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

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

Benchmarking a targeted 16S ribosomal RNA gene enrichment approach to reconstruct ancient microbial communities.

The taxonomic characterization of ancient microbiomes is a key step in the rapidly growing field of paleomicrobiology. While PCR amplification of the 16S ribosomal RNA (rRNA) gene is a widely used technique in modern microbiota studies, this method has systematic biases when applied to ancient microbial DNA. Shotgun metagenomic sequencing has proven to be the most effective method in reconstructing taxonomic profiles of ancient dental calculus samples. Nevertheless, shotgun sequencing approaches come with inherent limitations that could be addressed through hybridization enrichment capture. When employed together, shotgun sequencing and hybridization capture have the potential to enhance the characterization of ancient microbial communities. Here, we develop, test, and apply a hybridization enrichment capture technique to selectively target 16S rRNA gene fragments from the libraries of ancient dental calculus samples generated with shotgun techniques. We simulated data sets generated from hybridization enrichment capture, indicating that taxonomic identification of fragmented and damaged 16S rRNA gene sequences was feasible. Applying this enrichment approach to 15 previously published ancient calculus samples, we observed a 334-fold increase of ancient 16S rRNA gene fragments in the enriched samples when compared to unenriched libraries. Our results suggest that 16S hybridization capture is less prone to the effects of background contamination than 16S rRNA amplification, yielding a higher percentage of on-target recovery. While our enrichment technique detected low abundant and rare taxa within a given sample, these assignments may not achieve the same level of specificity as those achieved by unenriched methods.

Windows into the past: recent scientific techniques in dental analysis.

Teeth are the hardest and most chemically stable tissues in the body, are well-preserved in archaeological remains and, being resistant to decomposition in the soil, survive long after their supporting structures have deteriorated. It has long been recognised that visual and radiographic examination of teeth can provide considerable information relating to the lifestyle of an individual. This paper examines the latest scientific approaches that have become available to investigate recent and ancient teeth. These techniques include DNA analysis, which can be used to determine the sex of an individual, indicate familial relationships, study population movements, provide phylogenetic information and identify the presence of disease pathogens. A stable isotopic approach can shed light on aspects of diet and mobility and even research climate change. Proteomic analysis of ancient dental calculus can reveal specific information about individual diets. Synchrotron microcomputed tomography is a non-invasive technique which can be used to visualise physiological impactful events, such as parturition, menopause and diseases in cementum microstructure - these being displayed as aberrant growth lines.

Direct evidence of plant consumption in Neolithic Eastern Sudan from dental calculus analysis.

The Neolithic communities of Eastern Sudan combined intensive pastoralism with plant exploitation as their main subsistence strategies. However, to date, it remains unclear which plant species were part of the human diet during the Neolithic. This contribution presents direct data on plant consumption in Eastern Sudan from the Early to Late Neolithic, obtained through the analysis of microdebris inclusions in the dental calculus of 37 individuals, integrated by dentoalveolar pathology analysis of 78 individuals, from the sites UA53 (4th millennium BCE) and Mahal Teglinos (3rd-2nd millennium BCE), located in the Gash Delta/Kassala region. Dental calculus inclusions indicate a diverse intake of cereals, legumes, and tubers during the Middle Neolithic, thus supporting the hypothesis of high reliance on plant resources. Dentoalveolar pathologies, possibly related to the consumption of carbohydrate-rich foods, have also been recorded. For the Late Neolithic, consistent with the shift towards aridity that occurred in the Middle/Late Holocene, dental calculus exclusively indicates the exploitation of sorghum and tubers-species well adapted to arid conditions-showing how the Neolithic communities modified their subsistence in response to environmental changes. Evidence of plant processing techniques, such as cooking/heating, was also revealed from the dental calculus analysis.

First archaeological evidence for ginger consumption as a potential medicinal ingredient in a late medieval leprosarium at St Leonard, Peterborough, England.

