Search

The 18 O enrichment (Δ 18 O) of cellulose (Δ 18 O Cel ) is recognized as a unique archive of past climate and plant function. However, there is still uncertainty regarding the proportion of oxygen in cellulose (p ex ) that exchanges post-photosynthetically with medium water of cellulose synthesis. Particularly, recent research with C 3 grasses demonstrated that the Δ 18 O of leaf sucrose (Δ 18 O Suc , the parent substrate for cellulose synthesis) can be much higher than predicted from daytime Δ 18 O of leaf water (Δ 18 O LW ), which could alter conclusions on photosynthetic versus post-photosynthetic effects on Δ 18 O Cel via p ex . Here, we assessed p ex in leaves of perennial ryegrass (Lolium perenne) grown at different atmospheric relative humidity (RH) and CO 2 levels, by determinations of Δ 18 O Cel in leaves, Δ 18 O LGDZW (the Δ 18 O of water in the leaf growth-and-differentiation zone) and both Δ 18 O Suc and Δ 18 O LW (adjusted for ε bio , the biosynthetic fractionation between water and carbohydrates) as alternative proxies for the substrate for cellulose synthesis. Δ 18 O LGDZW was always close to irrigation water, and p ex was similar (0.53 ± 0.02 SE) across environments when determinations were based on Δ 18 O Suc . Conversely, p ex was erroneously and variably underestimated (range 0.02-0.44) when based on Δ 18 O LW . The photosynthetic signal fraction in Δ 18 O Cel is much more constant than hitherto assumed, encouraging leaf physiological reconstructions., (© 2024 The Authors. Plant, Cell & Environment published by John Wiley & Sons Ltd.)

Understanding the impact of human activities on the metabolic state of soil and aquatic environments is of paramount importance to implement measures for maintaining ecosystem services. Variations of natural abundance 18O/16O ratios in phosphate have been proposed as proxies for the holistic assessment of metabolic activity given the crucial importance of phosphoryl transfer reactions in fundamental biological processes. However, instrumental and procedural limitations inherent to oxygen isotope analysis in phosphate and organophosphorus compounds have so far limited the stable isotope-based evaluation of metabolic processes. Here, we discuss how recent developments in Orbitrap high resolution mass spectrometry enable measurements of 18O/16O ratios in phosphate and outline the critical mass spectrometry parameters for accurate and precise analysis. Subsequently, we evaluate the types of 18O kinetic isotope effects of phosphoryl transfer reactions and illustrate how novel analytical approaches will give rise to an improved understanding of 18O/16O ratio variations from biochemical processes affecting the microbial phosphorus metabolism., (Copyright 2024 Nora M. Bernet, Thomas B. Hofstetter. License: This work is licensed under a Creative Commons Attribution 4.0 International License.)

Photosynthesis and metabolism in plants involve oxygen as both a product and substrate. Oxygen is taken up during photorespiration and respiration and produced through water splitting during photosynthesis. To distinguish between processes that produce or consume O 2 in leaves, isotope mass separation and detection by mass spectrometry allows measurement of evolution and uptake of O 2 as well as CO 2 uptake. This chapter describes how to calculate the rate of Rubisco oxygenation and carboxylation from in vivo gas exchange of stable isotopes of 16 O 2 and 18 O 2 with a closed cuvette system for leaf discs and membrane inlet mass spectrometry., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Oxygen is both product and substrate of photosynthesis and metabolism in plants, by oxygen evolution through water splitting and uptake by photorespiration and respiration. It is important to investigate these processes simultaneously in leaves, especially in response to environmental variables, such as light and temperature. To distinguish between processes that evolve or take up O 2 in leaves in the light, in vivo gas exchange of stable isotopes of oxygen and membrane inlet mass spectrometry is used. A closed-cuvette system for gas exchange of leaf discs is described, using the stable isotopes 16 O 2 and 18 O 2 , with a semi-permeable membrane gas inlet and isotope mass separation and detection by mass spectrometry. Measurement of evolution and uptake, as well as CO 2 uptake, at a range of light levels allows composition of a light response curve, here described for French bean (Phaseolus vulgaris) and maize (Zea mays) leaf discs., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Quantitative proteomics approaches based on stable isotopic labeling and mass spectrometry have been widely applied to disease research, drug target discovery, biomarker identification, and systems biology. One of the notable stable isotopic labeling approaches is trypsin-catalyzed 18 O/ 16 O labeling, which has its own advantages of low sample consumption, simple labeling procedure, cost-effectiveness, and absence of chemical reactions that potentially generate by-products. In this chapter, a protocol for 18 O/ 16 O labeling-based quantitative proteomics approach is described with an application to the identification of proteomic biomarkers of acetaminophen (APAP)-induced hepatotoxicity in rats. The protocol involves first the extraction of proteins from liver tissues of control and APAP-treated rats and digestion into peptides by trypsin. After cleaning of the peptides by solid-phase extraction, equal amounts of peptides from the APAP treatment and the control groups are then subject to trypsin-catalyzed 18 O/ 16 O labeling. The labeled peptides are combined and fractionated by off-line strong cation exchange liquid chromatography (SCXLC), and each fraction is then analyzed by nanoflow reversed-phase LC coupled online with tandem mass spectrometry (RPLC-MS/MS) for identification and quantification of differential protein expression between APAP-treated rats and controls. The protocol is applicable to quantitative proteomic analysis for a variety of biological samples., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The oxygen isotope composition (δ 18 O) of tree-ring cellulose is used to evaluate tree physiological responses to climate, but their interpretation is still limited due to the complexity of the isotope fractionation pathways. We assessed the relative contribution of seasonal needle and xylem water δ 18 O variations to the intra-annual tree-ring cellulose δ 18 O signature of larch trees at two sites with contrasting soil water availability in the Swiss Alps. We combined biweekly δ 18 O measurements of soil water, needle water, and twig xylem water with intra-annual δ 18 O measurements of tree-ring cellulose, xylogenesis analysis, and mechanistic and structural equation modeling. Intra-annual cellulose δ 18 O values resembled source water δ 18 O mean levels better than needle water δ 18 O. Large parts of the rings were formed under high proportional exchange with unenriched xylem water (p ex ). Maximum p ex values were achieved in August and imprinted on sections at 50-75% of the ring. High p ex values were associated with periods of high atmospheric evaporative demand (VPD). While VPD governed needle water δ 18 O variability, we estimated a limited Péclet effect at both sites. Due to a variable p ex , source water has a strong influence over large parts of the intra-annual tree-ring cellulose δ 18 O variations, potentially masking signals coming from needle-level processes., (© 2023 The Authors. New Phytologist © 2023 New Phytologist Foundation.)

Oxygen and hydrogen isotopes of cellulose in plant biology are commonly used to infer environmental conditions, often from time series measurements of tree rings. However, the covariation (or the lack thereof) between δ 18 O and δ 2 H in plant cellulose is still poorly understood. We compared plant water, and leaf and branch cellulose from dominant tree species across an aridity gradient in Northern Australia, to examine how δ 18 O and δ 2 H relate to each other and to mean annual precipitation (MAP). We identified a decline in covariation from xylem to leaf water, and onwards from leaf to branch wood cellulose. Covariation in leaf water isotopic enrichment (Δ) was partially preserved in leaf cellulose but not branch wood cellulose. Furthermore, whilst δ 2 H was well-correlated between leaf and branch, there was an offset in δ 18 O between organs that increased with decreasing MAP. Our findings strongly suggest that postphotosynthetic isotope exchange with water is more apparent for oxygen isotopes, whereas variable kinetic and nonequilibrium isotope effects add complexity to interpreting metabolic-induced δ 2 H patterns. Varying oxygen isotope exchange in wood and leaf cellulose must be accounted for when δ 18 O is used to reconstruct climatic scenarios. Conversely, comparing δ 2 H and δ 18 O patterns may reveal environmentally induced shifts in metabolism., (© 2023 The Authors. New Phytologist © 2023 New Phytologist Foundation.)

