[Changes in protein requirements of elderly people over 5 years assessed using the indicator amino acid oxidation method].

OBJECTIVE: To evaluate the changes in protein requirements of the elderly during the past five years. METHODS: Based on the previous study of protein requirements of 14 elderly in 2017, 4 of these elderly(70-80 y) were included as study participants and protein requirements were re-evaluated using the indicator amino acid oxidation method. There were seven protein levels: 0.1, 0.3, 0.6, 0.9, 1.2, 1.5 and 1.8 g/(kg·d). Maintenance diets were given for the first two days of each protein level. A stable isotope study was conducted on the day 3, using L-~(13)C-phenylalanine as an indicator on the basis of an amino acid rationed diet, which was orally ingested into the body along with the amino acid rationed diet, and breath and urine samples were collected when the metabolism of L-~(13)C-phenylalanine reached steady state in the body. By measuring the kinetic parameters of labeled amino acids in the samples, a nonlinear mixed-effects model was constructed for the protein intake to be tested and the oxidation rate of labeled amino acids. The mean protein requirement of the study population was determined by the protein intake corresponding to the inflection point of the curve. RESULTS: Based on the production rate of ~(13)CO_2 in exhaled breath of four elderly people at different protein levels, the mean protein requirement was 1.05(95%CI 0.51-1.60) g/(kg·d). The protein recommended nutrient intake was 1.31(95%CI 0.64-2.00) g/(kg·d) was estimated by applying the coefficient of variation of the mean protein requirement to derive the recommended nutrient intake. CONCLUSION: Protein requirements in the elderly have increased over a five-year period and sarcopenia may be the main cause of increased protein requirements.

Shepherding the past: High-resolution data on Neolithic Southern Iberian livestock management at Cueva de El Toro (Antequera, Málaga).

The feeding strategies of the first domesticated herds had to manage the risks arising from the novelty of livestock practices in territories often distant from the animals' primary habitats. The Iberian Peninsula is characterised by a great diversity of environments, which undoubtedly influenced these dynamics. At the beginning of the Neolithic period these led the possibility to combine diverse livestock farming practices based on different animal feeding habits. This variability is also consistent with the rythms of adoption of domesticated animals, being later on the northern area. In order to address this issue, this work focuses on the dietary regimes of early sheep herds from southern Iberia, an area for which information is currently scarce. This study utilises high-resolution radiocarbon dating and stable isotope data on teeth to investigate sheep husbandry management strategies in Cueva de El Toro (Antequera, Málaga). The radiocarbon dates on the analysed remains evidenced they were deposited at the site over a short period, supporting the recurrent use of the cave. The sequential analysis of oxygen and carbon isotopes in tooth enamel reveals distinct livestock management strategies, reproduction patterns, feeding habits, and mobility during this short period. This variability demonstrates that livestock management practices in the western Mediterranean are more diverse than previously considered. Furthermore, these findings support the hypothesis that early Neolithic communities in the southern Iberian Peninsula were able to adopt different feeding strategies within the same herd, depending on their ecological and productive needs.

The influence of biopsy site and pregnancy on stable isotope ratios in humpback whale skin.

RATIONALE: Stable isotope analysis (SIA) of free-swimming mysticetes using biopsies is often limited in sample size and uses only one sample per individual, failing to capture both intra-individual variability and the influence of demographic and physiological factors on isotope ratios. METHODS: We applied SIA of δ13C and δ15N to humpback whale (Megaptera novaeangliae) biopsies taken during the foraging season along the western Antarctic Peninsula to quantify intra-individual variation from repeatedly sampled individuals, as well as to determine the effect of biopsy collection site, sex, and pregnancy on isotope ratios. RESULTS: There was substantial variability in δ13C from multiple biopsies taken from the same individuals, though δ15N was much more consistent. Side of the body (left versus right) and biopsy location (dorsal, anterior, ventral, and posterior) did marginally affect the isotopic composition of δ15N but not δ13C. Pregnancy had a significant effect on both δ13C and δ15N, where pregnant females were depleted in both when compared to non-pregnant females and males. CONCLUSIONS: These results indicate that isotopic signatures are influenced by multiple endogenous and exogenous factors and emphasize value in accounting for intra-individual variability and pregnancy status within a sampled population. Placed within an ecological context, the endogenous variability in δ13C observed here may be informative for future isotopic analyses.

