Palaeoproteomic investigation of an ancient human skeleton with abnormal deposition of dental calculus.

Detailed investigation of extremely severe pathological conditions in ancient human skeletons is important as it could shed light on the breadth of potential interactions between humans and disease etiologies in the past. Here, we applied palaeoproteomics to investigate an ancient human skeletal individual with severe oral pathology, focusing our research on bacterial pathogenic factors and host defense response. This female skeleton, from the Okhotsk period (i.e., fifth to thirteenth century) of Northern Japan, poses relevant amounts of abnormal dental calculus deposition and exhibits oral dysfunction due to severe periodontal disease. A shotgun mass-spectrometry analysis identified 81 human proteins and 15 bacterial proteins from the calculus of the subject. We identified two pathogenic or bioinvasive proteins originating from two of the three "red complex" bacteria, the core species associated with severe periodontal disease in modern humans, as well as two additional bioinvasive proteins of periodontal-associated bacteria. Moreover, we discovered defense response system-associated human proteins, although their proportion was mostly similar to those reported in ancient and modern human individuals with lower calculus deposition. These results suggest that the bacterial etiology was similar and the host defense response was not necessarily more intense in ancient individuals with significant amounts of abnormal dental calculus deposition.

Evolutionary biological perspectives on current social issues of breastfeeding and weaning.

Breastfeeding and weaning are actively studied from evolutionary, medical, and social research perspectives because of their close association with infant mortality, lifetime health, and human population dynamics. Each discipline benefits from an interdisciplinary exchange of knowledge regarding the bases, processes, and consequences of these phenomena. However, current social issues related to breastfeeding and weaning have received little attention from an evolutionary biology perspective. We address this gap by reviewing current social issues related to human breastfeeding and weaning in an evolutionary framework. This approach helps build a conceptual framework with the goal of better understanding ultimate causes of or influences on these current social issues. The six social issues reviewed here fall into three categories: the spatiotemporal constraints of breastfeeding, abuse of breast milk as valuable material, and mismatch in breastfeeding practices. Some of these issues have an evolutionary basis. We analyze the structure of these social issues and discuss their possible solutions in terms of extension of the trade-off theory in evolutionary biology. Our discussion on the current social issues in breastfeeding and weaning highlights the effectiveness of an approach rooted in evolutionary theory and biological anthropology.

Carbon and nitrogen stable isotopic data of premodern human skeletons from mainland Japan and the Ryukyu islands.

This dataset consists of carbon and nitrogen stable isotope ratios of bulk collagen extracted from 229 human skeletons from premodern Japan. All samples were derived from different individuals excavated from mainland Japan and the Ryukyu Islands. Most of the skeletal individuals were identified, sexed, and aged by physical anthropologists. Collagen samples were extracted from bone or root portion of tooth dentin. Collagen samples were measured by an elemental analyzer coupled to stable isotope ratio mass spectrometry. Stable isotope ratios are the useful proxy of palaeodiet, and this dataset can be used for dietary reconstruction of premodern people living in the Japan archipelago.

Carbon and nitrogen stable isotopic offsets between diet and hair/feces in captive orangutans.

Estimating stable isotopic offset values is crucial for dietary reconstructions. Although research into stable isotope ecology of wild nonhuman primates is increasing overall, only a minority of studies involve laboratory experiments. This study is the first to report the carbon and nitrogen stable isotopic offset values in hair and feces of orangutans. During an experiment lasting 1 week, the weight of each consumed food item was recorded for each of six captive Bornean orangutan (Pongo pygmaeus) individuals. The food, hair, and fecal samples were collected for a few days, and their stable carbon and nitrogen isotope ratios were measured using an elemental analyzer/isotope ratio mass spectrometer. Subsamples of feces were treated with ethanol during the preservation process. Monte Carlo analyses showed that the 95% confidence intervals (CIs) of the carbon and nitrogen offset values between hair and diet were +0.9‰ to +3.9‰ and +2.3‰ to +4.5‰, respectively. The 95% CIs of the carbon and nitrogen offset values between feces and diet were -3.7‰ to -0.9‰ and +0.3‰ to +2.7‰, respectively. The effect of ethanol treatment on the stable isotope ratios of feces was unclear and inconclusive. The computed offset values of hair in captive orangutans are similar to those reported in other nonhuman primates, although those of feces showed greater interspecies variations. The offset values estimated in this study contribute to isotopic studies into the feeding ecology of free-ranging orangutans who are critically endangered in most wild settings.

