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Pleistocene Pongo teeth show substantial variation in size and morphology, fueling taxonomic debates about the paleodiversity of the genus. We investigated prominent features of the enamel-dentine-junction junction (EDJ)-phylogenetically informative internal structures-of 71 fossil Pongo lower molars from various sites by applying geometric morphometrics and conducted paleoproteomic analyses from enamel proteins to attempt to identify extinct orangutan species. Forty-three orangutan lower molars representing Pongo pygmaeus and Pongo abelii were included for comparison. The shape of the EDJ was analyzed by placing five landmarks on the tip of the main dentine horns, and 142 semilandmarks along the marginal ridges connecting the dentine horns. Paleoproteomic analyses were conducted on 15 teeth of Late Pleistocene Pongo using high-resolution tandem mass spectrometry. The geometric morphometric results show variations in EDJ shape regarding aspects of the height and position of the dentine horns and connecting ridges. Despite the issue of molar position and sample size, modern molars are distinguished from fossil counterparts by their elongated tooth outline and narrowly positioned dentine horns. Proteomic results show that neither a distinction of P. pygmaeus and P. abelii, nor a consistent allocation of fossil specimens to extant species is feasible. Based on the EDJ shape, the (late) Middle to Late Pleistocene Pongo samples from Vietnam share the same morphospace, supporting the previous allocation to P. devosi, although substantial overlap with Chinese fossils could also indicate close affinities with P. weidenreichi. The hypothesis that both species represent one chronospecies cannot be ruled out. Two fossil specimens, one from Tam Hay Marklot (Laos, Late Pleistocene), and another from Sangiran (Java, Early to Middle Pleistocene), along with some specimens within the Punung sample (Java), exhibit affinities with Pongo abelii. The Punung fossils might represent a mix of early Late Pleistocene and later specimens (terminal Pleistocene to Holocene) related to modern Pongo. The taxonomy and phylogeny of the complete Punung sample needs to be further investigated., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Kubat 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.)

Leather was one of the most important materials of nomadic Scythians, used for clothing, shoes, and quivers, amongst other objects. However, our knowledge regarding the specific animal species used in Scythian leather production remains limited. In this first systematic study, we used palaeoproteomics methods to analyse the species in 45 samples of leather and two fur objects recovered from 18 burials excavated at 14 different Scythian sites in southern Ukraine. Our results demonstrate that Scythians primarily used domesticated species such as sheep, goat, cattle, and horse for the production of leather, while the furs were made of wild animals such as fox, squirrel and feline species. The surprise discovery is the presence of two human skin samples, which for the first time provide direct evidence of the ancient Greek historian Herodotus' claim that Scythians used the skin of their dead enemies to manufacture leather trophy items, such as quiver covers. We argue that leather manufacture is not incompatible with a nomadic lifestyle and that Scythians possessed sophisticated leather production technologies that ensured stable supply of this essential material., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Brandt 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.)

Marine historical ecology provides a means to establish baselines to inform current fisheries management. Groupers (Epinephelidae) are key species for fisheries in the Mediterranean, which have been heavily overfished. Species abundance and distribution prior to the 20th century in the Mediterranean remains poorly known. To reconstruct the past biogeography of Mediterranean groupers, we investigated whether Zooarchaeology by Mass Spectrometry (ZooMS) can be used for identifying intra-genus grouper bones to species level. We discovered 22 novel, species-specific ZooMS biomarkers for groupers. Applying these biomarkers to Kinet Höyük, a Mediterranean archaeological site, demonstrated 4000 years of regional Epinephelus aeneus dominance and resiliency through millennia of fishing pressures, habitat degradation and climatic changes. Combining ZooMS identifications with catch size reconstructions revealed the Epinephelus aeneus capacity for growing 30 cm larger than hitherto documented, revising the maximum Total Length from 120 to 150 cm. Our results provide ecological baselines for a key Mediterranean fishery which could be leveraged to define and assess conservation targets., Competing Interests: None of the authors have any competing interests to declare., (© 2023 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

In 2018, a well-constructed cist-type grave was discovered at Ba`ja, a Neolithic village (7,400-6,800 BCE) in Southern Jordan. Underneath multiple grave layers, an 8-year-old child was buried in a fetal position. Over 2,500 beads were found on the chest and neck, along with a double perforated stone pendant and a delicately engraved mother-of-pearl ring discovered among the concentration of beads. The first was found behind the neck, and the second on the chest. The meticulous documentation of the bead distribution indicated that the assemblage was a composite ornament that had gradually collapsed, partly due to the burying position. Our aim was to challenge time degradation and to reimagine the initial composition in order to best explore the significance of this symbolic category of material culture, not as mere group of beads, but as an ornamental creation with further aesthetic, artisanal and socioeconomic implications. The reconstruction results exceeded our expectations as it revealed an imposing multi-row necklace of complex structure and attractive design. Through multiple lines of evidence, we suggest that the necklace was created at Ba`ja, although significant parts of beads were made from exotic shells and stones, including fossil amber, an unprecedented material never attested before for this period. The retrieval of such an ornament from life and its attribution to a young dead child highlights the significant social status of this individual. Beyond the symbolic functions related to identity, the necklace is believed to have played a key role in performing the inhumation rituals, understood as a public event gathering families, relatives, and people from other villages. In this sense, the necklace is not seen as belonging completely to the realm of death but rather to the world of the living, materializing a collective memory and shared moments of emotions and social cohesion., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Alarashi 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.)

"Protect and restore ecosystems and biodiversity" is the second official aim of the current UN Ocean Decade (2021 to 2030) calling for the identification and protection of critical marine habitats. However, data to inform policy are often lacking altogether or confined to recent times, preventing the establishment of long-term baselines. The unique insights gained from combining bioarchaeology (palaeoproteomics, stable isotope analysis) with contemporary data (from satellite tracking) identified habitats which sea turtles have been using in the Eastern Mediterranean over five millennia. Specifically, our analysis of archaeological green turtle ( Chelonia mydas ) bones revealed that they likely foraged on the same North African seagrass meadows as their modern-day counterparts. Here, millennia-long foraging habitat fidelity has been directly demonstrated, highlighting the significance (and long-term dividends) of protecting these critical coastal habitats that are especially vulnerable to global warming. We highlight the potential for historical ecology to inform policy in safeguarding critical marine habitats.

