Application of stable isotopically labelled (SIL) molecules in Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI-MSI) over a series of time points allows the temporal and spatial dynamics of biochemical reactions to be tracked in a biological system. However, these large kinetic MSI datasets and the inherent variability of biological replicates presents significant challenges to the rapid analysis of the data. In addition, manual annotation of downstream SIL metabolites involves human input to carefully analyse the data based on prior knowledge and personal expertise. To overcome these challenges to the analysis of spatiotemporal MALDI-MSI data and improve the efficiency of SIL metabolite identification, a bioinformatics pipeline has been developed and demonstrated by analysing normal bovine lens glucose metabolism as a model system. The pipeline consists of spatial alignment to mitigate the impact of sample variability and ensure spatial comparability of the temporal data, dimensionality reduction to rapidly map regional metabolic distinctions within the tissue, and metabolite annotation coupled with pathway enrichment modules to summarise and display the metabolic pathways induced by the treatment. This pipeline will be valuable for the spatial metabolomics community to analyse kinetic MALDI-MSI datasets, enabling rapid characterisation of spatio-temporal metabolic patterns from tissues of interest.

The essential microelement zinc is absorbed in the small intestine mainly by the zinc transporter ZIP4, a representative member of the Zrt/Irt-like protein (ZIP) family. ZIP4 is reportedly upregulated in many cancers, making it a promising oncology drug target. To date, there have been no reports on the turnover number of ZIP4, which is a crucial missing piece of information needed to better understand the transport mechanism. In this work, we used a non-radioactive zinc isotope, 70Zn, and inductively coupled plasma mass spectrometry (ICP-MS) to study human ZIP4 (hZIP4) expressed in HEK293 cells. Our data showed that 70Zn can replace the radioactive 65Zn as a tracer in kinetic evaluation of hZIP4 activity. This approach, combined with the quantification of the cell surface expression of hZIP4 using biotinylation or surface-bound antibody, allowed us to estimate the apparent turnover number of hZIP4 to be in the range of 0.08-0.2 s-1. The turnover numbers of the truncated hZIP4 variants are significantly smaller than that of the full-length hZIP4, confirming a crucial role for the extracellular domain in zinc transport. Using 64Zn and 70Zn, we measured zinc efflux during the cell-based transport assay and found that it has little effect on the zinc import analysis under these conditions. Finally, we demonstrated that use of laser ablation (LA) ICP-TOF-MS on samples applied to a solid substrate significantly increased the throughput of the transport assay. We envision that the approach reported here can be applied to the studies of metal transporters beyond the ZIP family.

Limpets are a key taxon regulating the benthic community structure and serving as prey for various predators in rocky shores, however, their role in food web dynamics is still unclear. To determine environmental factors influencing the limpet diet, carbon and nitrogen stable isotopes in limpets and food sources were analyzed on three different coastal habitats. Sediment organic matter contributed the most (86 %) to the limpet diet in bedrocks around tidal flats with abundant sediment supply from the terrestrial matter inflow and the sediment resuspension. Microphytobenthos and macroalgae were the main food sources (57 % and 20 %, respectively) for the limpets around beaches, where benthic flora was abundant. Limpets in bedrocks, erosional habitat, primarily consumed relatively abundant phytoplankton (33 %) and microphytobenthos (28 %). Contrary to previous studies, habitat type, rather than latitude or seawater characteristics, was the most important factor determining the limpet diet. This result also suggests that limpets are non-selective scraper that consume abundant food sources.

