The chance discovery of a 1.5–3.5 years old mummified girl presents a unique opportunity to further our understanding of health and disease among children in 19th Century San Francisco. This study focuses on carbon and nitrogen stable isotope signatures in serial samples of hair that cover the last 14 months of her life. Results suggest an initial omnivorous diet with little input from marine resources or C4 plants. Around six months before death δ15N starts a steady increase, with a noticeable acceleration just two months before she died. The magnitude of δ15N change, +1.5‰ in total, is consi ✉ * E-mail: jweerkens@ucdavis.edu 18 9 2017 12 9 e0184921 e0184921 22 9 2017 © 2017 Eerkens et al This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract The chance discovery of a 1.5–3.5 years old mummified girl presents a unique opportunity to further our understanding of health and disease among children in 19 th Century San Francisco. This study focuses on carbon and nitrogen stable isotope signatures in serial samples of hair that cover the last 14 months of her life. This paper describes the results of high resolution stable carbon and nitrogen isotope analyses on segments of hair, with the goal of providing insight into the girl’s diet and cause of death. In particular, we aim to examine whether she died of sudden causes, for example, due to an accident or acute infectious disease, or a more drawn-out chronic illness. “Miranda Eve” The mummified girl was dubbed “Miranda Eve” by the home owner. This name was also used in popular media coverage reporting her discovery. Until genetic analyses (currently underway) confirm the true identity of the girl, we use that moniker to refer to her as well. We also attempt to characterize whether death resulting from this cause would be instantaneous (within a day; as in a gunshot wound to the head), acute (1–7 days; as in a virulent illness), or protracted (over weeks to months; as in a chronic illness). In comparison to today, where accidents (e.g., drownings, car collisions) are the top cause of death among children, and deaths due to infectious disease are rare [ 15 ], accidents comprised a minority (less than 4%) and infectious diseases the vast majority from 1861–1876. Stable isotopes in hair Human hair is composed mainly of keratin proteins that are synthesized within the body and secreted at follicle sites across the body. The effect has been observed, for example, in human hair of anorexic patients or individuals suffering serious nutritional deprivation [ 17 , 20 – 21 ], as well as in non-human animals [ 18 , 24 – 25 ], and is sometimes referred to as a “starvation signature.” By contrast, carbon isotopes in keratin reflect the source of dietary carbon, in particular C3 (e.g., wheat, rice; depleted in 13 C) vs C4 (e.g., maize, sorghum; enriched in 13 C) plants, animals that are fed C3 or C4 plants, and/or marine foods (enriched in 13 C) [ 22 ]. Because human hair grows sequentially over time, and is chemically stable once keratinized, it is possible to trace isotopically distinct sources of C and N used to synthesize hair. By sampling hair at different points along the shaft, we can trace changes in nutrient sources, including periods of undernourishment. Hair samples from Miranda Eve were pulled from the scalp, preserving the root. Hair was examined under the microscope, and any visible foreign matter removed (e.g., foreign fibers). Samples were washed of residual surface materials by sonicating in deionized water. Eight bundles of approximately 10–15 strands each (80–120 total hair strands) were aligned at the root and cut into 5 mm serial sections using conventional scissors and freeze-dried. The last two sections were combined into a single 10mm long analyte to meet minimum weight requirements. Samples were then washed again in a 2:1 chloroform-methanol solution to remove lipids Approximately 0.8mg of hair from the final 39 serial samples (last 20cm of hair growth) was submitted for δ 13 C and δ 15 N analysis at the Stable Isotope Facility at UC Davis using a PDZ Europa ANCA-GSL elemental analyzer interfaced to a PDZ Europa 20–20 isotope ratio mass spectrometer (Sercon Ltd., Cheshire, UK). Instrument precision is 0.1‰ and 0.2‰, respectively, for the two isotope measures based on repeated analyses of international standards. Individual human hairs follow a cycle including an anagen phase where the hair is actively growing, and a telogen phase where the hair is still in place in the scalp but no longer growing. The slight increase in δ 13 C associated with this final phase may represent the introduction of small amounts of food, liquids (e.g., soup, broth), or medicines containing oils or carbohydrates that were incorporated in the production of hair, or may represent catabolization of internal sources of carbon. Similar isotopic patterns have been observed in sequential dental tissues of famine victims in Ireland [ 39 ] Overall, isotopic data are inconsistent with an accident or acute disease (e.g., small pox) as a cause of death. Under such a scenario, we would expect little change in Miranda Eve’s diet in the months before death. However, unsanitary conditions, and poor understanding regarding the transmission of pathogens from person to person, could have exposed Miranda Eve, like many children, to one or more infectious agents that contributed to her death. In the final analysis, our examinations of cemetery records and stable isotope signatures in Miranda Eve’s hair underscore the dramatic effects that modern sanitation practices and medical science have had on human lives. Miranda Eve’s early death, while undoubtedly tragic for her family, was unfortunately a common experience for 19 th century families across the United States (and beyond).