The Neolithic burial of Grotta di Pietra Sant'Angelo (CS) represents a unique archaeological finding for the prehistory of Southern Italy. The unusual placement of the inhumation at a rather high altitude and far from inhabited areas, the lack of funerary equipment and the prone deposition of the body find limited similarities in coeval Italian sites. These elements have prompted wider questions on mortuary customs during the prehistory of Southern Italy. This atypical case requires an interdisciplinary approach aimed to build an integrated bioarchaeological profile of the individual. The paleopathological investigation of the skeletal remains revealed the presence of numerous markers that could be associated with craft activities, suggesting possible interpretations of the individual's lifestyle. CT analyses, carried out on the maxillary bones, showed the presence of a peculiar type of dental wear, but also a good density of the bone matrix. Biomolecular and micromorphological analyses of dental calculus highlight the presence of a rich Neolithic-like oral microbiome, the composition of which is consistent with the presence pathologies. Finally, paleogenomic data obtained from the individual were compared with ancient and modern Mediterranean populations, including unpublished high-resolution genome-wide data for 20 modern inhabitants of the nearby village of San Lorenzo Bellizzi, which provided interesting insights into the biodemographic landscape of the Neolithic in Southern Italy., (© 2023. The Author(s).)

The application of mass spectrometry-based proteomics to artworks provides accurate and detailed characterization of protein-based materials used in their production. This is highly valuable to plan conservation strategies and reconstruct the artwork's history. In this work, the proteomic analysis of canvas paintings from the Danish Golden Age led to the confident identification of cereal and yeast proteins in the ground layer. This proteomic profile points to a (by-)product of beer brewing, in agreement with local artists' manuals. The use of this unconventional binder can be connected to the workshops within the Royal Danish Academy of Fine Arts. The mass spectrometric dataset generated from proteomics was also processed with a metabolomics workflow. The spectral matches observed supported the proteomic conclusions, and, in at least one sample, suggested the use of drying oils. These results highlight the value of untargeted proteomics in heritage science, correlating unconventional artistic materials with local culture and practices.

The systematics of Madagascar's extinct elephant birds remains controversial due to large gaps in the fossil record and poor biomolecular preservation of skeletal specimens. Here, a molecular analysis of 1000-year-old fossil eggshells provides the first description of elephant bird phylogeography and offers insight into the ecology and evolution of these flightless giants. Mitochondrial genomes from across Madagascar reveal genetic variation that is correlated with eggshell morphology, stable isotope composition, and geographic distribution. The elephant bird crown is dated to ca. 30 Mya, when Madagascar is estimated to have become less arid as it moved northward. High levels of between-clade genetic variation support reclassifying Mullerornis into a separate family. Low levels of within-clade genetic variation suggest there were only two elephant bird genera existing in southern Madagascar during the Holocene. However, we find an eggshell collection from Madagascar's far north that represents a unique lineage of Aepyornis. Furthermore, divergence within Aepyornis coincides with the aridification of Madagascar during the early Pleistocene ca. 1.5 Ma, and is consistent with the fragmentation of populations in the highlands driving diversification and the evolution of extreme gigantism over shorts timescales. We advocate for a revision of their taxonomy that integrates palaeogenomic and palaeoecological perspectives., (© 2023. The Author(s).)

Previously, we showed that authentic peptide sequences could be obtained from 3.8-Ma-old ostrich eggshell (OES) from the site of Laetoli, Tanzania (Demarchi et al., 2016). Here, we show that the same sequences survive in a >6.5 Ma OES recovered from a palaeosteppe setting in northwestern China. The eggshell is thicker than those observed in extant species and consistent with the Liushu Struthio sp. ootaxon. These findings push the preservation of ancient proteins back to the Miocene and highlight their potential for paleontology, paleoecology, and evolutionary biology., Competing Interests: BD, MM, ZL, TD, MC, JC No competing interests declared, (© 2022, Demarchi et al.)

Transforming growth factor-β (TGF-β) signaling is essential for numerous biologic functions. It is a highly conserved pathway found in all metazoans including the nematode Caenorhabditis elegans, which has also been pivotal in identifying many components. Utilizing a comparative evolutionary approach, we explored TGF-β signaling in nine nematode species and revealed striking variability in TGF-β gene frequency across the lineage. Of the species analyzed, gene duplications in the DAF-7 pathway appear common with the greatest disparity observed in Pristionchus pacificus. Specifically, multiple paralogues of daf-3, daf-4 and daf-7 were detected. To investigate this additional diversity, we induced mutations in 22 TGF-β components and generated corresponding double, triple, and quadruple mutants revealing both conservation and diversification in function. Although the DBL-1 pathway regulating body morphology appears highly conserved, the DAF-7 pathway exhibits functional divergence, notably in some aspects of dauer formation. Furthermore, the formation of the phenotypically plastic mouth in P. pacificus is partially influenced through TGF-β with the strongest effect in Ppa-tag-68. This appears important for numerous processes in P. pacificus but has no known function in C. elegans. Finally, we observe behavioral differences in TGF-β mutants including in chemosensation and the establishment of the P. pacificus kin-recognition signal. Thus, TGF-β signaling in nematodes represents a stochastic genetic network capable of generating novel functions through the duplication and deletion of associated genes., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Recent improvements in the analysis of ancient biomolecules from human remains and associated dental calculus have provided new insights into the prehistoric diet and genetic diversity of our species. Here we present a multi-omics study, integrating metagenomic and proteomic analyses of dental calculus, and human ancient DNA analysis of the petrous bones of two post-Last Glacial Maximum (LGM) individuals from San Teodoro cave (Italy), to reconstruct their lifestyle and the post-LGM resettlement of Europe. Our analyses show genetic homogeneity in Sicily during the Palaeolithic, representing a hitherto unknown Italian genetic lineage within the previously identified Villabruna cluster. We argue that this lineage took refuge in Italy during the LGM, followed by a subsequent spread to central-western Europe. Analysis of dental calculus showed a diet rich in animal proteins which is also reflected on the oral microbiome composition. Our results demonstrate the power of this approach in the study of prehistoric humans and will enable future research to reach a more holistic understanding of the population dynamics and ecology., (© 2022. The Author(s).)

The realization that ancient biomolecules are preserved in "fossil" samples has revolutionized archaeological science. Protein sequences survive longer than DNA, but their phylogenetic resolution is inferior; therefore, careful assessment of the research questions is required. Here, we show the potential of ancient proteins preserved in Pleistocene eggshell in addressing a longstanding controversy in human and animal evolution: the identity of the extinct bird that laid large eggs which were exploited by Australia's indigenous people. The eggs had been originally attributed to the iconic extinct flightless bird Genyornis newtoni (†Dromornithidae, Galloanseres) and were subsequently dated to before 50 ± 5 ka by Miller et al. [ Nat. Commun. 7, 10496 (2016)]. This was taken to represent the likely extinction date for this endemic megafaunal species and thus implied a role of humans in its demise. A contrasting hypothesis, according to which the eggs were laid by a large mound-builder megapode (Megapodiidae, Galliformes), would therefore acquit humans of their responsibility in the extinction of Genyornis . Ancient protein sequences were reconstructed and used to assess the evolutionary proximity of the undetermined eggshell to extant birds, rejecting the megapode hypothesis. Authentic ancient DNA could not be confirmed from these highly degraded samples, but morphometric data also support the attribution of the eggshell to Genyornis . When used in triangulation to address well-defined hypotheses, paleoproteomics is a powerful tool for reconstructing the evolutionary history in ancient samples. In addition to the clarification of phylogenetic placement, these data provide a more nuanced understanding of the modes of interactions between humans and their environment.

