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Tropical peatlands are carbon-dense ecosystems that are significant sources of atmospheric methane (CH4). Recent work has demonstrated the importance of trees as an emission pathway for CH4 from the peat to the atmosphere. However, there remain questions over the processes of CH4 production in these systems and how they relate to substrate supply. Principally, these questions relate to the relative contribution of recent photosynthetically fixed carbon, released as root exudates, versus carbon substrate supply from the slowly decomposing peat matrix to CH4 emissions within these ecosystems. Here, we examined the role of root inputs in regulating CH4 production inferred from soil emissions using a combination of in situ tree girdling, in situ13C natural abundance labelling via stem injections, and a 13CO2 labelling of transplanted plants of two contrasting plant functional types, a broadleaved evergreen tree, and a canopy palm. Girdling of broadleaved evergreen trees reduced CH4 fluxes by up to 67%. Stem injections of trees and palms with a natural abundance label resulted in significant isotopic enrichment of CH4 fluxes, reinforcing the link between root carbon inputs and peat CH4 fluxes. Ex situ13CO2 labelling of plants resulted in significant 13C enrichment of peat CH4 fluxes. Taken together, our results demonstrate for the first time that plant root exudates make a substantial contribution to CH4 production in tropical peatlands. [ABSTRACT FROM AUTHOR]

Despite considerable experimental effort, the physiological mechanisms governing temperate tree species' water and carbon dynamics before the onset of the growing period remain poorly understood. We applied 2H‐enriched water during winter dormancy to the soil of four potted European tree species. After 8 weeks of chilling, hydrogen isotopes in stem, twig and bud water were measured six times during 2 consecutive weeks of forcing conditions (Experiment 1). Additionally, we pulse‐labelled above‐ground plant tissues using 2H‐enriched water vapour and 13C‐enriched CO2 7 days after exposure to forcing conditions to trace atmospheric water and carbon uptake (Experiment 2). Experiment 1 revealed soil water incorporation into the above‐ground organs of all species during the chilling phase and significant species‐specific differences in water allocation during the forcing conditions, which we attributed to differences in structural traits. Experiment 2 illustrated water vapour incorporation into all above‐ground tissue of all species. However, the incorporation of carbon was found for evergreen saplings only. Our results suggest that temperate trees take up and reallocate soil water and absorb atmospheric water to maintain sufficient above‐ground tissue hydration during winter. Therefore, our findings provide new insights into the water allocation dynamics of temperate trees during early spring. Summary statement: Our stable isotope labelling study demonstrates that tree saplings take up soil and atmospheric water to hydrate above‐ground tissues before budburst. However, no carbon uptake was found for the investigated deciduous species during this period. [ABSTRACT FROM AUTHOR]

The response to acute myotoxic injury requires stimulation of local repair mechanisms in the damaged tissue. However, satellite cells in muscle distant from acute injury have been reported to enter a functional state between quiescence and active proliferation. Here, we asked whether protein flux rates are altered in muscle distant from acute local myotoxic injury and how they compare to changes in gene expression from the same tissue. Broad and significant alterations in protein turnover were observed across the proteome in the limb contralateral to injury during the first 10 days after. Interestingly, mRNA changes had almost no correlation with directly measured protein turnover rates. In summary, we show consistent and striking changes in protein flux rates in muscle tissue contralateral to myotoxic injury, with no correlation between changes in mRNA levels and protein synthesis rates. This work motivates further investigation of the mechanisms, including potential neurological factors, responsible for this distant effect. Key points: Previous literature demonstrates that stem cells of uninjured muscle respond to local necrotic muscle tissue damage and regeneration.We show that muscle tissue that was distant from a model of local necrotic damage had functional changes at both the gene expression and the protein turnover level.However, these changes in distant tissue were more pronounced during the earlier stages of tissue regeneration and did not correlate well with each other.The results suggest communication between directly injured tissue and non‐affected tissues that are distant from injury, which warrants further investigation into the potential of this mechanism as a proactive measure for tissue regeneration from damage. [ABSTRACT FROM AUTHOR]

Here we report the efficient synthetic access to 13C/15N‐labelled pseudouridine phosphoramidites, which were incorporated into a binary H/ACA box guide RNA/product complex comprising 77 nucleotides (nts) in total and into a 75 nt E. coli tRNAGly. The stable isotope (SI) labelled pseudouridines were produced via a highly efficient chemo‐enzymatic synthesis. 13C/15N labelled uracils were produced via chemical synthesis and enzymatically converted to pseudouridine 5′‐monophosphate (ΨMP) by using YeiN, a Ψ‐5′‐monophosphate C‐glycosidase. Removal of the 5′‐phosphate group yielded the desired pseudouridine nucleoside (Ψ), which was transformed into a phosphoramidite building suitable for RNA solid phase synthesis. A Ψ ‐building block carrying both a 13C and a 15N label was incorporated into a product RNA and the complex formation with a 63 nt H/ACA box RNA could be observed via NMR. Furthermore, the SI labelled pseudouridine building block was used to determine imino proton bulk water exchange rates of a 75 nt E. coli tRNAGly CCmnm5U, identifying the TΨC‐loop 5‐methyluridine as a modifier of the exchange rates. The efficient synthetic access to SI‐labelled Ψ building blocks will allow the solution and solid‐state NMR spectroscopic studies of Ψ containing RNAs and will facilitate the mass spectrometric analysis of Ψ‐modified nucleic acids. [ABSTRACT FROM AUTHOR]

The field of metabolomics has gained tremendous interest in recent years. Whether the goal is to discover biomarkers related to certain pathologies or to better understand the impact of a drug or contaminant, numerous studies have demonstrated how crucial it is to understand variations in metabolism. Detailed knowledge of metabolic variabilities can lead to more effective treatments, as well as faster or less invasive diagnostics. Exploratory approaches are often employed in metabolomics, using relative quantitation to look at perturbations between groups of samples. Most metabolomics studies have been based on metabolite profiling using relative quantitation, with very few studies using an approach for absolute quantitation. Using accurate quantitation facilitates the comparison between different studies, as well as enabling longitudinal studies. In this review, we discuss the most widely used techniques for quantitative metabolomics using mass spectrometry (MS). Various aspects will be addressed, such as the use of external and/or internal standards, derivatization techniques, in vivo isotopic labelling, or quantitative MS imaging. The principles, as well as the associated limitations and challenges, will be described for each approach. [ABSTRACT FROM AUTHOR]