Leprosy was one of the most outwardly visible diseases in the European Middle Ages, a period during which leprosaria were founded to provide space for the sick. The extant documentary evidence for leprosy hospitals, especially in relation to diet, therapeutic, and medical care, is limited. However, human dental calculus stands to be an important source of information as it provides insight into the substances people were exposed to and accumulated in their bodies during their lives. In the present study, microremains and DNA were analysed from the calculus of individuals buried in the late medieval cemetery of St Leonard, a leprosarium located in Peterborough, England. The results show the presence of ginger (Zingiber officinale), a culinary and medicinal ingredient, as well as evidence of consumption of cereals and legumes. This research suggests that affected individuals consumed ingredients mentioned in medieval medical textbooks that were used to treat regions of the body typically impacted by leprosy. To the authors' knowledge, this is the first study which has identified Zingiber officinale in human dental calculus in England or on the wider European continent.

Periodontal status among 12-year-old schoolchildren: a population-based cross-sectional study in Quito, Ecuador.

This study aimed to estimate the prevalence and extent of bleeding on probing and calculus in 12-year-old schoolchildren of Quito, Ecuador, and evaluate the associated factors. We conducted an epidemiological survey with a representative sample of 1,100 12-year-old schoolchildren from public schools in the urban area of Quito, Ecuador. We assessed the periodontal health using the Community Periodontal Index (CPI). The prevalence and extent of the periodontal condition was based on the presence of at least one site with bleeding on probing (BOP), and the presence of dental calculus was also evaluated. We used univariate and multiple multilevel Poisson regression analyses to verify the association between the independent variables and the number of sextants with BOP and calculus. The prevalence of BOP and calculus was 92% and 69.9%, respectively. The adjusted mean of the affected sextants was 4.3 and 2.2 for BOP and calculus, respectively. The mother's schooling and malocclusion were associated with the number of sextants with bleeding. The mother's schooling and dental caries experience were associated with calculus. Gingival bleeding and the presence of dental calculus are highly prevalent in 12-year-old schoolchildren from Quito. Gingival bleeding is associated with maternal education and malocclusion, and dental calculus is associated with maternal education and dental caries.

Subgingival scaling and root planing during minimally invasive periodontal surgery: A randomized controlled split-mouth trial.

BACKGROUND: The purpose of this randomized, controlled split-mouth study was to evaluate a videoscope as a visual adjunct to scaling and root planing when utilized in combination with minimally invasive surgery. METHODS: Twenty-five pairs (89 interproximal surfaces) of periodontally hopeless teeth planned for extraction were scaled and root planed with minimal surgical access using surgical loupes (control) or adjunctive use of a videoscope (test). Teeth were extracted with minimal trauma, stained with methylene blue, and photographed with a digital microscope for analysis. The primary outcome of residual calculus was calculated as a percentage of the total interproximal area of interest. Secondary outcomes included treatment time, as well as residual calculus according to probing depth, tooth location, and treatment date. Data were analyzed using Student's paired t-tests, two-way analyses of variance, and Spearman's correlation tests. RESULTS: Residual calculus area was 2.61% on control and 2.71% on test surfaces with no significant difference between groups. Subgroup analysis showed no difference in residual calculus between groups at moderate or deep sites. Treatment time per surface was significantly longer in the test group compared to the control group. Treatment order, tooth location, and operator experience did not significantly affect the primary outcome. CONCLUSIONS: Though the videoscope provided excellent visual access, it did not improve the efficacy of root planing for flat interproximal surfaces during minimally invasive periodontal surgery. Small amounts of calculus remain after instrumentation even with minimal surgical access and when root surfaces appear visually clean and tactilely smooth.

Co-occurrence of periodontal lesions and dental wear in incisor and masticatory teeth in two sheep flocks in Brazil.

BACKGROUND: Periodontitis and tooth wear are multifactorial diseases with distinct etiopathogenesis that affect the health, feed efficiency and welfare of sheep. METHODS: This study evaluated the co-occurrence of tooth wear and periodontal lesions in 129 ewes from two Brazilian flocks, clinically classified the lesions and presence of dental calculus, and identified potential pathogens in the dental biofilm of 63 ewes by polymerase chain reaction. RESULTS: Of the 129 ewes included in the study, 75 presented periodontal lesions, while all animals presented tooth wear and dental calculus. Of the animals with periodontal lesions, 16.2% had lesions in incisor teeth and 52.7% in masticatory teeth. Regarding excessive tooth wear, 38.6% had severe wear on the incisor teeth and 89.1% on the masticatory teeth. Ewes older than 36 months had a higher frequency of periodontal lesions in incisor teeth (p < 0.001) and a greater amount of dental calculus (p < 0.001), but there was no association between tooth wear and animal age. Fusobacterium nucleatum, Tannerella forsythia and Fusobacterium necrophorum predominated in periodontal lesions. LIMITATION: This study is limited by the small sample size and lack of diagnostic imaging to assess periodontal disease. CONCLUSION: The co-occurrence of periodontal lesions and excessive dental wear involving both the incisor and masticatory teeth suggests that although the two diseases have different aetiologies, they likely have common risk factors.

