During the Late Neolithic (ca. 3500–2900 cal BC), the Rioja Alavesa region of north‐central Spain witnessed great demographic pressure and social unrest, manifested as widespread violent conflict. Drawing upon the ethnographic literature, it is possible that this situation impacted upon child‐rearing practices, both through food shortages and differential parental investment, favouring male infants. Here, carbon (δ13C) and nitrogen (δ15N) stable isotope measurements from bone collagen of 17 juveniles and from 163 serial microsamples of dentine from first and second molars of seven adults from the site of Alto de la Huesera are used to examine breastfeeding, weaning, and childhood diets. Bone and dentine collagen δ13C and δ15N isotope values both decrease from infancy to early childhood and increase slightly towards adolescence, but dentine provides a more time‐sensitive means of monitoring dietary changes. High δ13C and δ15N values compatible with exclusive breastfeeding are detected up to ca. 1 year, with a significantly shorter duration among males, suggesting differential sex‐related parental strategies from infancy. This is tentative given the small number of individuals being compared, but does suggest that further work would be worthwhile. A gradual decline in both δ13C and δ15N, compatible with the weaning process, is then observed up to ca. 4 years in both sexes. This delayed cessation of nursing is interpreted as a possible response to food shortage. With regard to postweaning patterns, shifts to lower δ13C and δ15N values in females at around age 9–11, and a general progressive increase in both isotope values from childhood to adolescence, are detected. These could be linked either with differential protein intake due to social age‐related nutritional practices or to physiological demand. The comparison between bone and dentine values shows differences between survivors and nonsurvivors in both isotopes, so that assessments based on deceased children may be biased by their potentially compromised health status.

The coexistence of cultural identities and their interaction is a fundamental topic of social sciences that is not easily addressed in prehistory. Differences in mortuary treatment can help approach this issue. Here, we present a multi-isotope study to track both diet and mobility through the life histories of 32 broadly coeval Late Neolithic individuals interred in caves and in megalithic graves of a restricted region of northern Iberia. The results show significant differences in infant- and child-rearing practices, in subsistence strategies, and in landscape use between burial locations. From this, we posit that the presence of communities with distinct lifestyles and cultural backgrounds is a primary reason for Late Neolithic variability in burial location in Western Europe and provides evidence of an early “them and us” scenario. We argue that this differentiation could have played a role in the building of lasting structures of socioeconomic inequality and, occasionally, violent conflict.

Tooth dentine serial sampling followed by isotope analyses allows detection of shifts in an individual's diet during the periods of tooth formation, providing information on breastfeeding, weaning, and childhood nutrition. Current sampling methods, however, do not fully capture the potential resolution of dentine increments because of the difficulties caused by the conical growth pattern, and changes in growth rate during tooth development resulting in uncertain timing. Here, we present an imaging‐assisted microsampling approach that takes the biological growth pattern of dentine into account in an effort to improve temporal resolution. We used high‐resolution light microscopy images of first molar longitudinal thin sections to generate an accurate optical reference of growth pattern and formulated a new scheme to assign collected microsamples to age. This allowed us to track dietary changes over more precisely confined and shorter time periods, providing greater detail and resolution for the breastfeeding and weaning process. We applied our method to track early life dietary history in four individuals from a small cemetery in Alsace, France, dated to the early 5th century AD (the Late Antique/Migration period). Even within this short timescale, 13C/12C and 15N/14N ratio sequences suggest variability in diets and weaning periods, and hint at early maternal mobility.