Species-specific responses of C and N allocation to N addition: evidence from dual 13C and 15N labeling in three tree species.

Soil nitrogen (N) availability affects plant carbon (C) utilization. However, it is unclear how various tree functional types respond to N addition in terms of C assimilation, allocation, and storage. Here, a microcosm experiment with dual 13C and 15N labeling was conducted to study the effects of N addition (i.e., control, 0 g N kg-1; moderate N addition, 1.68 g N kg-1; and high N addition, 3.36 g N kg-1 soil) on morphological traits, on changes in nonstructural carbohydrates (NSC) in different organs, as well as on C and N uptake and allocation in three European temperate forest tree species (i.e., Acer pseudoplatanus, Picea abies and Abies alba). Our results demonstrated that root N uptake rates of the three tree species increased by N addition. In A. pseudoplatanus, N uptake by roots, N allocation to aboveground organs, and aboveground biomass allocation significantly improved by moderate and high N addition. In A. alba, only the high N addition treatment considerably raised aboveground N and C allocation. In contrast, biomass as well as C and N allocation between above and belowground tissues were not altered by N addition in P. abies. Meanwhile, NSC content as well as C and N coupling (represented by the ratio of relative 13C and 15N allocation rates in organs) were affected by N addition in A. pseudoplantanus and P. abies but not in A. alba. Overall, A. pseudoplatanus displayed the highest sensitivity to N addition and the highest N requirement among the three species, while P. abies had a lower N demand than A. alba. Our findings highlight that the responses of C and N allocation to soil N availability are species-specific and vary with the amount of N addition.

Local circulation of elites punctuated by transregional mobility enabled steppe political consolidation in the Xiongnu nomadic state.

The Xiongnu polity (ca. 200 BC- 150 AD) emerged out of indigenous community-centered socio-political structures to forge a powerful state that commanded the Mongolian steppe and beyond. Underpinned by a highly mobile pastoralist population, accustomed to seasonally rhythmic moves and embedded in an equestrian culture that facilitated rapid transport over long-distances, it remains unclear precisely how the movement of commoners, local aristocrats and regional elites abetted the formation and organization of Xiongnu state structures. Here, we evaluate Xiongnu movement and dietary intake through multi-stable isotopic analyses of tooth enamel from directly dated Xiongnu intermediate elites recovered from the mortuary center of Baga Gazaryn Chuluu-a prominent granite outcrop set in the Gobi Desert. Carbon isotope (δ13C) analysis indicates millet was consumed by some individuals, but whether or not this C4 cultivar contributed to the diets of most elites remains ambiguous in this C3/C4 desert-steppe environment. The effectiveness of oxygen isotopes (δ18O) to establish mobility appears much reduced in steppe environments, where geospatially sensitive information appears disrupted by extraordinary seasonality in meteoric water oxygen isotopes, pronounced oxygen isotopic variation in potential drinking water sources, and culturally mediated drinking practices. Most revealing, strontium isotopes (87Sr/86Sr) indicate circulation of local elites around this central place and beyond, a mobility format that helped leaders cement their own position through political consolidation of spatially dispersed mobile pastoralist communities. The consistent presence at Baga Gazaryn Chuluu of extra-local intermediate elites also points toward the importance of transregional mobility in binding together the Xiongnu polity over the vast distances of the eastern steppe.

Growth and carbon turnover of Piaractus mesopotamicus Holmberg, 1887 (Osteicthyes: Characidae): contribution of extruded feed and natural food.

