 136, no. 2 (2004): 3245–54. http://dx.doi.org/10.1104/pp.104.048470.
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Hemmerling, Christin, Zhipeng Li, Lingling Shi, Johanna Pausch, and Liliane Ruess. "Flux of Root-Derived Carbon into the Nematode Micro-Food Web: A Comparison of Grassland and Agroforest." Agronomy 12, no. 4 (2022): 976. http://dx.doi.org/10.3390/agronomy12040976.
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Carbon (C) cycling is crucial to agroecosystem functioning. Important determinants for the belowground C flow are soil food webs, with microorganisms and microfaunal grazers, i.e., nematodes, as key biota. The present study investigates the incorporation of plant-derived C into the nematode micro-food web under two different cropping systems, grassland (ryegrass (Lolium perenne L.) and white clover (Trifolium repens L.)) and agroforest (willow (Salix schwerinii Wolf and Salix viminalis L)). To quantify the C flux from the plant into the soil micro-food web, grass and willow were pulse-labeled with 13CO2 and the incorporation of 13C into the nematode trophic groups was monitored 3, 7, 14 and 28 days after labeling. The natural stable isotope signals (13C/12C, 15N/14N) were analyzed to determine the structure of the nematode micro-food web. The natural isotopic δ15N signal revealed different trophic levels for omnivores and predators in grassland and agroforest soils. The incorporation of plant C into nematode tissue was detectable three days after 13CO2 labeling with the highest and fastest C allocation in plant feeders in grassland, and in fungal feeders in agroforest soil. C flux dynamics between the aboveground vegetation and belowground micro-food web varied with cropping system. This demonstrates that crop-specific translocation of C affects the multitrophic interactions in the root environment, which in turn can alter soil nutrient cycling.
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Couté, Yohann, Céline Hernandez, Ron D. Appel, Jean-Charles Sanchez, and Abelardo Margolles. "Labeling of Bifidobacterium longum Cells with 13C-Substituted Leucine for Quantitative Proteomic Analyses." Applied and Environmental Microbiology 73, no. 17 (2007): 5653–56. http://dx.doi.org/10.1128/aem.00667-07.
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ABSTRACT Stable isotope labeling of amino acids in cell culture was used for Bifidobacterium longum. A comprehensive proteomic strategy was developed and validated by designing an appropriate semidefined medium that allows stable replacement of natural leucine by [13C6]leucine. Using this strategy, proteins having variations of at least 50% in their expression rates can be quantified with great confidence.
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Brossard, N., M. Croset, J. Lecerf, et al. "Metabolic fate of an oral tracer dose of [13C]docosahexaenoic acid triglycerides in the rat." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 270, no. 4 (1996): R846—R854. http://dx.doi.org/10.1152/ajpregu.1996.270.4.r846.
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The appearance of 13C in rat lipoprotein, blood cells, and brain lipids was followed as a function of time after the ingestion of triglycerides (TG) containing [13C]22:6n-3. The time course of 13C abundance in 22:6n-3 of various lipid pools, measured by gas chromatography combustion-isotope mass spectrometry, established precursor-product relationships within lipids. The [13C]22:6n-3 was rapidly incorporated into very low density lipoprotein-chylomicron-TG and unesterified fatty acids bound to albumin, with a concomitant maximal appearance at 3 h and further decline. Lysophosphatidylcholines (lysoPC) bound to albumin were also enriched in [13C]22:6n-3, and their labeling appeared to be mainly due to hepatic secretion at the earliest time points. From 12 h postingestion, the synthesis of [13C]22:6n-3-lysoPC was twice as high as that of unesterified [13C]22:6n-3, making lysoPC a potential source of 22:6n-3 supply for tissues. The labeling of platelets, red blood cells, and brain phospholipids presented different kinetics, presumably involving the two lipid forms of [13C]22:6n-3 bound to albumin, to different extents. We conclude that [13C]22:6n-3 esterified in TG is rapidly redistributed within blood lipoproteins and the albumin fraction and that its incorporation in lipid species bound to albumin influences its uptake by target tissues.
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Sitarska, Agnieszka, Lukasz Skora, Julia Klopp, et al. "Affordable uniform isotope labeling with 2H, 13C and 15N in insect cells." Journal of Biomolecular NMR 62, no. 2 (2015): 191–97. http://dx.doi.org/10.1007/s10858-015-9935-6.
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Maxfield, P. J., E. R. C. Hornibrook, and R. P. Evershed. "Estimating High-Affinity Methanotrophic Bacterial Biomass, Growth, and Turnover in Soil by Phospholipid Fatty Acid 13C Labeling." Applied and Environmental Microbiology 72, no. 6 (2006): 3901–7. http://dx.doi.org/10.1128/aem.02779-05.
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ABSTRACT A time series phospholipid fatty acid (PLFA) 13C-labeling study was undertaken to determine methanotrophic taxon, calculate methanotrophic biomass, and assess carbon recycling in an upland brown earth soil from Bronydd Mawr (Wales, United Kingdom). Laboratory incubations of soils were performed at ambient CH4 concentrations using synthetic air containing 2 parts per million of volume of 13CH4. Flowthrough chambers maintained a stable CH4 concentration throughout the 11-week incubation. Soils were analyzed at weekly intervals by gas chromatography (GC), GC-mass spectrometry, and GC-combustion-isotope ratio mass spectrometry to identify and quantify individual PLFAs and trace the incorporation of 13C label into the microbial biomass. Incorporation of the 13C label was seen throughout the experiment, with the rate of incorporation decreasing after 9 weeks. The δ13C values of individual PLFAs showed that 13C label was incorporated into different components to various extents and at various rates, reflecting the diversity of PLFA sources. Quantitative assessments of 13C-labeled PLFAs showed that the methanotrophic population was of constant structure throughout the experiment. The dominant 13C-labeled PLFA was 18:1ω7c, with 16:1ω5 present at lower abundance, suggesting the presence of novel type II methanotrophs. The biomass of methane-oxidizing bacteria at optimum labeling was estimated to be about 7.2 � 106 cells g−1 of soil (dry weight). While recycling of 13C label from the methanotrophic biomass must occur, it is a slower process than initial 13CH4 incorporation, with only about 5 to 10% of 13C-labeled PLFAs reflecting this process. Thus, 13C-labeled PLFA distributions determined at any time point during 13CH4 incubation can be used for chemotaxonomic assessments, although extended incubations are required to achieve optimum 13C labeling for methanotrophic biomass determinations.