Recent methodological advancements in determining the nonexchangeable hydrogen isotopic composition (δ 2 H ne ) of plant carbohydrates make it possible to disentangle the drivers of hydrogen isotope ( 2 H) fractionation processes in plants. Here, we investigated the influence of phylogeny on the δ 2 H ne of twig xylem cellulose and xylem water, as well as leaf sugars and leaf water, across 73 Northern Hemisphere tree and shrub species growing in a common garden. 2 H fractionation in plant carbohydrates followed distinct phylogenetic patterns, with phylogeny reflected more in the δ 2 H ne of leaf sugars than in that of twig xylem cellulose. Phylogeny had no detectable influence on the δ 2 H ne of twig or leaf water, showing that biochemistry, not isotopic differences in plant water, caused the observed phylogenetic pattern in carbohydrates. Angiosperms were more 2 H-enriched than gymnosperms, but substantial δ 2 H ne variations also occurred at the order, family, and species levels within both clades. Differences in the strength of the phylogenetic signals in δ 2 H ne of leaf sugars and twig xylem cellulose suggest that the original phylogenetic signal of autotrophic processes was altered by subsequent species-specific metabolism. Our results will help improve 2 H fractionation models for plant carbohydrates and have important consequences for dendrochronological and ecophysiological studies., (© 2023 The Authors New Phytologist © 2023 New Phytologist Foundation.)

Natural bioactive compounds (NBCs) are regarded as candidates for many medical applications widely. Due to the complicated structure and biosynthesis source, only a few NBCs were supplied with commercial isotopic labeled standards. This shortage resulted in poor quantitation reliability in bio-samples for most NBCs, considering the remarkable matrix effects. NBCs metabolism and distribution studies would be restricted consequently. Those properties played critical roles in drug discovery and development. In this study, a fast, convenient, widely adopting 16 O/ 18 O exchange reaction was optimized for stable, available, affordable NBCs 18 O-labeled standards preparation. With 18 O- labeled internal standard, a UPLC-MRM-based NBCs pharmacokinetics analysis strategy was formed. Pharmacokinetics of caffeic acid with Hyssopus Cuspidatus Boriss extract (SXCF) dosed mice was carried out by established strategy. Compared with traditional external standards quantitation, adapting 18 O-labeled internal standards, both accuracy and precision were enhanced significantly. Thus, the platform built by this work would accelerate the pharmaceutical research with NBCs, by providing a reliable, wide-adapted, affordable, isotopic internal standard-based bio-samples NBCs absolute quantitation strategy., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Cephalopod carbonate geochemistry underpins studies ranging from Phanerozoic, global-scale change to outcrop-scale paleoecological reconstructions. Interpreting these data hinges on assumed similarity to model organisms, such as Nautilus, and generalization from other molluscan biomineralization processes. Aquarium rearing and capture of wild Nautilus suggest shell carbonate precipitates quickly (35 μm/day) in oxygen isotope equilibrium with seawater. Other components of Nautilus shell chemistry are less well-studied but have potential to serve as proxies for paleobiology and paleoceanography. To calibrate the geochemical response of cephalopod δ15Norg, δ13Corg, δ13Ccarb, δ18Ocarb, and δ44/40Cacarb to modern anthropogenic environmental change, we analyzed modern, historical, and subfossil Nautilus macromphalus from New Caledonia. Samples span initial human habitation, colonialization, and industrial pCO2 increase. This sampling strategy is advantageous because it avoids the shock response that can affect geochemical change in aquarium experiments. Given the range of living depths and more complex ecology of Nautilus, however, some anthropogenic signals, such as ocean acidification, may not have propagated to their living depths. Our data suggest some environmental changes are more easily preserved than others given variability in cephalopod average living depth. Calculation of the percent respired carbon incorporated into the shell using δ13Corg, δ13Ccarb, and Suess-effect corrected δ13CDIC suggests an increase in the last 130 years that may have been caused by increasing carbon dioxide concentration or decreasing oxygen concentration at the depths these individuals inhabited. This pattern is consistent with increasing atmospheric CO2 and/or eutrophication offshore of New Caledonia. We find that δ44/40Ca remains stable across the last 130 years. The subfossil shell from a cenote may exhibit early δ44/40Ca diagenesis. Questions remain about the proportion of dietary vs ambient seawater calcium incorporation into the Nautilus shell. Values of δ15N do not indicate trophic level change in the last 130 years, and the subfossil shell may show diagenetic alteration of δ15N toward lower values. Future work using historical collections of Sepia and Spirula may provide additional calibration of fossil cephalopod geochemistry., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Linzmeier et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Stable isotope abundances convey valuable information about plant physiological processes and underlying environmental controls. Central gaps in our mechanistic understanding of hydrogen isotope abundances impede their widespread application within the plant and biogeosciences. To address these gaps, we analysed intramolecular deuterium abundances in glucose of Pinus nigra extracted from an annually resolved tree-ring series (1961-1995). We found fractionation signals (i.e. temporal variability in deuterium abundance) at glucose H 1 and H 2 introduced by closely related metabolic processes. Regression analysis indicates that these signals (and thus metabolism) respond to drought and atmospheric CO 2 concentration beyond a response change point. They explain ≈ 60% of the whole-molecule deuterium variability. Altered metabolism is associated with below-average yet not exceptionally low growth. We propose the signals are introduced at the leaf level by changes in sucrose-to-starch carbon partitioning and anaplerotic carbon flux into the Calvin-Benson cycle. In conclusion, metabolism can be the main driver of hydrogen isotope variation in plant glucose., (© 2022 The Authors New Phytologist © 2022 New Phytologist Foundation.)

An expression was earlier derived for the non-steady state isotopic composition of a leaf when the composition of the water entering the leaf was not necessarily the same as that of the water being transpired (Farquhar and Cernusak 2005). This was relevant to natural conditions because the associated time constant is typically sufficiently long to ensure that the leaf water composition and fluxes of the isotopologues are rarely steady. With the advent of laser-based measurements of isotopologues, leaves have been enclosed in cuvettes and time courses of fluxes recorded. The enclosure modifies the time constant by effectively increasing the resistance to the one-way gross flux out of the stomata because transpiration increases the vapour concentration within the chamber. The resistance is increased from stomatal and boundary layer in series, to stomata, boundary layer and chamber resistance, where the latter is given by the ratio of leaf area to the flow rate out of the chamber. An apparent change in concept from one-way to net flux, introduced by Song, Simonin, Loucos and Barbour (2015) is resolved, and shown to be unnecessary, but the value of their data is reinforced., (© 2021 John Wiley & Sons Ltd.)

The doubly labeled water (DLW) method measures total energy expenditure (TEE) in free-living subjects. Several equations are used to convert isotopic data into TEE. Using the International Atomic Energy Agency (IAEA) DLW database (5,756 measurements of adults and children), we show considerable variability is introduced by different equations. The estimated rCO 2 is sensitive to the dilution space ratio (DSR) of the two isotopes. Based on performance in validation studies, we propose a new equation based on a new estimate of the mean DSR. The DSR is lower at low body masses (<10 kg). Using data for 1,021 babies and infants, we show that the DSR varies non-linearly with body mass between 0 and 10 kg. Using this relationship to predict DSR from weight provides an equation for rCO 2 over this size range that agrees well with indirect calorimetry (average difference 0.64%; SD = 12.2%). We propose adoption of these equations in future studies., Competing Interests: The authors declare no competing interests., (© 2021 The Authors.)