Piaractus mesopotamicus, is a fish usually farmed in semi-intensive systems with access to natural food and supplementary feed. This study evaluates effects of feed allowance on the productive performance, carbon turnover and proportions of nutrient (carbon) contribution of feed and natural food for the growth of pacu. Juvenile fish were stocked in fiberglass tanks and fed to 100, 75, 50, 25, 0% apparent satiety (ApS), with a practical, extruded (C4 photosynthetic pathway) feed in a randomized design trial (n=3); plankton production for simulated semi-intensive farming system condition was induced by chemical fertilization. A control treatment was set up in tanks devoid of natural food. Data on muscle stable carbon isotope ratios were used to study carbon turnover using a relative growth-based model. Low variation of the δ13C impaired fitting a turnover model curve for the 0 and 25 % ApS treatments. Fish of the 100% and 75% ApS treatments reached circa 95% and 82.85% of the carbon turnover, respectively. Extruded feed was the main nutrient source for the growth of pacu in the semi-intensive, simulated farming condition. The current study contributes to the knowledge of the relationship between feeding rates and carbon turnover rates in the pacu muscle.

Metabolic coupling between soil aerobic methanotrophs and denitrifiers in rice paddy fields.

Paddy fields are hotspots of microbial denitrification, which is typically linked to the oxidation of electron donors such as methane (CH4) under anoxic and hypoxic conditions. While several anaerobic methanotrophs can facilitate denitrification intracellularly, whether and how aerobic CH4 oxidation couples with denitrification in hypoxic paddy fields remains virtually unknown. Here we combine a ~3300 km field study across main rice-producing areas of China and 13CH4-DNA-stable isotope probing (SIP) experiments to investigate the role of soil aerobic CH4 oxidation in supporting denitrification. Our results reveal positive relationships between CH4 oxidation and denitrification activities and genes across various climatic regions. Microcosm experiments confirm that CH4 and methanotroph addition promote gene expression involved in denitrification and increase nitrous oxide emissions. Moreover, 13CH4-DNA-SIP analyses identify over 70 phylotypes harboring genes associated with denitrification and assimilating 13C, which are mostly belonged to Rubrivivax, Magnetospirillum, and Bradyrhizobium. Combined analyses of 13C-metagenome-assembled genomes and 13C-metabolomics highlight the importance of intermediates such as acetate, propionate and lactate, released during aerobic CH4 oxidation, for the coupling of CH4 oxidation with denitrification. Our work identifies key microbial taxa and pathways driving coupled aerobic CH4 oxidation and denitrification, with important implications for nitrogen management and greenhouse gas regulation in agroecosystems.

Using Carbon Stable Isotopes to Study C3 and C4 Photosynthesis: Models and Calculations.

Stable carbon isotopes are a powerful tool to study photosynthesis. Initial applications consisted of determining isotope ratios of plant biomass using mass spectrometry. Subsequently, theoretical models relating C isotope values to gas exchange characteristics were introduced and tested against instantaneous online measurements of 13C photosynthetic discrimination. Beginning in the twenty-first century, laser absorption spectroscopes with sufficient precision for determining isotope mixing ratios became commercially available. This has allowed collection of large data sets at lower cost and with unprecedented temporal resolution. More data and accompanying knowledge have permitted refinement of 13C discrimination model equations, but often at the expense of increased model complexity and difficult parametrization. This chapter describes instantaneous online measurements of 13C photosynthetic discrimination, provides recommendations for experimental setup, and presents a thorough compilation of equations available to researchers. We update our previous 2018 version of this chapter by including recently improved descriptions of (photo)respiratory processes and associated fractionations. We discuss the capabilities and limitations of the diverse 13C discrimination model equations and provide guidance for selecting the model complexity needed for different applications.

Amino acid-specific isotopes reveal changing five-dimensional niche segregation in Pacific seabirds over 50 years.