Earlier spring growth onset in temperate forests is a visible effect of global warming that alters global water and carbon cycling. Consequently, it becomes crucial to accurately predict the future spring phenological shifts in vegetation under different climate warming scenarios. However, current phenological models suffer from a lack of physiological insights of tree dormancy and are rarely experimentally validated. Here, we sampled twig cuttings of five deciduous tree species at two climatically different locations (270 and 750 m a.s.l. ~ 2.3 °C difference) throughout the winter of 2019–20. Twig budburst success, thermal time to budburst, bud water content and short-term 2H-labelled water uptake into buds were quantified to link bud dormancy status with vascular water transport efficacy, with the objective of establishing connections between the dormancy status of buds and their effectiveness in vascular water transport. We found large differences in the dormancy status between species throughout the entire investigation period, likely reflecting species-specific environmental requirements to initiate and release dormancy, whereas only small differences in the dormancy status were found between the two studied sites. We found strong 2H-labelled water uptake into buds during leaf senescence, followed by a sharp decrease, which we ascribed to the initiation of endodormancy. However, surprisingly, we did not find a progressive increase in 2H-labelled water uptake into buds as winter advanced. Nonetheless, all examined tree species exhibited a consistent relationship between bud water content and dormancy status. Our results suggest that short-term 2H-labelled water uptake may not be a robust indicator of dormancy release, yet it holds promise as a method for tracking the induction of dormancy in deciduous trees. By contrast, bud water content emerges as a cost-effective and more reliable indicator of dormancy release. [ABSTRACT FROM AUTHOR]

/Objective: The extent of exocrine pancreatic insufficiency (EPI) in the paediatric population with acute pancreatitis (AP) is unknown. The primary objective was to use a 6 h stable-isotope breath test to determine the prevalence of EPI in children with AP. The secondary objective was to determine the diagnostic ability of a 4 h abbreviated breath test in the detection of EPI. 13C-mixed triglyceride (MTG) breath test was used to measure fat digestibility in 12 children with AP and 12 normal children. EPI was diagnosed based on a cumulative dose percentage recovery (cPDR) cut-off value < 26.8% present in literature. To reduce the test burden, the diagnostic accuracy of an abbreviated 4 h test was evaluated, using a cPDR cut-off that was the 2.5th percentile of its distribution in control children. The cPDR of cases was significantly lower than that of controls (27.71 ± 7.88% vs 36.37 ± 4.70%, p = 0.005). The cPDR during acute illness was not significantly different to that at 1 month follow up (24.69 ± 6.83% vs 26.98 ± 11.10%, p = 0.52). The 4 h and 6 h breath test results correlated strongly (r = 0.93, p < 0.001) with each other. The new 4 h test had 87.5% sensitivity and 93.8% specificity for detecting EPI. Two-thirds (66.7%) of this sample of children with AP had EPI during admission, which persisted at 1 month follow up. The 4 h abbreviated 13C-MTG breath test has good diagnostic ability to detect EPI in children and may improve its clinical utility in this age group. [ABSTRACT FROM AUTHOR]

Background and Aims Plant nitrogen (N) acquisition via arbuscular mycorrhizal fungi (AMF) serves as a dominant pathway in the N nutrition of many plants, but the functional impact of AMF in acquisition of N by wetland plants has not been well quantified. Subtropical lake–wetland ecosystems are characterized by seasonal changes in the water table and low N availability in soil. Yet, it is unclear whether and how AMF alters the N acquisition pattern of plants for various forms of N and how this process is influenced by soil water conditions. Methods We performed a pot study with Carex thunbergii that were either colonized by AMF or not colonized and also subjected to different water conditions. We used 15N labelling to track plant N uptake. Key Results Colonization by AMF had little effect on the biomass components of C. thunbergii but did significantly affect the plant functional traits and N acquisition in ways that were dependent on the soil water conditions. The N uptake rate of AMF-colonized plants was significantly lower than that of the non-colonized plants in conditions of low soil water. A decreased NO3− uptake rate in AMF-colonized plants reduced the N:P ratio of the plants. Although C. thunbergii predominantly took up N in the form of NO3−, higher water availability increased the proportion of N taken up as NH4+, irrespective of the inoculation status. Conclusions These results emphasize the importance of AMF colonization in controlling the N uptake strategies of plants and can improve predictions of N budget under the changing water table conditions in this subtropical wetland ecosystem. [ABSTRACT FROM AUTHOR]

Identifying metabolites in model organisms is critical for many areas of biology, including unravelling disease aetiology or elucidating functions of putative enzymes. Even now, hundreds of predicted metabolic genes in Saccharomyces cerevisiae remain uncharacterized, indicating that our understanding of metabolism is far from complete even in well-characterized organisms. While untargeted high-resolution mass spectrometry (HRMS) enables the detection of thousands of features per analysis, many of these have a non-biological origin. Stable isotope labelling (SIL) approaches can serve as credentialing strategies to distinguish biologically relevant features from background signals, but implementing these experiments at large scale remains challenging. Here, we developed a SIL-based approach for high-throughput untargeted metabolomics in S. cerevisiae, including deep-48 well format-based cultivation and metabolite extraction, building on the peak annotation and verification engine (PAVE) tool. Aqueous and nonpolar extracts were analysed using HILIC and RP liquid chromatography, respectively, coupled to Orbitrap Q Exactive HF mass spectrometry. Of the approximately 37,000 total detected features, only 3–7% of the features were credentialed and used for data analysis with open-source software such as MS-DIAL, MetFrag, Shinyscreen, SIRIUS CSI:FingerID, and MetaboAnalyst, leading to the successful annotation of 198 metabolites using MS2 database matching. Comparable metabolic profiles were observed for wild-type and sdh1Δ yeast strains grown in deep-48 well plates versus the classical shake flask format, including the expected increase in intracellular succinate concentration in the sdh1Δ strain. The described approach enables high-throughput yeast cultivation and credentialing-based untargeted metabolomics, providing a means to efficiently perform molecular phenotypic screens and help complete metabolic networks. [ABSTRACT FROM AUTHOR]