Rationale: Tracing isotopically labeled water into proteins allows for the detection of species-specific metabolic activity in complex communities. However, a stress response may alter the newly synthesized proteins., Methods: We traced 18-oxygen from heavy water into proteins of Escherichia coli K12 grown from permissive to retardant temperatures. All samples were analyzed using UPLC/Orbitrap Q-Exactive-MS/MS operating in positive electrospray ionization mode., Results: We found that warmer temperatures resulted in significantly (P-value < 0.05) higher incorporation of 18-oxygen as seen by both substrate utilization as relative isotope abundance (RIA) and growth as labeling ratio (LR). However, the absolute number of peptides with incorporation of 18-oxygen showed no significant correlation to temperature, potentially caused by the synthesis of different proteins at low temperatures, namely, proteins related to cold stress response., Conclusions: Our results unveil the species-specific cold stress response of E. coli K12 that could be misinterpreted as general growth; this is why the quantity as RIA and LR but also the quality as absolute number of peptides with incorporation (relative abundance, RA) and their function must be considered to fully understand the activity of microbial communities., (© 2020 John Wiley & Sons, Ltd.)

The oxygen isotope signature of sulphate (δ 18 O sulphate ) is increasingly used to study nutritional fluxes and sulphur transformation processes in a variety of natural environments. However, mechanisms controlling the δ 18 O sulphate signature in soil-plant systems are largely unknown. The objective of this study was to determine key factors, which affect δ 18 O sulphate values in soil and plants. The impact of an 18 O-water isotopic gradient and different types of fertilizers was investigated in a soil incubation study and a radish (Raphanus sativus L.) greenhouse growth experiment. Water provided 31-64% of oxygen atoms in soil sulphate formed via mineralization of organic residues (green and chicken manures) while 49% of oxygen atoms were derived from water during oxidation of elemental sulphur. In contrast, δ 18 O sulphate values of synthetic fertilizer were not affected by soil water. Correlations between soil and plant δ 18 O sulphate values were controlled by water δ 18 O values and fertilizer treatments. Additionally, plant δ 34 S data showed that the sulphate isotopic composition of plants is a function of S assimilation. This study documents the potential of using compound-specific isotope ratio analysis for investigating and tracing fertilization strategies in agricultural and environmental studies., (© 2020 John Wiley & Sons Ltd.)

The Craig-Gordon type (C-G) leaf water isotope enrichment models assume a homogeneous distribution of enriched water across the leaf surface, despite observations that Δ 18 O can become increasingly enriched from leaf base to tip. Datasets of this 'progressive isotope enrichment' are limited, precluding a comprehensive understanding of (a) the magnitude and variability of progressive isotope enrichment, and (b) how progressive enrichment impacts the accuracy of C-G leaf water model predictions. Here, we present observations of progressive enrichment in two conifer species that capture seasonal and diurnal variability in environmental conditions. We further examine which leaf water isotope models best capture the influence of progressive enrichment on bulk needle water Δ 18 O. Observed progressive enrichment was large and equal in magnitude across both species. The magnitude of this effect fluctuated seasonally in concert with vapour pressure deficit, but was static in the face of diurnal cycles in meteorological conditions. Despite large progressive enrichment, three variants of the C-G model reasonably successfully predicted bulk needle Δ 18 O. Our results thus suggest that the presence of progressive enrichment does not impact the predictive success of C-G models, and instead yields new insight regarding the physiological and anatomical mechanisms that cause progressive isotope enrichment., (© 2020 John Wiley & Sons Ltd.)

Water enriched with oxygen-18 (H 2 18 O) is a potential tracer for evaluating the sources of glucose and glycogen synthesis since it is incorporated into specific sites of glucose-6-phosphate via specific enzyme-mediated exchange/addition mechanisms. Unlike 2 H, 18 O does not experience significant isotope effects for any of these processes. Therefore, H 2 18 O might provide more precise estimates of endogenous carbohydrate synthesis compared with deuterated water provided that positional 18 O enrichments of glucose can be measured. As a proof of concept, H 2 18 O was incorporated into a well characterized hemolysate model of sugar phosphate metabolism and 13 C NMR was applied to quantify positional 18 O enrichment of glucose-6-phosphate oxygens. Human erythrocyte hemolysate preparations were incubated overnight at 37 °C with a buffer containing sugar phosphate precursors and 20% (n = 5) and 80% (n = 1) H 2 18 O. Enrichment of glucose-6-phosphate was analyzed by 13 C NMR analysis of 18 O-shifted versus unshifted signals following derivatization to monoacetone glucose (MAG). 13 C NMR MAG spectra from hemolysate revealed resolved 18 O-shifted signals in Positions 1-5. Mean 18 O enrichments were 16.4 ± 1.6% (Position 1), 13.3 ± 1.3% (Position 2), 4.1 ± 1.1% (Position 3), 12.6 ± 0.8% (Position 4), 10.7 ± 1.4% (Position 5), and no detectable enrichment of Position 6. No 18 O-shifted glucose-6-phosphate signals were detected in preparations containing sugar phosphate precursors only. H 2 18 O is incorporated into Positions 1-5 of glucose-6-phosphate in accordance with spontaneous aldose hydration and specific enzymatic reaction mechanisms. This provides a basis for its deployment as a tracer for glucose and glycogen biosynthesis., (© 2020 John Wiley & Sons, Ltd.)

On Conch Reef, Florida Keys, USA we examined the effects of reef hydrography and topography on the patterns of stable isotope values (δ 18 O and δ 13 C) in the benthic green alga, Halimeda tuna. During the summer, benthic temperatures show high-frequency fluctuations (2 to 8 °C) associated with internal waves that advected cool, nutrient-rich water across the reef. The interaction between local water flow and reef morphology resulted in a highly heterogenous physical environment even within isobaths that likely influenced the growth regime of H. tuna. Variability in H. tuna isotopic values even among closely located individuals suggest biological responses to the observed environmental heterogeneity. Although isotopic composition of reef carbonate material can be used to reconstruct past temperatures (T(°C) = 14.2-3.6 (δ 18 O Halimeda  - δ 18 O seawater ); r 2  = 0.92), comparing the temperatures measured across the reef with that predicted by an isotopic thermometer suggests complex interactions between the environment and Halimeda carbonate formation at temporal and spatial scales not normally considered in mixed sediment samples. The divergence in estimated range between measured and predicted temperatures demonstrates the existence of species- and location-specific isotopic relationships with physical and environmental factors that should be considered in contemporary as well as ancient reef settings.

