Age estimates for hominin fossils and the onset of the Upper Palaeolithic at Denisova Cave.

Denisova Cave in the Siberian Altai (Russia) is a key site for understanding the complex relationships between hominin groups that inhabited Eurasia in the Middle and Late Pleistocene epoch. DNA sequenced from human remains found at this site has revealed the presence of a hitherto unknown hominin group, the Denisovans1,2, and high-coverage genomes from both Neanderthal and Denisovan fossils provide evidence for admixture between these two populations3. Determining the age of these fossils is important if we are to understand the nature of hominin interaction, and aspects of their cultural and subsistence adaptations. Here we present 50 radiocarbon determinations from the late Middle and Upper Palaeolithic layers of the site. We also report three direct dates for hominin fragments and obtain a mitochondrial DNA sequence for one of them. We apply a Bayesian age modelling approach that combines chronometric (radiocarbon, uranium series and optical ages), stratigraphic and genetic data to calculate probabilistically the age of the human fossils at the site. Our modelled estimate for the age of the oldest Denisovan fossil suggests that this group was present at the site as early as 195,000 years ago (at 95.4% probability). All Neanderthal fossils-as well as Denisova 11, the daughter of a Neanderthal and a Denisovan4-date to between 80,000 and 140,000 years ago. The youngest Denisovan dates to 52,000-76,000 years ago. Direct radiocarbon dating of Upper Palaeolithic tooth pendants and bone points yielded the earliest evidence for the production of these artefacts in northern Eurasia, between 43,000 and 49,000 calibrated years before present (taken as AD 1950). On the basis of current archaeological evidence, it may be assumed that these artefacts are associated with the Denisovan population. It is not currently possible to determine whether anatomically modern humans were involved in their production, as modern-human fossil and genetic evidence of such antiquity has not yet been identified in the Altai region.

Trends in deamidation across archaeological bones, ceramics and dental calculus.

The accumulation of post-translational modifications (PTMs) in proteins throughout the lifecycle has been studied for decades, particularly more so with the advent of soft-ionization mass spectrometry-based proteomic techniques. However, particular PTMs, such as the deamidations of asparagine and glutamine residues, continue to accumulate in proteins that remain into the forensic, archaeological, and palaeontological records. The accurate measurement of these ancient 'molecular timers' has been proposed as a method to not only differentiate between exogenous and endogenous proteins within complex mixtures (i.e., contamination), but also as a method of providing relative age estimations into geological time. In this study we explored the extent to which deamidation varies with chronological age across different proteins in bones, as well as investigated differences between proteins across dental calculus and archaeological ceramics. We also analysed the relationships between the observed extent of deamidation and the protein primary structure. We found that collagen obtained from archaeological bones showed a chronological dependence on the extent of deamidation observed, but only when they were from similar environments, supporting prior suggestions about 'thermal age' being a major influence on the deamidation observed. Our study on non-collagenous proteins (NCPs) in archaeological bones showed that while biglycan, and to a lesser extent chondroadherin, showed positive correlations between geological age and the extent of deamidation, others including fetuin-A and serum albumin did not. However, despite the well-known dependence of deamidation on the three-dimensional structure of the peptides, we were unable to find any clear correlation between the structural motifs of the peptides in archaeological bones and the extent of deamidation observed. Our analysis of a set of food proteins obtained from Neolithic archaeological ceramics in Çatalhöyük also showed similar deamidation levels irrespective of the protein structure. Overall, our results suggest that deamidation in archaeological samples could be useful for obtaining additional information beyond identification of species and tissue type, be that as a measure of protein endogeneity and potential contamination, or a measure of protein degradation, or as an indicator of thermal age and for relative dating; however, further research needs to be undertaken to understand why particular proteins are better for this than others, going beyond simple consideration of their secondary structure.

Comparing the Use of Magnetic Beads with Ultrafiltration for Ancient Dental Calculus Proteomics.

Over the past two decades, proteomic analysis has greatly developed in application to the field of biomolecular archaeology, coinciding with advancements in LC-MS/MS instrumentation sensitivity and improvements in sample preparation methods. Recently, human dental calculus has received much attention for its well-preserved proteomes locked in mineralized dental plaque which stores information on human diets and the oral microbiome otherwise invisible to other biomolecular approaches. Maximizing proteome recovery in ancient dental calculus, available only in minute quantities and irreplaceable after destructive analysis, is of paramount importance. Here, we compare the more traditional ultrafiltration-based and acetone precipitation approaches with the newer paramagnetic bead approach in order to test the influence of demineralization acid on recovered proteome complexity obtained from specimens as well as the sequence coverages matched for significant proteins. We found that a protocol utilizing EDTA combined with paramagnetic beads increased proteome complexity, in some cases doubling the number of unique peptides and number of proteins matched, compared to protocols involving the use of HCl and either acetone precipitation or ultrafiltration. Although the increase in the number of proteins was almost exclusively of bacterial origin, a development that has implications for the study of diseases within these ancient populations, an increase in the peptide number for the dairy proteins ß-lactoglobulin and casein was also observed reflecting an increase in sequence coverage for these dietary proteins of interest. We also consider structural explanations for the discrepancies observed between these two key dietary proteins preserved in archaeological dental calculus.

