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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.)

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.)

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.)

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.

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.)

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.)

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.)

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.

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.

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.)

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.)

The study aims to assess variability in leaf water isotopic enrichment occurring in the field under natural conditions. We focused on seasonal variation and difference between sun-exposed and shaded leaves. Isotopic composition (δ 18 O, δ 2 H) of leaf water was monitored in a beech tree (Fagus sylvatica L.) growing in the forest-meadow ecotone together with δ 18 O ( 2 H) of water compartments which are in close relation to this signal, namely twig and soil water. The sampling was carried out in approximately two-week intervals during five consecutive vegetation seasons. The δ 18 O ( 2 H) data showed a distinct seasonal pattern and a consistency in relative differences between the seasons and sample categories. Leaf water was the most isotopically enriched water compartment. The leaf water enrichment decreased toward the autumn reflecting the change in δ 18 O ( 2 H) of source water and evaporative demands. The soil and twig water isotopic signal was depleted against current precipitation as it partly retained the isotopic signature from winter precipitation however the seasonal pattern of soil and twig water followed that of precipitation. No significant differences between sun-exposed and shaded samples were detected. Nevertheless, the observed strong seasonal pattern of isotope composition of leaf, twig and soil water should be taken into account when using leaf water enrichment for further calculations or modeling., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

The oxygen-18 isotopic ( 18 O) composition in CO 2 provides an important insight into the variation of rate in isotopic fractionation reaction regulated by carbonic anydrase (CA) metalloenzyme. This work aims to employ an 18 O-isotope ratio-based analytical method for quantitative estimation of CA activity in erythrocytes for clinical testing purposes. Here, a new method has been developed that contains the measurements of 18 O/ 16 O isotope ratios during oxygen-18 isotopic exchange between 12 C 16 O 16 O and H 2 18 O of an in vitro biochemical reaction controlled by erythrocytes CA and estimation of enzymatic activity of CA from the isotopic composition of CO 2 . We studied the enrichments of 18 O-isotope of CO 2 with increments of CA activities during isotopic fractionation reaction. To check the influence of subject-specific body temperature, pH, H 2 18 O, and cellular produced CO 2 on this reaction, we performed the in vitro experiments in closed containers with variations of those parameters. Finally, we mimicked the exchange reaction at 5% [CO 2 ], 5‰ [H 2 18 O], pH of 7.4, and temperature of 37 °C to create the physiological environment equivalent to that of the human body and monitored the exchange kinetics with variations of CA activities, and subsequently, we derived the quantitative relation between the 18 O-isotope of CO 2 and CA activity in erythrocytes. This assay may be applicable for rapid and simple quantification of carbonic anhydrase activity which is very important to prevent the carbonic-anhydrase-associated disorders in human.

The aim of this study was to monitor seasonal changes in stable isotopes of pool freshwater and harp seal ( Phoca groenlandica ) body water, and to study whether these potential seasonal changes might bias results obtained using the doubly labelled water (DLW) method when measuring energy expenditure in animals with access to freshwater. Seasonal changes in the background levels of deuterium and oxygen-18 in the body water of four captive harp seals and in the freshwater pool in which they were kept were measured over a time period of 1 year. The seals were offered daily amounts of capelin and kept under a seasonal photoperiod of 69°N. Large seasonal variations of deuterium and oxygen-18 in the pool water were measured, and the isotope abundance in the body water showed similar seasonal changes to the pool water. This shows that the seals were continuously equilibrating with the surrounding water as a result of significant daily water drinking. Variations in background levels of deuterium and oxygen-18 in freshwater sources may be due to seasonal changes in physical processes such as precipitation and evaporation that cause fractionation of isotopes. Rapid and abrupt changes in the background levels of deuterium and oxygen-18 may complicate calculation of energy expenditure by use of the DLW method. It is therefore strongly recommended that analysis of seasonal changes in background levels of isotopes is performed before the DLW method is applied on (free-ranging) animals, and to use a control group in order to correct for changes in background levels., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