The Greater Glasgow & Clyde NHS Trust Community Respiratory Response Team was established to manage patients with chronic respiratory disease at home during the COVID-19 pandemic. The team aimed to avert hospital admission while maximally utilising remote consultations. This observational study analysed outcomes of the triage pathway used, use of remote consultations, hospital admissions and mortality among patients managed by the team. Patients' electronic health records were retrospectively reviewed. Rates of emergency department attendance, hospital admission and death within 28 days of referral were compared across triage pathways. Segmented linear regression was carried out for emergency admissions in Greater Glasgow and Clyde pre- and post- Community Respiratory Response Team implementation, using emergency admissions for chronic obstructive pulmonary disease in the rest of Scotland as control and adjusting for all-cause emergency admissions. The triage category correlated with hospital admission and death. The red pathway had the highest proportion attending the emergency department (21%), significantly higher than the amber and green pathways (p = 0.03 and p = 0.004, respectively). The highest number of deaths were in the blue "end-of-life" pathway (p < 0.001). 87% of interactions were undertaken remotely. Triage severity appropriately led to targeted home visits. No nosocomial COVID-19 infections occurred among patients or staff. The Community Respiratory Response Team was associated with a significant decrease in emergency admissions (RR = 0.96 for each additional month under the Poisson model) compared to the counterfactual if the service had not been in place, suggesting a benefit in reducing secondary care pressures. The Community Respiratory Response Team effectively managed patients with chronic respiratory disease in the community, with an associated reduction in secondary care pressures during the COVID-19 pandemic., (© 2022. The Author(s).)

Fur is known from contemporary written sources to have been a key commodity in the Viking Age. Nevertheless, the fur trade has been notoriously difficult to study archaeologically as fur rarely survives in the archaeological record. In Denmark, fur finds are rare and fur in clothing has been limited to a few reports and not recorded systematically. We were therefore given access to fur from six Danish high status graves dated to the Viking Age. The fur was analysed by aDNA and palaeoproteomics methods to identify the species of origin in order to explore the Viking Age fur trade. Endogenous aDNA was not recovered, but fur proteins (keratins) were analysed by MALDI-TOF-MS and LC-MS/MS. We show that Viking Age skin clothing were often composites of several species, showing highly developed manufacturing and material knowledge. For example, fur was produced from wild animals while leather was made of domesticates. Several examples of beaver fur were identified, a species which is not native to Denmark, and therefore indicative of trade. We argue that beaver fur was a luxury commodity, limited to the elite and worn as an easily recognisable indicator of social status., Competing Interests: The authors have declared that no competing interests exist.

Purpose: Few studies have examined the importance of improving the sexual health delivery system beyond HIV among trans women. We assessed survey data from the National HIV Behavioral Surveillance Transgender Woman (NHBS-Trans) Study in San Francisco to characterize the utilization of sexual health services among HIV-negative trans women and to explore opportunities to improve sexual health services for trans women., Methods: Trans women were recruited through respondent-driven sampling from July 2019 to February 2020. The analytic sample was restricted to 116 HIV-negative trans women. We identified trends in data using chi-squared tests to assess significance between sexual risk behavior and the use of preventative sexual health services and built logistic regression models to assess the relationships between sexual risk behaviors and sexually transmitted infection (STI) testing., Results: The majority of sample was trans women of color with most identifying as Latinx (42.2%). Over half were low income (56%), and majority had been homeless in past 12 months (62.9%). The prevalence of condomless receptive anal sex was 52.6% with about two-thirds (62.1%) recently having an STI test. Participants who engaged in recent condomless receptive anal sex had more than fivefold greater odds of having a recent STI test compared to their counterparts who did not (adjusted odds ratio [aOR] 5.60, 95% confidence interval [CI] 1.83-17.11; p =0.003). We also found age- and education-related disparities in STI testing., Conclusion: This study characterized the utilization of sexual health services among HIV-negative trans women and identified important disparities in STI testing. We discuss opportunities to strengthen sexual health care delivery systems., Competing Interests: No competing financial interests exist., (Copyright 2022, Mary Ann Liebert, Inc., publishers.)

We investigated the effects of environmental factors on average daily milk yield and day-to-day variation in milk yield of barn-housed Scottish dairy cows milked with an automated milking system. An incomplete Wood gamma function was fitted to derive parameters describing the milk yield curve including initial milk yield, inclining slope, declining slope, peak milk yield, time of peak, persistency (time in which the cow maintains high yield beyond the peak), and predicted total lactation milk yield (PTLMY). Lactation curves were fitted using generalized linear mixed models incorporating the above parameters (initial milk yield, inclining and declining slopes) and both the indoor and outdoor weather variables (temperature, humidity, and temperature-humidity index) as fixed effects. There was a higher initial milk yield and PTLMY in multiparous cows, but the incline slope parameter and persistency were greatest in primiparous cows. Primiparous cows took 54 d longer to attain a peak yield (mean ± standard error) of 34.25 ± 0.58 kg than multiparous (47.3 ± 0.45 kg); however, multiparous cows yielded 2,209 kg more PTLMY. The best models incorporated 2-d lagged minimum temperature. However, effect of temperature was minimal (primiparous decreased milk yield by 0.006 kg/d and multiparous by 0.001 kg/d for each degree increase in temperature). Both primiparous and multiparous cows significantly decreased in day-to-day variation in milk yield as temperature increased (primiparous cows decreased 0.05 kg/d for every degree increase in 2-d lagged minimum temperature indoors, which was greater than the effect in multiparous cows of 0.008 kg/d). Though the model estimates for both indoor and outdoor were different, a similar pattern of the average daily milk yield and day-to-day variation in milk yield and milk yield's dependence on environmental factors was observed for both primiparous and multiparous cows. In Scotland, primiparous cows were more greatly affected by the 2-d lagged minimum temperature compared with multiparous cows. After peak lactation had been reached, primiparous and multiparous cows decreased milk yield as indoor and outdoor minimum temperature increased., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Archaeologists increasingly use large radiocarbon databases to model prehistoric human demography (also termed paleo-demography). Numerous independent projects, funded over the past decade, have assembled such databases from multiple regions of the world. These data provide unprecedented potential for comparative research on human population ecology and the evolution of social-ecological systems across the Earth. However, these databases have been developed using different sample selection criteria, which has resulted in interoperability issues for global-scale, comparative paleo-demographic research and integration with paleoclimate and paleoenvironmental data. We present a synthetic, global-scale archaeological radiocarbon database composed of 180,070 radiocarbon dates that have been cleaned according to a standardized sample selection criteria. This database increases the reusability of archaeological radiocarbon data and streamlines quality control assessments for various types of paleo-demographic research. As part of an assessment of data quality, we conduct two analyses of sampling bias in the global database at multiple scales. This database is ideal for paleo-demographic research focused on dates-as-data, bayesian modeling, or summed probability distribution methodologies., (© 2022. The Author(s).)