Collagen Sequence Analysis Reveals Evolutionary History of Extinct West Indies Nesophontes (Island-Shrews).

Ancient biomolecule analyses are proving increasingly useful in the study of evolutionary patterns, including extinct organisms. Proteomic sequencing techniques complement genomic approaches, having the potential to examine lineages further back in time than achievable using ancient DNA, given the less stringent preservation requirements. In this study, we demonstrate the ability to use collagen sequence analyses via proteomics to assist species delimitation as a foundation for informing evolutionary patterns. We uncover biogeographic information of an enigmatic and recently extinct lineage of Nesophontes across their range on the Caribbean islands. First, evolutionary relationships reconstructed from collagen sequences reaffirm the affinity of Nesophontes and Solenodon as sister taxa within Solenodonota. This relationship helps lay the foundation for testing geographical isolation hypotheses across islands within the Greater Antilles, including movement from Cuba toward Hispaniola. Second, our results are consistent with Cuba having just two species of Nesophontes (N. micrus and N. major) that exhibit intrapopulation morphological variation. Finally, analysis of the recently described species from the Cayman Islands (N. hemicingulus) indicates that it is a closer relative to N. major rather than N. micrus as previously speculated. This proteomic sequencing improves our understanding of the origin, evolution, and distribution of this extinct mammal lineage, particularly with respect to the approximate timing of speciation. Such knowledge is vital for this biodiversity hotspot, where the magnitude of recent extinctions may obscure true estimates of species richness in the past.

Mutations in the Epithelial Cadherin-p120-Catenin Complex Cause Mendelian Non-Syndromic Cleft Lip with or without Cleft Palate.

Non-syndromic cleft lip with or without cleft palate (NS-CL/P) is one of the most common human birth defects and is generally considered a complex trait. Despite numerous loci identified by genome-wide association studies, the effect sizes of common variants are relatively small, with much of the presumed genetic contribution remaining elusive. We report exome-sequencing results in 209 people from 72 multi-affected families with pedigree structures consistent with autosomal-dominant inheritance and variable penetrance. Herein, pathogenic variants are described in four genes encoding components of the p120-catenin complex (CTNND1, PLEKHA7, PLEKHA5) and an epithelial splicing regulator (ESRP2), in addition to the known CL/P-associated gene, CDH1, which encodes E-cadherin. The findings were also validated in a second cohort of 497 people with NS-CL/P, comprising small families and singletons with pathogenic variants in these genes identified in 14% of multi-affected families and 2% of the replication cohort of smaller families. Enriched expression of each gene/protein in human and mouse embryonic oro-palatal epithelia, demonstration of functional impact of CTNND1 and ESRP2 variants, and recapitulation of the CL/P spectrum in Ctnnd1 knockout mice support a causative role in CL/P pathogenesis. These data show that primary defects in regulators of epithelial cell adhesion are the most significant contributors to NS-CL/P identified to date and that inherited and de novo single gene variants explain a substantial proportion of NS-CL/P.

Double Mandibular Osteotomy for Access to High-Carotid Pathology.

BACKGROUND: Anecdotal experience demonstrates the existence of patients with superiorly located carotid stenosis, neoplasms, or aneurysms where the mandible obstructs effective surgical access using standard techniques. As carotid pathology extends anatomically beyond the limits of standard operative technique, additional exposure becomes paramount to safely and effectively address the lesion. Double mandibular osteotomy (DMO) is one of several techniques to obtain additional exposure to high-carotid pathology; however, there is no large series to address the outcomes of patients undergoing this procedure. METHODS: A retrospective case series was performed for all patients undergoing surgery for carotid pathology from 2011-2019 that could not be approached with standard cervical incision. The primary predictor variable was high-anatomic carotid pathology necessitating DMO. The primary outcome variable was early and late complications sustained by patients. RESULTS: Fifteen patients met study criteria and underwent 16 DMOs to access high-carotid pathology including carotid stenosis (n = 8 patients), carotid aneurysm (n = 2 patients), and carotid body tumor (n = 8 patients). Two patients had dual ipsilateral pathology with one patient having both carotid artery stenosis and aneurysm, and the other patient diagnosed with carotid artery stenosis and carotid body tumor. One patient had bilateral carotid artery stenosis, each requiring high anatomic exposure for treatment. Early complications occurred in 8 patients. Five patients experienced significant dysphagia requiring enteral feeding, and 2 patients developed malocclusion directly related to the double mandibular osteotomy. One patient experienced contralateral cortical watershed infarcts. Late complications included one patient developing osteomyelitis of the mandible, and this patient also developed distal mandibular segment screw exposure. The comparison of the outcome groups for categorical predictor variables using Fisher's exact test detected no statistically significant differences for gender, hypertension, hyperlipidemia, type 2 diabetes, chronic obstructive pulmonary disease, tobacco use, chronic kidney disease, or cerebrovascular disease. For the continuous variable comparisons, independent-samples t-tests detected no difference between the complication groups for age, operative time, or years of follow-up. No significant differences were found between the groups for body mass index or intraoperative blood loss. CONCLUSIONS: The double mandibular osteotomy provides excellent exposure and surgical access to the distal internal carotid artery for repair of vascular pathology with acceptable outcomes and long-term complications compared with previously reported techniques. Because of the early complications realized with the DMO, we recommend the procedure for symptomatic patients with a high risk of failing medical therapy alone and not appropriate for endovascular treatment as well as those patients with tumors requiring surgical intervention.