Faecal proteomics as a novel method to study mammalian behaviour and physiology.

Mammalian faeces can be collected noninvasively during field research and provide valuable information on the ecology and evolution of the source individuals. Undigested food remains, genome/metagenome, steroid hormones, and stable isotopes obtained from faecal samples provide evidence on diet, host/symbiont genetics, and physiological status of the individuals. However, proteins in mammalian faeces have hardly been studied, which hinders the molecular investigations into the behaviour and physiology of the source individuals. Here, we apply mass spectrometry-based proteomics to faecal samples (n = 10), collected from infant, juvenile, and adult captive Japanese macaques (Macaca fuscata), to describe the proteomes of the source individual, of the food it consumed, and its intestinal microbes. The results show that faecal proteomics is a useful method to: (i) investigate dietary changes along with breastfeeding and weaning, (ii) reveal the taxonomic and histological origin of the food items consumed, and (iii) estimate physiological status inside intestinal tracts. These types of insights are difficult or impossible to obtain through other molecular approaches. Most mammalian species are facing extinction risk and there is an urgent need to obtain knowledge on their ecology and evolution for better conservation strategy. The faecal proteomics framework we present here is easily applicable to wild settings and other mammalian species, and provides direct evidence of their behaviour and physiology.

Blurred time resolution of tooth dentin serial sections.

OBJECTIVES: The growth of tooth dentin is incremental, so its formation represents a dietary record in early life. With archeological skeletons, applying sequential stable isotope analysis to the horizontal sections of tooth dentin has revealed weaning patterns and dietary changes that took place during childhood. However, the assignment of ages to dentin serial sections (DSSs) is problematic due to the changing extension rate and oblique growth layers of dentin, and these effects have not been quantified. This study presents a mathematical model for investigating the corresponding age range of the horizontal DSSs of human permanent incisors, canines, and molars. METHODS: Parameters describing the tooth dentin microstructure were taken from previous studies, and dentin growth patterns were modeled. The model was implemented as the R package MDSS. RESULTS: The developed model shows that the true corresponding age of the sections differed by a few years on average from the estimated age with equal temporal divisions, that the model gave values extending across a wide range, and that these differences become large for sections formed at older ages. The stable isotope ratio of the sections presented an aggregate representation of possibly complex dietary changes across a few years, and dietary changes over short times, such as several months, could not be accurately reconstructed even when using a finer horizontal sectioning method. CONCLUSIONS: These results demonstrate that DSSs correspond to an unexpectedly wider (i.e., three to four times) and different (i.e., -2 to 0.5 years on average) age range than previously assumed and that complicated patterns of dietary change blur in the isotopic trajectory of the sections. Alternative experimental methods, such as imaging-assisted oblique sampling, should be used to retrieve an accurate and precise sequential dietary record from tooth dentin.

Ancient Beringian paleodiets revealed through multiproxy stable isotope analyses.

The earliest Native Americans have often been portrayed as either megafaunal specialists or generalist foragers, but this debate cannot be resolved by studying the faunal record alone. Stable isotope analysis directly reveals the foods consumed by individuals. We present multi-tissue isotope analyses of two Ancient Beringian infants from the Upward Sun River site (USR), Alaska (~11,500 years ago). Models of fetal bone turnover combined with seasonally-sensitive taxa show that the carbon and nitrogen isotope composition of USR infant bone collagen reflects maternal diets over the summer. Using comparative faunal isotope data, we demonstrate that although terrestrial sources dominated maternal diets, salmon was also important, supported by carbon isotope analysis of essential amino acids and bone bioapatite. Tooth enamel samples indicate increased salmon use between spring and summer. Our results do not support either strictly megafaunal specialists or generalized foragers but indicate that Ancient Beringian diets were complex and seasonally structured.

Societal perceptions and lived experience: Infant feeding practices in premodern Japan.