Mollusc shells represent excellent systems for the preservation and retrieval of genuine biomolecules from archaeological or palaeontological samples. As a consequence, the post-mortem breakdown of intracrystalline mollusc shell proteins has been extensively investigated, particularly with regard to its potential use as a "molecular clock" for geochronological applications. But despite seventy years of ancient protein research, the fundamental aspects of diagenesis-induced changes to protein structures and sequences remain elusive. In this study we investigate the degradation of intracrystalline proteins by performing artificial degradation experiments on the shell of the thorny oyster, Spondylus gaederopus, which is particularly important for archaeological research. We used immunochemistry and tandem mass tag (TMT) quantitative proteomics to simultaneously track patterns of structural loss and of peptide bond hydrolysis. Powdered and bleached shell samples were heated in water at four different temperatures (80, 95, 110, 140 °C) for different time durations. The structural loss of carbohydrate and protein groups was investigated by immunochemical techniques (ELLA and ELISA) and peptide bond hydrolysis was studied by tracking the changes in protein/peptide relative abundances over time using TMT quantitative proteomics. We find that heating does not induce instant organic matrix decay, but first facilitates the uncoiling of cross-linked structures, thus improving matrix detection. We calculated apparent activation energies of structural loss: E a (carbohydrate groups) = 104.7 kJ/mol, E a (protein epitopes) = 104.4 kJ/mol, which suggests that secondary matrix structure degradation may proceed simultaneously with protein hydrolysis. While prolonged heating at 110 °C (10 days) results in complete loss of the structural signal, surviving peptide sequences were still observed. Eight hydrolysis-prone peptide bonds were identified in the top scoring shell sequence, the uncharacterised protein LOC117318053 from Pecten maximus. Interestingly, these were not the expected "weak" bonds based on published theoretical stabilities calculated for peptides in solution. This further confirms that intracrystalline protein degradation patterns are complex and that the overall microchemical environment plays an active role in protein stability. Our TMT approach represents a major stepping stone towards developing a model for studying protein diagenesis in biomineralised systems., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Hepatitis C infections continue to rise among marginalized communities, including among transgender people. Efforts to eliminate hepatitis C from San Francisco require successful identification of active HCV infections among transgender women and subsequent treatment of infection. This secondary analysis of the National HIV Behavioral Surveillance Transgender Women (NHBS-Trans) Study aims to identify areas of improvement in the hepatitis C care cascade and associated barriers that preclude successful treatment. One hundred and eighty (89.6%) trans women reported being previously screened for HCV, 47 (26.1%) reported being diagnosed with HCV, twenty-eight of the 47 (59.6%) who reported HCV diagnosis also reported that they received HCV treatment, with and 24 of the 28 (85.7%) reported completing their treatment. Overall, we detected HCV antibodies among 23.9% of participants and detected HCV RNA among 6.0%. This suggests that despite improvements in screening for HCV, active cases may not be successfully treated. Efforts to reduce barriers to HCV care should be prioritized, with heightened consideration for trans-specific needs., (© 2021 John Wiley & Sons Ltd.)

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., (© 2021 The Authors. Molecular Ecology Resources published by John Wiley & Sons Ltd.)

The domestic dog has inhabited the anthropogenic niche for at least 15 000 years, but despite their impact on human strategies, the lives of dogs and their interactions with humans have only recently become a subject of interest to archaeologists. In the Arctic, dogs rely exclusively on humans for food during the winter, and while stable isotope analyses have revealed dietary similarities at some sites, deciphering the details of provisioning strategies have been challenging. In this study, we apply zooarchaeology by mass spectrometry (ZooMS) and liquid chromatography tandem mass spectrometry to dog palaeofaeces to investigate protein preservation in this highly degradable material and obtain information about the diet of domestic dogs at the Nunalleq site, Alaska. We identify a suite of digestive and metabolic proteins from the host species, demonstrating the utility of this material as a novel and viable substrate for the recovery of gastrointestinal proteomes. The recovered proteins revealed that the Nunalleq dogs consumed a range of Pacific salmon species (coho, chum, chinook and sockeye) and that the consumed tissues derived from muscle and bone tissues as well as roe and guts. Overall, the study demonstrated the viability of permafrost-preserved palaeofaeces as a unique source of host and dietary proteomes.

The origins, prevalence and nature of dairying have been long debated by archaeologists. Within the last decade, new advances in high-resolution mass spectrometry have allowed for the direct detection of milk proteins from archaeological remains, including ceramic residues, dental calculus, and preserved dairy products. Proteins recovered from archaeological remains are susceptible to post-excavation and laboratory contamination, a particular concern for ancient dairying studies as milk proteins such as beta-lactoglobulin (BLG) and caseins are potential laboratory contaminants. Here, we examine how site-specific rates of deamidation (i.e., deamidation occurring in specific positions in the protein chain) can be used to elucidate patterns of peptide degradation, and authenticate ancient milk proteins. First, we characterize site-specific deamidation patterns in modern milk products and experimental samples, confirming that deamidation occurs primarily at low half-time sites. We then compare this to previously published palaeoproteomic data from six studies reporting ancient milk peptides. We confirm that site-specific deamidation rates, on average, are more advanced in BLG  recovered from ancient dental calculus and pottery residues. Nevertheless, deamidation rates displayed a high degree of variability, making it challenging to authenticate samples with relatively few milk peptides. We demonstrate that site-specific deamidation is a useful tool for identifying modern contamination but highlight the need for multiple lines of evidence to authenticate ancient protein data.