Ubiquitination is one of the major post-translational modifications and entails conjugation of ubiquitin molecules to target proteins. To make free ubiquitin molecules available for conjugation, in cells ubiquitin is not only synthesized de novo, but is also provided by cleaving off existing conjugated ubiquitin molecules, so-called deubiquitination reaction. Therefore, intracellular ubiquitin molecules are thought to be recycled, but the recycling frequency remains elusive. The main reason for the lack of such mechanistic details is that the original and recycled ubiquitin molecules are indistinguishable in their chemical and physical properties. To tackle this issue, here we applied 18 O-labeling to trace how ubiquitin is recycled in a simultaneous ubiquitination/deubiquitination reaction (ubiquitin cycle reaction). Because deubiquitination is a hydrolysis reaction, the two 16 O atoms of the C-terminal carboxy group of a ubiquitin molecule can be exchanged with 18 O atoms by deubiquitination in 18 O-labeled aqueous solution. By using quantitative mass spectrometry, we detected 18 O atom incorporation into the C-terminal carboxy group of ubiquitin in the course of a deubiquitination reaction, in addition, we were able to quantify the 18 O-incorporation in a ubiquitin cycle reaction. Unexpectedly, kinetic analysis suggested that ubiquitination reactivity was accelerated in the presence of a deubiquitinating enzyme. Collectively, we have established a quantitative method to trace ubiquitin cycle reactions by analyzing deubiquitination-associated 18 O-incorporation into ubiquitin., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

According to Organisation Internationale de la vigne et du vin (OIV) standards, when analysing the stable isotope ratio of deuterium to hydrogen D/H at the methyl (I) and methylene (II) site of ethanol from concentrated must, a dilution with tap water is needed in order to carry out the alcoholic fermentation. This dilution causes a partial transfer of water hydrogens to the sugar, and this affects the (D/H) I and (D/H) II isotopic values of ethanol, which need to be normalised through specific equations based on the analysis of water δ 18 O or δ 2 H. The aim of this study was to evaluate the effectiveness and correctness of these equations experimentally. Grape, cane, and beet sugar, as well as grape must were diluted with water with increasing H and O stable isotope ratios, fermented, and analysed. SNIF-NMR and IRMS techniques were applied following the respective OIV methods. The equations based on the δ 2 H analysis of the diluted sugar/must solutions proved to be reliable in all the cases, although it is not an OIV standard. When using the equations based on the values of δ 18 O of the diluted solution, data normalisation was reliable only in cases where the water used for dilution had not undergone isotopic fractionation due, for example, to evaporation. In these cases, δ 2 H should be analysed.

Unprocessed C-terminal lysine (C-term Lys) is one of the most common causes for the formation of basic variants in therapeutic monoclonal antibodies (mAbs). Although the C-term Lys variants are routinely quantified by a LC-MS-based peptide mapping method using the relative MS responses from both C-terminal peptides (with and without Lys), this approach often leads to overestimation of Lys-containing peptide due to the intrinsic difference in ionization efficiency. Herein, we report an 18 O-labeling assisted LC-MS method, which takes advantage of the carboxypeptidase B-catalyzed Lys removal and 18 O-labeling to achieve improved accuracy of C-term Lys quantitation. The fidelity of this method was first demonstrated using synthetic peptide mixture standards that mimic a wide range of C-term Lys levels. Finally, the newly developed method was applied in a case study where C-term Lys variants in mAb samples manufactured from different processes were accurately quantified and compared. This new method provides a valuable solution for studies where accurate C-term Lys levels are needed to assist decision-making and root-cause investigation.

Most developments in synthetic biology try to depart from life as we know it, attempting to create orthogonal constructions. Here, following a variational principle, I try to explore how slight changes in the buildup of cells reveal critical features of life's physics. In a first section, I suggest that we use stable isotopes of the atoms of life to see how living cells fare, beginning with life in heavy water. Subsequently, isotopes of the other main biogenic atoms are suggested as an extension of the variational principle, despite their likely very small influence on the course of biological activity. Finally, two atoms of the second row of Mendeleev's table, boron and fluorine are explored as a further extension of the principle. The use of the former is still in its infancy, whereas the latter, based on existing fluorinases, could open the door to a more general use of halogens in synthetic biology., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Water wires are critical for the functioning of many membrane proteins, as in channels that conduct water, protons, and other ions. Here, in liquid crystalline lipid bilayers under symmetric environmental conditions, the selective hydrogen bonding interactions between eight waters comprising a water wire and a subset of 26 carbonyl oxygens lining the antiparallel dimeric gramicidin A channel are characterized by 17 O NMR spectroscopy at 35.2 T (or 1,500 MHz for 1 H) and computational studies. While backbone 15 N spectra clearly indicate structural symmetry between the two subunits, single site 17 O labels of the pore-lining carbonyls report two resonances, implying a break in dimer symmetry caused by the selective interactions with the water wire. The 17 O shifts document selective water hydrogen bonding with carbonyl oxygens that are stable on the millisecond timescale. Such interactions are supported by density functional theory calculations on snapshots taken from molecular dynamics simulations. Water hydrogen bonding in the pore is restricted to just three simultaneous interactions, unlike bulk water environs. The stability of the water wire orientation and its electric dipole leads to opposite charge-dipole interactions for K + ions bound at the two ends of the pore, thereby providing a simple explanation for an ∼20-fold difference in K + affinity between two binding sites that are ∼24 Å apart. The 17 O NMR spectroscopy reported here represents a breakthrough in high field NMR technology that will have applications throughout molecular biophysics, because of the acute sensitivity of the 17 O nucleus to its chemical environment., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Temperature influences microbiological growth and catabolic rates. Between 15 and 35 °C the growth rate and cell specific sulfate reduction rate of the sulfate reducing bacterium Desulfococcus multivorans increased with temperature. Sulfur isotope fractionation during sulfate reduction decreased with increasing temperature from 27.2 ‰ at 15 °C to 18.8 ‰ at 35 °C which is consistent with a decreasing reversibility of the metabolic pathway as the catabolic rate increases. Oxygen isotope fractionation, in contrast, decreased between 15 and 25 °C and then increased again between 25 and 35 °C, suggesting increasing reversibility in the first steps of the sulfate reducing pathway at higher temperatures. This points to a decoupling in the reversibility of sulfate reduction between the steps from the uptake of sulfate into the cell to the formation of sulfite, relative to the whole pathway from sulfate to sulfide. This observation is consistent with observations of increasing sulfur isotope fractionation when sulfate reducing bacteria are living near their upper temperature limit. The oxygen isotope decoupling may be a first signal of changing physiology as the bacteria cope with higher temperatures., (© FEMS 2020.)

The resource-poor, isolated islands of Wallacea have been considered a major adaptive obstacle for hominins expanding into Australasia. Archaeological evidence has hinted that coastal adaptations in Homo sapiens enabled rapid island dispersal and settlement; however, there has been no means to directly test this proposition. Here, we apply stable carbon and oxygen isotope analysis to human and faunal tooth enamel from six Late Pleistocene to Holocene archaeological sites across Wallacea. The results demonstrate that the earliest human forager found in the region c. 42,000 years ago made significant use of coastal resources prior to subsequent niche diversification shown for later individuals. We argue that our data provides clear insights into the huge adaptive flexibility of our species, including its ability to specialize in the use of varied environments, particularly in comparison to other hominin species known from Island Southeast Asia.