OBJECTIVES: A change in how children were treated and valued occurred in premodern Japan, as popularized ideas of an inheritance-based family system led to more careful and affectionate child-rearing practices by lower social-status groups. A number of books were written, advising that breastfeeding should last approximately 3 years. The objective of this study is to reconstruct and compare breastfeeding and weaning practices before and after the transition, to illuminate the impact of documented changes in child-rearing practices on subadults' lived experience. MATERIALS AND METHODS: Carbon and nitrogen stable isotopic data were obtained from 40 subadult skeletons excavated from the Sakai Kango Toshi 871 (SKT871) site (late 17th-19th century, Osaka, Japan). Isotopic results from SKT871 were compared with previously reported results from the Hitotsubashi site (AD 1657-1683, Tokyo, Japan). Hitotsubashi and SKT871 represent urban populations of lower status before and after the transition of societal perception of subadults. RESULTS: The most probable age at the end of weaning reconstructed in SKT871 was 1.9 years (1.4-2.7 years with a 95% credible interval) and was lower than that in Hitotsubashi (2.1-4.1 years with a 95% credible interval). DISCUSSION: The age at the end of weaning became younger after the transition of societal perception toward subadults, and this younger weaning age is inconsistent with written recommendations for the duration of weaning in premodern Japan. It is possible that an increased need for inheritors under the inheritance-based family system led to earlier weaning and shorter inter-birth intervals, but authorities recommended an ideal practice of a longer breastfeeding period.

Palaeoproteomic identification of breast milk protein residues from the archaeological skeletal remains of a neonatal dog.

Accurate postmortem estimation of breastfeeding status for archaeological or forensic neonatal remains is difficult. Confident identification of milk-specific proteins associated with these remains would provide direct evidence of breast milk consumption. We used liquid chromatography coupled to tandem mass spectrometry (MS) to confidently identify beta-lactoglobulin-1 (LGB1) and whey acidic protein (WAP), major whey proteins associated with a neonatal dog (Canis lupus familiaris) skeleton (430-960 cal AD), from an archaeological site in Hokkaido, Japan. The age at death of the individual was estimated to be approximately two weeks after birth. Protein residues extracted from rib and vertebra fragments were analyzed and identified by matching tandem MS spectra against the dog reference proteome. A total of 200 dog protein groups were detected and at least one peptide from canine LGB1 and two peptides from canine WAP were confidently identified. These milk proteins most probably originated from the mother's breast milk, ingested by the neonate just before it died. We suggest the milk diffused outside the digestive apparatus during decomposition, and, by being absorbed into the bones, it partially preserved. The result of this study suggests that proteomic analysis can be used for postmortem reconstruction of the breastfeeding status at the time of death of neonatal mammalian, by analyzing their skeletal archaeological remains. This method is also applicable to forensic and wildlife studies.

Post-weaning diet in archaeological human populations: A meta-analysis of carbon and nitrogen stable isotope ratios of child skeletons.

OBJECTIVES: Childhood is a unique stage in human life history, in which subadults have completed their weaning process but are still dependent on older individuals for survival. Although the importance of food provisioning during childhood has been intensively discussed, childhood diet in the past has rarely been studied in a systematic manner. METHODS: In this study, a meta-analysis of carbon and nitrogen stable isotope ratios of post-weaning children (PWC) in Holocene human populations around the world is presented. The isotope ratios of PWC were standardized with those of adult females and males in the same population, and they were analyzed in terms of the difference in subsistence. RESULTS: Results of this study indicate that diets of PWC and adults were generally similar (most differences were within the range of ±1‰), which is consistent with the universal feature of food provisioning to PWC in humans. In hunter-gatherer populations, there is no significant difference between PWC and adult isotope ratios. In non-hunter-gatherer populations, however, PWC probably consumed significantly larger proportions of foods from lower trophic levels than did the adults, and such foods would be terrestrial C3 plants. CONCLUSIONS: Potential factors relating to the dietary differences among PWC and adults are presented from a perspective of balance between food provisioning and self-acquisition by PWC. Significant isotopic differences between PWC and adults in non-hunter-gatherer populations revealed in this study have implications for declined health during the subsistence transition in Holocene, isotopic studies using human tooth enamel, and "δ15 N dip" of subadults after weaning.

Carbon and nitrogen stable isotopic offsets between diet and hair/feces in captive chimpanzees.