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Hungate, Bruce A., Rebecca L. Mau, Egbert Schwartz, et al. "Quantitative Microbial Ecology through Stable Isotope Probing." Applied and Environmental Microbiology 81, no. 21 (2015): 7570–81. http://dx.doi.org/10.1128/aem.02280-15.
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ABSTRACTBacteria grow and transform elements at different rates, and as yet, quantifying this variation in the environment is difficult. Determining isotope enrichment with fine taxonomic resolution after exposure to isotope tracers could help, but there are few suitable techniques. We propose a modification tostableisotopeprobing (SIP) that enables the isotopic composition of DNA from individual bacterial taxa after exposure to isotope tracers to be determined. In our modification, after isopycnic centrifugation, DNA is collected in multiple density fractions, and each fraction is sequenced separately. Taxon-specific density curves are produced for labeled and nonlabeled treatments, from which the shift in density for each individual taxon in response to isotope labeling is calculated. Expressing each taxon's density shift relative to that taxon's density measured without isotope enrichment accounts for the influence of nucleic acid composition on density and isolates the influence of isotope tracer assimilation. The shift in density translates quantitatively to isotopic enrichment. Because this revision to SIP allows quantitative measurements of isotope enrichment, we propose to call it quantitative stable isotope probing (qSIP). We demonstrated qSIP using soil incubations, in which soil bacteria exhibited strong taxonomic variations in18O and13C composition after exposure to [18O]water or [13C]glucose. The addition of glucose increased the assimilation of18O into DNA from [18O]water. However, the increase in18O assimilation was greater than expected based on utilization of glucose-derived carbon alone, because the addition of glucose indirectly stimulated bacteria to utilize other substrates for growth. This example illustrates the benefit of a quantitative approach to stable isotope probing.
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MacGregor, Barbara J., Henricus T. S. Boschker, and Rudolf Amann. "Comparison of rRNA and Polar-Lipid-Derived Fatty Acid Biomarkers for Assessment of 13C-Substrate Incorporation by Microorganisms in Marine Sediments." Applied and Environmental Microbiology 72, no. 8 (2006): 5246–53. http://dx.doi.org/10.1128/aem.00423-06.
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ABSTRACT We determined whether a recently developed method to isolate specific small-subunit (SSU) rRNAs can be used in 13C-labeling studies to directly link community structure and function in natural ecosystems. Replicate North Sea sediment cores were incubated at the in situ temperature following addition of 13C-labeled acetate, propionate, amino acids, or glucose. Eukaryotic and bacterial SSU rRNAs were separated from total RNA by means of biotin-labeled oligonucleotide probes and streptavidin-coated paramagnetic beads, and the 13C content of the isolated rRNA was determined by elemental analysis-isotope ratio mass spectrometry. The SSU rRNA yield with the bead-capture protocol was improved by using helper probes. Incorporation of label into bacterial SSU rRNA was detectable after 2 h of incubation. The labeling was always much greater in bacterial SSU rRNA than in eukaryotic SSU rRNA, suggesting that bacteria were the main consumers of the 13C-labeled compounds. Similar results were obtained with the 13C-labeled polar-lipid-derived fatty acid (PLFA) approach, except that more label was detected in bacterial PLFA than in bacterial SSU rRNA. This may be attributable to the generally slow growth of sediment microbial populations, which results in low ribosome synthesis rates and relatively few ribosomes per cell. We discuss possible ways to improve the probe-capture protocol and the sensitivity of the 13C analysis of the captured SSU rRNA.
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Zou, J., L. Zhao, S. Xu, et al. "Livestock exclosure with consequent vegetation changes alters photo-assimilated carbon cycling in a <i>Kobresia</i> meadow." Biogeosciences Discussions 10, no. 11 (2013): 17633–61. http://dx.doi.org/10.5194/bgd-10-17633-2013.
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Abstract. Livestock exclosure has been widely used as an approach for grassland restoration. However, the effects of exclosure on grassland are controversial and can depend on many factors, such as the grassland ecosystem types, evolutionary history and so on. In this study, we conduct field experiments to investigate the variations of ecosystem function in response to livestock exclosure in a Kobresia humilis meadow under six years grazing exclosure on the Qinghai-Tibetan plateau. We focused on two ecosystem functions: plant community structure and ecosystem carbon cycling. The plant aboveground productivity, plant diversity and the composition of plant functional groups of the meadow were addressed as the indicators of the plant community structure. The 13C isotope pulse labeling technique was applied to evaluate the alterations of ecosystem carbon cycling during the short-term. The results showed that the plant community structure was changed after being fenced for six years, with significantly decreased aboveground productivity, species loss and varied composition of the four plant functional groups (grasses, sedges, legumes and forbs). Using the pulse labeling technique, we found a lower cycling rate of 13C in the plant–soil system of the fenced plots compared with the grazed sites during the first 4 days after labeling. A higher proportion of 13C amount recovered in the plant–soil system were migrated into soil as root exudates immediately after labeling at both fenced and control grazed sites, with significantly lower proportion in the fenced site, coinciding with the lower loss of 13C in soil respiration. Thirty-two days after labeling, 37% of recovered 13C remained in the soil of the fenced plots, with significant differences compared to the grazed plots (47%). In addition, less 13C (5% vs. 7%) was lost by soil respiration in the fenced plots during the chase period of 32 d. Overall, our study suggested that livestock exclosure had negative effects on the two ecosystem functions investigated, and the effects on 13C cycling and sequestrations in the soil were in response to variations in community structures, especially the suppression of forbs and legumes in the fenced site.