Thick filaments from some striated muscles are regulated by phosphorylation of myosin regulatory light chains (RLCs). A tarantula thick filament quasi-atomic model achieved by cryo-electron microscopy has advanced our understanding on how this regulation occurs. In native thick filaments, an asymmetric intramolecular interaction between the actin-binding region of one myosin head ("blocked") and the converter region of the other head ("free") switches both heads off, establishing the myosin interacting-heads motif (IHM). This structural finding, together with motility assays, sequence analysis, and mass spectrometry (MS) observations have suggested a cooperative phosphorylation activation (CPA) mechanism for thick filament activation. In the CPA mechanism, some myosin free heads are phosphorylated constitutively in Ser35 by protein kinase C (PKC) and -under Ca 2+ control - others (free or blocked) heads temporally on Ser45 by myosin light chain kinase (MLCK), in a way that explains both force development and post-tetanic potentiation in tarantula striated muscle. We tested this model using MS to verify if Ca 2+ -activation phosphorylates de novo un-phosphorylated Ser35 heads. For this purpose, we standardized an approach based on 18 O isotopic ATP labeling to accurately detect by MS-MS the RLC phosphorylation under Ca 2+ -activation. MS spectra showed de novo 18 O incorporation only on Ser45 but not on Ser35. As the constitutive Ser35 phosphorylation cannot be dephosphorylated, this result suggests that the number of RLCs on free heads with constitutively phosphorylated Ser35 does remain constant on Ca 2+ -activation supporting that the myosin has a basal activation and force modulation or potentiation is controlled by MLCK Ser45 phosphorylation., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The 18 O signature of atmospheric water vapour (δ 18 O V ) is known to be transferred via leaf water to assimilates. It remains, however, unclear how the 18 O-signal transfer differs among plant species and growth forms. We performed a 9-hr greenhouse fog experiment (relative humidity ≥ 98%) with 18 O-depleted water vapour (-106.7‰) on 140 plant species of eight different growth forms during daytime. We quantified the 18 O-signal transfer by calculating the mean residence time of O in leaf water (MRT LW ) and sugars (MRT Sugars ) and related it to leaf traits and physiological drivers. MRT LW increased with leaf succulence and thickness, varying between 1.4 and 10.8 hr. MRT Sugars was shorter in C 3 and C 4 plants than in crassulacean acid metabolism (CAM) plants and highly variable among species and growth forms; MRT Sugars was shortest for grasses and aquatic plants, intermediate for broadleaf trees, shrubs, and herbs, and longest for conifers, epiphytes, and succulents. Sucrose was more sensitive to δ 18 O V variations than other assimilates. Our comprehensive study shows that plant species and growth forms vary strongly in their sensitivity to δ 18 O V variations, which is important for the interpretation of δ 18 O values in plant organic material and compounds and thus for the reconstruction of climatic conditions and plant functional responses., (© 2019 John Wiley & Sons Ltd.)

Isotopic fractionation factors against 15 N and 18 O during anammox (anaerobic ammonia oxidization by nitrite) are critical for evaluating the importance of this process in natural environments. We performed batch incubation experiments with an anammox-dominated biomass to investigate nitrogen (N) and oxygen (O) isotopic fractionation factors during anammox and also examined apparent isotope fractionation factors during anammox in an actual wastewater treatment plant. We conducted one incubation experiment with high δ 18 O of water to investigate the effects of water δ 18 O. The N isotopic fractionation factors estimated from incubation experiments and the wastewater treatment plant were similar to previous values. We also found that the N isotopic effect ( 15 ε NXR of -77.8 to -65.9‰ and 15 Δ NXR of -31.3 to -30.4‰) and possibly O isotopic effect ( 18 ε NXR of -20.6‰) for anaerobic nitrite oxidation to nitrate were inverse. We applied the estimated isotopic fractionation factors to the ordinary differential equation model to clarify whether anammox induces deviations in the δ 18 O vs δ 15 N of nitrate from a linear trajectory of 1, similar to heterotrophic denitrification. Although this deviation has been attributed to nitrite oxidation, the O isotopic fractionation factor for anammox is crucial for obtaining a more detailed understanding of the mechanisms controlling this deviation. In our model, anammox induced the trajectory of the δ 18 O vs δ 15 N of nitrate during denitrification to less than one, which strongly indicates that this deviation is evidence of nitrite oxidation by anammox under denitrifying conditions.

Theoretical models of photosynthetic isotopic discrimination of CO 2 ( 13 C and 18 O) are commonly used to estimate mesophyll conductance ( g m ). This requires making simplifying assumptions and assigning parameter values so that g m can be solved for as the residual term. Uncertainties in g m estimation occur due to measurement noise and assumptions not holding, including parameter uncertainty and model parametrization. Uncertainties in the 13 C model have been explored previously, but there has been little testing undertaken to determine the reliability of g m estimates from the 18 O model ( g m18 ). In this study, we exploited the action of carbonic anhydrase in equilibrating CO 2 with leaf water and manipulated the observed photosynthetic discrimination (Δ 18 O) by changing the oxygen isotopic composition of the source gas CO 2 and water vapor. We developed a two-source δ 18 O method, whereby two measurements of Δ 18 O were obtained for a leaf with its gas-exchange characteristics otherwise unchanged. Measurements were performed in broad bean ( Vicia faba ) and Algerian oak ( Quercus canariensis ) in response to light and vapor pressure deficit. Despite manipulating the Δ 18 O by over 100‰, in most cases we observed consistency in the calculated g m18 , providing confidence in the measurements and model theory. Where there were differences in g m18 estimates between source-gas measurements, we explored uncertainty associated with two model assumptions (the isotopic composition of water at the sites of CO 2 -water exchange, and the humidity of the leaf internal airspace) and found evidence for both. Finally, we provide experimental guidelines to minimize the sensitivity of g m18 estimates to measurement errors. The two-source δ 18 O method offers a flexible tool for model parameterization and provides an opportunity to refine our understanding of leaf water and CO 2 fluxes., (© 2019 American Society of Plant Biologists. All Rights Reserved.)

Natural abundance of stable nitrogen (N) and oxygen (O) isotopes are invaluable biogeochemical tracers for assessing the N transformations in the environment. To fully exploit these tracers, the N and O isotope effects ( 15 ε and 18 ε) associated with the respective nitrogen transformation processes must be known. However, the N and O isotope effects of anaerobic ammonium oxidation (anammox), one of the major fixed N sinks and NO 3 - producers, are not well known. Here, we report the dual N and O isotope effects associated with anammox by three different anammox bacteria including "Ca. Scalindua japonica", a putative marine species, which were measured in continuous enrichment culture experiments. All three anammox species yielded similar N isotope effects of NH 4 + oxidation to N 2 ( 15 ε NH4→N2 ) ranging from 30.9‰ to 32.7‰ and inverse kinetic isotope effects of NO 2 - oxidation to NO 3 - ( 15 ε NO2→NO3  = -45.3‰ to -30.1‰). In contrast, 15 ε NO2→N2 (NO 2 - reduction to N 2 ) were significantly different among three species, which is probably because individual anammox bacteria species might possess different types of nitrite reductase. We also report the combined O isotope effects for NO 2 - oxidation ( 18 E NO2→NO3 ) by anammox bacteria. These obtained dual N and O isotopic effects could provide significant insights into the contribution of anammox bacteria to the fixed N loss and NO 2 - reoxidation (N recycling) in various natural environments.

Phosphorus (P) nutrition of beech ecosystems depends on soil processes, plant internal P cycling and P acquisition. P uptake of trees in the field is currently not validated due to the lack of an experimental approach applicable in natural forests. Application of radiolabelled tracers such as 33 P and 32 P is limited to special research sites and not allowed in natural environments. Moreover, only one stable isotope of P, namely 31 P, exists. One alternative tool to measure P acquisition in the field could be the use of 18 O-labelled 31 P-phosphate ( 31 P 18 O 4 3- ). Phosphate (P i ) uptake rates calculated from the 18 O enrichment of dried root material after application of 31 P i 18 O 4 3- via nutrient solution was always lower compared to 33 P incorporation, did not show increasing rates of P i uptake at P deficiency under controlled conditions, and did not reveal seasonal fluctuations in the field. Consequently, a clear correlation between 33 P-based and 18 O-based P i uptake by roots could not be established. Comparison of P i  uptake rates achieved from 33 P-P i and 18 O-P i application led to the conclusion of high P i metabolism in roots after P i uptake. The replacement of 18 O by 16 O from water in 18 O-P i during root influx, but most probably after P i uptake into roots, due to metabolic activities, indicates high and fast turnover of P i . Hence, the use of 18 O-P i as an alternative tool to estimate P i acquisition of trees in the field must consider the increase of 18 O abundance in root water that was disregarded in dried root material., (© 2018 German Society for Plant Sciences and The Royal Botanical Society of the Netherlands.)

