Threatened North African seagrass meadows have supported green turtle populations for millennia.

"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.

Human-like enamel growth in Homo naledi.

OBJECTIVES: A modern pattern (rate and duration) of dental development occurs relatively recently during human evolution. Given the temporal overlap of Homo naledi with the first appearance of fossil Homo sapiens in Africa, this small-bodied and small-brained hominin presents an opportunity to elucidate the evolution of enamel growth in the hominin clade. Here we conduct the first histological study of two permanent mandibular canines and one permanent maxillary first molar, representing three individuals attributed to H. naledi. We reconstruct the rate and duration of enamel growth and compare these findings to those reported for other fossil hominins and recent humans. MATERIALS AND METHODS: Thin sections of each tooth were produced using standard histological methods. Daily and longer period incremental markings were measured to reconstruct enamel secretion and extension rates, Retzius periodicity, canine crown and molar cusp formation time. RESULTS: Daily enamel secretion rates overlapped with those from recent hominins. Canine crown formation time is similar to that observed in recent Europeans but is longer than canine formation times reported for most other hominins including Australopithecus and H. neanderthalensis. The extended period of canine formation appears to be due to a relatively tall enamel crown and a sustained slow rate of enamel extension in the cervical portion of the crown. A Retzius periodicity of 11 days for the canines, and nine days for the molar, in H. naledi parallel results found in recent humans. An 11-day periodicity has not been reported for Late Pleistocene Homo (H. erectus, H. neanderthalensis) and is rarely found in Australopithecus and Paranthropus species. DISCUSSION: Enamel growth of H. naledi is most similar to recent humans though comparative data are limited for most fossil hominin species. The high Retzius periodicity values do not follow expectations for a small-brained hominin.

Sequential trypsin and ProAlanase digestions unearth immunological protein biomarkers shrouded by skeletal collagen.

This study investigates the efficacy of proteomic analysis of human remains to identify active infections in the past through the detection of pathogens and the host response to infection. We advance leprosy as a case study due to the sequestering of sufferers in leprosaria and the suggestive skeletal lesions that can result from the disease. Here we present a sequential enzyme extraction protocol, using trypsin followed by ProAlanase, to reduce the abundance of collagen peptides and in so doing increase the detection of non-collagenous proteins. Through our study of five individuals from an 11th to 18th century leprosarium, as well as four from a contemporaneous non-leprosy associated cemetery in Barcelona, we show that samples from 2 out of 5 leprosarium individuals extracted with the sequential digestion methodology contain numerous host immune proteins associated with modern leprosy. In contrast, individuals from the non-leprosy associated cemetery and all samples extracted with a trypsin-only protocol did not. Through this study, we advance a palaeoproteomic methodology to gain insights into the health of archaeological individuals and take a step toward a proteomics-based method to study immune responses in past populations.

Deep-time phylogenetic inference by paleoproteomic analysis of dental enamel.

In temperate and subtropical regions, ancient proteins are reported to survive up to about 2 million years, far beyond the known limits of ancient DNA preservation in the same areas. Accordingly, their amino acid sequences currently represent the only source of genetic information available to pursue phylogenetic inference involving species that went extinct too long ago to be amenable for ancient DNA analysis. Here we present a complete workflow, including sample preparation, mass spectrometric data acquisition and computational analysis, to recover and interpret million-year-old dental enamel protein sequences. During sample preparation, the proteolytic digestion step, usually an integral part of conventional bottom-up proteomics, is omitted to increase the recovery of the randomly degraded peptides spontaneously generated by extensive diagenetic hydrolysis of ancient proteins over geological time. Similarly, we describe other solutions we have adopted to (1) authenticate the endogenous origin of the protein traces we identify, (2) detect and validate amino acid variation in the ancient protein sequences and (3) attempt phylogenetic inference. Sample preparation and data acquisition can be completed in 3-4 working days, while subsequent data analysis usually takes 2-5 days. The workflow described requires basic expertise in ancient biomolecules analysis, mass spectrometry-based proteomics and molecular phylogeny. Finally, we describe the limits of this approach and its potential for the reconstruction of evolutionary relationships in paleontology and paleoanthropology.

Grouping groupers in the Mediterranean: Ecological baselines revealed by ancient proteins.

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.

Comparing extraction method efficiency for high-throughput palaeoproteomic bone species identification.