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Azemtsop Matanfack, Georgette, Aikaterini Pistiki, Petra Rösch, and Jürgen Popp. "Raman Stable Isotope Probing of Bacteria in Visible and Deep UV-Ranges." Life 11, no. 10 (2021): 1003. http://dx.doi.org/10.3390/life11101003.
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Raman stable isotope probing (Raman-SIP) is an excellent technique that can be used to access the overall metabolism of microorganisms. Recent studies have mainly used an excitation wavelength in the visible range to characterize isotopically labeled bacteria. In this work, we used UV resonance Raman spectroscopy (UVRR) to evaluate the spectral red-shifts caused by the uptake of isotopes (13C, 15N, 2H(D) and 18O) in E. coli cells. Moreover, we present a new approach based on the extraction of labeled DNA in combination with UVRR to identify metabolically active cells. The proof-of-principle study on E. coli revealed heterogeneities in the Raman features of both the bacterial cells and the extracted DNA after labeling with 13C, 15N, and D. The wavelength of choice for studying 18O- and deuterium-labeled cells is 532 nm is, while 13C-labeled cells can be investigated with visible and deep UV wavelengths. However, 15N-labeled cells are best studied at the excitation wavelength of 244 nm since nucleic acids are in resonance at this wavelength. These results highlight the potential of the presented approach to identify active bacterial cells. This work can serve as a basis for the development of new techniques for the rapid and efficient detection of active bacteria cells without the need for a cultivation step.
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Wang, Shizong, Anja Miltner, and Karolina M. Nowak. "Identification of degradation routes of metamitron in soil microcosms using 13C-isotope labeling." Environmental Pollution 220 (January 2017): 927–35. http://dx.doi.org/10.1016/j.envpol.2016.10.078.
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Pan, Xinli, Nicole Domin, Sebastian Schieferdecker, Hirokazu Kage, Martin Roth, and Markus Nett. "Herpetopanone, a diterpene from Herpetosiphon aurantiacus discovered by isotope labeling." Beilstein Journal of Organic Chemistry 13 (November 17, 2017): 2458–65. http://dx.doi.org/10.3762/bjoc.13.242.
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The genome of the predatory bacterium Herpetosiphon aurantiacus 114-95T harbors a number of biosynthesis genes, including four terpene cyclase genes. To identify the terpenes biosynthesized from H. aurantiacus 114-95T, we fed the strain with 13C-labeled glucose and, subsequently, searched for characteristic mass shifts in its metabolome. This approach led to the discovery of a new natural product, of which the isotope pattern is indicative for a diterpene originating from the methylerythritol phosphate pathway. After large-scale fermentation of H. aurantiacus 114-95T, the putative diterpene was isolated in sufficient quantity to enable NMR-based structure elucidation. The compound, for which the name herpetopanone is proposed, features a rare octahydro-1H-indenyl skeleton. Herpetopanone bears resemblance to cadinane-type sesquiterpenes from plants, but is structurally entirely unprecedented in bacteria. Based on its molecular architecture, a possible biosynthetic pathway is postulated.
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Lamichhane, Hari Prasad. "Intensity Revival of Weak Symmetric Infrared Band is Possible in Ubiquinone Molecules through the Asymmetric Site-specific Isotope Labeling." Himalayan Physics 5 (July 1, 2015): 39–46. http://dx.doi.org/10.3126/hj.v5i0.12838.
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Modifications of Infrared (IR) spectral bands of ubiquinone molecule (UQ1) upon site-specific 13C labeling at the C5 or C6 position are studied in CCl4 using Gaussian 03. Polarizable continuum model (PCM) has been used to optimize the UQ1 molecule in solvent. The unlabeled neutral ubiquinone molecule consists of three intense IR bands in the frequency region between 1700 cm-1 to 1550 cm-1. The symmetric fourth band in this spectral region does not appear in the spectrum because of very weak intensity. However, site-specific 13C labeling at C5 or C6 position removes the molecular symmetry and hence there appear four equivalent IR bands in the spectral region thus considered. This observation explains why there appears an extra spectral band in the experimental spectra observed by Brudler, R. et al [1].The Himalayan Physics Year 5, Vol. 5, Kartik 2071 (Nov 2014)Page: 39-46
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Kimura, Yukihiro, Michie Imanishi, Yong Li, et al. "Identification of metal-sensitive structural changes in the Ca2+-binding photocomplex from Thermochromatium tepidum by isotope-edited vibrational spectroscopy." Journal of Chemical Physics 156, no. 10 (2022): 105101. http://dx.doi.org/10.1063/5.0075600.
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Calcium ions play a dual role in expanding the spectral diversity and structural stability of photocomplexes from several Ca2+-requiring purple sulfur phototrophic bacteria. Here, metal-sensitive structural changes in the isotopically labeled light-harvesting 1 reaction center (LH1-RC) complexes from the thermophilic purple sulfur bacterium Thermochromatium ( Tch.) tepidum were investigated by perfusion-induced attenuated total reflection (ATR) Fourier transform infrared (FTIR) spectroscopy. The ATR-FTIR difference spectra induced by exchanges between native Ca2+ and exogenous Ba2+ exhibited interconvertible structural and/or conformational changes in the metal binding sites at the LH1 C-terminal region. Most of the characteristic Ba2+/Ca2+ difference bands were detected even when only Ca ions were removed from the LH1-RC complexes, strongly indicating the pivotal roles of Ca2+ in maintaining the LH1-RC structure of Tch. tepidum. Upon 15N-, 13C- or 2H-labeling, the LH1-RC complexes exhibited characteristic 15N/14N-, 13C/12C-, or 2H/1H-isotopic shifts for the Ba2+/Ca2+ difference bands. Some of the 15N/14N or 13C/12C bands were also sensitive to further 2H-labelings. Given the band frequencies and their isotopic shifts along with the structural information of the Tch. tepidum LH1-RC complexes, metal-sensitive FTIR bands were tentatively identified to the vibrational modes of the polypeptide main chains and side chains comprising the metal binding sites. Furthermore, important new IR marker bands highly sensitive to the LH1 BChl a conformation in the Ca2+-bound states were revealed based on both ATR-FTIR and near-infrared Raman analyses. The present approach provides valuable insights concerning the dynamic equilibrium between the Ca2+- and Ba2+-bound states statically resolved by x-ray crystallography.