The stable carbon (δ 13 C) and oxygen (δ 18 O) isotope compositions in plant matter reflect photosynthetic and transpirative conditions in plants, respectively. However, the nature of hydrogen isotope composition (δ 2 H) and what it reflects of plant performance is poorly understood. Using durum wheat ( Triticum turgidum var durum ), this study evaluated the effect of different water and nitrogen growing field conditions on transpiration and how this effect influenced the performance of δ 2 H in autotrophic (flag leaf), mixotrophic (ears), and heterotrophic (grains and roots) organs. Moreover, δ 2 H was compared with the δ 13 C and δ 18 O in the same organs. Isotope compositions were analyzed in dry matter, the water-soluble fraction, and in water from different tissues of a set of genotypes. Similar to δ 13 C, the δ 2 H correlated negatively with stomatal conductance, whereas no correlation was observed for δ 18 O. Moreover, δ 2 H was not only affected by changes in transpiration but also by photosynthetic reactions, probably as a consequence of NADPH formation in autotrophic organs. Compared with the δ 2 H of stem water, plant δ 2 H was strongly diminished in photosynthetic organs such as the flag leaves, whereas it strongly increased in heterotrophic organs such as grains and roots. In heterotrophic organs, δ 2 H was associated with postphotosynthetic effects because there are several processes that lead to 2 H-enrichment of carbohydrates. In summary, δ 2 H exhibited specific features that inform about the water conditions of the wheat crop, together with the photosynthetic characteristics of the plant part considered. Moreover, correlations of δ 2 H with grain yield illustrate that this isotope can be used to assess plant performance under different growing conditions., (© 2019 American Society of Plant Biologists. All Rights Reserved.)

The 'two-water-worlds' hypothesis is based on stable isotope differences in stream, soil and xylem waters in dual isotope space. It postulates no connectivity between bound and mobile soil waters, and preferential plant water uptake of bound soil water sources. We tested the pool-weighted impact of isotopically distinct water pools for hydrological cycling, the influence of species-specific water use and the degree of ecohydrological separation. We combined stable isotope analysis (δ 18 O and δ 2 H) of ecosystem water pools of precipitation, groundwater, soil and xylem water of two distinct species (Quercus suber, Cistus ladanifer) with observations of soil water contents and sap flow. Shallow soil water was evaporatively enriched during dry-down periods, but enrichment faded strongly with depth and upon precipitation events. Despite clearly distinct water sources and water-use strategies, both species displayed a highly opportunistic pattern of root water uptake. Here we offer an alternative explanation, showing that the isotopic differences between soil and plant water vs groundwater can be fully explained by spatio-temporal dynamics. Pool weighting the contribution of evaporatively enriched soil water reveals only minor annual impacts of these sources to ecosystem water cycling (c. 11% of bulk soil water and c. 14% of transpired water)., (© 2019 The Authors. New Phytologist © 2019 New Phytologist Trust.)

Migration is an integral feature of modern mysticete whale ecology, and the demands of migration may have played a key role in shaping mysticete evolutionary history. Constraining when migration became established and assessing how it has changed through time may yield valuable insight into the evolution of mysticete whales and the oceans in which they lived. However, there are currently few data which directly assess prehistoric mysticete migrations. Here we show that calcite δ 18 O profiles of two species of modern whale barnacles (coronulids) accurately reflect the known migration routes of their host whales. We then analyze well-preserved fossil coronulids from three different locations along the eastern Pacific coast, finding that δ 18 O profiles from these fossils exhibit trends and ranges similar to modern specimens. Our results demonstrate that migration is an ancient behavior within the humpback and gray whale lineages and that multiple Pleistocene populations were undertaking migrations of an extent similar to those of the present day., Competing Interests: The authors declare no conflict of interest.

The internal CO 2 gradient imposed by mesophyll conductance (g m ) reduces substrate availability for C 3 photosynthesis. With several assumptions, estimates of g m can be made from coupled leaf gas exchange with isoflux analysis of carbon ∆ 13 C-g m and oxygen in CO 2 , coupled with transpired water (H 2 O) ∆ 18 O-g m to partition g m into its biochemical and anatomical components. However, these assumptions require validation under changing leaf temperatures. To test these assumptions, we measured and modeled the temperature response (15-40°C) of ∆ 13 C-g m and ∆ 18 O-g m along with leaf biochemistry in the C 3 grass Panicum bisulcatum, which has naturally low carbonic anhydrase activity. Our study suggests that assumptions regarding the extent of isotopic equilibrium (θ) between CO 2 and H 2 O at the site of exchange, and that the isotopic composition of the H 2 O at the sites of evaporation ( δ w - e 18 ) and at the site of exchange ( δ w - ce 18 ) are similar, may lead to errors in estimating the ∆ 18 O-g m temperature response. The input parameters for ∆ 13 C-g m appear to be less sensitive to temperature. However, this needs to be tested in species with diverse carbonic anhydrase activity. Additional information on the temperature dependency of cytosolic and chloroplastic pH may clarify uncertainties used for ∆ 18 O-g m under changing leaf temperatures., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

Environmental fluctuations are recorded in a variety of sedimentary archives of lacustrine depositional systems. Geochemical signals recovered from bottom sediments in closed-basin lakes are among the most sensitive paleoenvironmental indicators and are commonly used in reconstructing lake evolution. Microbialites (i.e., organosedimentary deposits accreted through microbial trapping and binding of detrital sediment or in situ mineral precipitation on organics [Palaios, 2, 1987, 241]), however, have been largely overlooked as paleoenvironmental repositories. Here, we investigate concentrically laminated mineralized microbialites from Laguna Negra, a high-altitude (4,100 m above sea level) hypersaline, closed-basin lake in northwestern Argentina, and explore the potential for recovery of environmental signals from these unique sedimentary archives. Spatial heterogeneity in hydrological regime helps define zones inside Laguna Negra, each with their own morphologically distinct microbialite type. Most notably, platey microbialites (in Zone 3A) are precipitated by evaporative concentration processes, while discoidal oncolites (in Zone 3C) are interpreted result from fluid mixing and biologically mediated nucleation. This spatial heterogeneity is reflected in petrographically distinct carbonate fabrics: micritic, botryoidal, and isopachous. Fabric type is interpreted to reflect a combination of physical and biological influences during mineralization, and paired C-isotope measurement of carbonate and organic matter supports ecological differences as a dominant control on C-isotopic evolution between zones. Laminae of Laguna Negra microbialites preserve a range of δ 13 C carb from +5.75‰ to +18.25‰ and δ 18 O carb from -2.04‰ to +9.28‰. Temporal trends of lower carbon and oxygen isotopic compositions suggest that the influence of CO 2 degassing associated with evaporation has decreased over time. Combined, these results indicate that microbialite archives can provide data that aid in interpretation of both lake paleohydrology and paleoenvironmental change., (© 2018 John Wiley & Sons Ltd.)