Abstract Methane (CH4) formation by vegetation has been studied intensively over the last 15 years. However, reported CH4 emissions vary by several orders of magnitude, thus making global estimates difficult. Moreover, the mechanism(s) for CH4 formation by plants is (are) largely unknown. Here, we introduce a new approach for making CH4 formation by plants clearly visible. By application of 13C‐labeled dimethyl sulfoxide (DMSO) onto the leaves of tobacco plants (Nicotiana tabacum) and Chinese silver grass (Miscanthus sinensis) the effect of light and dark conditions on CH4 formation of this pathway was examined by monitoring stable carbon isotope ratios of headspace CH4 (δ13C‐CH4 values). Both plant species showed increasing headspace δ13C‐CH4 values while exposed to light. Higher light intensities increased CH4 formation rates in N. tabacum but decreased rates for M. sinensis. In the dark no formation of CH4 could be detected for N. tabacum, while M. sinensis still produced ~50% of CH4 compared to that during light exposure. Our findings suggest that CH4 formation is clearly dependent on light conditions and plant species and thus indicate that DMSO is a potential precursor of vegetative CH4. The novel isotope approach has great potential to investigate, at high temporal resolution, physiological, and environmental factors that control pathway‐specific CH4 emissions from plants.

The major benefits of integrating ion mobility (IM) into LC–MS methods for small molecules are the additional separation dimension and especially the use of IM-derived collision cross sections (CCS) as an additional ion-specific identification parameter. Several large CCS databases are now available, but outliers in experimental interplatform IM-MS comparisons are identified as a critical issue for routine use of CCS databases for identity confirmation. We postulate that different routine external calibration strategies applied for traveling wave (TWIM-MS) in comparison to drift tube (DTIM-MS) and trapped ion mobility (TIM-MS) instruments is a critical factor affecting interplatform comparability. In this study, different external calibration approaches for IM-MS were experimentally evaluated for 87 steroids, for which TWCCSN2, DTCCSN2 and TIMCCSN2 are available. New reference CCSN2 values for commercially available and class-specific calibrant sets were established using DTIM-MS and the benefit of using consolidated reference values on comparability of CCSN2 values assessed. Furthermore, use of a new internal correction strategy based on stable isotope labelled (SIL) internal standards was shown to have potential for reducing systematic error in routine methods. After reducing bias for CCSN2 between different platforms using new reference values (95% of TWCCSN2 values fell within 1.29% of DTCCSN2 and 1.12% of TIMCCSN2 values, respectively), remaining outliers could be confidently classified and further studied using DFT calculations and CCSN2 predictions. Despite large uncertainties for in silico CCSN2 predictions, discrepancies in observed CCSN2 values across different IM-MS platforms as well as non-uniform arrival time distributions could be partly rationalized. [ABSTRACT FROM AUTHOR]

Biotransformation reactions that xenobiotics undergo during their metabolism are crucial for their proper excretion from the body, but can also be a source of toxicity, especially in the case of reactive metabolite formation. Unstable, reactive metabolites are capable of covalent binding to proteins, and have often been linked to liver damage and other undesired side effects. A common technique to assess the formation of reactive metabolites employs trapping them in vitro with glutathione and characterizing the resulting adducts by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Some endogenous compounds, however, can interfere with xenobiotic metabolites of interest, making the analysis more difficult. This study demonstrates the usefulness of isotope-labeled compounds to detect and elucidate the structures of both stable metabolites and trapped adducts of three estrogen analogs using an untargeted LC-MS/MS workflow. The metabolism of estradiol, estrone and ethinyl estradiol was investigated. Unlabeled and deuterated versions of these three compounds were incubated with human or rat liver microsomes in the presence of two different trapping agents, namely glutathione and N-acetylcysteine. The detection of closely eluting deuterated peaks allowed us to confirm the formation of several known metabolites, as well as many previously uncharacterized ones. The structure of each adduct was elucidated by the detailed analysis of high-resolution MS/MS spectra for elucidating fragmentation pathways with accurate mass measurements. The use of isotopic labeling was crucial in helping confirm many metabolites and adduct structures, as well as removing endogenous interferences. [ABSTRACT FROM AUTHOR]

Methane (CH4) formation by vegetation has been studied intensively over the last 15 years. However, reported CH4 emissions vary by several orders of magnitude, thus making global estimates difficult. Moreover, the mechanism(s) for CH4 formation by plants is (are) largely unknown.Here, we introduce a new approach for making CH4 formation by plants clearly visible. By application of 13C‐labeled dimethyl sulfoxide (DMSO) onto the leaves of tobacco plants (Nicotiana tabacum) and Chinese silver grass (Miscanthus sinensis) the effect of light and dark conditions on CH4 formation of this pathway was examined by monitoring stable carbon isotope ratios of headspace CH4 (δ13C‐CH4 values).Both plant species showed increasing headspace δ13C‐CH4 values while exposed to light. Higher light intensities increased CH4 formation rates in N. tabacum but decreased rates for M. sinensis. In the dark no formation of CH4 could be detected for N. tabacum, while M. sinensis still produced ~50% of CH4 compared to that during light exposure.Our findings suggest that CH4 formation is clearly dependent on light conditions and plant species and thus indicate that DMSO is a potential precursor of vegetative CH4. The novel isotope approach has great potential to investigate, at high temporal resolution, physiological, and environmental factors that control pathway‐specific CH4 emissions from plants. [ABSTRACT FROM AUTHOR]

Stable isotope labelling is state‐of‐the‐art in quantitative mass spectrometry, yet often accessing the required standards is cumbersome and very expensive. Here, a unifying synthetic concept for 18O‐labelled phosphates is presented, based on a family of modified 18O2‐phosphoramidite reagents. This toolbox offers access to major classes of biologically highly relevant phosphorylated metabolites as their isotopologues including nucleotides, inositol phosphates, ‐pyrophosphates, and inorganic polyphosphates. 18O‐enrichment ratios >95 % and good yields are obtained consistently in gram‐scale reactions, while enabling late‐stage labelling. We demonstrate the utility of the 18O‐labelled inositol phosphates and pyrophosphates by assignment of these metabolites from different biological matrices. We demonstrate that phosphate neutral loss is negligible in an analytical setup employing capillary electrophoresis electrospray ionisation triple quadrupole mass spectrometry. [ABSTRACT FROM AUTHOR]