Trans women have been understudied in the Hepatitis C virus (HCV) epidemic, yet data suggest they may be at elevated risk of the disease. Using data collected from the Centers for Disease Control and Prevention's (CDC) National HIV Behavioral Surveillance (NHBS) survey, we measured HCV seropositivity, viremia, and associated risk factors for HCV infection among trans women in San Francisco from June 2019 to February 2020. Respondent-driven sampling (RDS) was used to obtain a diverse, community-based sample of 201 trans women, of whom 48 (23.9%, 95% CI 17.9% - 30.0%) were HCV seropositive. HCV seropositivity significantly increased with increasing age (adjusted prevalence ratio [APR] 1.04 per year, 95% CI 1.01-1.07) and history of injection drug use (APR 4.44, 95% CI 2.15-9.18). We also found that many had HCV viremia as twelve (6.0% of the total sample, 95% CI 2.7% - 9.3%) were RNA-positive for HCV. Trans women are highly impacted by HCV and could benefit from access to regular and frequent HCV screening and treatment access. HCV screening could be offered regularly in trans-specific health services, in the community, in jails and prisons, and integrated syringe exchange programs where treatment access or referral are also available., Competing Interests: The authors have declared that no competing interests exist.

We used palaeoproteomics and peptide mass fingerprinting to obtain secure species identifications of key specimens of early domesticated fauna from South Africa, dating to ca. 2000 BP. It can be difficult to distinguish fragmentary remains of early domesticates (sheep) from similar-sized local wild bovids (grey duiker, grey rhebok, springbok-southern Africa lacks wild sheep) based on morphology alone. Our analysis revealed a Zooarchaeology by Mass Spectrometry (ZooMS) marker (m/z 1532) present in wild bovids and we demonstrate through LC-MS/MS that it is capable of discriminating between wild bovids and caprine domesticates. We confirm that the Spoegrivier specimen dated to 2105 ± 65 BP is indeed a sheep. This is the earliest directly dated evidence of domesticated animals in southern Africa. As well as the traditional method of analysing bone fragments, we show the utility of minimally destructive sampling methods such as PVC eraser and polishing films for successful ZooMS identification. We also show that collagen extracted more than 25 years ago for the purpose of radiocarbon dating can yield successful ZooMS identification. Our study demonstrates the importance of developing appropriate regional frameworks of comparison for future research using ZooMS as a method of biomolecular species identification.

Background: The surge of syphilis infections in the United States continues. This study examined the sexual networks of early syphilis cases to determine whether repeat infection, demographics, or behaviors differed among network components (groups of connected persons)., Methods: We examined a retrospective cohort of all early syphilis cases in Central Indiana reported from January, 1, 2016, to January 24, 2018, in the state's reporting database. Cases were linked to their sexual partners and analyzed using UCINET and NetDraw. Attributes of network members were derived from disease intervention specialist interviews. Repeat cases were defined as those who also had ≥1 early syphilis infection at any time between 2011 and 2017. The resulting network included 1140 unique individuals, 436 cases and 704 contacts. We compared members of the main component, that is, the largest one, with the rest of the network using multiple logistic regression and network analyses., Results: The network formed 197 separate components. The main component contained 473 individuals (41% of the cohort). Main component members were more likely to have repeat early syphilis, gonorrhea, Black race, male sex, and a history of methamphetamine use, and be HIV positive by multiple logistic regression. Results from multiple centrality measures indicated persons who might be most effective at disseminating information or fragmenting the main component., Conclusions: Network analysis identified a group of linked individuals most likely to be reinfected with syphilis in Central Indiana. Further investigation is needed to determine whether engaging such a high-risk group could better focus resources and decrease infections., Competing Interests: Conflict of Interest and Sources of Funding: The authors report no conflicts of interest., (Copyright © 2020 American Sexually Transmitted Diseases Association. All rights reserved.)

Mineralized dental plaque (calculus) has proven to be an excellent source of ancient biomolecules. Here we present a Mycobacterium leprae genome (6.6-fold), the causative agent of leprosy, recovered via shotgun sequencing of sixteenth-century human dental calculus from an individual from Trondheim, Norway. When phylogenetically placed, this genome falls in branch 3I among the diversity of other contemporary ancient strains from Northern Europe. Moreover, ancient mycobacterial peptides were retrieved via mass spectrometry-based proteomics, further validating the presence of the pathogen. Mycobacterium leprae can readily be detected in the oral cavity and associated mucosal membranes, which likely contributed to it being incorporated into this individual's dental calculus. This individual showed some possible, but not definitive, evidence of skeletal lesions associated with early-stage leprosy. This study is the first known example of successful multi-omics retrieval of M. leprae from archaeological dental calculus. Furthermore, we offer new insights into dental calculus as an alternative sample source to bones or teeth for detecting and molecularly characterizing M. leprae in individuals from the archaeological record. This article is part of the theme issue 'Insights into health and disease from ancient biomolecules'.

The extensive peat bogs of Southern Scandinavia have yielded rich Mesolithic archaeological assemblages, with one of the most iconic artefacts being the bone point. Although great in number they remain understudied. Here we present a combined investigation of the typology, protein-based species composition, and absolute chronology of Maglemosian bone points. The majority of the bone points are made from cervids and bovines. However, changes both in species composition and barb morphology can be directly linked to a paucity of finds lasting nearly 600 years in Southern Scandinavia around 10,300 cal BP. We hypothesize that this hiatus was climate-driven and forced hunter-gatherers to abandon the lakes. Furthermore, the marked change in bone points coincides with a change in lithic technology. We, therefore, propose that the Maglemose culture in Southern Scandinavia is fundamentally divided into an Early Complex and a Late Complex.

Ancient protein analysis is providing new insights into the evolutionary relationships between hominin fossils across the Pleistocene. Protein identification commonly relies on the proteolysis of a protein extract using a single protease, trypsin. As with modern proteome studies, alternative or additional proteases have the potential to increase both proteome size and protein sequence recovery. This could enhance the recovery of phylogenetic information from ancient proteomes. Here we identify 18 novel hominin bone specimens from the Kleine Feldhofer Grotte using MALDI-TOF MS peptide mass fingerprinting of collagen type I. Next, we use one of these hominin bone specimens and three Late Pleistocene Equidae specimens identified in a similar manner and present a comparison of the bone proteome size and protein sequence recovery obtained after using nanoLC-MS/MS and parallel proteolysis using six different proteases, including trypsin. We observe that the majority of the preserved bone proteome is inaccessible to trypsin. We also observe that for proteins recovered consistently across several proteases, protein sequence coverage can be increased significantly by combining peptide identifications from two or more proteases. Our results thereby demonstrate that the proteolysis of Pleistocene proteomes by several proteases has clear advantages when addressing evolutionary questions in palaeoproteomics. SIGNIFICANCE: Maximizing proteome and protein sequence recovery of ancient skeletal proteomes is important when analyzing unique hominin fossils. As with modern proteome studies, palaeoproteomic analysis of Pleistocene bone and dentine samples has almost exclusively used trypsin as its only protease, despite the demonstrated advantages of alternative proteases to increase proteome recovery in modern proteome studies. We demonstrate that Pleistocene bone proteomes can be significantly expanded by using additional proteases beside trypsin, and that this also improves sequence coverage of individual proteins. The use of several alternative proteases beside trypsin therefore has major benefits to maximize the phylogenetic information retrieved from ancient skeletal proteomes., Competing Interests: Declaration of Competing Interest The authors declare there is no conflict of interest., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