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Zou, J., L. Zhao, S. Xu, et al. "Field <sup>13</sup>CO<sub>2</sub> pulse labeling reveals differential partitioning patterns of photoassimilated carbon in response to livestock exclosure in a <i>Kobresia</i> meadow." Biogeosciences 11, no. 16 (2014): 4381–91. http://dx.doi.org/10.5194/bg-11-4381-2014.
Full textAbstract:
Abstract. Livestock exclosure has been widely used as an approach for grassland restoration. However, the effects of exclosures on grasslands are controversial and can depend on many factors, such as the grassland ecosystem types, evolutionary history and so on. In this study, we conduct field experiments to investigate the variations of the ecosystem function in response to livestock exclosure in a Kobresia humilis meadow with 6 years of grazing exclosure on the Qinghai–Tibetan Plateau. We focused on two ecosystem functions: plant community structure and ecosystem carbon cycling. The plant aboveground productivity, plant diversity and the composition of plant functional groups of the meadow were addressed as the indicators of the plant community structure. The 13C isotope pulse labeling technique was applied to evaluate the alterations of ecosystem carbon cycling during a short term. The results showed that the plant community structure was changed after being fenced in for 6 years, with significantly decreased aboveground productivity, species loss and varied composition of the four plant functional groups (grasses, sedges, legumes and forbs). Using the pulse labeling technique, we found a lower cycling rate of 13C in the plant–soil system of the fenced plots compared with the grazed sites during the first 24 h after labeling. A higher proportion of recovered 13C in the plant–soil system migrated into the soil as root exudates immediately after labeling at both fenced and control grazed sites, with a significantly lower proportion in the fenced site, coinciding with the lower proportion of 13C lost from soil respiration. Thirty-two days after labeling, 37% of the recovered 13C remained in the soil of the fenced plots, with significant differences compared to in the grazed plots (47%). In addition, less 13C (5 vs. 7%) was lost by soil respiration in the fenced plots during the chase period of 32 days. Overall, our study suggests that livestock exclosures have negative effects on the plant community structure and partitioning patterns of the photoassimilated carbon in the Kobresia meadow, and the effects on photoassimilated carbon cycling are likely to result from the variations of community structures in the ecosystem.
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Olenginski, Lukasz T., and Theodore K. Dayie. "Quantifying the effects of long-range 13C-13C dipolar coupling on measured relaxation rates in RNA." Journal of Biomolecular NMR 75, no. 4-5 (2021): 203–11. http://dx.doi.org/10.1007/s10858-021-00368-8.
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AbstractSelective stable isotope labeling has transformed structural and dynamics analysis of RNA by NMR spectroscopy. These methods can remove 13C-13C dipolar couplings that complicate 13C relaxation analyses. While these phenomena are well documented for sites with adjacent 13C nuclei (e.g. ribose C1′), less is known about so-called isolated sites (e.g. adenosine C2). To investigate and quantify the effects of long-range (> 2 Å) 13C-13C dipolar interactions on RNA dynamics, we simulated adenosine C2 relaxation rates in uniformly [U-13C/15N]-ATP or selectively [2-13C]-ATP labeled RNAs. Our simulations predict non-negligible 13C-13C dipolar contributions from adenosine C4, C5, and C6 to C2 longitudinal (R1) relaxation rates in [U-13C/15N]-ATP labeled RNAs. Moreover, these contributions increase at higher magnetic fields and molecular weights to introduce discrepancies that exceed 50%. This will become increasingly important at GHz fields. Experimental R1 measurements in the 61 nucleotide human hepatitis B virus encapsidation signal ε RNA labeled with [U-13C/15N]-ATP or [2-13C]-ATP corroborate these simulations. Thus, in the absence of selectively labeled samples, long-range 13C-13C dipolar contributions must be explicitly taken into account when interpreting adenosine C2 R1 rates in terms of motional models for large RNAs.
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Dusny, Christian, and Andreas Schmid. "The Metabolic Flux Probe (MFP)—Secreted Protein as a Non-Disruptive Information Carrier for 13C-Based Metabolic Flux Analysis." International Journal of Molecular Sciences 22, no. 17 (2021): 9438. http://dx.doi.org/10.3390/ijms22179438.
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Novel cultivation technologies demand the adaptation of existing analytical concepts. Metabolic flux analysis (MFA) requires stable-isotope labeling of biomass-bound protein as the primary information source. Obtaining the required protein in cultivation set-ups where biomass is inaccessible due to low cell densities and cell immobilization is difficult to date. We developed a non-disruptive analytical concept for 13C-based metabolic flux analysis based on secreted protein as an information carrier for isotope mapping in the protein-bound amino acids. This “metabolic flux probe” (MFP) concept was investigated in different cultivation set-ups with a recombinant, protein-secreting yeast strain. The obtained results grant insight into intracellular protein turnover dynamics. Experiments under metabolic but isotopically nonstationary conditions in continuous glucose-limited chemostats at high dilution rates demonstrated faster incorporation of isotope information from labeled glucose into the recombinant reporter protein than in biomass-bound protein. Our results suggest that the reporter protein was polymerized from intracellular amino acid pools with higher turnover rates than biomass-bound protein. The latter aspect might be vital for 13C-flux analyses under isotopically nonstationary conditions for analyzing fast metabolic dynamics.