Objectives: Stable isotope analysis of sequential dentine samples is a potentially powerful method to reveal insights into early life-histories of individuals in the past. Dentine incremental growth structures are complex, however, and current approaches that apply horizontal sectioning of demineralized tooth halves or quarters risk combining multiple growth layers and may include unwanted cementum or secondary dentine. They also require destruction of large parts of a tooth. Here, we present a less destructive and relatively straightforward protocol that reduces damage, increases temporal resolution, and improves the accuracy of age-alignment between individuals. Material and methods: We outline a protocol that includes the sampling of small (1 mm diameter) cylindrical plug transects from a thin section, along with an age-alignment scheme predicated on average growth rates for dentine areas. Results and discussion: The proposed protocol is readily applicable and more anatomically sensitive than horizontal slicing. Micro-samples are smaller (in both length and depth), hence minimizing temporal overlap and avoid directions that may contravene growth pattern. They completely avoid areas where secondary and tertiary dentine or cementum can be deposited. Age-alignment is improved by using growth ratios of anatomical tooth zones. Conclusion: This method minimizes destruction, enables finer temporal resolution and facilitates data comparison. It can be readily combined with fluorescence imaging-based or other pre-screening methods of dentine collagen preservation.

Cellular reduced glutathione (GSH) levels have been identified as an essential determinant in zinc-induced cytotoxicity. However, cytotoxic effects of zinc have also been observed without depletion of GSH stores. In a previous study, the intracellular activity of GSSG reductase (GR) has come into focus (Walther et al. 2000, Biol Trace Elem Res 78:163-177). In the present paper we have tried to address this issue more deeply by inhibiting the activity of cellular GR without any appreciable decreases of cellular glutathione. In three pulmonary cell lines, GR activity was inhibited in a dose-dependent manner by the alkylating agent carmustine (BCNU), a known inhibitor of GR. Cells were pretreated with BCNU for 14 h, followed by exposure to various concentrations of zinc chloride. Then we determined the incorporation of radiolabelled methionine (to assess protein synthesis), and measured the GSH and oxidized glutathione (GSSG) levels. Additionally, GR activity of controls was measured. IC(50) values for zinc-induced inhibition of methionine incorporation, as well as GSH contents, was strongly correlated to the decreased GR activity. These results firmly suggest that GR is an important factor in the event chain of zinc cytotoxicity. Together with the results from our previously cited study where impaired regeneration of GSH levels were accompanied by a decrease in total cellular glutathione (GSH + GSSG) we conclude that GSSG itself is an important effector in zinc cytotoxicity.

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Figures: 3 Supplementary Material Highlights: • Diagenetic changes and the degree of degradation of collagen in tooth dentine can be effectively monitored by fluorescence microscopy. • Autofluorescence can be used to map suitable areas for sampling. • Fluorescence imaging is an effective screening method to predict collagen quality and quantity ahead of sampling.

Instead of the conventional approach to sectioning tooth dentine horizontally for isotope analysis, a new serial sampling strategy has been developed recently. This new method considers the developmental direction of growth lines in dentine when samples are sectioned. It can therefore provide more precise and detailed information about an individual's dietary history, including breastfeeding/weaning patterns, infant feeding experiences, and dietary shifts throughout the early life of an individual. In order to enhance our understanding of human groups in ancient Sichuan, it is practical and necessary to apply this new technique to archaeological samples from this region.In this study, carbon and nitrogen isotope compositions of bone and serial dentinal collagen from two Yingpanshan human individuals (M32 and M40) were analysed and compared, in order to reveal the dietary history during their lifetime. Results indicate that their long‐term averaged diets after childhood consisted of both C3 and C4‐type foods, predominantly the former. The contribution of animal protein to the childhood diet of individual M40, between the ages of 3.0 and 7.5, was similar to his long‐term averaged diet after childhood. He had also consumed more millets during the childhood. By comparison, individual M32 consumed more animal protein and likely also more C3‐type foods between the ages of 4.5 and 13.5 than after childhood. Little variations in δ15N dentinal collagen of each individual suggest that the relative consumption of animal to plant foods in their respective diets was more or less consistent in their childhood. However, the larger variations in the δ13C values of dentine samples show that the proportion of C3 and C4‐plant foods in their respective diets varied at different phases in their childhood. The variations are probably related to environmental factors and/or human behaviour. The dietary history and weaning practices revealed by using this technique also indicate that the two individuals might have been non‐locals or received some foods that originated from the outside.