Exploring Biological and Geological Age-related Changes through Variations in Intra- and Intertooth Proteomes of Ancient Dentine.

Proteomic analyses are becoming more widely used in archeology not only due to the greater preservation of proteins in ancient specimens than DNA but also because they can offer different information, particularly relating to compositional preservation and potentially a means to estimate biological and geological age. However, it remains unclear to what extent different burial environments impact these aspects of proteome decay. Teeth have to date been much less studied than bone but are ideal to explore how proteins decay with time due to the negligible turnover that occurs in dentine relative to bone. We investigated the proteome variability and deamidation levels of different sections of molar teeth from archeological bovine mandibles as well as their mandibular bone. We obtained a greater yield of proteins from the crown of the teeth but did not find differences between the different molars analyzed within each mandible. We also obtained the best variety of protein from a well-preserved mandible that was not the youngest one in terms of chronological age, showing the influence of the preservation conditions on the final proteomic outcome. Intriguingly, we also noticed an increase in abundance levels of fetuin-A in biologically younger mandibles as reported previously, but the opposite trend in tooth dentine. Interestingly, we observed higher glutamine deamidation levels in teeth from the geologically oldest mandible despite it being the biologically youngest specimen, showing that the archeological age strongly impacts on the level of deamidations observed, much more so than biological aging. This indicates that the glutamine deamidation ratio of selected peptides may act as a good predictor of the relative geochronological age of archeological specimens.

Targeted amplification of delivery to cell surface receptors by dendrimer self-assembly.

Nanometer-scale architectures assembled on cell surface receptors from smaller macromolecular constituents generated a large amplification of fluorescence. A targeted dendrimer was synthesized from a cystamine-core G4 PAMAM dendrimer, and contained an anti-BrE3 monoclonal antibody as the targeting group, several fluorophores and an average of 12 aldehyde moieties as complementary bio-orthogonal reactive sites for the covalent assembly. A cargo dendrimer, derived from a PAMAM G4 dendrimer, contained several fluorophores as the cargo for delivery and five hydrazine moieties as complimentary bio-orthogonal reactive sites. The system is designed to be flexible and allow for facile incorporation of a variety of targeting ligands.

An infant burial from Arma Veirana in northwestern Italy provides insights into funerary practices and female personhood in early Mesolithic Europe.

The evolution and development of human mortuary behaviors is of enormous cultural significance. Here we report a richly-decorated young infant burial (AVH-1) from Arma Veirana (Liguria, northwestern Italy) that is directly dated to 10,211-9910 cal BP (95.4% probability), placing it within the early Holocene and therefore attributable to the early Mesolithic, a cultural period from which well-documented burials are exceedingly rare. Virtual dental histology, proteomics, and aDNA indicate that the infant was a 40-50 days old female. Associated artifacts indicate significant material and emotional investment in the child's interment. The detailed biological profile of AVH-1 establishes the child as the earliest European near-neonate documented to be female. The Arma Veirana burial thus provides insight into sex/gender-based social status, funerary treatment, and the attribution of personhood to the youngest individuals among prehistoric hunter-gatherer groups and adds substantially to the scant data on mortuary practices from an important period in prehistory shortly following the end of the last Ice Age.

Ancient Beringian paleodiets revealed through multiproxy stable isotope analyses.

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

Comparing ancient DNA survival and proteome content in 69 archaeological cattle tooth and bone samples from multiple European sites.