Stable isotope labelling in combination with high-resolution mass spectrometry approaches are increasingly used to analyze both metabolite and protein modification dynamics. To enable correct estimation of the resulting dynamics, it is critical to correct the measured values for naturally occurring stable isotopes, a process commonly called isotopologue correction or deconvolution. While the importance of isotopologue correction is well recognized in metabolomics, it has received far less attention in proteomics approaches. Although several tools exist that enable isotopologue correction of mass spectrometry data, the majority is tailored for the analysis of low molecular weight metabolites. We here present PICor which has been developed for isotopologue correction of complex isotope labelling experiments in proteomics or metabolomics and demonstrate the importance of appropriate correction for accurate determination of protein modifications dynamics, using histone acetylation as an example. [ABSTRACT FROM AUTHOR]

The desirable wine aroma compounds 3-sulfanylhexan-1-ol (3SH) and 3-sulfanylhexyl acetate (3SHA) are released during fermentation from non-volatile precursors present in the grapes. This work explores the relative contribution of four precursors (E -2-hexenal, 3- S -glutathionylhexan-1-ol, 3- S -glutathionylhexanal, and 3- S -cysteinylhexan-1-ol) to 3SH and 3SHA. Through the use of isotopically labelled analogues of these precursors in defined fermentation media, new insights into the role of each precursor have been identified. E -2-Hexenal was shown to contribute negligible amounts of thiols, while 3- S -glutathionylhexan-1-ol was the main precursor of both 3SH and 3SHA. The glutathionylated precursors were both converted to 3SHA more efficiently than 3- S -cysteinylhexan-1-ol. Interestingly, 3- S -glutathionylhexanal generated 3SHA without detectable concentrations of 3SH, suggesting possible differences in the way this precursor is metabolised compared to 3- S -glutathionylhexan-1-ol and 3- S -cysteinylhexan-1-ol. We also provide the first evidence for chemical conversion of 3- S -glutathionylhexan-1-ol to 3- S -(γ-glutamylcysteinyl)-hexan-1-ol in an oenological system. [Display omitted] • First evidence of chemical degradation of Glut3SH in wine-like conditions. • Glut3SHal behaves similarly to Glut3SH in terms of conversion to 3SHA. • Cys3SH is not well converted to 3SHA in this model system. • Glut3SH is the main precursor to 3SH and 3SHA in wine-like conditions. • E-2-Hexenal is not a meaningful precursor to 3SH during fermentation. [ABSTRACT FROM AUTHOR]

The metabolism of Legionella pneumophila strain Paris was elucidated during different time intervals of growth within its natural host Acanthamoeba castellanii. For this purpose, the amoebae were supplied after bacterial infection (t =0 h) with 11 mM [U-13C6]glucose or 3 mM [U-13C3]serine, respectively, during 0−17 h, 17−25 h, or 25−27 h of incubation. At the end of these time intervals, bacterial and amoebal fractions were separated. Each of these fractions was hydrolyzed under acidic conditions. 13C-Enrichments and isotopologue distributions of resulting amino acids and 3-hydroxybutyrate were determined by gas chromatography – mass spectrometry. Comparative analysis of the labelling patterns revealed the substrate preferences, metabolic pathways, and relative carbon fluxes of the intracellular bacteria and their amoebal host during the time course of the infection cycle. Generally, the bacterial infection increased the usage of exogenous glucose via glycolysis by A. castellanii. In contrast, carbon fluxes via the amoebal citrate cycle were not affected. During the whole infection cycle, intracellular L. pneumophila incorporated amino acids from their host into the bacterial proteins. However, partial bacterial de novo biosynthesis from exogenous 13C-Ser and, at minor rates, from 13C-glucose could be shown for bacterial Ala, Asp, Glu, and Gly. More specifically, the catabolic usage of Ser increased during the post-exponential phase of intracellular growth, whereas glucose was utilized by the bacteria throughout the infection cycle and not only late during infection as assumed on the basis of earlier in vitro experiments. The early usage of 13C-glucose by the intracellular bacteria suggests that glucose availability could serve as a trigger for replication of L. pneumophila inside the vacuoles of host cells.

The upside-down jellyfish Cassiopea engages in symbiosis with photosynthetic microalgae that facilitate uptake and recycling of inorganic nutrients. By contrast to most other symbiotic cnidarians, algal endosymbionts in Cassiopea are not restricted to the gastroderm but are found in amoebocyte cells within the mesoglea. While symbiont-bearing amoebocytes are highly abundant, their role in nutrient uptake and cycling in Cassiopea remains unknown. By combining isotopic labelling experiments with correlated scanning electron microscopy, and Nano-scale secondary ion mass spectrometry (NanoSIMS) imaging, we quantified the anabolic assimilation of inorganic carbon and nitrogen at the subcellular level in juvenile Cassiopea medusae bell tissue. Amoebocytes were clustered near the sub-umbrella epidermis and facilitated efficient assimilation of inorganic nutrients. Photosynthetically fixed carbon was efficiently translocated between endosymbionts, amoebocytes and host epidermis at rates similar to or exceeding those observed in corals. The Cassiopea holobionts efficiently assimilated ammonium, while no nitrate assimilation was detected, possibly reflecting adaptation to highly dynamic environmental conditions of their natural habitat. The motile amoebocytes allow Cassiopea medusae to distribute their endosymbiont population to optimize access to light and nutrients, and transport nutrition between tissue areas. Amoebocytes thus play a vital role for the assimilation and translocation of nutrients in Cassiopea, providing an interesting new model for studies of metabolic interactions in photosymbiotic marine organisms. [ABSTRACT FROM AUTHOR]