Paleoserological detection of Coronavirus antigens in dental calculus of human remains dating from the beginning of the 19th century, French Ardennes.

OBJECTIVE: Vanishing viral RNA restricts our ability to detect ancient pathogens, so, we used paleo serological approaches to trace the dynamics of the Coronavirus in ancient populations. MATERIALS AND METHODS: We investigated 10 ancient dental calculus samples collected from a cemetery dated to the beginning of the 19th century and excavated in Charleville-Mézières. After paleoserum samples were extracted from dental calculus, paleoserology using mini-line-blot incorporating one alpha-Coronavirus (Coronavirus 229 E) and two beta-Coronavirus (Coronavirus OC 43, SARS-CoV-2) antigens and controls was completed by an automated Western blotting assay. RESULTS: Once appropriate controls had validated the data, mini-line-blot detected antibodies against the two beta-Coronavirus antigens in individuals US1300 and US1339, automated Western blotting confirming one beta-Coronavirus antigen for individual US1300 and an additional individual US1326. DISCUSSION: Combing mini-line blot and automated Western blot assays made it possible to detect immunoreactive immunoglobulin tracing circulation of Coronavirus in France at the very beginning of the 19th century.

Ancient human dental calculus metadata collection and sampling strategies: Recommendations for best practices.

OBJECTIVES: Ancient human dental calculus is a unique, nonrenewable biological resource encapsulating key information about the diets, lifestyles, and health conditions of past individuals and populations. With compounding calls its destructive analysis, it is imperative to refine the ways in which the scientific community documents, samples, and analyzes dental calculus so as to maximize its utility to the public and scientific community. MATERIALS AND METHODS: Our research team conducted an IRB-approved survey of dental calculus researchers with diverse academic backgrounds, research foci, and analytical specializations. RESULTS: This survey reveals variation in how metadata is collected and utilized across different subdisciplines and highlights how these differences have profound implications for dental calculus research. Moreover, the survey suggests the need for more communication between those who excavate, curate, and analyze biomolecular data from dental calculus. DISCUSSION: Challenges in cross-disciplinary communication limit researchers' ability to effectively utilize samples in rigorous and reproducible ways. Specifically, the lack of standardized skeletal and dental metadata recording and contamination avoidance procedures hinder downstream anthropological applications, as well as the pursuit of broader paleodemographic and paleoepidemiological inquiries that rely on more complete information about the individuals sampled. To provide a path forward toward more ethical and standardized dental calculus sampling and documentation approaches, we review the current methods by which skeletal and dental metadata are recorded. We also describe trends in sampling and contamination-control approaches. Finally, we use that information to suggest new guidelines for ancient dental calculus documentation and sampling strategies that will improve research practices in the future.

Obesity Phenotypes and Dental Calculus in Young Adults: CHIEF Oral Health Study.