Hutchison's niche theory suggests that coexisting competing species occupy non-overlapping hypervolumes, which are theoretical spaces encompassing more than three dimensions, within an n-dimensional space. The analysis of multiple stable isotopes can be used to test these ideas where each isotope can be considered a dimension of niche space. These hypervolumes may change over time in response to variation in behaviour or habitat, within or among species, consequently changing the niche space itself. Here, we use isotopic values of carbon and nitrogen of ten amino acids, as well as sulphur isotopic values, to produce multi-isotope models to examine niche segregation among an assemblage of five coexisting seabird species (ancient murrelet Synthliboramphus antiquus, double-crested cormorant Phalacrocorax auritus, Leach's storm-petrel Oceanodrama leucorhoa, rhinoceros auklet Cerorhinca monocerata, pelagic cormorant Phalacrocorax pelagicus) that inhabit coastal British Columbia. When only one or two isotope dimensions were considered, the five species overlapped considerably, but segregation increased in more dimensions, but often in complex ways. Thus, each of the five species occupied their own isotopic hypervolume (niche), but that became apparent only when factoring the increased information from sulphur and amino acid specific isotope values, rather than just relying on proxies of δ15N and δ13C alone. For cormorants, there was reduction of niche size for both species consistent with a decline in their dominant prey, Pacific herring Clupea pallasii, from 1970 to 2006. Consistent with niche theory, cormorant species showed segregation across time, with the double-crested demonstrating a marked change in diet in response to prey shifts in a higher dimensional space. In brief, incorporating multiple isotopes (sulfur, PC1 of δ15N [baselines], PC2 of δ15N [trophic position], PC1 and PC2 of δ13C) metrics allowed us to infer changes and differences in food web topology that were not apparent from classic carbon-nitrogen biplots.

Enhanced dietary reconstruction of Korean prehistoric populations by combining δ13C and δ15N amino acids of bone collagen.

Compound specific stable isotope analysis of amino acids (CSIA-AA) is a powerful tool for determining dietary behaviors in complex environments and improving dietary reconstructions. Here, we conducted CSIA-AA on human (n = 32) and animal (n = 13) remains from two prehistoric archaeological sites (Mumun, Imdang) to assess in more detail the dietary sources consumed by prehistoric Korean populations. Results of estimated trophic position (TP) using Δ15NGlx-Phe show that the Imdang individuals consumed aquatic resources, as well as terrestrial resources. Principal component analysis (PCA) using δ13C and δ15N essential amino acid (EAA) values show that the Imdang humans closely cluster with game birds and terrestrial herbivores, whilst the Mumun humans closely cluster with C4 plants. Quantitative estimation by a Bayesian mixing model (MixSIAR) indicates that the Imdang humans derived a large proportion of their proteins from terrestrial animals and marine fish, whereas the main protein sources for the Mumun humans were C4 plants and terrestrial animals. Additionally, the comparison between the EAA and bulk isotope models shows that there is a tendency to overestimate the consumption of plant proteins when using bulk isotopic data. Our CSIA-AA approach reveals that in prehistoric Korea there were clear differences in human diets through time. This study adds to a growing body of literature that demonstrates the potential of CSIA-AA to provide more accurate estimations of protein consumption in mixed diets than previous bulk isotopic studies.

Stable Carbon Isotope Ratios (δ13C Values [‰]) of Individual Sterols in the Oils of C3, C4, and CAM Plants.

The compound-specific determination of δ13C values [‰] by gas chromatography interfaced with isotope ratio mass spectrometry (GC-IRMS) is a powerful analytical method to indicate minute but relevant variations in the 13C/12C ratio of sample compounds. In this study, the δ13C values [‰] of individual sterols were measured in eleven different oils of C3, C4, and CAM plants (n = 33) by GC-IRMS. For this purpose, a suitable acetylation method was developed for sterols. Nine of the eleven phytosterols identified by GC with mass spectrometry (GC/MS) could be measured by GC-IRMS. The δ13C values [‰] of individual sterols and squalene of C3 plant oils were between 3‰ and >16‰ more negative (lighter in carbon) than in C4 and CAM oils. We also showed that the blending of C4 oils into C3 oils (exemplarily conducted with one olive and one corn oil) would be precisely determined by means of the δ13C value [‰] of ß-sitosterol.

Linking African herbivore community enamel isotopes and environments: challenges, opportunities, and paleoecological implications.