High-throughput proteomic analysis of archaeological skeletal remains provides information about past fauna community compositions and species dispersals in time and space. Archaeological skeletal remains are a finite resource, however, and therefore it becomes relevant to optimize methods of skeletal proteome extraction. Ancient proteins in bone specimens can be highly degraded and consequently, extraction methods for well-preserved or modern bone might be unsuitable for the processing of highly degraded skeletal proteomes. In this study, we compared six proteomic extraction methods on Late Pleistocene remains with variable levels of proteome preservation. We tested the accuracy of species identification, protein sequence coverage, deamidation, and the number of post-translational modifications per method. We find striking differences in obtained proteome complexity and sequence coverage, highlighting that simple acid-insoluble proteome extraction methods perform better in highly degraded contexts. For well-preserved specimens, the approach using EDTA demineralization and protease-mix proteolysis yielded a higher number of identified peptides. The protocols presented here allowed protein extraction from ancient bone with a minimum number of working steps and equipment and yielded protein extracts within three working days. We expect further development along this route to benefit large-scale screening applications of relevance to archaeological and human evolution research.

Palaeoproteomics identifies beaver fur in Danish high-status Viking Age burials - direct evidence of fur trade.

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.

The degradation of intracrystalline mollusc shell proteins: A proteomics study of Spondylus gaederopus.

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: Ea (carbohydrate groups) = 104.7 kJ/mol, Ea (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.

Palaeoproteomics confirm earliest domesticated sheep in southern Africa ca. 2000 BP.

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.

Multi-protease analysis of Pleistocene bone proteomes.

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.

The putative tumour suppressor protein Latexin is secreted by prostate luminal cells and is downregulated in malignancy.

Loss of latexin (LXN) expression negatively correlates with the prognosis of several human cancers. Despite association with numerous processes including haematopoietic stem cell (HSC) fate, inflammation and tumour suppression, a clearly defined biological role for LXN is still lacking. Therefore, we sought to understand LXN expression and function in the normal and malignant prostate to assess its potential as a therapeutic target. Our data demonstrate that LXN is highly expressed in normal prostate luminal cells but downregulated in high Gleason grade cancers. LXN protein is both cytosolic and secreted by prostate cells and expression is directly and potently upregulated by all-trans retinoic acid (atRA). Whilst overexpression of LXN in prostate epithelial basal cells did not affect cell fate, LXN overexpression in the luminal cancer line LNCaP reduced plating efficiency. Transcriptome analysis revealed that LXN overexpression had no direct effects on gene expression but had significant indirect effects on important genes involved in both retinoid metabolism and IFN-associated inflammatory responses. These data highlight a potential role for LXN in retinoid signaling and inflammatory pathways. Investigating the effects of LXN on immune cell function in the tumour microenvironment (TME) may reveal how observed intratumoural loss of LXN affects the prognosis of many adenocarcinomas.

A 5700 year-old human genome and oral microbiome from chewed birch pitch.

The rise of ancient genomics has revolutionised our understanding of human prehistory but this work depends on the availability of suitable samples. Here we present a complete ancient human genome and oral microbiome sequenced from a 5700 year-old piece of chewed birch pitch from Denmark. We sequence the human genome to an average depth of 2.3× and find that the individual who chewed the pitch was female and that she was genetically more closely related to western hunter-gatherers from mainland Europe than hunter-gatherers from central Scandinavia. We also find that she likely had dark skin, dark brown hair and blue eyes. In addition, we identify DNA fragments from several bacterial and viral taxa, including Epstein-Barr virus, as well as animal and plant DNA, which may have derived from a recent meal. The results highlight the potential of chewed birch pitch as a source of ancient DNA.

Spontaneous development of Epstein-Barr Virus associated human lymphomas in a prostate cancer xenograft program.

Prostate cancer research is hampered by the lack of in vivo preclinical models that accurately reflect patient tumour biology and the clinical heterogeneity of human prostate cancer. To overcome these limitations we propagated and characterised a new collection of patient-derived prostate cancer xenografts. Tumour fragments from 147 unsupervised, surgical prostate samples were implanted subcutaneously into immunodeficient Rag2-/-γC-/- mice within 24 hours of surgery. Histologic and molecular characterisation of xenografts was compared with patient characteristics, including androgen-deprivation therapy, and exome sequencing. Xenografts were established from 47 of 147 (32%) implanted primary prostate cancers. Only 14% passaged successfully resulting in 20 stable lines; derived from 20 independent patient samples. Surprisingly, only three of the 20 lines (15%) were confirmed as prostate cancer; one line comprised of mouse stroma, and 16 were verified as human donor-derived lymphoid neoplasms. PCR for Epstein-Barr Virus (EBV) nuclear antigen, together with exome sequencing revealed that the lymphomas were exclusively EBV-associated. Genomic analysis determined that 14 of the 16 EBV+ lines had unique monoclonal or oligoclonal immunoglobulin heavy chain gene rearrangements, confirming their B-cell origin. We conclude that the generation of xenografts from tumour fragments can commonly result in B-cell lymphoma from patients carrying latent EBV. We recommend routine screening, of primary outgrowths, for latent EBV to avoid this phenomenon.