Stable isotopes in tree rings are increasingly used as proxies for climatic and ecophysiological changes. However, uncertainties remain about the strength and consistency of their response to environmental variation at different temporal (i.e. seasonal to inter-decadal) scales. We developed 5 yr of intra-seasonal and 62 yr of early- and late-wood δ 13 C and δ 18 O series of Smith fir (Abies georgei var. smithii) on the southeastern Tibetan Plateau, and used a process-based forward model to examine the relative importance of environmental and physiological controls on the isotopic data. In this temperate high-altitude region, the response, both δ 18 O and δ 13 C, is primarily to variations in relative humidity, but by different processes. In δ 18 O, the response is via source water δ 18 O but also arises from leaf water 18 O enrichment. In δ 13 C, the response is via changes in stomatal conductance but is modified by carry-over effects from prior periods. We conclude that tree-ring δ 18 O may be a more robust climate proxy than δ 13 C, and δ 13 C may be more suited to studies of site-related physiological responses to the local environment., (© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.)

Rationale: Potential post-mortem alteration to the oxygen isotope composition of biogenic silica is critical to the validity of palaeoclimate reconstructions based on oxygen isotope ratios (δ 18 O values) from sedimentary silica. We calculate the degree of oxygen isotope alteration within freshly cultured diatom biogenic silica in response to heating and storing in the laboratory., Methods: The experiments used freshly cultured diatom silica. Silica samples were either stored in water or dried at temperatures between 20 °C and 80 °C. The mass of affected oxygen and the associated silica-water isotope fractionation during alteration were calculated by conducting parallel experiments using endmember waters with δ 18 O values of -6.3 to -5.9 ‰ and -36.3 to -35.0 ‰. Dehydroxylation and subsequent oxygen liberation were achieved by stepwise fluorination with BrF 5 . The 18 O/ 16 O ratios were measured using a ThermoFinnigan MAT 253 isotope ratio mass spectrometer., Results: Significant alterations in silica δ 18 O values were observed, most notably an increase in the δ 18 O values following drying at 40-80 °C. Storage in water for 7 days between 20 and 80 °C also led to significant alteration in δ 18 O values. Mass balance calculations suggest that the amount of affected oxygen is positively correlated with temperature. The estimated oxygen isotope fractionation during alteration is an inverse function of temperature, consistent with the extrapolation of models for high-temperature silica-water oxygen isotope fractionation., Conclusions: Routinely used preparatory methods may impart significant alterations to the δ 18 O values of biogenic silica, particularly when dealing with modern cultured or field-collected material. The significance of such processes within natural aquatic environments is uncertain; however, there is potential that similar processes also affect sedimentary diatoms, with implications for the interpretation of biogenic silica-hosted δ 18 O palaeoclimate records., (© 2017 The Authors. Rapid Communications in Mass Spectrometry Published by John Wiley & Sons Ltd.)

Closely related taxa are, on average, more similar in terms of their physiology, morphology and ecology than distantly related ones. How this biological similarity affects geochemical signals, and their interpretations, has yet to be tested in an explicitly evolutionary framework. Here we compile and analyze planktonic foraminiferal size-specific stable carbon and oxygen isotope values (δ 13 C and δ 18 O, respectively) spanning the last 107 million years. After controlling for dominant drivers of size-δ 13 C and size-δ 18 O trends, such as geological preservation, presence of algal photosymbionts, and global environmental changes, we identify that shared evolutionary history has shaped the evolution of species-specific vital effects in δ 13 C, but not in δ 18 O. Our results lay the groundwork for using a phylogenetic approach to correct species δ 13 C vital effects through time, thereby reducing systematic biases in interpretations of long-term δ 13 C records-a key measure of holistic organismal biology and of the global carbon cycle.

Multiannual time series of (palaeo)hydrological information can be reconstructed from the oxygen isotope composition of cellulose (δ 18 O Cel ) in biological archives, for example, tree rings, but our ability to temporally resolve information at subannual scale is limited. We capitalized on the short and predictable leaf appearance interval (2.4 d) of a perennial C 4 grass (Cleistogenes squarrosa), to assess its potential for providing highly time-resolved δ 18 O Cel records of vapour pressure deficit (VPD). Plants grown at low (0.63 kPa) or high (1.58 kPa) VPD were swapped between VPD environments and exposed to the new environment for 7 d with simultaneous 13 CO 2 labelling. Then, leaves were sampled by age/position along individual tillers. Five leaves at different developmental stages were growing simultaneously. The period of most-active leaf elongation, from 10 to 90% of final length, lasted 6.6 d, and ~80% of all carbon and oxygen incorporation in whole-leaf cellulose occurred within 7 d. Cellulose deposition stopped at (or shortly after) full leaf expansion. The direction of change, low-to-high or high-to-low VPD, had no differential effect on new oxygen and carbon incorporation in cellulose. Successive leaves produced by tillers of C. squarrosa provide a δ 18 O Cel record useful for reconstructions of short-term hydrological dynamics., (© 2017 John Wiley & Sons Ltd.)