Paleoenvironmental reconstructions of fossil sites based on isotopic analyses of enamel typically rely on data from multiple herbivore taxa, with the assumption that this dietary spectrum represents the community's isotopic range and provides insights into local or regional vegetation patterns. However, it remains unclear how representative the sampled taxa are of the broader herbivore community and how well these data correspond to specific ecosystems. Verifying these underlying assumptions is essential to refining the utility of enamel isotopic values for paleoenvironmental reconstructions. This study explores potential links between modern herbivore community carbon isotopic enamel spectra, biome types, and climate in sub-Saharan Africa. This region is one of the most comprehensively isotopically sampled areas globally and is of particular relevance to hominin evolution. Our extensive data compilation reveals that published enamel isotopic data from sub-Saharan Africa typically sample only a small percentage of the taxa documented at most localities and that some biome types (e.g., subtropical savannas) are dramatically overrepresented relative to others (e.g., forests) in these modern data sets. Multiple statistical analyses, including linear models and cluster analyses, revealed weak relationships of associated mammalian herbivore enamel isotopic values, biome type, and climate parameters. These results confound any simple assumptions about how community isotopic profiles map onto specific environments, highlighting the need for more precise strategic approaches in extending isotopic frameworks into the past for paleoecological reconstructions. Developing more refined modern analogs will ultimately allow us to more accurately characterize the isotopic spectra of paleo-communities and link isotopic dietary signatures to specific ecosystems.

Controlling effects of terrestrial organic matter on metal contamination and toxicity risks in port sediments.

The contents of metals, total carbon, total nitrogen (TN), total organic carbon (TOC), and stable isotope composition (δ13Corg and δ15N) of sediment organic matter (SOM) were investigated to explore the sources and spatial distribution of metals and SOM in the surface sediments (Kaohsiung Port, Taiwan). Results showed that TOC and metals in estuarine sediments are high, gradually decreasing toward the port entrances. The δ13Corg, δ15N, and TOC/TN ratios indicate that SOM comes mainly from terrestrial sources. This study proposes a befitting model between metal pollution and toxicity risk index and SOM sources in port sediments by combining stable isotope composition, correlation matrix, and multiple linear regression analysis. The model indicates that the degree of metal pollution and toxicity risk in sediments are mainly affected by TOCterr content and SOM source. The results help to understand the influence of organic matter sources in port sediments on metal concentration distribution.

Anaerobic biotransformation of hexachlorocyclohexane isomers in aqueous condition: Dual CCl isotope fractionation and impact on microbial community compositions.

Hexachlorocyclohexane (HCH) isomers are persistent organic pollutants (POPs) with high toxicity, lipid solubility, chemical stability. Despite the current ban on usage of Lindane, residual contamination cannot be ignored, and HCH are frequently detected in groundwater and threaten human health. Cultures capable of degrading α-HCH, ß-HCH, γ-HCH, and δ-HCH individually have been enriched in anoxic aqueous conditions. Compound-Specific Isotope Analysis (CSIA) was applied to examine the transformation mechanisms of different HCH isomers by the four enrichment cultures. 16S rRNA sequencing techniques were employed to examine the community composition of the enrichment cultures and detect changes in these communities resulting from adding individual HCH isomers. The results indicated that the ability of the enrichment cultures for dichloroelimination of HCH isomers was inconsistent. During dichloroelimination, different bond cleavage mode of ß- and δ-HCH led to distinct isotopic effects. HCH isomers had significant impact on the microbial community, while different microbial communities showed comparable isotopic effects during the transformation of a specific HCH isomer. In addition, bacteria in the phyla Proteobacteria and Firmicutes were proposed as the dominant dechlorinators. This study provides a novel perspective on the mode of bond cleavage during HCH dichloroelimination and the effect of HCH on microbial communities, which could potentially support the evaluation of HCH transformation by CSIA and their effects on the microecosystems of groundwater.

Tracking the transformation of persistent organic pollutants in food webs using multi element isotope and enantiomer fractionation.