Warming in the Arctic accelerates top‐soil decomposition and deep‐soil permafrost thaw. This may lead to an increase in plant‐available nutrients throughout the active layer soil and near the permafrost thaw front. For nitrogen (N) limited high arctic plants, increased N availability may enhance growth and alter community composition, importantly affecting the ecosystem carbon balance. However, the extent to which plants can take advantage of this newly available N may be constrained by the following three factors: vertical distribution of N within the soil profile, timing of N‐release, and competition with other plants and microorganisms. Therefore, we investigated species‐ and depth‐specific plant N uptake in a high arctic tundra, northeastern Greenland. Using stable isotopic labelling (15N‐NH4+), we simulated autumn N‐release at three depths within the active layer: top (10 cm), mid (45 cm) and deep‐soil near the permafrost thaw front (90 cm). We measured plant species‐specific N uptake immediately after N‐release (autumn) and after 1 year, and assessed depth‐specific microbial N uptake and resource partitioning between above‐ and below‐ground plant parts, microorganisms and soil. We found that high arctic plants actively foraged for N past the peak growing season, notably the graminoid Kobresia myosuroides. While most plant species (Carex rupestris, Dryas octopetala, K. myosuroides) preferred top‐soil N, the shrub Salix arctica also effectively acquired N from deeper soil layers. All plants were able to obtain N from the permafrost thaw front, both in autumn and during the following growing season, demonstrating the importance of permafrost‐released N as a new N source for arctic plants. Finally, microbial N uptake markedly declined with depth, hence, plant access to deep‐soil N pools is a competitive strength. In conclusion, plant species‐specific competitive advantages with respect to both time‐ and depth‐specific N‐release may dictate short‐ and long‐term plant community changes in the Arctic and consequently, larger‐scale climate feedbacks. [ABSTRACT FROM AUTHOR]

Rubber trees (Hevea brasiliensis) are the main source of natural rubber, extracted from latex, which exudes from the trunk after tapping. Tapped trees require large amounts of carbon (C) to regenerate the latex after its collection. Knowing the contribution of C sources involved in latex biosynthesis will help in understanding how rubber trees face this additional C demand. Whole crown 13CO2 pulse labelling was performed on 4-year-old rubber trees in June, when latex production was low, and in October, when it was high. 13C content was quantified in the foliage, phloem sap, wood, and latex. In both labelling periods, 13C was recovered in latex just after labelling, indicating that part of the carbohydrate was directly allocated to latex. However, significant amounts of 13C were still recovered in latex after 100 d and the peak was reached significantly later than in phloem sap, demonstrating the contribution of a reserve pool as a source of latex C. The contribution of new photosynthates to latex regeneration was faster and higher when latex metabolism was well established, in October, than in June. An improved understanding of C dynamics and the source–sink relationship in rubber tree is crucial to adapt tapping system practices and ensure sustainable latex production. [ABSTRACT FROM AUTHOR]

Aims: Here we assessed N and P uptake of four grassland species grown together in response to a short-term drought event along a soil P gradient. Methods: We used 15N and 32P tracers to examine uptake of N and P by the grasses Bothriochloa macra, Themeda triandra, Lolium perenne and Microlaena stipoides grown together in pots with initial available P levels of 3, 8, 12, 20 mg P kg−1 soil. Soil moisture in half the pots was reduced from 60 to 30% water holding capacity during a 7-day period to simulate drought. Results: Plant P uptake was strongly reduced by drought in all species across all P levels, much more so than N uptake, indicating decoupling in N and P uptake. Soil available P (Bray method) was not affected by drought, suggesting that the reduced P uptake with drought was due to reduced soil P mobility. Plant competition for N and P changed with drought and soil P levels, where relatively more N and P was taken up with drought by M. stipoides at the lowest soil P level. Conclusions: We showed that greater reductions in P than in N uptake and shifts in N and P uptake among species caused by a short-term drought have strong consequences for plant growth. [ABSTRACT FROM AUTHOR]

Arbuscular mycorrhizal fungi are obligate symbionts of land plants; furthermore, some of the species harbor endobacteria. Although the molecular approach increased our knowledge of the diversity and origin of the endosymbiosis and its metabolic possibilities, experiments to address the functions of the fungal host have been limited. In this study, a C flow of the fungus to the bacteria was investigated. Onion seedlings colonized with Gigaspora margarita, possessing Candidatus Glomeribacter gigasporarum (CaGg, Gram-negative, resides in vacuole) and Candidatus Moeniiplasma glomeromycotorum (CaMg, Gram-positive, resides in the cytoplasm,) were labelled with 13CO2. The 13C localization within the mycorrhiza was analyzed using high-resolution secondary ion mass spectrometry (SIMS). Correlative TEM-SIMS analysis of the fungal cells revealed that the 13C/12C ratio of CaGg was the lowest among CaMg and mitochondria and was the highest in the cytoplasm. By contrast, the plant cells, mitochondria, plastids, and fungal cytoplasm, which are contributors to the host, showed significantly higher 13C enrichment than the host cytoplasm. The C allocation patterns implied that CaMg has a greater impact than CaGg on G. margarita, but both seemed to be less burdensome to the host fungus in terms of C cost.

Comparative glycosylation analysis of biopharmaceuticals requires the development of methods that deliver the necessary throughput, support structural elucidation and relative quantitation of glycans released from therapeutics. The current study presents the development and applicability assessment of a twoplex approach using light and heavy isotopolouges of 3-aminobenzenesulfonic acid (3-ASA) under wet labeling conditions followed by UHPLC-MS analysis in data dependent acquisition mode. Excellent labelling efficiency, >90%, was achieved for both the light and heavy variants of the reagent. Glycan distributions of two human IgG lots labeled by light and heavy isotopolouges were identical, demonstrating no labeling bias introduced by either of the isotopologues. Peak area distributions of glycan profiles of two human IgG lots were compared to 2-aminobenzamide (2-AB) and RapiFluor-MS protocols. The comparison led to identical results in peak area distribution across the three dyes, but differences in chromatographic selectivity attributed to the different tags. MS1 based relative quantitation was further validated by releasing glycans from the same lot of human IgG, with glycan pools obtained labeled with light and heavy isotopologues separately, followed by mixing and clean-up of the same amount of light and heavy labeled glycan pools. MS analyses of each glycan resulted in a ratio of light and heavy XIC in the range of 0.97 ≤ x ≤ 1.05, demonstrating the method is amenable for the relative quantitation of glycans. Excellent correlation between the relative quantitation data of N-glycans from two human IgG N-glycan pools using the twoplex approach and ratios from peak area distribution calculated from the fluorescent chromatogram was observed (r = 0.986), further corroborating the reliability of the method and its potential applicability in the biopharmaceutical industry. Highly informative HCD-MS2 spectra dominated mostly by Y- and Z-type single and double glycosidic fragment ions facilitate structural interpretation of the oligosaccharides. Image 1 • New wet labeling workflow for glycan preparation. • Introduction of 3-ASA as a promising new tag for glycan labeling. • Demonstrating applicability of 3-ASA isotopologues in comparative glycomics. • Data dependent acquisition enables relative quantitation of glycans released from two samples and structural information. [ABSTRACT FROM AUTHOR]