RATIONALE: Estimation of the stable isotopic offsets between tissue and diet is important for dietary reconstructions. Although stable isotopic studies of chimpanzees (Pan troglodytes) are increasing, the isotopic offsets in chimpanzees have never been studied. In this study, the carbon and nitrogen stable isotopic offset values in hair and feces were measured for 13 captive chimpanzees for the first time. METHODS: All consumed food items and quantities were recorded for each individual for 1 week. Food samples were typically collected three times, hair was collected 3 weeks after the experimental week, and feces were collected ad libitum during the experimental week. The stable carbon and nitrogen isotope ratios were measured using elemental analyzer-isotope ratio mass spectrometry (EA-IRMS). RESULTS: As the results of Monte Carlo analysis, the estimated carbon and nitrogen offsets between the hair and diet were +3.0 to +3.9‰ and +2.8 to +3.7‰, respectively, for the 95% confidence intervals (CIs). The 95% CIs of the carbon and nitrogen offset values between the feces and diet were -1.6 to 0.0‰ and +1.2 to +2.7‰, respectively. CONCLUSIONS: These offset values are generally consistent with those of the other primate species reported in previous studies. However, potential variations in the offset values due to dietary and physiological factors should be studied in detail in the future. Copyright © 2016 John Wiley & Sons, Ltd.

Nitrogen fixation and nifH diversity in human gut microbiota.

It has been hypothesized that nitrogen fixation occurs in the human gut. However, whether the gut microbiota truly has this potential remains unclear. We investigated the nitrogen-fixing activity and diversity of the nitrogenase reductase (NifH) genes in the faecal microbiota of humans, focusing on Papua New Guinean and Japanese individuals with low to high habitual nitrogen intake. A (15)N2 incorporation assay showed significant enrichment of (15)N in all faecal samples, irrespective of the host nitrogen intake, which was also supported by an acetylene reduction assay. The fixed nitrogen corresponded to 0.01% of the standard nitrogen requirement for humans, although our data implied that the contribution in the gut in vivo might be higher than this value. The nifH genes recovered in cloning and metagenomic analyses were classified in two clusters: one comprising sequences almost identical to Klebsiella sequences and the other related to sequences of Clostridiales members. These results are consistent with an analysis of databases of faecal metagenomes from other human populations. Collectively, the human gut microbiota has a potential for nitrogen fixation, which may be attributable to Klebsiella and Clostridiales strains, although no evidence was found that the nitrogen-fixing activity substantially contributes to the host nitrogen balance.

Association between sex inequality in animal protein intake and economic development in the Papua New Guinea highlands: The carbon and nitrogen isotopic composition of scalp hair and fingernail.

OBJECTIVES: People in the Papua New Guinea Highlands consume sweet potatoes as their dietary staple; consumption of animal protein is limited. In such societies with marginal protein intake, the intra-household allocation of animal protein in terms of sex or age is of importance. The objective of this study was to investigate how the allocation pattern of protein-rich foods by sex and age is associated with economic development in the Papua New Guinea Highlands. METHODS: The carbon and nitrogen isotopic compositions of fingernails, collected in 1995 in two areas (Tari and Port Moresby [the national capital where Tari migrants resided]), and of scalp hair, collected in 2007, 2012, and 2013 in three areas of different degree of economic development (Levani, Tari, and Goroka) were analyzed. RESULTS: Analysis of fingernail samples showed that δ(15)N was lower in rural communities than in the urban migrant community, while a sex difference in δ(15)N (higher in males than in females) was found in the former but not in the latter community. Age was not associated with either δ(15)N or δ(13)C values. The analysis of scalp hair samples showed that δ(15)N values were lowest in Levani, the least developed area. Furthermore, there were statistically significant sex differences in δ(15)N values in Levani but not in Tari and Goroka. Age was not associated with either δ(15)N or δ(13)C values. DISCUSSION: The sex inequality in animal protein consumption seems to have decreased as the communities in the Papua New Guinea Highlands have experienced economic development.

Association of protein intakes and variation of diet-scalp hair nitrogen isotopic discrimination factor in Papua New Guinea highlanders.