In order to track the transformation of persistent organic pollutants (POPs) in food webs, field experiments were conducted at two sites using stable isotope and enantiomer fractionation concepts. The enantiomers of α-hexachlorocyclohexane (α-HCH) were selected as representative compounds for POPs. Isotope and enantiomer fractionation allowed the characterization of α-HCH enantiomer biotransformation processes along trophic levels of the food web - from soil and plants to animal livers, fat tissues and milk. The enrichment of heavy isotopes in soils, plants and sediments as well as the changes of enantiomer fractionation indicate that the biotransformation of α-HCH occurred in these compartments. Moreover, the increase of carbon and chlorine isotopic compositions as well as the changes of enantiomer fractionation of liver, fat tissues and milk demonstrated that the overall HCH exposure was much higher than estimates based on concentration levels, while the isotope and enantiomer fractionation revealed the enantiomer specific enantiomer uptake across the blood-brain barriers. Dual element isotope analysis suggested that complex transformation processes have occurred along the potential food web from the HCH sources over different environmental compartments to animal livers, fat tissues and milk. The results imply that the analyses of stable isotope compositions and concentrations has potential to reconstruct the exposure of higher organisms to POPs.

Partial mycoheterotrophy in the leafless orchid Eulophia zollingeri specialized on wood-decaying fungi.

Although the absence of normal leaves is often considered a sign of full heterotrophy, some plants remain at least partially autotrophic despite their leafless habit. Leafless orchids with green stems and capsules probably represent a late evolutionary stage toward full mycoheterotrophy and serve as valuable models for understanding the pathways leading to this nutritional strategy. In this study, based on molecular barcoding and isotopic analysis, we explored the physiological ecology of the leafless orchid Eulophia zollingeri, which displays green coloration, particularly during its fruiting phase. Although previous studies had shown that E. zollingeri, in its adult stage, is associated with Psathyrellaceae fungi and exhibits high 13C isotope signatures similar to fully mycoheterotrophic orchids, it remained uncertain whether this symbiotic relationship is consistent throughout the orchid's entire life cycle and whether the orchid relies exclusively on mycoheterotrophy for its nutrition during the fruiting season. Our study has demonstrated that E. zollingeri maintains a specialized symbiotic relationship with Psathyrellaceae fungi throughout all life stages. However, isotopic analysis and chlorophyll data have shown that the orchid also engages in photosynthesis to meet its carbon needs, particularly during the fruiting stage. This research constitutes the first discovery of partial mycoheterotrophy in leafless orchids associated with saprotrophic non-rhizoctonia fungi.

Effects of HCO3- and CO2 conversion rates on carbon assimilation strategies in marine microalgae: Implication by stable carbon isotope analysis of fatty acids.

Marine microalgae are an essential component of marine plankton and critical primary producers, playing a vital role in marine ecosystems. The seawater carbonate system is a dynamic equilibrium system, and changes in any component can alter the carbonate balance. In CO2-concentrating mechanisms (CCMs), carbonic anhydrase (CA) regulates CO2 concentration by catalyzing the interconversion between CO2 and HCO3-. Therefore, limiting the activity of extracellular carbonic anhydrase (exCA) alters the rate at which carbonate equilibrium is reached and further affects the carbon assimilation process in microalgae. In this study, two different microalgae, Phaeodactylum tricornutum and Nannochloropsis oceanica, were selected to investigate the effects of changes in the carbonate system on photosynthetic carbon assimilation in microalgae by inhibiting exCA activity with acetazolamide (AZ). Inhibition of exCA activity reduces specific growth rates and photosynthetic efficiency of microalgae. The total alkalinity, HCO3- concentration, and CO2 concentration of the cultures increased with the decrease of pH, but the changes of the ribulose 1,5- bisphosphate carboxylase/oxygenase (Rubisco) activities of the two microalgae were different. In addition, the two microalgae possessed different lipid and carbohydrate synthesis strategies, but both restricted triacylglycerol (TAG) synthesis. Meanwhile, the microalgal cells had to utilize more 13CO2 when HCO3- and CO2 conversion rates were limited and restricted. This led to the continuous accumulation of 13C in fatty acids and the elevation of δ13CFAs. In conclusion, our study provides a new perspective on the role of microalgae in correlating carbonate changes with photosynthetic carbon assimilation strategies under mechanistic constraints on inorganic carbon utilization.

Current methods for hyperpolarized [1-13C]pyruvate MRI human studies.