Spatial patterns of leaf water isotopes are challenging to predict because of the intricate link between vein and lamina water. Many models have attempted to predict these patterns, but to date, most have focused on monocots with parallel veins. These provide a simple system to study, but do not represent the majority of plant species. Here, a new protocol is developed using a Picarro induction module coupled to a cavity ringdown spectrometer to obtain maps of the leaf water isotopes ( 18 O and 2 H). The technique is applied to Colocasia esculenta leaves. The results are compared with isotope ratio mass spectrometry. In C. esculenta, a large enrichment in the radial direction is observed, but not in the longitudinal direction. The string-of-lakes model fails to predict the observed patterns, while the Farquhar-Gan model is more successful, especially when enrichment is accounted for along the radial direction. Our results show that reticulate-veined leaves experience a larger enrichment along the axis of the secondary veins than along the midrib. We hypothesize that this is due to the lower major/minor vein ratio that leads to longer pathways between major veins and sites of evaporation., (© 2017 John Wiley & Sons Ltd.)

Purpose: Parameter identifiability and confidence intervals were determined using a profile likelihood (PL) analysis method in a quantification model of the cerebral metabolic rate of oxygen consumption (CMRO 2 ) with direct 17 O MRI., Methods: Three-dimensional dynamic 17 O MRI datasets of the human brain were acquired after inhalation of 17 O 2 gas with the help of a rebreathing system, and CMRO 2 was quantified with a pharmacokinetic model. To analyze the influence of the different model parameters on the identifiability of CMRO 2 , PLs were calculated for different settings of the model parameters. In particular, the 17 O enrichment fraction of the inhaled 17 O 2 gas, α, was investigated assuming a constant and a linearly varying model. Identifiability was analyzed for white and gray matter, and the dependency on different priors was studied., Results: Prior knowledge about only one α-related parameter was sufficient to resolve the CMRO 2 nonidentifiability, and CMRO 2 rates (0.72-0.99 µmol/g tissue /min in white matter, 1.02-1.78 µmol/g tissue /min in gray matter) are in a good agreement with the results of 15 O positron emission tomography studies. Nonconstant α values significantly improved model fitting., Conclusion: The profile likelihood analysis shows that CMRO 2 can be measured reliably in 17 O gas MRI experiment if the 17 O enrichment fraction is used as prior information for the model calculations. Magn Reson Med 78:1157-1167, 2017. © 2016 International Society for Magnetic Resonance in Medicine., (© 2016 International Society for Magnetic Resonance in Medicine.)

Rationale: Fungal denitrifiers can contribute substantially to N 2 O emissions from arable soil and show a distinct site preference for N 2 O (SP(N 2 O)). This study sought to identify another process-specific isotopic tool to improve precise identification of N 2 O of fungal origin by mass spectrometric analysis of the N 2 O produced., Methods: Three pure bacterial and three fungal species were incubated under denitrifying conditions in treatments with natural abundance and stable isotope labelling to analyse the N 2 O produced. Combining different applications of isotope ratio mass spectrometry enabled us to estimate the oxygen (O) exchange accelerated by denitrifying enzymes and the ongoing microbial pathway in parallel. This experimental set-up allowed the determination of δ 18 O(N 2 O) values and isotopic fractionation of O, as well as SP(N 2 O) values, as a perspective to differentiate between microbial denitrifiers., Results: Oxygen exchange during N 2 O production was lower for bacteria than for fungi, differed between species, and depended also on incubation time. Apparent O isotopic fractionation during denitrification was in a similar range for bacteria and fungi, but application of the fractionation model indicated that different enzymes in bacteria and fungi were responsible for O exchange. This difference was associated with different isotopic fractionation for bacteria and fungi., Conclusions: δ 18 O(N 2 O) values depend on isotopic fractionation and isotopic fractionation may differ between processes and organism groups. By comparing SP(N 2 O) values, O exchange and the isotopic signature of precursors, we propose here a novel tool for differentiating between different sources of N 2 O., (Copyright © 2017 John Wiley & Sons, Ltd.)