OBJECTIVES: We present new nitrogen isotopic discrimination factor between diets and scalp hairs (Δ(15) NHair-Diet : δ(15) NHair - δ(15) NDiet ) for indigenous residents in three communities in the Papua New Guinea Highlands who consumed various amounts and qualities of protein. The Δ(15) N is important for precise evaluation of the dietary habits of human populations; in both contemporary and traditional lifestyles. Several hypotheses have been proposed regarding factors that affect Δ(15) N values, based largely on observations from animal feeding experiments. However, variations and factors controlling Δ(15) N in humans are not well understood, mainly due to the difficulty of controlling the diets of participants. MATERIALS AND METHODS: These residents were studied because they have maintained relatively traditional dietary habits, which allow quantitative recording of diets. Δ(15) N was estimated by comparing hair δ(15) N values to mean dietary δ(15) N values calculated from the recorded intake of each food item and their δ(15) N values. RESULTS: The results showed that: i) there was a significant difference in Δ(15) N among study locations (3.9 ± 0.9‰ for most urbanized, 5.2 ± 1.0‰ for medium and 5.0 ± 0.9‰ for least urbanized communities; range = 1.2-7.3‰ for all participants); and ii) estimated Δ(15) N values were negatively correlated with several indicators of animal protein intake (% nitrogen in diet: range = 0.9-7.6%). DISCUSSION: We hypothesize that a combination of several factors, which presumably included urea recycling and amino acid and protein recycling and/or de novo synthesis during metabolic processes, altered the Δ(15) N values of the participants.

Weaning age in an expanding population: stable carbon and nitrogen isotope analysis of infant feeding practices in the Okhotsk culture (5th-13th centuries AD) in Northern Japan.

OBJECTIVE: The Okhotsk people were sedentary hunter-gatherer-fishers who lived and prospered in Sakhalin, Hokkaido, and the Kurile Islands during the 5th to 13th centuries AD. They expanded rapidly along the northeastern coast of Hokkaido. We reconstructed infant feeding practices of the Moyoro population of the Okhotsk culture in eastern Hokkaido, Japan. METHODS: Stable isotope ratios in 58 subadult human skeletons were measured. RESULTS: The results suggest that complementary foods with a relatively low carbon isotope ratio were consumed during and after weaning, as observed in ethnographic descriptions of northern human populations such as the Ainu and isotopically suggested in ancient northern hunter-gatherer-fisher populations. Nitrogen isotope ratios of subadults showed that the age at the end of weaning in the Moyoro population was 1.8 (1.4-2.2 in 95% credible interval) years, which is earlier than that in other northern hunter-gatherer-fisher populations. CONCLUSIONS: Because weaning age is one of the most important determinants of fertility, a shorter breastfeeding period suggests increased fertility. Furthermore, better nutrition would further promote the population increase, and thus populations of the Okhotsk culture could expand into new regions. These findings are consistent with recent emerging evidence of great contributions of the Okhotsk to the formation of later Ainu populations and culture.

Infant feeding practice in medieval Japan: stable carbon and nitrogen isotope analysis of human skeletons from Yuigahama-minami.

A longer breastfeeding duration provides various positive effects in subadult health because of abundant immunological factors and nutrients in human breast milk, and decreases the natural fertility of a population through lactational amenorrhea. In this study, we measured stable carbon and nitrogen isotope ratios in the bone collagen of three adults and 45 subadults from the Yuigahama-minami site (from 12th to 14th century) in Kamakura, the early medieval capital of Japan. Marine foods, C3 -based terrestrial foods, and freshwater fish are the primarily protein sources for adults. The changes in the nitrogen isotope ratios of subadults suggest that the relative dietary protein contribution from breast milk started to decrease from 1.1 years of age and ended at 3.8 years. The age at the end of weaning in the Yuigahama-minami population was greater than that in the typical non-industrial populations, a premodern population in the Edo period Japan, and medieval populations in the UK. Skeletons of townspeople from medieval Kamakura indicate severe nutritional stress (e.g., enamel hypoplasia and cribra orbitalia), yet this longer duration of breastfeeding did not compensate adverse effects for nutritional deficiency. The longer breastfeeding period may have been a consequence of complementary food shortage and bad health of subadults. Kamakura experienced urbanization and population increase in the early medieval period. The younger age-at-death distribution and high nutritional stresses in the Yuigahama-minami population and later weaning, which is closely associated with longer inter-birth interval for mothers, suggests that Kamakura developed and increased its population by immigration during urbanization.

Reconstruction of breastfeeding and weaning practices using stable isotope and trace element analyses: A review.