MRI with hyperpolarized (HP) 13C agents, also known as HP 13C MRI, can measure processes such as localized metabolism that is altered in numerous cancers, liver, heart, kidney diseases, and more. It has been translated into human studies during the past 10 years, with recent rapid growth in studies largely based on increasing availability of HP agent preparation methods suitable for use in humans. This paper aims to capture the current successful practices for HP MRI human studies with [1-13C]pyruvate-by far the most commonly used agent, which sits at a key metabolic junction in glycolysis. The paper is divided into four major topic areas: (1) HP 13C-pyruvate preparation; (2) MRI system setup and calibrations; (3) data acquisition and image reconstruction; and (4) data analysis and quantification. In each area, we identified the key components for a successful study, summarized both published studies and current practices, and discuss evidence gaps, strengths, and limitations. This paper is the output of the "HP 13C MRI Consensus Group" as well as the ISMRM Hyperpolarized Media MR and Hyperpolarized Methods and Equipment study groups. It further aims to provide a comprehensive reference for future consensus, building as the field continues to advance human studies with this metabolic imaging modality.

Finding balance: Tree-ring isotopes differentiate between acclimation and stress-induced imbalance in a long-term irrigation experiment.

Scots pine (Pinus sylvestris L.) is a common European tree species, and understanding its acclimation to the rapidly changing climate through physiological, biochemical or structural adjustments is vital for predicting future growth. We investigated a long-term irrigation experiment at a naturally dry forest in Switzerland, comparing Scots pine trees that have been continuously irrigated for 17 years (irrigated) with those for which irrigation was interrupted after 10 years (stop) and non-irrigated trees (control), using tree growth, xylogenesis, wood anatomy, and carbon, oxygen and hydrogen stable isotope measurements in the water, sugars and cellulose of plant tissues. The dendrochronological analyses highlighted three distinct acclimation phases to the treatments: irrigated trees experienced (i) a significant growth increase in the first 4 years of treatment, (ii) high growth rates but with a declining trend in the following 8 years and finally (iii) a regression to pre-irrigation growth rates, suggesting the development of a new growth limitation (i.e. acclimation). The introduction of the stop treatment resulted in further growth reductions to below-control levels during the third phase. Irrigated trees showed longer growth periods and lower tree-ring δ13 C values, reflecting lower stomatal restrictions than control trees. Their strong tree-ring δ18 O and δ2 H (O-H) relationship reflected the hydrological signature similarly to the control. On the contrary, the stop trees had lower growth rates, conservative wood anatomical traits, and a weak O-H relationship, indicating a physiological imbalance. Tree vitality (identified by crown transparency) significantly modulated growth, wood anatomical traits and tree-ring δ13 C, with low-vitality trees of all treatments performing similarly regardless of water availability. We thus provide quantitative indicators for assessing physiological imbalance and tree acclimation after environmental stresses. We also show that tree vitality is crucial in shaping such responses. These findings are fundamental for the early assessment of ecosystem imbalances and decline under climate change.

Efficient palladium-catalyzed electrocarboxylation enables late-stage carbon isotope labelling.

Carbon isotope labelling of bioactive molecules is essential for accessing the pharmacokinetic and pharmacodynamic properties of new drug entities. Aryl carboxylic acids represent an important class of structural motifs ubiquitous in pharmaceutically active molecules and are ideal targets for the installation of a radioactive tag employing isotopically labelled CO2. However, direct isotope incorporation via the reported catalytic reductive carboxylation (CRC) of aryl electrophiles relies on excess CO2, which is incompatible with carbon-14 isotope incorporation. Furthermore, the application of some CRC reactions for late-stage carboxylation is limited because of the low tolerance of molecular complexity by the catalysts. Herein, we report the development of a practical and affordable Pd-catalysed electrocarboxylation setup. This approach enables the use of near-stoichiometric 14CO2 generated from the primary carbon-14 source Ba14CO3, facilitating late-stage and single-step carbon-14 labelling of pharmaceuticals and representative precursors. The proposed isotope-labelling protocol holds significant promise for immediate impact on drug development programmes.