Although oxygen consumption is a key factor in metabolic phenotyping, its assessment in tumors remains critical, as current technologies generally display poor specificity. The objectives of this study were to explore the feasibility of direct 17 O nuclear magnetic resonance (NMR) spectroscopy to assess oxygen metabolism in tumors and its modulations. To investigate the impact of hypometabolism induction in the murine fibrosarcoma FSAII tumor model, we monitored the oxygen consumption of normothermic (37°C) and hypothermic (32°C) tumor-bearing mice. Hypothermic animals showed an increase in tumor pO 2 (measured by electron paramagnetic resonance oximetry) contrary to normothermic animals. This was related to a decrease in oxygen consumption rate (assessed using 17 O magnetic resonance spectroscopy (MRS) after the inhalation of 17 O 2 -enriched gas). This study highlights the ability of direct 17 O MRS to measure oxygen metabolism in tumors and modulations of tumor oxygen consumption rate., (Copyright © 2017 John Wiley & Sons, Ltd.)

In this initial work, the in vivo degradation of 17 O-labeled glucose was studied during cellular glycolysis. To monitor cellular glucose metabolism, direct 17 O-magnetic resonance spectroscopy (MRS) was used in the mouse brain at 9.4 T. Non-localized spectra were acquired with a custom-built transmit/receive (Tx/Rx) two-turn surface coil and a free induction decay (FID) sequence with a short TR of 5.4 ms. The dynamics of labeled oxygen in the anomeric 1-OH and 6-CH 2 OH groups was detected using a Hankel-Lanczos singular value decomposition (HLSVD) algorithm for water suppression. Time-resolved 17 O-MRS (temporal resolution, 42/10.5 s) was performed in 10 anesthetized (1.25% isoflurane) mice after injection of a 2.2 M solution containing 2.5 mg/g body weight of differently labeled 17 O-glucose dissolved in 0.9% physiological saline. From a pharmacokinetic model fit of the H 2 17 O concentration-time course, a mean apparent cerebral metabolic rate of 17 O-labeled glucose in mouse brain of CMR Glc  = 0.07 ± 0.02 μmol/g/min was extracted, which is of the same order of magnitude as a literature value of 0.26 ± 0.06 μmol/g/min reported by 18 F-fluorodeoxyglucose ( 18 F-FDG) positron emission tomography (PET). In addition, we studied the chemical exchange kinetics of aqueous solutions of 17 O-labeled glucose at the C1 and C6 positions with dynamic 17 O-MRS. In conclusion, the results of the exchange and in vivo experiments demonstrate that the C6- 17 OH label in the 6-CH 2 OH group is transformed only glycolytically by the enzyme enolase into the metabolic end-product H 2 17 O, whereas C1- 17 OH ends up in water via direct hydrolysis as well as glycolysis. Therefore, dynamic 17 O-MRS of highly labeled 17 O-glucose could provide a valuable non-radioactive alternative to FDG PET in order to investigate glucose metabolism., (Copyright © 2017 John Wiley & Sons, Ltd.)

Almost no δ 18 O data are available for leaf carbohydrates, leaving a gap in the understanding of the δ 18 O relationship between leaf water and cellulose. We measured δ 18 O values of bulk leaf water (δ 18 O LW ) and individual leaf carbohydrates (e.g. fructose, glucose and sucrose) in grass and tree species and δ 18 O of leaf cellulose in grasses. The grasses were grown under two relative humidity (rH) conditions. Sucrose was generally 18 O-enriched compared with hexoses across all species with an apparent biosynthetic fractionation factor (ε bio ) of more than 27‰ relative to δ 18 O LW , which might be explained by isotopic leaf water and sucrose synthesis gradients. δ 18 O LW and δ 18 O values of carbohydrates and cellulose in grasses were strongly related, indicating that the leaf water signal in carbohydrates was transferred to cellulose (ε bio  = 25.1‰). Interestingly, damping factor p ex p x , which reflects oxygen isotope exchange with less enriched water during cellulose synthesis, responded to rH conditions if modelled from δ 18 O LW but not if modelled directly from δ 18 O of individual carbohydrates. We conclude that δ 18 O LW is not always a good substitute for δ 18 O of synthesis water due to isotopic leaf water gradients. Thus, compound-specific δ 18 O analyses of individual carbohydrates are helpful to better constrain (post-)photosynthetic isotope fractionation processes in plants., (© 2017 John Wiley & Sons Ltd.)