Ancient DNA (aDNA) is the most informative biomolecule extracted from skeletal remains at archaeological sites, but its survival is unpredictable and its extraction and analysis is time consuming, expensive and often fails. Several proposed methods for better understanding aDNA survival are based upon the characterisation of some aspect of protein survival, but these are typically non-specific; proteomic analyses may offer an attractive method for understanding preservation processes. In this study, in-depth proteomic (LC-Orbitrap-MS/MS) analyses were carried out on 69 archaeological bovine bone and dentine samples from multiple European archaeological sites and compared with mitochondrial aDNA and amino acid racemisation (AAR) data. Comparisons of these data, including estimations of the relative abundances for seven selected non-collagenous proteins, indicate that the survival of aDNA in bone or dentine may correlate with the survival of some proteins, and that proteome complexity is a more useful predictor of aDNA survival than protein abundance or AAR. The lack of a strong correlation between the recovery of aDNA and the proteome abundance may indicate that the survival of aDNA is more closely linked to its ability to associate with bone hydroxyapatite crystals rather than to associate with proteins. SIGNIFICANCE: Ancient biomolecule survival remains poorly understood, even with great advancements in 'omics' technologies, both in genomics and proteomics. This study investigates the survival of ancient DNA in relation to that of proteins, taking into account proteome complexity and the relative protein abundances to improve our understanding of survival mechanisms. The results show that although protein abundance is not necessarily directly related to aDNA survival, proteome complexity appears to be.

Adhesive use in oral and maxillofacial surgery.

Presently, tissue adhesives and sealants have limited use in oral and maxillofacial surgical procedures. Skin closure occurs regularly with cyanoacrylate adhesives. Sealing of dural tears in conjunction with dural closure has been shown to be very successful. With the development of more head and neck reconstructive procedures and cosmetic procedures, demand will increase for better surgical adhesives. Clinical trials are beginning for newly developed adhesives with the chemical characterizations, the safe reabsorptive profile, and the adhesive strength necessary to benefit oral and maxillofacial surgery patients in the near future. Adhesives for bone fixation, while in early development, also show a promising chemical profile and will be of significant benefit to oral and maxillofacial surgical patients.

The effects of Le Fort I osteotomies on velopharyngeal and speech functions in cleft patients.

PURPOSE: Orthognathic surgery alters or even worsens symptoms of velopharyngeal insufficiency in cleft patients. The goal of this study was to evaluate how advancing the maxilla would affect the speech and articulation disorders of these patients. PATIENTS AND METHODS: This was a retrospective study in which we compiled and evaluated the speech scores of 54 cleft lip and palate patients who underwent maxillary advancement between 1981 and 2001. Although 34 individuals underwent an isolated Le Fort I advancement, 20 patients had a combined Le Fort I advancement/mandibular setback operation. The following variables were recorded from both preoperative and postoperative speech evaluations: presence of a pharyngeal flap at the time of surgery, oronasal fistulas, nasality, 7 different articulation errors, velopharyngeal function assessment, and overall speech score. Preoperative and postoperative changes in the data were analyzed using the McNemar test and paired t test. RESULTS: A decrease in competent velopharyngeal function mechanisms was noted postoperatively (42% to 18%), increased borderline incompetence (9% to 22%), and complete velopharyngeal insufficiency (13% to 20%). Speech scores deteriorated significantly (P <.05), whereas articulation defects insignificantly (P =.146) improved after surgery (84% to 73%), with those related to the anterior dentition (P =.064) showing the greatest change (64% to 47%). The frequency of hyponasality decreased after surgery. The number of cases of mild to moderate hypernasality increased. CONCLUSION: This study confirms previous findings that patients with clefts of the lip and palate or palate alone are predisposed to velopharyngeal function alteration after maxillary advancement, particularly with borderline function preoperatively. However, the results show that surgical correction of skeletal relationships and occlusion may translate into improvements in certain aspects of speech disorders.

Recovery after third molar surgery: clinical and health-related quality of life outcomes.

PURPOSE: The study goal was to assess both clinical and health-related quality of life (HRQOL) outcomes after third molar surgery. METHODS: Patients who were having 4 third molars removed were enrolled in a prospective clinical trial. Baseline data were recorded that included demographics, the patient's and surgeon's assessment of third molar conditions, and details of the surgical procedure. After surgery, clinical data were collected that detailed healing and any treatment that was rendered. Each patient was given an HRQOL instrument to complete on each postsurgery day for 14 days; the instrument was designed to assess a patient's perception of recovery in 4 main categories: pain, lifestyle, oral function, and other symptoms related to the procedure. RESULTS: Recovery data were available for 630 of 740 enrolled patients. The median age of the 630 patients was 21 years, and the median operation time was 30 minutes. Recovery for most HRQOL measures occurred within 5 days after surgery. However, recovery from pain to the criterion of "little or none" was delayed relative to other HRQOL measures. Twenty-two percent of patients were treated for delayed healing after surgery. CONCLUSIONS: Having both clinical and HRQOL data on recovery after third molar surgery could assist the surgeon when informing prospective patients about what to expect after surgery to remove third molars.