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Zorn, H., M. Fischer-Zorn, and R. G. Berger. "A Labeling Study To Elucidate the Biosynthesis of 4-(4-Hydroxyphenyl)-Butan-2-one (Raspberry Ketone) by Nidula niveo-tomentosa." Applied and Environmental Microbiology 69, no. 1 (2003): 367–72. http://dx.doi.org/10.1128/aem.69.1.367-372.2003.
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ABSTRACT Submerged cells of the basidiomycete Nidula niveo-tomentosa, a microbial producer of 4-(4-hydroxyphenyl)-butan-2-one, were supplemented with 13C-labeled l-phenylalanines and with [1-13C]glucose. Labeled transformation products were detected by a novel method of analyzing stable isotope-labeled metabolites, gas chromatography (GC) coupled to an atomic emission detector, and by GC-mass spectrometry. A benzoate moiety was side chain elongated according to the poly-β-keto scheme. The presence of an acetyl coenzyme A-carboxylase inhibitor shifted the spectrum of products to benzyl compounds. Hence, the fungal pathway differs from the one established for plant tissues.
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Hasenour, Clinton M., Martha L. Wall, D. Emerson Ridley, et al. "Mass spectrometry-based microassay of 2H and 13C plasma glucose labeling to quantify liver metabolic fluxes in vivo." American Journal of Physiology-Endocrinology and Metabolism 309, no. 2 (2015): E191—E203. http://dx.doi.org/10.1152/ajpendo.00003.2015.
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Mouse models designed to examine hepatic metabolism are critical to diabetes and obesity research. Thus, a microscale method to quantitatively assess hepatic glucose and intermediary metabolism in conscious, unrestrained mice was developed. [13C3]propionate, [2H2]water, and [6,6-2H2]glucose isotopes were delivered intravenously in short- (9 h) and long-term-fasted (19 h) C57BL/6J mice. GC-MS and mass isotopomer distribution (MID) analysis were performed on three 40-μl arterial plasma glucose samples obtained during the euglycemic isotopic steady state. Model-based regression of hepatic glucose and citric acid cycle (CAC)-related fluxes was performed using a comprehensive isotopomer model to track carbon and hydrogen atom transitions through the network and thereby simulate the MIDs of measured fragment ions. Glucose-6-phosphate production from glycogen diminished, and endogenous glucose production was exclusively gluconeogenic with prolonged fasting. Gluconeogenic flux from phospho enolpyruvate (PEP) remained stable, whereas that from glycerol modestly increased from short- to long-term fasting. CAC flux [i.e., citrate synthase ( V CS)] was reduced with long-term fasting. Interestingly, anaplerosis and cataplerosis increased with fast duration; accordingly, pyruvate carboxylation and the conversion of oxaloacetate to PEP were severalfold higher than V CS in long-term fasted mice. This method utilizes state-of-the-art in vivo methodology and comprehensive isotopomer modeling to quantify hepatic glucose and intermediary fluxes during physiological stress in mice. The small plasma requirements permit serial sampling without stress and the affirmation of steady-state glucose kinetics. Furthermore, the approach can accommodate a broad range of modeling assumptions, isotope tracers, and measurement inputs without the need to introduce ad hoc mathematical approximations.
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Tang, Yinjie J., Adam L. Meadows, James Kirby, and Jay D. Keasling. "Anaerobic Central Metabolic Pathways in Shewanella oneidensis MR-1 Reinterpreted in the Light of Isotopic Metabolite Labeling." Journal of Bacteriology 189, no. 3 (2006): 894–901. http://dx.doi.org/10.1128/jb.00926-06.
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ABSTRACT It has been proposed that during growth under anaerobic or oxygen-limited conditions, Shewanella oneidensis MR-1 uses the serine-isocitrate lyase pathway common to many methylotrophic anaerobes, in which formaldehyde produced from pyruvate is condensed with glycine to form serine. The serine is then transformed through hydroxypyruvate and glycerate to enter central metabolism at phosphoglycerate. To examine its use of the serine-isocitrate lyase pathway under anaerobic conditions, we grew S. oneidensis MR-1 on [1-13C]lactate as the sole carbon source, with either trimethylamine N-oxide (TMAO) or fumarate as an electron acceptor. Analysis of cellular metabolites indicated that a large percentage (>70%) of lactate was partially oxidized to either acetate or pyruvate. The 13C isotope distributions in amino acids and other key metabolites indicate that under anaerobic conditions, although glyoxylate synthesized from the isocitrate lyase reaction can be converted to glycine, a complete serine-isocitrate pathway is not present and serine/glycine is, in fact, oxidized via a highly reversible degradation pathway. The labeling data also suggest significant activity in the anapleurotic (malic enzyme and phosphoenolpyruvate carboxylase) reactions. Although the tricarboxylic acid (TCA) cycle is often observed to be incomplete in many other anaerobes (absence of 2-oxoglutarate dehydrogenase activity), isotopic labeling supports the existence of a complete TCA cycle in S. oneidensis MR-1 under certain anaerobic conditions, e.g., TMAO-reducing conditions.
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Seemann, Janina. "The use of 13C and 15N isotope labeling techniques to assess heterotrophy of corals." Journal of Experimental Marine Biology and Ecology 442 (April 2013): 88–95. http://dx.doi.org/10.1016/j.jembe.2013.01.004.
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McAtee, Allison G., Lara J. Jazmin, and Jamey D. Young. "Application of isotope labeling experiments and 13C flux analysis to enable rational pathway engineering." Current Opinion in Biotechnology 36 (December 2015): 50–56. http://dx.doi.org/10.1016/j.copbio.2015.08.004.
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Guthertz, Nicolas, Julia Klopp, Aurélie Winterhalter, César Fernández, and Alvar D. Gossert. "Auto-inducing media for uniform isotope labeling of proteins with 15N, 13C and 2H." Journal of Biomolecular NMR 62, no. 2 (2015): 169–77. http://dx.doi.org/10.1007/s10858-015-9931-x.
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Moiz, Bilal, Jonathan Garcia, Sarah Basehore, et al. "13C Metabolic Flux Analysis Indicates Endothelial Cells Attenuate Metabolic Perturbations by Modulating TCA Activity." Metabolites 11, no. 4 (2021): 226. http://dx.doi.org/10.3390/metabo11040226.