The phylogenetic relationships between hominins of the Early Pleistocene epoch in Eurasia, such as Homo antecessor, and hominins that appear later in the fossil record during the Middle Pleistocene epoch, such as Homo sapiens, are highly debated 1-5 . For the oldest remains, the molecular study of these relationships is hindered by the degradation of ancient DNA. However, recent research has demonstrated that the analysis of ancient proteins can address this challenge 6-8 . Here we present the dental enamel proteomes of H. antecessor from Atapuerca (Spain) 9,10 and Homo erectus from Dmanisi (Georgia) 1 , two key fossil assemblages that have a central role in models of Pleistocene hominin morphology, dispersal and divergence. We provide evidence that H. antecessor is a close sister lineage to subsequent Middle and Late Pleistocene hominins, including modern humans, Neanderthals and Denisovans. This placement implies that the modern-like face of H. antecessor-that is, similar to that of modern humans-may have a considerably deep ancestry in the genus Homo, and that the cranial morphology of Neanderthals represents a derived form. By recovering AMELY-specific peptide sequences, we also conclude that the H. antecessor molar fragment from Atapuerca that we analysed belonged to a male individual. Finally, these H. antecessor and H. erectus fossils preserve evidence of enamel proteome phosphorylation and proteolytic digestion that occurred in vivo during tooth formation. Our results provide important insights into the evolutionary relationships between H. antecessor and other hominin groups, and pave the way for future studies using enamel proteomes to investigate hominin biology across the existence of the genus Homo.

Background and Objective: Home mechanical ventilation (HMV) is used in heterogeneous conditions underlying chronic hypercapnic respiratory failure, but there are sparse data on long-term clinical outcomes. The aim was to systematically analyse the time and the circumstances of death on HMV., Methods: All-cause mortality data of HMV patients were prospectively collected between 2008 and 2018 in a large tertiary centre. Data were categorised into diagnostic groups including neuromuscular disease (NMD), chest wall disease (CWD), chronic obstructive pulmonary disease (COPD), obesity hypoventilation syndrome (OHS), overlap syndrome of COPD and OSA (overlap) and other group. The primary outcome was time-to-death from initiation of HMV., Results: 1210 deaths were recorded over a 10-year period. Median time-to-death was 19.5 [6-55] months and differed between groups (Kruskal Wallis p < 0.001). CWD (98.5 [23.5-120] months) and slowly progressive NMD (64.5 [28-120] months) had the longest time-to-death on HMV, while OHS (33 [13-75] months) and overlap syndrome (30.5 [14.5-68.5] months) had a longer median time-to-death than COPD (19.5 [7-42.5] months) and motor neurone disease (7 [3-14] months). Daily adherence to HMV of greater than 4 h/night was associated with better outcomes (10 [3-24] vs. 30 [10-76] months; p < 0.001). 43% with confirmed location of death died outside the hospital., Conclusions: The time-to-death on home mechanical ventilation varies widely across disease groups with chronic respiratory failure and seems to be associated with daily usage time., Trial Registration: researchregistry.com UIN: researchregistry4122., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

We present the analysis of an osseous finger ring from a predominantly early Neolithic context in Denmark. To characterize the artefact and identify the raw material used for its manufacture, we performed micro-computed tomography scanning, zooarchaeology by mass spectrometry (ZooMS) peptide mass fingerprinting, as well as protein sequencing by liquid chromatography tandem mass spectrometry (LC-MS/MS). We conclude that the ring was made from long bone or antler due to the presence of osteons (Haversian canals). Subsequent ZooMS analysis of collagen I and II indicated that it was made from Alces alces or Cervus elaphus material. We then used LC-MS/MS analysis to refine our species identification, confirming that the ring was made from Cervus elaphus , and to examine the rest of the proteome. This study demonstrates the potential of ancient proteomics for species identification of prehistoric artefacts made from osseous material., Competing Interests: We declare we have no competing interests., (© 2020 The Authors.)

Background: Sepsis is a global health priority. Interventions to reduce the burden of sepsis need to be both effective and cost-effective. We performed a systematic review of the literature on health economic evaluations of sepsis treatments in critically ill adult patients and summarised the evidence for cost-effectiveness., Methods: We systematically searched MEDLINE, Embase, and the Cochrane Library using thesaurus (e.g. MeSH) and free-text terms related to sepsis and economic evaluations. We included all articles that reported, in any language, an economic evaluation of an intervention for the management of sepsis in critically ill adult patients. Data extracted included study details, intervention details, economic evaluation methodology, and outcomes. Included studies were appraised for reporting quality using the Consolidated Health Economic Evaluation Reporting Standards (CHEERS) checklist., Results: We identified 50 records representing 46 economic evaluations for a variety of interventions including antibiotics ( n = 5), fluid therapy ( n = 2), early goal-directed therapy and other resuscitation protocols ( n = 8), immunoglobulins ( n = 2), and interventions no longer in clinical use such as monoclonal antibodies ( n = 7) and drotrecogin alfa ( n = 13). Twelve (26%) evaluations were of excellent reporting quality. Incremental cost-effectiveness ratios (ICERs) ranged from dominant (lower costs and higher effectiveness) for early goal-directed therapy, albumin, and a multifaceted sepsis education program to dominated (higher costs and lower effectiveness) for polymerase chain reaction assays (LightCycler SeptiFast testing MGRADE®, SepsiTest™, and IRIDICA BAC BSI assay). ICERs varied widely across evaluations, particularly in subgroup analyses., Conclusions: There is wide variation in the cost-effectiveness of sepsis interventions. There remain important gaps in the literature, with no economic evaluations identified for several interventions routinely used in sepsis. Given the high economic and social burden of sepsis, high-quality economic evaluations are needed to increase our understanding of the cost-effectiveness of these interventions in routine clinical practice and to inform decision makers., Trial Registration: PROSPERO CRD42018095980., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s). 2020.)