Leaf carbon and oxygen isotope ratios can potentially provide a time-integrated proxy for stomatal conductance (gs) and transpiration rate (E), and can be used to estimate transpiration efficiency (TE). In this study, we found significant relationships of bulk leaf carbon isotopic signature (δ13CBL) and bulk leaf oxygen enrichment above source water (Δ18OBL) with gas exchange and TE in the model C4 grasses Setaria viridis and S. italica. Leaf δ13C had strong relationships with E, gs, water use, biomass, and TE. Additionally, the consistent difference in δ13CBL between well-watered and water-limited plants suggests that δ13CBL is effective in separating C4 plants with different availability of water. Alternatively, the use of Δ18OBL as a proxy for E and TE in S. viridis and S. italica was problematic. First, the oxygen isotopic composition of source water, used to calculate leaf water enrichment (Δ18OLW), was variable with time and differed across water treatments. Second, water limitations changed leaf size and masked the relationship of Δ18OLW and Δ18OBL with E. Therefore, the data collected here suggest that δ13CBL but not Δ18OBL may be an effective proxy for TE in C4 grasses., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Transplantation of macroencapsulated tissue-engineered grafts (TEGs) is being investigated as a treatment for type 1 diabetes, but there is a critical need to measure TEG viability both in vitro and in vivo. Oxygen deficiency is the most critical issue preventing widespread implementation of TEG transplantation and delivery of supplemental oxygen (DSO) has been shown to enhance TEG survival and function in vivo. In this study, we demonstrate the first use of oxygen-17 magnetic resonance spectroscopy ( 17 O-MRS) to measure the oxygen consumption rate (OCR) of TEGs and show that in addition to providing therapeutic benefits to TEGs, DSO with 17 O 2 can also enable measurements of TEG viability. Macroencapsulated TEGs containing βTC3 murine insulinoma cells were prepared with three fractional viabilities and provided with 17 O 2 . Cellular metabolism of 17 O 2 into nascent mitochondrial water (H 2 17 O) was monitored by 17 O-MRS and, from the measured data, OCR was calculated. For comparison, OCR was simultaneously measured on a separate, but equivalent sample of cells with a well-established stirred microchamber technique. OCR measured by 17 O-MRS agreed well with measurements made in the stirred microchamber device. These studies confirm that 17 O-MRS can quantify TEG viability noninvasively. Biotechnol. Bioeng. 2017;114: 1118-1121. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Mesophyll conductance (g m ) is an important factor limiting rates of C 3 photosynthesis. However, its role in C 4 photosynthesis is poorly understood because it has been historically difficult to estimate. We use two methods to derive the temperature responses of g m in C 4 species. The first (Δ 18 O) combines measurements of gas exchange with models and measurements of 18 O discrimination. The second method (in vitro V pmax ) derives g m by retrofitting models of C 4 photosynthesis and 13 C discrimination with gas exchange, kinetic constants and in vitro V pmax measurements. The two methods produced similar g m for Setaria viridis and Zea mays. Additionally, we present the first temperature response (10-40°C) of C 4 g m in S. viridis, Z. mays and Miscanthus × giganteus. Values for g m at 25°C ranged from 2.90 to 7.85 μmol m -2  s -1  Pa -1 . Our study demonstrated that: the two described methods are suitable to calculate g m in C 4 species; g m values in C 4 are similar to high-end values reported for C 3 species; and g m increases with temperature analogous to reports for C 3 species and the response is species specific. These results improve our mechanistic understanding of C 4 photosynthesis., (© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.)