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Disrupted endothelial metabolism is linked to endothelial dysfunction and cardiovascular disease. Targeted metabolic inhibitors are potential therapeutics; however, their systemic impact on endothelial metabolism remains unknown. In this study, we combined stable isotope labeling with 13C metabolic flux analysis (13C MFA) to determine how targeted inhibition of the polyol (fidarestat), pentose phosphate (DHEA), and hexosamine biosynthetic (azaserine) pathways alters endothelial metabolism. Glucose, glutamine, and a four-carbon input to the malate shuttle were important carbon sources in the baseline human umbilical vein endothelial cell (HUVEC) 13C MFA model. We observed two to three times higher glutamine uptake in fidarestat and azaserine-treated cells. Fidarestat and DHEA-treated HUVEC showed decreased 13C enrichment of glycolytic and TCA metabolites and amino acids. Azaserine-treated HUVEC primarily showed 13C enrichment differences in UDP-GlcNAc. 13C MFA estimated decreased pentose phosphate pathway flux and increased TCA activity with reversed malate shuttle direction in fidarestat and DHEA-treated HUVEC. In contrast, 13C MFA estimated increases in both pentose phosphate pathway and TCA activity in azaserine-treated cells. These data show the potential importance of endothelial malate shuttle activity and suggest that inhibiting glycolytic side branch pathways can change the metabolic network, highlighting the need to study systemic metabolic therapeutic effects.
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Blackwell, Barbara A., J. David Miller, and Marc E. Savard. "Production of Carbon 14-Labeled Fumonisin in Liquid Culture." Journal of AOAC INTERNATIONAL 77, no. 2 (1994): 506–11. http://dx.doi.org/10.1093/jaoac/77.2.506.
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Abstract A method for the production and purification of radiolabeled fumonisin that involves the addition of 14C-acetate to liquid cultures of Fusarium moniliforme in shake flasks is reported. Stable isotope 13C labeling studies were carried out using specifically enriched acetate and several amino acids to determine the location of labeled carbon atoms in the radiolabeled fumonisin that was also produced (650 μCi/mmol). These experiments determined that the 14C was distributed throughout the molecule making it useful for studies of fumonisin residues in animal products. Additionally, the 13C studies indicated that the biosynthesis of fumonisin involves the addition of methionine, glutarate, and serine or alanine to the hydrocarbon backbone. These data best fit the hypothesis that this backbone is polyketide in origin as opposed to being a modified lipid.
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Poeaknapo, Chotima, Ursula Fisinger, Meinhart H. Zenk, and Jürgen Schmidt. "Evaluation of the mass spectrometric fragmentation of codeine and morphine after 13C-isotope biosynthetic labeling." Phytochemistry 65, no. 10 (2004): 1413–20. http://dx.doi.org/10.1016/j.phytochem.2004.05.005.
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Zhou, Mingxin, and Yibo Li. "Long-Term Nitrogen Addition Regulates Plant-Soil 15N–13C Coupling Through Species Traits and Temporal-Spatial Dynamics in a Temperate Forest." Forests 16, no. 7 (2025): 1046. https://doi.org/10.3390/f16071046.
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Nitrogen deposition is a critical driver of plant-soil interactions in forest ecosystems. However, the species-specific coordination of nitrogen uptake and carbon assimilation—traced using 15N- and 13C-labeled compounds—under varying nitrogen forms, depths, and time points remains poorly understood. We conducted a dual-isotope (15NH4Cl, K15NO3, and Na213CO3) labeling experiment in a temperate secondary forest to investigate nutrient uptake and carbon assimilation in three understory species—Carex siderosticta, Maianthemum bifolium, and Oxalis acetosella—across three nitrogen treatments (control, low N, and high N), two soil depths (0–5 cm and 5–15 cm), and two post-labeling time points (24 h and 72 h). We quantified 15N uptake and 13C assimilation in above- and belowground plant tissues, as well as 15N and 13C retention in soils. C. siderosticta exhibited the highest total 15N uptake (2.2–6.9 μg N m−2 aboveground; 1.4–4.1 μg N m−2 belowground) and 13C assimilation (58.4–111.2 mg C m−2 aboveground; 17.6–39.2 mg C m−2 belowground) under high ammonium at 72 h. High nitrogen input significantly enhanced the coupling between plant biomass and nutrient assimilation (R2 > 0.9), and increased 15N-TN and 13C-SOC retention in the surface soil layer (13,200–17,400 μg N kg−1; 30,000–44,000 μg C kg−1). Multifactorial analysis revealed significant interactions among nitrogen treatment, form, depth, and time. These findings demonstrate that ammonium-based enrichment promotes nutrient acquisition and carbon assimilation in responsive species and enhances surface soil C—N retention, highlighting the integrative effects of nitrogen form, species traits, and spatial–temporal dynamics on forest biogeochemistry.
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Habib, El-Sayed E., J. Neel Scarsdale, and Kevin A. Reynolds. "Biosynthetic Origin of Hygromycin A." Antimicrobial Agents and Chemotherapy 47, no. 7 (2003): 2065–71. http://dx.doi.org/10.1128/aac.47.7.2065-2071.2003.