Gigantopithecus blacki was a giant hominid that inhabited densely forested environments of Southeast Asia during the Pleistocene epoch 1 . Its evolutionary relationships to other great ape species, and the divergence of these species during the Middle and Late Miocene epoch (16-5.3 million years ago), remain unclear 2,3 . Hypotheses regarding the relationships between Gigantopithecus and extinct and extant hominids are wide ranging but difficult to substantiate because of its highly derived dentognathic morphology, the absence of cranial and post-cranial remains 1,3-6 , and the lack of independent molecular validation. We retrieved dental enamel proteome sequences from a 1.9-million-year-old G. blacki molar found in Chuifeng Cave, China 7,8 . The thermal age of these protein sequences is approximately five times greater than that of any previously published mammalian proteome or genome. We demonstrate that Gigantopithecus is a sister clade to orangutans (genus Pongo) with a common ancestor about 12-10 million years ago, implying that the divergence of Gigantopithecus from Pongo forms part of the Miocene radiation of great apes. In addition, we hypothesize that the expression of alpha-2-HS-glycoprotein, which has not been previously observed in enamel proteomes, had a role in the biomineralization of the thick enamel crowns that characterize the large molars in Gigantopithecus 9,10 . The survival of an Early Pleistocene dental enamel proteome in the subtropics further expands the scope of palaeoproteomic analysis into geographical areas and time periods previously considered incompatible with the preservation of substantial amounts of genetic information.

The sequencing of ancient DNA has enabled the reconstruction of speciation, migration and admixture events for extinct taxa 1 . However, the irreversible post-mortem degradation 2 of ancient DNA has so far limited its recovery-outside permafrost areas-to specimens that are not older than approximately 0.5 million years (Myr) 3 . By contrast, tandem mass spectrometry has enabled the sequencing of approximately 1.5-Myr-old collagen type I 4 , and suggested the presence of protein residues in fossils of the Cretaceous period 5 -although with limited phylogenetic use 6 . In the absence of molecular evidence, the speciation of several extinct species of the Early and Middle Pleistocene epoch remains contentious. Here we address the phylogenetic relationships of the Eurasian Rhinocerotidae of the Pleistocene epoch 7-9 , using the proteome of dental enamel from a Stephanorhinus tooth that is approximately 1.77-Myr old, recovered from the archaeological site of Dmanisi (South Caucasus, Georgia) 10 . Molecular phylogenetic analyses place this Stephanorhinus as a sister group to the clade formed by the woolly rhinoceros (Coelodonta antiquitatis) and Merck's rhinoceros (Stephanorhinus kirchbergensis). We show that Coelodonta evolved from an early Stephanorhinus lineage, and that this latter genus includes at least two distinct evolutionary lines. The genus Stephanorhinus is therefore currently paraphyletic, and its systematic revision is needed. We demonstrate that sequencing the proteome of Early Pleistocene dental enamel overcomes the limitations of phylogenetic inference based on ancient collagen or DNA. Our approach also provides additional information about the sex and taxonomic assignment of other specimens from Dmanisi. Our findings reveal that proteomic investigation of ancient dental enamel-which is the hardest tissue in vertebrates 11 , and is highly abundant in the fossil record-can push the reconstruction of molecular evolution further back into the Early Pleistocene epoch, beyond the currently known limits of ancient DNA preservation.

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.

The living tree sloths Choloepus and Bradypus are the only remaining members of Folivora, a major xenarthran radiation that occupied a wide range of habitats in many parts of the western hemisphere during the Cenozoic, including both continents and the West Indies. Ancient DNA evidence has played only a minor role in folivoran systematics, as most sloths lived in places not conducive to genomic preservation. Here we utilize collagen sequence information, both separately and in combination with published mitochondrial DNA evidence, to assess the relationships of tree sloths and their extinct relatives. Results from phylogenetic analysis of these datasets differ substantially from morphology-based concepts: Choloepus groups with Mylodontidae, not Megalonychidae; Bradypus and Megalonyx pair together as megatherioids, while monophyletic Antillean sloths may be sister to all other folivorans. Divergence estimates are consistent with fossil evidence for mid-Cenozoic presence of sloths in the West Indies and an early Miocene radiation in South America.

We thank Mr [...].

Several human dental traits typical of modern humans appear to be associated with the prolonged period of development that is a key human attribute. Understanding when, and in which early hominins, these dental traits first appeared is thus of strong interest. Using x-ray multiresolution synchrotron phase-contrast microtomography, we quantify dental growth and development in an archaic Homo juvenile from the Xujiayao site in northern China dating to 161,000-224,000 years or 104,000-125,000 years before present. Despite the archaic morphology of Xujiayao hominins, most aspects of dental development of this juvenile fall within modern human ranges (e.g., prolonged crown formation time and delayed first molar eruption). For its estimated age-at-death (6.5 years), its state of dental development is comparable to that of equivalently aged modern children. These findings suggest that several facets of modern human dental growth and development evolved in East Asia before the appearance of fully modern human morphology.

The composition of ancient oral microbiomes has recently become accessible owing to advanced biomolecular methods such as metagenomics and metaproteomics, but the utility of metaproteomics for such analyses is less explored. Here, we use quantitative metaproteomics to characterize the dental calculus associated with the remains of 21 humans retrieved during the archeological excavation of the medieval (ca. 1100-1450 CE) cemetery of Tjærby, Denmark. We identify 3671 protein groups, covering 220 bacterial species and 81 genera across all medieval samples. The metaproteome profiles of bacterial and human proteins suggest two distinct groups of archeological remains corresponding to health-predisposed and oral disease-susceptible individuals, which is supported by comparison to the calculus metaproteomes of healthy living individuals. Notably, the groupings identified by metaproteomics are not apparent from the bioarchaeological analysis, illustrating that quantitative metaproteomics has the potential to provide additional levels of molecular information about the oral health status of individuals from archeological contexts.

The range of Ixodes scapularis is expanding in Ontario, increasing the risk of Lyme disease. As an effective public health response requires accurate information on disease distribution and areas of risk, this study aims to establish the geographic distribution of I. scapularis and its associated pathogen, B. burgdorferi , in northwestern Ontario. We assessed five years of active and passive tick surveillance data in northwestern Ontario. Between 2013 and 2017, 251 I. scapularis were submitted through passive surveillance. The submission rate increased over time, and the proportion infected with B. burgdorferi was 13.5%. Active tick surveillance from 2014 to 2016 found few I. scapularis specimens. In 2017, 102 I. scapularis were found in 10 locations around the city of Kenora; 60% were infected with B. burgdorferi , eight tested positive for A. phagocytophilum , and one for POWV. I. scapularis ticks were found in 14 locations within the Northwestern Health Unit area, with seven locations containing B. burgdorferi -positive ticks. We found abundant I. scapularis populations in the southern portion of northwestern Ontario and northward expansion is expected. It is recommended that I. scapularis populations continue to be monitored and mitigation strategies should be established for rural northern communities.

The analysis of lipids (fats, oils and waxes) absorbed within archaeological pottery has revolutionized the study of past diets and culinary practices. However, this technique can lack taxonomic and tissue specificity and is often unable to disentangle signatures resulting from the mixing of different food products. Here, we extract ancient proteins from ceramic vessels from the West Mound of the key early farming site of Çatalhöyük in Anatolia, revealing that this community processed mixes of cereals, pulses, dairy and meat products, and that particular vessels may have been reserved for specialized foods (e.g., cow milk and milk whey). Moreover, we demonstrate that dietary proteins can persist on archaeological artefacts for at least 8000 years, and that this approach can reveal past culinary practices with more taxonomic and tissue-specific clarity than has been possible with previous biomolecular techniques.