Bivalves are often employed for biomonitoring contaminants in marine environments; however, in these large-scale programs, unavoidably, using multiple species presents a significant challenge. Interspecies differences in contaminant bioaccumulation can complicate data interpretation, and direct comparisons among species may result in misleading conclusions. Here, we propose a robust framework based on toxicokinetic measurements that accounts for interspecies differences in bioaccumulation. Specifically, via a recently developed double stable isotope tracer technique, we determined the toxicokinetics of cadmium (Cd)─a metal known for its high concentrations in bivalves and significant interspecies bioaccumulation variability─in six widespread bivalve species including mussels (Perna viridis, Mytilus unguiculatus, Mytilus galloprovincialis) and oysters (Magallana gigas, Magallana hongkongensis, Magallana angulata). Results show that oysters generally have higher Cd uptake rate constants (ku: 1.18-3.09 L g-1 d-1) and lower elimination rate constants (ke: 0.008-0.017 d-1) than mussels (ku: 0.21-0.64 L g-1 d-1; ke: 0.018-0.037 d-1). The interspecies differences in tissue Cd concentrations are predominantly due to Cd uptake rather than elimination. Utilizing toxicokinetic parameters to back-calculate Cd concentrations in seawater, we found that the ranking of Cd contamination levels at the six sites markedly differs from those based on tissue Cd concentrations. We propose that this approach will be useful for interpreting data from past and future biomonitoring programs.

Omega-3 long-chain polyunsaturated fatty acids (n3-LCPUFAs) are produced primarily in aquatic ecosystems and are considered essential nutrients for predators given their structural role in vertebrates\' cerebral tissues. Alarmingly, with urbanization, many aquatic animals now rely on anthropogenic foods lacking n3-LCPUFAs. In this study undertaken in Newfoundland (Canada), we tested whether recent or longer term diet explains the cerebral fatty acid composition of ring-billed gulls (Larus delawarensis), a seabird that now thrives in cities. During the breeding season, cerebral levels of n3-LCPUFAs were significantly higher for gulls nesting in a natural habitat and foraging on marine food (mean ± s.d.: 32 ± 1% of total identified fatty acids) than for urban nesters exploiting rubbish (27 ± 1%). Stable isotope analysis of blood and feathers showed that urban and natural nesters shared similar diets in autumn and winter, suggesting that the difference in cerebral n3-LCPUFAs during the breeding season was owing to concomitant and transient differences in diet. We also experimentally manipulated gulls\' diets throughout incubation by supplementing them with fish oil rich in n3-LCPUFAs, a caloric control lacking n3-LCPUFAs, or nothing, and found evidence that fish oil increased urban nesters\' cerebral n3-LCPUFAs. These complementary analyses provide evidence that the brain of this seabird remains plastic during adulthood and responds to short-term dietary changes.

Golden cuttlefish play a significant role in the food web of the East and Yellow Seas and are a valuable fishery resource in Chinese coastal waters. Samples of golden cuttlefish were obtained from the northern East China Sea between September 2021 and March 2022, and stable isotope methods were utilized in this study to examine the variations in the forage ecology of golden cuttlefish. Our findings reveal dynamic shifts in carbon and nitrogen stable isotopes (δ13C and δ15N), highlighting intricate foraging strategies tailored to growth and environmental changes. A notable trend emerges: an initial growth-linked rise in δ13C and δ15N enrichment, followed by seasonal fluctuations mirroring seasonal food availability. The ontogenetic niche evolution displays striking habitat shifts and trophic level escalation in small mantle length stages, transitioning to niche overlap and subtle trophic shifts later on. Sex-specific differences emerge, with females occupying higher trophic levels than males in most samples. This comprehensive study underscores the complexity and adaptability of golden cuttlefish feeding ecology, inviting further inquiry into their intricate relationships within the marine ecosystem.

Amino acids form the building blocks of body protein. Dietary protein sources provide the amino acids needed, but protein sources vary widely in amio acid composition. To ensure humans can meet body demands for amino acids, amino acid intake recommendations are provided by the Dietary Reference Intakes (DRI) and by Food and Agriculture Organization/World Health Organization/United Nations University (FAO/WHO/UNU). Current amino acid intake recommendations, however, are based on data collected predominantly from young adult males. The development of the minimally invasive indicator amino acid oxidation (IAAO) method has permitted the evaluation of amino acid requirements in various vulnerable populations. The purpose of this review is to discuss recent amino acid requirement studies in school-age children, pregnant females and the elderly determined using the IAAO technique. These requirements will help to inform evidence-based recommendations that will help to guide dietary guidelines.