The destruction of mitochondria through macroautophagy (autophagy) has been recognised as a major route of mitochondrial protein degradation since its discovery more than 50 years ago, but fundamental questions remain unanswered. First, how much mitochondrial protein turnover occurs through auto-phagy? Mitochondrial proteins are also degraded by nonautophagic mechanisms, and the proportion of mitochondrial protein turnover that occurs through autophagy is still unknown. Second, does auto-phagy degrade mitochondrial proteins uniformly or selectively? Autophagy was originally thought to degrade all mitochondrial proteins at the same rate, but recent work suggests that mitochondrial autophagy may be protein selective. To investigate these questions, we used a proteomics-based approach in the fruit fly Drosophila melanogaster, comparing mitochondrial protein turnover rates in autophagy-deficient Atg7 mutants and controls. We found that ~35% of mitochondrial protein turnover occurred via autophagy. Similar analyses using parkin mutants revealed that parkin-dependent mitophagy accounted for ~25% of mitochondrial protein turnover, suggesting that most mitochondrial autophagy specifically eliminates dysfunctional mitochondria. We also found that our results were incompatible with uniform autophagic turnover of mitochondrial proteins and consistent with protein-selective autophagy. In particular, the autophagic turnover rates of individual mitochondrial proteins varied widely, and only a small amount of the variation could be attributed to tissue differences in mitochondrial composition and autophagy rate. Furthermore, analyses comparing autophagy-deficient and control human fibroblasts revealed diverse autophagy-dependent turnover rates even in homogeneous cells. In summary, our work indicates that autophagy acts selectively on mitochondrial proteins, and that most mitochondrial protein turnover occurs through non-autophagic processes. Abbreviations:Atg5: Autophagy-related 5 (Drosophila); ATG5: autophagy related 5 (human); Atg7: Autophagy-related 7 (Drosophila); ATG7: autophagy related 7 (human); DNA: deoxyribonucleic acid; ER: endoplasmic reticulum; GFP: green fluorescent protein; MS: mass spectrometry; park: parkin (Drosophila); Pink1: PTEN-induced putative kinase 1 (Drosophila); PINK1: PTEN-induced kinase 1 (human); PRKN: parkin RBR E3 ubiquitin protein ligase (human); RNA: ribonucleic acid; SD: standard deviation; Ub: ubiquitin/ubiquitinated; WT: wild-type; YME1L: YME1 like ATPase (Drosophila); YME1L1: YME1 like 1 ATPase (human) [ABSTRACT FROM AUTHOR]

• Confirm the synthesis of MCs by cyanobacteria after chlorosis mechanism. • Comparison of the priority of the use of different nitrogen sources by algae cells • Level of MC-LY increased under nitrogen-limited conditions of M. aeruginosa. In this study, the biosynthesis of microcystins (MCs) was investigated after long-term nitrogen-starved conditions in cyanobacterium Microcystis aeruginosa. The results demonstrated that the algal cells were able to survive in a non-growing state with nitrogen starvation for more than one month. The physiological properties of the algal cells were studied to elucidate the mechanisms of viability under nitrogen-deprivation conditions. After the state of nitrogen chlorosis, new toxins could be resynthesized and tracked using 15N-stable isotope-labelled nitrogen. Nitrogen starvation of nutritionally replete cells resulted in a significant increase of microcystin-LY (MC-LY), thereby suggesting that MC-LY may undergo catabolism to provide nitrogen or that MC-LY may be produced to play an important role in the cell in response to nitrogen deprivation. The rank order of different types of nitrogen in algal cells assimilation was N-ammonium > N-urea > N-nitrate > N-alanine. The relationship between the production of toxin variants and various environmental conditions is an interesting issue for future research and may help improve the understanding of the ecological role of cyanobacterial toxins. [ABSTRACT FROM AUTHOR]

• CD4+ and CD8+ memory T-cells from bone marrow, spleen and lymph nodes have similar turnover rates. • Memory T-cells are continuously self-renewing and recirculating between blood, bone marrow, spleen and lymph nodes. • A dynamic model for memory T-cell maintenance. The maintenance and dynamics of memory T-cells in the bone marrow are a matter of ongoing debate. It has been suggested that memory T-cells in the bone marrow are maintained as long-lived, quiescent cells. We have recently shown that memory T-cells isolated from goat bone marrow undergo self-renewal and recirculate via the blood and lymph. Using the well-established memory T-cell markers CD44 and CD62L we here show very similar results in mice. This provides further support for the concept that memory T-cells are continuously self-renewing and recirculating between blood, bone marrow, spleen and lymph nodes. [ABSTRACT FROM AUTHOR]

Legume–rhizobia symbioses play a major role in food production for an ever growing human population. In this symbiosis, dinitrogen is reduced ("fixed") to ammonia by the rhizobial nitrogenase enzyme complex and is secreted to the plant host cells, whereas dicarboxylic acids derived from photosynthetically produced sucrose are transported into the symbiosomes and serve as respiratory substrates for the bacteroids. The symbiosome membrane contains high levels of SST1 protein, a sulfate transporter. Sulfate is an essential nutrient for all living organisms, but its importance for symbiotic nitrogen fixation and nodule metabolism has long been underestimated. Using chemical imaging, we demonstrate that the bacteroids take up 20‐fold more sulfate than the nodule host cells. Furthermore, we show that nitrogenase biosynthesis relies on high levels of imported sulfate, making sulfur as essential as carbon for the regulation and functioning of symbiotic nitrogen fixation. Our findings thus establish the importance of sulfate and its active transport for the plant–microbe interaction that is most relevant for agriculture and soil fertility. This article provides a comprehensive explanation for the importance of the nodule‐specific sulfate transporter and the role of sulfate by dissecting the sulfur distribution across the nodule tissue and directly linking sulfate incorporation to nitrogenase biosynthesis. [ABSTRACT FROM AUTHOR]