Archaeological dental calculus has emerged as a rich source of ancient biomolecules, including proteins. Previous analyses of proteins extracted from ancient dental calculus revealed the presence of the dietary milk protein β-lactoglobulin, providing direct evidence of dairy consumption in the archaeological record. However, the potential for calculus to preserve other food-related proteins has not yet been systematically explored. Here we analyse shotgun metaproteomic data from 100 archaeological dental calculus samples ranging from the Iron Age to the post-medieval period (eighth century BC to nineteenth century AD) in England, as well as 14 dental calculus samples from contemporary dental patients and recently deceased individuals, to characterize the range and extent of dietary proteins preserved in dental calculus. In addition to milk proteins, we detect proteomic evidence of foodstuffs such as cereals and plant products, as well as the digestive enzyme salivary amylase. We discuss the importance of optimized protein extraction methods, data analysis approaches and authentication strategies in the identification of dietary proteins from archaeological dental calculus. This study demonstrates that proteomic approaches can robustly identify foodstuffs in the archaeological record that are typically under-represented due to their poor macroscopic preservation., (© 2018 The Authors.)

Ahead of display, a non-original layer was observed on the surface of a fragment of a wall painting by Ambrogio Lorenzetti (active 1319, died 1348/9). FTIR analysis suggested proteinaceous content. Mass spectrometry was used to better characterise this layer and revealed two protein components: sheep and cow glue and chicken and duck egg white. Analysis of post-translational modifications detected several photo-oxidation products, which suggest that the egg experienced prolonged exposure to UV light and was likely applied long before the glue layer. Additionally, glycation products detected may indicate naturally occurring glycoprotein degradation or reaction with a carbohydrate material such as starch, identified by ATR-FTIR in a cross-section of a sample taken from the painting. Palaeoproteomics is shown to provide detailed characterisation of organic layers associated with mural paintings and therefore aids reconstruction of the conservation history of these objects., (© 2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Proteomic analysis of dental calculus is emerging as a powerful tool for disease and dietary characterisation of archaeological populations. To better understand the variability in protein results from dental calculus, we analysed 21 samples from three Roman-period populations to compare: 1) the quantity of extracted protein; 2) the number of mass spectral queries; and 3) the number of peptide spectral matches and protein identifications. We found little correlation between the quantity of calculus analysed and total protein identifications, as well as no systematic trends between site location and protein preservation. We identified a wide range of individual variability, which may be associated with the mechanisms of calculus formation and/or post-depositional contamination, in addition to taphonomic factors. Our results suggest dental calculus is indeed a stable, long-term reservoir of proteins as previously reported, but further systematic studies are needed to identify mechanisms associated with protein entrapment and survival in dental calculus.

Objectives: Recently, studies have interpreted regular spacing and average number of perikymata between dental enamel defects in orangutans to reflect seasonal episodes of physiological stress. To estimate the amount of time between developmental defects (enamel hypoplasia), studies have relied on perikymata counts. Unfortunately, perikymata are frequently not continuously visible between defects, significantly reducing data sets. A method is presented here for estimating the number of perikymata between defects using standard perikymata profiles (SPP) that allow the number of perikymata between all pairs of defects across a tooth to be analyzed. The SPP method should allow the entire complement of defects to be analyzed within the context of an individual's crown formation time., Materials and Methods: The average number of perikymata were established per decile and charted to create male and female Pongo pygmaeus SPPs. The position of the beginning of each defect was recorded for lower canines from males (n = 6) and females (n = 17). The number of perikymata between defects estimated by the SPP was compared to the actual count (where perikymata were continuously visible)., Results: The number of perikymata between defects estimated by the SPPs was accurate within three perikymata and highly correlated with the actual counts, significantly increasing the number of analyzable defect pairs., Conclusion: SPPs allow all defect pairs to be included in studies of defect timing, not just those with continuously visible perikymata. Establishing an individual's entire complement of dental defects makes it possible to calculate the regularity (and potential seasonality) of defects., (© 2017 Wiley Periodicals, Inc.)

Objectives: This study investigates where the Xujiayao juvenile (I(1) and C(1) ) fits into the array of perikymata distribution patterns found within the genus Homo., Materials and Methods: In addition to the I(1) and the C(1) of the Xujiayao juvenile, this study includes samples of early Homo (H. rudolfensis and H. erectus), Neandertals, early modern humans (Qafzeh), and recent modern humans from Southern Africa, Newcastle (UK), and North America (Inupiaq, AK). Three sets of analyses were undertaken, including a comparison of percentage of perikymata in the cervical half of the crown, repeated measures analysis of the percentage of total perikymata in each decile, and canonical variates analysis using both total perikymata number and the percentage of perikymata in the cervical half of the crown., Results: The I(1) and C(1) of early Homo and Neandertals have a lower percentage of perikymata in the cervical half of the crown than modern human samples. Repeated measures analysis reveals clear distinctions in the distribution of perikymata between the modern human and fossil samples. Canonical variates analysis suggests greater differences between modern humans and the fossil samples than within the fossil samples, and classifies the Xujiayao teeth among modern humans., Discussion: The present study further clarifies variation of perikymata distribution patterns within the genus Homo. The perikymata distribution of the Xujiayao juvenile tends to be more similar to that of modern humans than to either early Homo or Neandertals., (© 2015 Wiley Periodicals, Inc.)

Milk is a major food of global economic importance, and its consumption is regarded as a classic example of gene-culture evolution. Humans have exploited animal milk as a food resource for at least 8500 years, but the origins, spread, and scale of dairying remain poorly understood. Indirect lines of evidence, such as lipid isotopic ratios of pottery residues, faunal mortality profiles, and lactase persistence allele frequencies, provide a partial picture of this process; however, in order to understand how, where, and when humans consumed milk products, it is necessary to link evidence of consumption directly to individuals and their dairy livestock. Here we report the first direct evidence of milk consumption, the whey protein β-lactoglobulin (BLG), preserved in human dental calculus from the Bronze Age (ca. 3000 BCE) to the present day. Using protein tandem mass spectrometry, we demonstrate that BLG is a species-specific biomarker of dairy consumption, and we identify individuals consuming cattle, sheep, and goat milk products in the archaeological record. We then apply this method to human dental calculus from Greenland's medieval Norse colonies, and report a decline of this biomarker leading up to the abandonment of the Norse Greenland colonies in the 15(th) century CE.