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ABSTRACT Hygromycin A, an antibiotic produced by Streptomyces hygroscopicus, is an inhibitor of bacterial ribosomal peptidyl transferase. The antibiotic binds to the ribosome in a distinct but overlapping manner with other antibiotics and offers a different template for generation of new agents effective against multidrug-resistant pathogens. Reported herein are the results from a series of stable-isotope-incorporation studies demonstrating the biosynthetic origins of the three distinct structural moieties which comprise hygromycin A. Incorporation of [1-13C]mannose and intact incorporation of d-[1,2-13C2]glucose into the 6-deoxy-5-keto-d-arabino-hexofuranose moiety are consistent with a pathway in which mannose is converted to an activated l-fucose, via a 4-keto-6-deoxy-d-mannose intermediate, with a subsequent unusual mutation of the pyranose to the corresponding furanose. The aminocyclitol moiety was labeled by d-[1,2-13C2]glucose in a manner consistent with formation of myo-inositol and a subsequent unprecedented oxidation and transamination of the C-2 hydroxyl group to generate neo-inosamine-2. Incorporation of [carboxy- 13C]-4-hydroxybenzoic acid and intact incorporation of [2,3-13C2]propionate are consistent with a polyketide synthase-type decarboxylation condensation to generate the 3,4-dihydroxy-α-methylcinnamic acid moiety of hygromycin A. No labeling of hygromycin A was observed when [3-13C]tyrosine, [3-13C]phenylalanine, or [carboxy- 13C]benzoic acid was used, suggesting that the 4-hydroxybenzoic acid is derived directly from chorismic acid. Consistent with this hypothesis was the observation that hygromycin A titers could be reduced by addition of N-(phosphonomethyl)-glycine (an inhibitor of chorismic acid biosynthesis) and restored by coaddition of 4-hydroxybenzoic acid. The convergent biosynthetic pathway established for hygromycin A offers significant versatility for applying the techniques of combinatorial and directed biosynthesis to production of new antibiotics which target the ribosomal peptidyl transferase activity.
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Dai, Charles, Yoon-Mi Chung, Eric A. Klein, and Nima Sharifi. "A dual stable isotope method by LC-MS/MS to define patterns of androgen metabolism in localized versus advanced prostate cancer." Journal of Clinical Oncology 34, no. 2_suppl (2016): 40. http://dx.doi.org/10.1200/jco.2016.34.2_suppl.40.
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40 Background: Prior work has shown that androstenedione (AD) rather than testosterone (T) is the preferred substrate of 5α-reductase for dihydrotestosterone (DHT) synthesis in castration-resistant prostate cancer. However, patterns of metabolism in hormone-naive prostate cancer are still poorly defined. Previously, we reported on the utility of dual radioisotope labeling of steroid precursors to characterize androgen metabolism in localized prostate tissue. We now describe an alternative approach via stable isotope labeling and analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS), which has unique advantages over the former method. Methods: LNCaP cell lines and prostate tissue from patients undergoing radical prostatectomy for localized cancer were incubated in serum-free media, spiked with 13C-labeled AD and 2H-labeled T. Media was collected at 7, 24, and 48 hours of incubation. Steroids were extracted, separated, and then analyzed by way of LC-MS/MS to identify labeled metabolites of AD and T. Results: Incubation of labeled AD and T resulted in conversion over time to both 13C-labeled and 2H-labeled downstream metabolites, 5α-dione and DHT. Although both precursors contributed to 5α-dione and DHT formation, the steroid of origin could be determined on the basis of differential labeling. In ex-vivo tissue incubations, unlabeled 5α-dione was also observed, which was distinguishable from its stable isotopic form and most likely represents endogenous steroid. Conclusions: Both cell lines and tissue appear to metabolize labeled AD and T, and formation of DHT occurs through both precursors. Furthermore, the presence of endogenous 5α-dione suggests that alternative pathways of DHT synthesis, which bypass T, may be naturally accessible in the hormone-naïve setting. We propose a robust ex-vivo technique to readily track simultaneous pathways of DHT synthesis in prostate cancer. Future work will focus on defining metabolic phenotypes of localized prostate cancer and specific patterns of flux under different hormonal and pharmacologic conditions.
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Bontes, B. M., R. Pel, B. W. Ibelings, H. T. S. Boschker, J. J. Middelburg, and E. Van Donk. "The effects of biomanipulation on the biogeochemistry, carbon isotopic composition and pelagic food web relations of a shallow lake." Biogeosciences 3, no. 1 (2006): 69–83. http://dx.doi.org/10.5194/bg-3-69-2006.
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Abstract. In this study we investigated the effects of experimental biomanipulation on community structure, ecosystem metabolism, carbon biogeochemistry and stable isotope composition of a shallow eutrophic lake in the Netherlands. Three different biomanipulation treatments were applied. In two parts of the lake, isolated from the rest, fish was removed and one part was used as a reference treatment in which no biomanipulation was applied. Stable isotopes have proved useful to trace trophic interactions at higher food web levels but until now methodological limitations have restricted species specific isotope analysis in the plankton community. We applied a new approach based on the combination of fluorescence activated cell sorting (FACS) and isotope ratio mass spectrometry (IRMS) to trace carbon flow through the planktonic food web. With this method we aimed at obtaining group specific δ13C signatures of phytoplankton and to trace possible shifts in δ13C resulting from fish removal. Biomanipulation led to an increase in transparency and macrophyte biomass and decrease in phytoplankton abundance, but zooplankton numbers did not increase. Fish removal also resulted in high pH, high O2, low CO2 and more negative δ13CDIC values than expected, which is attributed to chemical enhanced diffusion with large negative fractionation. Despite high temporal variation we detected differences between the isotopic signatures of the primary producers and between the different treatments. The fractionation values of green algae (~21) and diatoms (~23) were similar and independent of treatment, while fractionation factors of filamentous cyanobacteria were variable between the treatments that differed in CO2 availability. 13C-labeling of the phytoplankton groups showed that biomanipulation led to increased growth rates of green algae and diatoms at the expense of cyanobacteria. Finally, consumers seemed generalists to the available food sources.
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Stuani, Lucille, Fabien Riols, Pierre Millard, et al. "Stable Isotope Labeling Highlights Enhanced Fatty Acid and Lipid Metabolism in Human Acute Myeloid Leukemia." International Journal of Molecular Sciences 19, no. 11 (2018): 3325. http://dx.doi.org/10.3390/ijms19113325.