Biogeochemical methods using stable isotopes and trace elements have been increasingly developed and applied to reconstruct modern and ancient breastfeeding and weaning practices of mammals, including humans, because they offer direct proxies for the dietary intake of subadults. Carbon, nitrogen, and oxygen isotopes have been used to evaluate breast milk lipid, protein, and water intake, respectively. Carbon and sulfur isotopes have been used to estimate the content of weaning foods. The elemental concentrations of Sr and Ba in subadult tissues differ because of the dietary change during the weaning process. For analyses, various tissues have been used, such as hair, nail, blood, and feces for modern mammals and bone and teeth for ancient ones. Of these, trace element analysis of tooth enamel offers a good opportunity for the reconstruction of breastfeeding and weaning practices of the more distant past at finer resolution, although further understanding of the metabolism of trace elements is necessary. There are various tissue- and element-specific advantages and disadvantages, and a combination of different proxies can illuminate practices from various viewpoints. Finally, applying the geochemical reconstruction of breastfeeding and weaning practices to human ecology, primatology, and paleoanthropology is important; basic studies of the underlying physiological mechanisms and technical improvements in the analyses will further highlight avenues for future research.

Stable isotopic reconstructions of adult diets and infant feeding practices during urbanization of the city of Edo in 17th century Japan.

The urbanization of the city of Edo, the capital of premodern Japan, has been assumed to be not as a result of natural increase but that of in-migration although this assumption has never been verified. To obtain information on natural fertility in Edo, we analyzed stable carbon and nitrogen isotopes in 46 adult and 84 subadult human skeletons excavated from the Hitotsubashi site (1657-1683 AD: the early Edo period), Tokyo, Japan and reconstructed their breastfeeding period, one of the most important determinants of fertility. Adult females are significantly more depleted in (15) N by 0.7‰ than adult males, suggesting a dietary differentiation between sexes and/or the effect of pregnancy. The changes in the nitrogen isotope ratios of subadults suggest that supplementary foods were introduced around the age of 0.2 years and weaning ended around 3.1 years, which agrees with descriptions in various historical documents of the period. The duration of breastfeeding in the Hitotsubashi population was relatively longer than those in modern industrial and traditional societies and four previously reported populations in medieval and in the industrial England. As later weaning closely associates with longer inter-birth interval for mothers, our data suggest a lower natural fertility for the Hitotsubashi population. Assuming that the proportion of married people was also lower in the major cities of the earlier Edo period, our results support the assumption that Edo developed and increased its population by attracting immigrants during urbanization.

Quantitative reconstruction of weaning ages in archaeological human populations using bone collagen nitrogen isotope ratios and approximate Bayesian computation.

BACKGROUND: Nitrogen isotope analysis of bone collagen has been used to reconstruct the breastfeeding practices of archaeological human populations. However, weaning ages have been estimated subjectively because of a lack of both information on subadult bone collagen turnover rates and appropriate analytical models. METHODOLOGY: Temporal changes in human subadult bone collagen turnover rates were estimated from data on tissue-level bone metabolism reported in previous studies. A model for reconstructing precise weaning ages was then developed using a framework of approximate Bayesian computation and incorporating the estimated turnover rates. The model is presented as a new open source R package, WARN (Weaning Age Reconstruction with Nitrogen isotope analysis), which computes the age at the start and end of weaning, (15)N-enrichment through maternal to infant tissue, and [Formula: see text] value of collagen synthesized entirely from weaning foods with their posterior probabilities. The model was applied to 39 previously reported Holocene skeletal populations from around the world, and the results were compared with weaning ages observed in ethnographic studies. CONCLUSIONS: There were no significant differences in the age at the end of weaning between the archaeological (2.80±1.32 years) and ethnographic populations. By comparing archaeological populations, it appears that weaning ages did not differ with the type of subsistence practiced (i.e., hunting-gathering or not). Most of [Formula: see text]-enrichment (2.44±0.90‰) was consistent with biologically valid values. The nitrogen isotope ratios of subadults after the weaning process were lower than those of adults in most of the archaeological populations (-0.48±0.61‰), and this depletion was greater in non-hunter-gatherer populations. Our results suggest that the breastfeeding period in humans had already been shortened by the early Holocene compared with those in extant great apes.