The aim of the present study was to determine the total energy expenditure, physical activity and dietary intake of men with type 2 diabetes mellitus and control participants without type 2 diabetes mellitus who were matched for age and body mass index. The participants in the present study were 12 well-controlled type 2 diabetes mellitus patients and 10 controls, aged 40-75 years, with a body mass index <30 kg/m 2 . Total energy expenditure under free-living conditions was assessed using the doubly labeled water method, and physical activity was measured using a triaxial accelerometer. Dietary intake was assessed using a self-recorded food intake diary during the measurement period. Participants were instructed to record their dietary intake over 3 days, including 2 weekdays. Total energy expenditure was not significantly different between the groups (P = 0.153), nor were energy (P = 0.969) or macronutrient intakes. In conclusion, when age and body mass index are matched, total energy expenditure and self-reported energy intake are not significantly different between type 2 diabetes mellitus patients and healthy controls., (© 2018 The Authors. Journal of Diabetes Investigation published by Asian Association for the Study of Diabetes (AASD) and John Wiley & Sons Australia, Ltd.)

Historical and future trends in net primary productivity (NPP) and its sensitivity to global change are largely unknown because of the lack of long-term, high-resolution data. Here we test whether annually resolved tree-ring stable carbon (δ 13 C) and oxygen (δ 18 O) isotopes can be used as proxies for reconstructing past NPP. Stable isotope chronologies from four sites within three distinct hydroclimatic environments in the eastern United States (US) were compared in time and space against satellite-derived NPP products, including the long-term Global Inventory Modeling and Mapping Studies (GIMMS3g) NPP (1982-2011), the newest high-resolution Landsat NPP (1986-2015), and the Moderate Resolution Imaging Spectroradiometer (MODIS, 2001-2015) NPP. We show that tree-ring isotopes, in particular δ 18 O, correlate strongly with satellite NPP estimates at both local and large geographical scales in the eastern US. These findings represent an important breakthrough for estimating interannual variability and long-term changes in terrestrial productivity at the biome scale.

As the Western Antarctic Peninsula (WAP) region responds to a warmer climate, the impacts of glacial meltwater on the Southern Ocean are expected to intensify. The Antarctic Peninsula fjord system offers an ideal system to understand meltwater's properties, providing an extreme in the meltwater's spatial gradient from the glacio-marine boundary to the WAP continental shelf. Glacial meltwater discharge in Arctic and Greenland fjords is typically characterized as relatively lower temperature, fresh and with high turbidity. During two cruises conducted in December 2015 and April 2016 in Andvord Bay, we found a water lens of low salinity and low temperature along the glacio-marine interface. Oxygen isotope ratios identified this water lens as a mixture of glacial ice and deep water in Gerlache Strait suggesting this is glacial meltwater. Conventional hydrographic measurements were combined with optical properties to effectively quantify its spatial extent. Fine suspended sediments associated with meltwater (nanoparticles of ~ 5nm) had a significant impact on the underwater light field and enabled the detection of meltwater characteristics and spatial distribution. In this study, we illustrate that glacial meltwater in Andvord Bay alters the inherent and apparent optical properties of the water column, and develop statistical models to predict the meltwater content from hydrographic and optical measurements. The predicted meltwater fraction is in good agreement with in-situ values. These models offer a potential for remote sensing and high-resolution detection of glacial meltwater in Antarctic waters. Furthermore, the possible influence of meltwater on phytoplankton abundance in the surface is highlighted; a significant correlation is found between meltwater fraction and chlorophyll concentration., Competing Interests: The authors have declared that no competing interests exist.

We describe a protocol for investigating microbial growth in environmental samples via stable isotope probing (SIP) with H 2 18 O. Water is a universal substrate for all microorganisms and replication is required for DNA to become labeled with 18 O. By measuring how much the DNA of each taxon becomes enriched with 18 O when an environmental sample is incubated with H 2 18 O, it is feasible to quantify that population's DNA replication rate, which is a proxy for growth.

Chip-SIP is a stable isotope probing (SIP) method for linking microbial identity and function in mixed communities and is capable of analyzing multiple isotopes ( 13 C, 15 N, and 18 O) simultaneously. This method uses a high-density microarray to separate taxon-specific 16S (or 18S) rRNA genes and a high sensitivity magnetic sector secondary ion mass spectrometer (SIMS) to determine the relative isotope incorporation of the rRNA at each probe location. Using a maskless array synthesizer (MAS), we synthesize multiple unique sequences to target hundreds of taxa at the ribosomal operational taxonomic unit (OTU) level on an array surface, and then analyze it with a NanoSIMS 50, using its high-spatial resolution imaging capability to generate isotope ratios for individual probes. The Chip-SIP method has been used in diverse systems, including surface marine and estuarine water, rhizosphere, and peat soils, to quantify taxon-specific relative incorporation of different substrates in complex microbial communities. Depending on the hypothesis and experimental design, Chip-SIP allows the user to compare the same community incorporating different substrates, different communities incorporating the same substrate(s), or quantify how a community responds to treatment effects, such as temperature or nutrient concentrations.

Quantitative stable isotope probing (qSIP) measures rates of taxon-specific element assimilation in intact microbial communities, utilizing substrates labeled with a heavy isotope.The laboratory protocol for qSIP is nearly identical to that for conventional stable isotope probing, with two key additions: (1) in qSIP, qPCR measurements are conducted on each density fraction recovered after isopycnic separation, and (2) in qSIP, multiple density fractions are sequenced spanning the entire range of densities over which nucleic acids were recovered. qSIP goes beyond identifying taxa assimilating a substrate, as it also allows for measuring that assimilation for each taxon within a given microbial community. Here, we describe an analysis process necessary to determine atom fraction excess of a heavy stable isotope added to an environmental sample for a given taxon's DNA.

Rationale: Variation in 18 O natural abundance can lead to errors in the calculation of total energy expenditure (TEE) when using the doubly labelled water (DLW) method. The use of Bayesian statistics allows a distribution to be assigned to 18 O natural abundance, thus allowing a best-fit value to be used in the calculation. The aim of this study was to calculate within-subject variation in 18 O natural abundance and apply this to our original working model for TEE calculation., Methods: Urine samples from a cohort of 99 women, dosed with 50 g of 20% 2 H 2 O, undertaking a 14-day breast milk intake protocol, were analysed for 18 O. The within-subject variance was calculated and applied to a Bayesian model for the calculation of TEE in a separate cohort of 36 women. This cohort of 36 women had taken part in a DLW study and had been dosed with 80 mg/kg body weight 2 H 2 O and 150 mg/kg body weight H 2 18 O., Results: The average change in the δ 18 O value from the 99 women was 1.14‰ (0.77) [0.99, 1.29], with the average within-subject 18 O natural abundance variance being 0.13‰ 2 (0.25) [0.08, 0.18]. There were no significant differences in TEE (9745 (1414), 9804 (1460) and 9789 (1455) kJ/day, non-Bayesian, Bluck Bayesian and modified Bayesian models, respectively) between methods., Conclusions: Our findings demonstrate that using a reduced natural variation in 18 O as calculated from a population does not impact significantly on the calculation of TEE in our model. It may therefore be more conservative to allow a larger variance to account for individual extremes., (© 2018 The Authors. Rapid Communications in Mass Spectrometry Published by John Wiley & Sons Ltd.)