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Background: In Acute Myeloid Leukemia (AML), a complete response to chemotherapy is usually obtained after conventional chemotherapy but overall patient survival is poor due to highly frequent relapses. As opposed to chronic myeloid leukemia, B lymphoma or multiple myeloma, AML is one of the rare malignant hemopathies the therapy of which has not significantly improved during the past 30 years despite intense research efforts. One promising approach is to determine metabolic dependencies in AML cells. Moreover, two key metabolic enzymes, isocitrate dehydrogenases (IDH1/2), are mutated in more than 15% of AML patient, reinforcing the interest in studying metabolic reprogramming, in particular in this subgroup of patients. Methods: Using a multi-omics approach combining proteomics, lipidomics, and isotopic profiling of [U-13C] glucose and [U-13C] glutamine cultures with more classical biochemical analyses, we studied the impact of the IDH1 R132H mutation in AML cells on lipid biosynthesis. Results: Global proteomic and lipidomic approaches showed a dysregulation of lipid metabolism, especially an increase of phosphatidylinositol, sphingolipids (especially few species of ceramide, sphingosine, and sphinganine), free cholesterol and monounsaturated fatty acids in IDH1 mutant cells. Isotopic profiling of fatty acids revealed that higher lipid anabolism in IDH1 mutant cells corroborated with an increase in lipogenesis fluxes. Conclusions: This integrative approach was efficient to gain insight into metabolism and dynamics of lipid species in leukemic cells. Therefore, we have determined that lipid anabolism is strongly reprogrammed in IDH1 mutant AML cells with a crucial dysregulation of fatty acid metabolism and fluxes, both being mediated by 2-HG (2-Hydroxyglutarate) production.
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Pervushin, Konstantin. "Impact of Transverse Relaxation Optimized Spectroscopy (TROSY) on NMR as a technique in structural biology." Quarterly Reviews of Biophysics 33, no. 2 (2000): 161–97. http://dx.doi.org/10.1017/s0033583500003619.
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1. Transverse relaxation and the molecular size limit in liquid state NMR 1612. TROSY: how does it work? 1632.1 Transverse relaxation in coupled spin systems 1632.2 The TROSY effect, relaxation due to remote protons and 2H isotope labeling 1653. Direct heteronuclear chemical shift correlations 1683.1 Single-Quantum [15N,1H]-TROSY 1683.2 Zero-Quantum [15N,1H]-TROSY 1713.3 Single-Quantum TROSY with aromatic 13C–1H moieties 1764. Resonance assignment and NOE spectroscopy of large biomolecules 1804.1 TROSY-based triple resonance experiments for 13C, 15N and 1HN backbone resonance assignment in uniformly 2H, 13C, 15N labeled proteins 1804.2 TROSY-type NOE spectroscopy 1865. Scalar coupling across hydrogen bonds observed by TROSY 1876. The use of TROSY for measurements of residual dipolar coupling constants 1907. Conclusions 1918. Acknowledgements 1919. References 191The application of nuclear magnetic resonance (NMR) spectroscopy for structure determination of proteins and nucleic acids (Wüthrich, 1986) with molecular mass exceeding 30 kDa is largely constrained by two factors, fast transverse relaxation of spins of interest and complexity of NMR spectra, both of which increase with increasing molecular size (Wagner, 1993b; Clore & Gronenborn, 1997, 1998b; Kay & Gardner, 1997). The good news is that neither of these factors represent a fundamental limit for the application of NMR techniques to protein structure determination in solution (Clore & Gronenborn, 1998a; Wüthrich, 1998; Wider & Wüthrich, 1999). In fact, in the past few years the size limitations imposed by these factors have been pushed up to 50–70 kDa by the use of 13C, 15N and 2H isotope labeling combined with selective reprotonation of individual chemical groups in conjunction with the use of triple-resonance experiments (Bax, 1994; Gardner et al. 1997; Gardner & Kay, 1998) and heteronuclear-resolved NMR (Fesik & Zuiderweg, 1988; Marion et al. 1989a; Otting & Wüthrich, 1990). Among the largest biomolecules whose 3D structure was solved by NMR are the 44 kDa trimeric ectodomain of simian immunodeficiency virus (SIV) gp41 (Caffrey et al. 1998) and 40–60 kDa particles of the elongation initiation factor 4E solubilized in CHAPS micelles (Matsuo et al. 1997; McGuire et al. 1998).
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Wang, Hehua, Juan Wang, Chaorong Ge, and Huaiying Yao. "Fungi Dominated the Incorporation of 13C-CO2 into Microbial Biomass in Tomato Rhizosphere Soil under Different CO2 Concentrations." Microorganisms 9, no. 10 (2021): 2121. http://dx.doi.org/10.3390/microorganisms9102121.
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An elevated CO2 (eCO2) fumigation experiment was carried out to study the influence of various CO2 concentrations on microorganisms involved in the incorporation of root-derived C in greenhouse soil systems. In this study, 400 and 800 µmol·mol−1 CO2 fumigation treatments were conducted during tomato planting. Phospholipid fatty acid (PLFA) profiling based on the stable isotope probing (SIP) technique was applied to trace active microorganisms. The absolute total abundance of 13C-PLFAs was much higher under eCO2 treatment. Most of the 13C-CO2 was incorporated into the 13C-PLFAs 18:2ω6,9 (fungi), 16:0 (general PLFA), 18:1ω9c (Gram-negative bacteria, G−) and i17:0 (Gram-positive bacteria, G+) via rhizodeposition from tomato under ambient CO2 (aCO2) and eCO2 treatments, suggesting similar responses of active microorganisms to different CO2 treatments. However, the fungi (characterized by the 13C-PLFA 18:2ω6,9) played a much more dominant role in the incorporation of root-derived C under eCO2. Actinomycetes, marked by the 13C-PLFA 10-Me-18:0, occurred only on labeling day 15 under the eCO2 treatment, indicating that the actinomycetes fed on both soil organic carbon and fresh rhizodeposition. It was indicated that eCO2 significantly affected microbial biomass and microbial community structures involved in the incorporation of 13C-CO2 via tomato root secretions, as supported by Adonis analysis and the Mantel test.