Abstract Neonatal mammals have increased disease susceptibility and sub-optimal vaccine responses. This raises problems in both humans and farm animals. The high prevalence of paratuberculosis in goats and the lack of an effective vaccine against it have a strong impact on the dairy sector, and calls for vaccines optimized for the neonatal immune system. We characterized the composition of the T-cell pool in neonatal kids and adult goats and quantified their turnover rates using in vivo deuterium labelling. From birth to adulthood, CD4+ T-cells were the predominant subset in the thymus and lymph nodes, while spleen and bone marrow contained mainly CD8+ lymphocytes. In blood, CD4+ T-cells were the predominant subset during the neonatal period, while CD8+ T-cells predominated in adults. We observed that thymic mass and cellularity increased during the first 5 months after birth, but decreased later in life. Deuterium labelling revealed that T-cell turnover rates in neonatal kids are considerably higher than in adult animals. Highlights • In goats he CD4/CD8 ratio in blood declines with age. • Throughout the life of a goat, CD4+ T-cells predominate in thymus and LN, while CD8+ T-cells predominate in spleen and BM. • Total leukocytes in thymus and bone marrow decrease, while they increase in lymph node and spleen from neonates to adults. • Thymic involution in goats does not occur during the first 5 months after birth, but becomes significant later in life. • T-cells in neonates have faster turnover rates than in adult goats. [ABSTRACT FROM AUTHOR]

Differences in the protein composition of fast- and slow-twitch muscle may be maintained by different rates of protein turnover. We investigated protein turnover rates in slow-twitch soleus and fast-twitch plantaris of male Wistar rats (body weight 412 ± 69 g). Animals were assigned to four groups (n = 3, in each), including a control group (0 d) and three groups that received deuterium oxide (D2O) for either 10 days, 20 days or 30 days. D2O administration was initiated by an intraperitoneal injection of 20 μL of 99% D2O-saline per g body weight, and maintained by provision of 4% (v/v) D2O in the drinking water available ad libitum. Soluble proteins from harvested muscles were analysed by liquid chromatography–tandem mass spectrometry and identified against the SwissProt database. The enrichment of D2O and rate constant (k) of protein synthesis was calculated from the abundance of peptide mass isotopomers. The fractional synthesis rate (FSR) of 44 proteins in soleus and 34 proteins in plantaris spanned from 0.58%/day (CO1A1: Collagen alpha-1 chain) to 5.40%/day NDRG2 (N-myc downstream-regulated gene 2 protein). Eight out of 18 proteins identified in both muscles had a different FSR in soleus than in plantaris (p < 0.05).

Bacterial metabolism is fundamental to survival and pathogenesis. We explore how Mycobacterium tuberculosis utilises amino acids as nitrogen sources, using a combination of bacterial physiology and stable isotope tracing coupled to mass spectrometry metabolomics methods. Our results define core properties of the nitrogen metabolic network from M. tuberculosis, such as: (i) the lack of homeostatic control of certain amino acid pool sizes; (ii) similar rates of utilisation of different amino acids as sole nitrogen sources; (iii) improved nitrogen utilisation from amino acids compared to ammonium; and (iv) co-metabolism of nitrogen sources. Finally, we discover that alanine dehydrogenase is involved in ammonium assimilation in M. tuberculosis, in addition to its essential role in alanine utilisation as a nitrogen source. This study represents the first in-depth analysis of nitrogen source utilisation by M. tuberculosis and reveals a flexible metabolic network with characteristics that are likely a product of evolution in the human host.

Abstract The male ejaculate contains a multitude of seminal fluid proteins (SFPs), many of which are key reproductive molecules, as well as sperm. However, the identification of SFPs is notoriously difficult and a detailed understanding of this complex phenotype has only been achieved in a few model species. We employed a recently developed proteomic method involving whole-organism stable isotope labelling coupled with proteomic and transcriptomic analyses to characterize ejaculate proteins in the seed beetle Callosobruchus maculatus. We identified 317 proteins that were transferred to females at mating, and a great majority of these showed signals of secretion and were highly male-biased in expression in the abdomen. These male-derived proteins were enriched with proteins involved in general metabolic and catabolic processes but also with proteolytic enzymes and proteins involved in protection against oxidative stress. Thirty-seven proteins showed significant homology with SFPs previously identified in other insects. However, no less than 92 C. maculatus ejaculate proteins were entirely novel, receiving no significant blast hits and lacking homologs in extant data bases, consistent with a rapid and divergent evolution of SFPs. We used 3D micro-tomography in conjunction with proteomic methods to identify 5 distinct pairs of male accessory reproductive glands and to show that certain ejaculate proteins were only recovered in certain male glands. Finally, we provide a tentative list of 231 candidate female-derived reproductive proteins, some of which are likely important in ejaculate processing and/or sperm storage. Graphical abstract Image 1 Highlights • Male ejaculate proteins are diverse and rapidly evolving reproductive proteins but their identification is challenging. • We use a proteomic method based on whole-organism stable isotope labelling and tandem mass spectrometry. • We identified 317 male derived proteins, of which 92 are novel proteins, and 231 candidate female reproductive proteins. • 3D micro-tomography unveiled 5 distinct pairs of male accessory reproductive glands. • Some proteins were recovered only in some glands, suggesting substantial differentiation across male glands. [ABSTRACT FROM AUTHOR]

Background To understand the mechanisms related to the ‘dynamical ordering’ of macromolecules and biological systems, it is crucial to monitor, in detail, molecular interactions and their dynamics across multiple timescales. Solution nuclear magnetic resonance (NMR) spectroscopy is an ideal tool that can investigate biophysical events at the atomic level, in near-physiological buffer solutions, or even inside cells. Scope of review In the past several decades, progress in solution NMR has significantly contributed to the elucidation of three-dimensional structures, the understanding of conformational motions, and the underlying thermodynamic and kinetic properties of biomacromolecules. This review discusses recent methodological development of NMR, their applications and some of the remaining challenges. Major conclusions Although a major drawback of NMR is its difficulty in studying the dynamical ordering of larger biomolecular systems, current technologies have achieved considerable success in the structural analysis of substantially large proteins and biomolecular complexes over 1 MDa and have characterised a wide range of timescales across which biomolecular motion exists. While NMR is well suited to obtain local structure information in detail, it contributes valuable and unique information within hybrid approaches that combine complementary methodologies, including solution scattering and microscopic techniques. General significance For living systems, the dynamic assembly and disassembly of macromolecular complexes is of utmost importance for cellular homeostasis and, if dysregulated, implied in human disease. It is thus instructive for the advancement of the study of the dynamical ordering to discuss the potential possibilities of solution NMR spectroscopy and its applications. This article is part of a Special Issue entitled “Biophysical Exploration of Dynamical Ordering of Biomolecular Systems” edited by Dr. Koichi Kato. [ABSTRACT FROM AUTHOR]