Here we report on a new nanoscale secondary ion mass spectrometry (nanoSIMS) approach based on enzyme-mediated oxygen isotope exchange, which combines the visualization of general metabolic activity in the cytoplasm with insights into the activity of enzymes related to polyphosphate (polyP) inclusions. The polyP-accumulating strain of the large sulfur bacterium Beggiatoa was used as a model organism. Beggiatoa cultures were grown under oxic and anoxic conditions when exposed to either low- or high-sulfide conditions, which are known to influence polyP metabolism in this strain. Subsequent incubation with 18 O-labeled water led to high 18 O enrichments above the natural background in the cytoplasm and polyP granules derived from enzymatically mediated oxygen isotope exchange. The relative importance of polyP under the different sulfide regimes became evident by an apparent continued metabolic activity at polyP inclusions under stressfully high sulfide concentrations, in contrast to a decreased general metabolic activity in the cytoplasm. This finding confirms the role of polyP as a critical component in bacterial stress response and maintenance of a survival metabolism. IMPORTANCE Microbial organisms exert a large influence on the environment as they directly affect the turnover of essential elements. This is particularly true for polyphosphate-accumulating large sulfur bacteria, which can either accumulate phosphate as polyphosphate or degrade it and release phosphate into the environment, depending on environmental conditions. This study presents a new approach to simultaneously visualize general metabolic activity and enzymatic activity at polyphosphate granules by incubation with 18 O-labeled water as the only stable isotope tracer. For this purpose, the well-studied Beggiatoa sp. strain 35Flor was used as a model organism and was exposed to different stress regimes. General metabolic activity was strongly impaired during high-stress regimes. In contrast, intense intracellular polyP cycling was not restricted to favorable or stressful conditions, highlighting the importance of polyP for general cell physiology, especially during hostile conditions. The nanoSIMS approach adds a new tool to study microorganisms involved in phosphorus cycling in the environment together with the identification of general metabolic activity., (Copyright © 2018 Langer et al.)

We developed novel approaches for using the isotope composition of tree-ring subdivisions to study seasonal dynamics in tree-climate relations. Across a 30-year time series, the δ 13 C and δ 18 O values of the earlywood (EW) cellulose in the annual rings of Pinus ponderosa reflected relatively high intrinsic water-use efficiencies and high evaporative fractionation of 18 O/ 16 O, respectively, compared with the false latewood (FLW), summerwood (SW), and latewood (LW) subdivisions. This result is counterintuitive, given the spring origins of the EW source water and midsummer origins of the FLW, SW, and LW. With the use of the Craig-Gordon (CG), isotope-climate model revealed that the isotope ratios in all of the ring subdivision are explained by the existence of seasonal lags, lasting several weeks, between the initial formation of tracheids and the production of cellulosic secondary cell walls during maturation. In contrast to some past studies, modification of the CG model according to conventional methods to account for mixing of needle water between fractionated and nonfractionated sources did not improve the accuracy of predictions. Our results reveal new potential in the use of tree-ring isotopes to reconstruct past intra-annual tree-climate relations if lags in cambial phenology are reconciled with isotope ratio observations and included in theoretical treatments., (© 2018 John Wiley & Sons Ltd.)

Stable isotope ratios in tree rings have become an important proxy for palaeoclimatology, particularly in temperate regions. Yet temperate forests are often characterized by heterogeneous stand structures, and the effects of stand dynamics on carbon (δ 13 C) and oxygen isotope ratios (δ 18 O) in tree rings are not well explored. In this study, we investigated long-term trends and offsets in δ 18 O and δ 13 C of Picea abies and Fagus sylvatica in relation to tree age, size, and distance to the upper canopy at seven temperate sites across Europe. We observed strong positive trends in δ 13 C that are best explained by the reconstructed dynamics of individual trees below the upper canopy, highlighting the influence of light attenuation on δ 13 C in shade-tolerant species. We also detected positive trends in δ 18 O with increasing tree size. However, the observed slopes are less steep and consistent between trees of different ages and thus can be more easily addressed. We recommend restricting the use of δ 13 C to years when trees are in a dominant canopy position to infer long-term climate signals in δ 13 C when relying on material from shade-tolerant species, such as beech and spruce. For such species, δ 18 O should be in principle the superior proxy for climate reconstructions., (© 2018 John Wiley & Sons Ltd.)

Anatomical adaptations to high-salinity environments in mangrove leaves may be recorded in leaf water isotopes. Recent studies observed lower 18 O enrichment (Δ L ) of leaf water with respect to source water in three mangrove species relative to adjacent freshwater trees, but the factors that govern this phenomenon remain unclear. To resolve this issue, we investigated leaf traits and Δ L in 15 species of true mangrove plants, 14 species of adjacent freshwater trees, and 4 species of semi-mangrove plants at five study sites along south-eastern coast of China. Our results confirm that Δ L was generally 3-4‰ lower for mangrove species than for adjacent freshwater or semi-mangrove species. We hypothesized that higher leaf water content (LWC) and lower leaf stomatal density (LS) both played important roles in reducing Δ L in mangroves relative to nearby freshwater plants. Both differences acted to elongate effective leaf mixing length (L) in mangroves by about 200% and lower stomatal conductance by about 30%. Péclet models based on both LWC and LS could accurately predict Δ L . Our findings highlight the potential species-specific anatomical determinants of Δ L (or L), which has important implications for the interpretation of environmental information from metabolites produced by leaf water isotopes in palaeoclimate research., (© 2018 John Wiley & Sons Ltd.)

We introduce a simple online 18 O-labeling protocol for protein samples that uses a parallelizing microbore hollow fiber enzyme reactor (mHFER) as an alternative tool for online proteolytic digestion. Online 18 O-labeling is performed by separately attaching two mHFERs in parallel to a 10-port switching valve with a high-pressure syringe pump and two syringes containing 16 O- or 18 O-water. 16 O-/ 18 O-labeled peptides are formed in this manner and simultaneously analyzed online using nanoflow liquid chromatography-tandem mass spectrometry (nLC-MS/MS) without any residual trypsin activity. The usefulness of a parallel mHFER platform (P-mHFER) in 18 O-labeling was tested using both cytochrome C and alpha-1-acid-glycoprotein to verify the incorporation level of two 18 O atoms into tryptic peptides and to provide a quantitative assessment with varied mixing ratios. Additionally, our 18 O-labeling approach was used to study the serum N-glycoproteome from lung cancer patients and controls to evaluate the applicability of lectin-based quantitative N-glycoproteomics. We successfully quantified 76 peptides (from 62 N-glycoproteins). Nineteen of these peptides from lung cancer serum were up-/down-regulated at least 2.5-fold compared to controls. As a result, the P-mHFER-based online 18 O-labeling platform presented here can be a simple and reproducible way to allow quantitative proteomic analysis of diverse proteome samples., (Copyright © 2018. Published by Elsevier B.V.)

Rationale: Otolith δ 18 O and δ 13 C values have been used extensively to reconstruct thermal and diet histories. Researchers have suggested that individual growth rate and size may have an effect on otolith isotope ratios and subsequently confound otolith-based thermal and diet reconstructions. As few explicit tests of the effect on fish in freshwater environments exist, here we determine experimentally the potential for related growth rate and size effects on otolith δ 18 O and δ 13 C values., Methods: Fifty Arctic charr were raised in identical conditions for two years after which their otoliths were removed and analyzed for their δ 18 O and δ 13 C values. The potential effects of final length and the Thermal Growth Coefficient (TGC) on otolith isotope ratios were tested using correlation and regression analysis to determine if significant effects were present and to quantify effects when present., Results: The analyses indicated that TGC and size had significant and similar positive non-linear relationships with δ 13 C values and explained 35% and 42% of the variability, respectively. Conversely, both TGC and size were found to have no significant correlation with otolith δ 18 O values. There was no significant correlation between δ 18 O and δ 13 C values., Conclusions: The investigation indicated the presence of linked growth rate and size effects on otolith δ 13 C values, the nature of which requires further study. Otolith δ 18 O values were unaffected by individual growth rate and size, confirming the applicability of these values to thermal reconstructions of fish habitat., (© 2018 John Wiley & Sons, Ltd.)