The oxygen isotope composition of cellulose (δ 18 O Cel ) archives hydrological and physiological information. Here, we assess previously unexplored direct and interactive effects of the δ 18 O of CO 2 (δ 18 O CO2 ), nitrogen (N) fertilizer supply and vapour pressure deficit (VPD) on δ 18 O Cel , 18 O-enrichment of leaf water (Δ 18 O LW ) and cellulose (Δ 18 O Cel ) relative to source water, and p ex p x , the proportion of oxygen in cellulose that exchanged with unenriched water at the site of cellulose synthesis, in a C 4 grass (Cleistogenes squarrosa). δ 18 O CO2 and N supply, and their interactions with VPD, had no effect on δ 18 O Cel , Δ 18 O LW , Δ 18 O Cel and p ex p x . Δ 18 O Cel and Δ 18 O LW increased with VPD, while p ex p x decreased. That VPD-effect on p ex p x was supported by sensitivity tests to variation of Δ 18 O LW and the equilibrium fractionation factor between carbonyl oxygen and water. N supply altered growth and morphological features, but not 18 O relations; conversely, VPD had no effect on growth or morphology, but controlled 18 O relations. The work implies that reconstructions of VPD from Δ 18 O Cel would overestimate amplitudes of VPD variation, at least in this species, if the VPD-effect on p ex p x is ignored. Progress in understanding the relationship between Δ 18 O LW and Δ 18 O Cel will require separate investigations of p ex and p x and of their responses to environmental conditions., (© 2016 John Wiley & Sons Ltd.)

The oxygen isotope composition of leaf water imparts a signal to a range of molecules in the atmosphere and biosphere, but has been notoriously difficult to measure in studies requiring a large number of samples as a consequence of the labour-intensive extraction step. We tested a method of direct equilibration of water in fresh leaf samples with CO2 , and subsequent oxygen isotope analysis on an optical spectrometer. The oxygen isotope composition of leaf water measured by the direct equilibration technique was strongly linearly related to that of cryogenically extracted leaf water in paired samples for a wide range of species with differing anatomy, with an R(2) of 0.95. The somewhat more enriched values produced by the direct equilibration method may reflect lack of full equilibration with unenriched water in the vascular bundles, but the strong relationship across a wide range of species suggests that this difference can be adequately corrected for using a simple linear relationship., (© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.)

The application of neural stem cell (NSC) research to neurodegenerative diseases has led to promising clinical trials. Currently, NSC therapy is most promising for Parkinson's disease (PD). We conducted behavioral tests and immunoassays for the profiling of a PD model in rats to assess the therapeutic effects of NSC treatments. Further, using a multiple sample comparison workflow, combined with (18) O-labeled proteome mixtures, we compared the differentially expressed proteins from control, PD, and NSC-treated PD rats. The results were analyzed bioinformatically and verified by Western blot. Based on our initial findings, we believe that the proteomic approach is a valuable tool in evaluating the therapeutic effects of NSC transplantation on neurodegenerative disorders., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The gastric pathogen Helicobacter pylori utilize glucose during metabolism, but the underlying mechanisms linking to oxygen-18 ((18)O) and carbon-13 ((13)C)-isotopic fractionations of breath CO2 during glucose metabolism are poorly understood. Using the excretion dynamics of (18)O/(16)O and (13)C/(12)C-isotope ratios of breath CO2, we found that individuals with Helicobacter pylori infections exhibited significantly higher isotopic enrichments of (18)O in breath CO2 during the 2h-glucose metabolism regardless of the isotopic nature of the substrate, while no significant enrichments of (18)O in breath CO2 were manifested in individuals without the infections. In contrast, the (13)C-isotopic enrichments of breath CO2 were significantly higher in individuals with Helicobacter pylori compared to individuals without infections in response to (13)C-enriched glucose uptake, whereas a distinguishable change of breath (13)C/(12)C-isotope ratios was also evident when Helicobacter pylori utilize natural glucose. Moreover, monitoring the (18)O and (13)C-isotopic exchange in breath CO2 successfully diagnosed the eradications of Helicobacter pylori infections following a standard therapy. Our findings suggest that breath (12)C(18)O(16)O and (13)C(16)O(16)O can be used as potential molecular biomarkers to distinctively track the pathogenesis of Helicobacter pylori and also for eradication purposes and thus may open new perspectives into the pathogen's physiology along with isotope-specific non-invasive diagnosis of the infection.