Background and aims: One means of reducing nitrate leaching in temperate farming is to include catch crops in crop rotations, which immobilize residual nitrogen (N) in their biomass. For an accurate quantification of the N stored in catch crops and subsequently released from residues, their total biomass, including roots and rhizodeposits has to be assessed. Methods: In a pot experiment under controlled conditions, oil and forage radish ( Raphanus sativus L . var. oleiformis Pers .) and winter turnip rape ( Brassica rapa L . var. silvestris [Lam.] Briggs) plants were leaf-labelled every five to seven days with N-urea (99at%) five times during the vegetation. At harvest, plants were separated into shoot, coarse, medium and fine roots by hand picking and wet sieving, respectively. The amount of N derived from rhizodeposition (NdfR) was calculated using two different calculation approaches. In addition to the pot experiment, a field experiment with unlabelled plants was set up to extrapolate the results from the pot experiment to the field scale, at the same time evaluating the influence of additional mineral N fertilization on biomass distribution. The contribution of rhizodeposition to total N in the field was estimated by extrapolating the root-N-to-rhizodeposition-N ratio of the pot experiment to the field experiment. Results: In the pot trial, between 4.6 and 10.3% of the total assimilated nitrogen of the catch crops was found as rhizodeposits, which is at the lower end of values from other studies on legumes and non-legumes. In the field experiment the shoot-to-root ratio was lower compared to the pot experiment. Thus, the contribution of rhizodeposition to total N under field conditions is substantially higher. Fertilization in the field trial mostly influenced the formation of above-ground plant biomass. Conclusions: Considering the rhizodeposition reveals, that the investigated catch crops store more N than previously assumed. As a consequence, catch crops have to be evaluated with a stronger focus on below-ground biomass to make sure the right amounts of N are considered for fertilization schemes in crop rotations. [ABSTRACT FROM AUTHOR]

Background: /Objective: The extent of exocrine pancreatic insufficiency (EPI) in the paediatric population with acute pancreatitis (AP) is unknown. The primary objective was to use a 6 h stable-isotope breath test to determine the prevalence of EPI in children with AP. The secondary objective was to determine the diagnostic ability of a 4 h abbreviated breath test in the detection of EPI., Methods: 13 C-mixed triglyceride (MTG) breath test was used to measure fat digestibility in 12 children with AP and 12 normal children. EPI was diagnosed based on a cumulative dose percentage recovery (cPDR) cut-off value < 26.8% present in literature. To reduce the test burden, the diagnostic accuracy of an abbreviated 4 h test was evaluated, using a cPDR cut-off that was the 2.5 th percentile of its distribution in control children., Results: The cPDR of cases was significantly lower than that of controls (27.71 ± 7.88% vs 36.37 ± 4.70%, p = 0.005). The cPDR during acute illness was not significantly different to that at 1 month follow up (24.69 ± 6.83% vs 26.98 ± 11.10%, p = 0.52). The 4 h and 6 h breath test results correlated strongly (r = 0.93, p < 0.001) with each other. The new 4 h test had 87.5% sensitivity and 93.8% specificity for detecting EPI., Conclusion: Two-thirds (66.7%) of this sample of children with AP had EPI during admission, which persisted at 1 month follow up. The 4 h abbreviated 13 C-MTG breath test has good diagnostic ability to detect EPI in children and may improve its clinical utility in this age group., (Copyright © 2023 IAP and EPC. Published by Elsevier B.V. All rights reserved.)

Over the last two decades, lipids have come to be understood as far more than merely components of cellular membranes and forms of energy storage, and are now also being implicated to play important roles in a variety of diseases, with lipid biomarker research one of the most widespread applications of lipidomic techniques both in research and in clinical settings. Stable isotope labelling has become a staple technique in the analysis of small molecule metabolism and dynamics, as it is the only experimental setup by which biosynthesis, remodelling and degradation of biomolecules can be directly measured. Using state-of-the-art analytical technologies such as chromatography-coupled high resolution tandem mass spectrometry, the stable isotope label can be precisely localized and quantified within the biomolecules. The application of stable isotope labelling to lipidomics is however complicated by the diversity of lipids and the complexity of the necessary data analysis. This article discusses key experimental aspects of stable isotope labelling in the field of mass spectrometry-based lipidomics, summarizes current applications and provides an outlook on future developments and potential.

Capillary electrophoresis (CE) offers excellent efficiency and orthogonality to liquid chromatographic (LC) separations for oligosaccharide structural analysis. Combination of CE with high resolution mass spectrometry (MS) for glycan analysis remains a challenging task due to the MS incompatibility of background electrolyte buffers and additives commonly used in offline CE separations. Here, a novel method is presented for the analysis of 2-aminobenzoic acid (2-AA) labelled glycans by capillary electrophoresis coupled to mass spectrometry (CE-MS). To ensure maximum resolution and excellent precision without the requirement for excessive analysis times, CE separation conditions including the concentration and pH of the background electrolyte, the effect of applied pressure on the capillary inlet and the capillary length were evaluated. Using readily available C stable isotopologues of 2-AA, the developed method can be applied for quantitative glycan profiling in a twoplex manner based on the generation of extracted ion electropherograms (EIE) for C 'light' and C 'heavy' 2-AA labelled glycan isotope clusters. The twoplex quantitative CE-MS glycan analysis platform is ideally suited for comparability assessment of biopharmaceuticals, such as monoclonal antibodies, for differential glycomic analysis of clinical material for potential biomarker discovery or for quantitative microheterogeneity analysis of different glycosylation sites within a glycoprotein. Additionally, due to the low injection volume requirements of CE, subsequent LC-MS analysis of the same sample can be performed facilitating the use of orthogonal separation techniques for structural elucidation or verification of quantitative performance. [ABSTRACT FROM AUTHOR]