Morphologically similar species that occupy resource-limited environments tend to differ in their ecological traits in order to coexist, which may result in differential exposure to environmental threats. For instance, partitioning of feeding resources may influence contaminant exposure and bioaccumulation in marine predators through different diets or foraging habitats. Here, we sampled three tropical seabird species breeding in sympatry in the southwestern Atlantic Ocean for blood and feather trace element concentrations (As, Hg, Cd, Pb), and assessed their foraging ecology with bio-logging (GPS tracks and time-depth recorders), analysis of regurgitated prey, and carbon and nitrogen stable isotope mixing models. Red-billed tropicbirds (Phaethon aethereus), brown (Sula leucogaster) and masked (S. dactylatra) boobies differed in their preferred foraging locations, the range of foraging trips, diving parameters and diets. In addition, interspecific differences were detected in blood or feathers for all trace elements analyzed, suggesting influence of the differences observed in diet and space use. Red-billed tropicbirds had the largest foraging range over the continental shelf and over the slope, suggesting lower exposure to continental sources of metals. Brown and masked boobies had higher Hg concentrations than tropicbirds, higher δ15N values (a proxy for trophic level), and δ15N correlated with Hg levels, suggesting biomagnification of Hg along the food chain. Nonetheless, red-billed tropicbirds showed comparable levels of As and Cd in blood or feathers, and higher levels of Pb in both tissues in comparison to boobies, which may suggest overall exposure of seabirds in the region, through their diets and foraging areas. Resource partitioning is critical for allowing coexistence of different seabird species in shared breeding sites where they act as central-place foragers. Nonetheless, in scenarios of environmental pollution, these species-specific strategies lead to differential bioaccumulation, and thus distinct effects of pollution on populations are expected.

Chlorinated pollutants may follow distinct degradation pathways in anaerobic environments compared to aerobic settings. However, the understanding of the behaviors and fate of dichlorodiphenyltrichloroethanes (DDTs) under anaerobic conditions remains limited. To address this knowledge gap, we conducted a study on flooded soil samples collected from three typical paddy fields in China using an integrated approach of enantiomer-specific analysis and compound-specific stable carbon isotope analysis. It is unexpected that the dichlorodiphenyldichloroethane /dichlorodiphenyldichloroethylene ratios (DDD/DDE=(o,p\'-DDD+p,p\'-DDD)/(o,p\'-DDE+p,p\'-DDE)) were below 1 in over 90 % of the samples. This might be attributed to the higher recalcitrance of p,p\'-DDE, which concentrations were found to be 36 times higher than p,p\'-DDD on average. There were 71.7 % of the samples showing enantiomeric fractions (EFs) of o,p\'-DDT below 0.5, indicating a preferential accumulation of the (-)-enantiomer. The δ13C values of the anaerobic metabolite o,p\'-DDD (-24.76 ± 1.35 ‰ to -34.39 ± 0.20 ‰) all deviated negatively from the initial product, while those of the aerobic metabolite o,p\'-DDE (-23.61 ± 0.48 ‰ to -38.95 ± 0.81 ‰) displayed either negative or positive deviations. This demonstrates that o,p\'-DDD is the primary metabolite of o,p\'-DDT under anaerobic conditions. However, no clear correlations were observed between the δ13C and EF of o,p\'-DDT. This study underscores the importance of such an integrated methodology in unraveling the fate and behaviors of DDTs in complex environmental systems.

Traditional studies of cellular metabolism have relied on the use of radioisotopes. These have clear disadvantages associated with safety and waste generation. Furthermore, detection of the labeled species by scintillation counting provides only a quantification of its presence or absence. The use of stable isotopes, by contrast, allows the application of powerful, orthogonal spectroscopic approaches such as nuclear magnetic resonance spectroscopy (NMR) and various mass spectrometric methods. Using stable isotope labeling to study heme metabolism requires integrating methods for (a) generating the heme in labeled forms, (b) cultivating and quantifying the organism of choice in chemically defined media, to which labeled compounds can be added, (c) recovering cellular components and/or spent growth media, and (d) analyzing these materials for the labeled species using spectroscopic and mass spectrometric methods. These methods are summarized here in the context of Bacteroides thetaiotaomicron, a generally nonpathogenic anaerobe and heme auxotroph.