AIM: The study aimed to examine the association of obesity phenotypes with dental calculus. BACKGROUND: Obesity has been recognized as a risk factor for kidney and gallbladder stones formation and periodontitis. OBJECTIVE: We have investigated the association between obesity, metabolic risk factors, and dental calculus, which is a sequela following periodontitis. METHODS: This study included 5,281 military members, aged 19-45 years, without antihypertensive medications in Taiwan. Obesity was defined as body mass index ≥27.5 kg/m2, and metabolic syndrome (MetS) was defined according to the modified ATP III criteria. Supragingival calculus in any teeth, except for impacted teeth and the third molar, was the outcome of interest. Multiple linear regression analysis with adjustments for age, sex, toxic substance use, brushing teeth frequency, and blood leukocyte counts, was used to determine the association of obesity with dental calculus numbers. Multiple logistic regression analysis was used to assess the association between obesity with or without MetS and the presence of any dental calculus. RESULTS: BMI was positively correlated to dental calculus numbers [ß and confidence intervals (CI) = 0.023 (0.014, 0.032)]. Compared to the obesity(-)/MetS(-) group, there were dosedependent associations for the obesity(-)/MetS(+), obesity(+)/MetS(-), and obesity(+)/MetS(+) groups with the presence of any dental calculus [odds ratios (ORs): 1.08 (0.76, 1.53), 1.31 (1.08, 1.58), and 1.51 (1.20, 1.90), respectively]. Of the metabolic risk factors, abdominal obesity and hypertension were independently associated with dental calculus [ORs: 1.33 (1.13, 1.55) and 1.30 (1.11, 1.52), respectively]. CONCLUSION: This study suggests general obesity as an independent risk factor for dental calculus formation, and MetS, particularly the components of abdominal obesity, and hypertension may also increase the prevalence of dental calculus. Diet control and regular exercise might be preventive measures for the development of both obesity and dental calculus.

The evidential value of dental calculus in the identification process.

DNA analysis-based identification is by far the gold standard in forensic genetics and it should be performed in every case involving human remains or unidentified bodies. Bones and teeth are the preferred source of human DNA for genetic analysis. However, there are cases where the nature of the proceedings and historical significance prevent the disruption of skeletal structure. The remains may also be heavily degraded. In such situations, forensic geneticists seek alternative sources of human DNA. Teeth calculus has proven to be a viable source of DNA for identification purposes. The aim of this study was to assess the concentration of human DNA in teeth calculus and evaluate the usefulness of teeth calculus as a DNA source in the identification process. Teeth calculus was collected from skeletons exhumed between 2021 and 2022 by the PBGOT (Polish Genetic Database of Victims of Totalitarianism) team from the former Stalag IID prisoner-of-war camp in Stargard. Genetic analyses included the determination of autosomal and Y-STR markers. The total concentration of human DNA was also evaluated in samples from teeth calculus and teeth taken from the same individuals. The pilot study included 22 skeletons with a sufficient amount of calculus for isolation (specified in the protocol). Samples were taken from the largest areas of calculus deposited on lingual surfaces of mandibular incisors. The prepared samples underwent DNA extraction. Our study demonstrated that teeth calculus is a source of human DNA for remains from the World War II period. The obtained DNA concentration allowed for the determination of STR markers. It was shown that teeth calculus contains human DNA in an amount suitable for preliminary identification analyses.

Ancient dental calculus reveals oral microbiome shifts associated with lifestyle and disease in Great Britain.

The prevalence of chronic, non-communicable diseases has risen sharply in recent decades, especially in industrialized countries. While several studies implicate the microbiome in this trend, few have examined the evolutionary history of industrialized microbiomes. Here we sampled 235 ancient dental calculus samples from individuals living in Great Britain (∼2200 BCE to 1853 CE), including 127 well-contextualized London adults. We reconstructed their microbial history spanning the transition to industrialization. After controlling for oral geography and technical biases, we identified multiple oral microbial communities that coexisted in Britain for millennia, including a community associated with Methanobrevibacter, an anaerobic Archaea not commonly prevalent in the oral microbiome of modern industrialized societies. Calculus analysis suggests that oral hygiene contributed to oral microbiome composition, while microbial functions reflected past differences in diet, specifically in dairy and carbohydrate consumption. In London samples, Methanobrevibacter-associated microbial communities are linked with skeletal markers of systemic diseases (for example, periostitis and joint pathologies), and their disappearance is consistent with temporal shifts, including the arrival of the Second Plague Pandemic. This suggests pre-industrialized microbiomes were more diverse than previously recognized, enhancing our understanding of chronic, non-communicable disease origins in industrialized populations.

Factors associated with oral health care behaviors of pregnant women in a northeastern province in Thailand: A hospital-based cross-sectional study.

BACKGROUND: Oral healthcare behavior leads to oral health status. Factors associated with oral healthcare behavior might affect oral hygiene in pregnant women, who are at high risk for gingivitis and dental caries. This study aimed to explore factors associated with oral healthcare behaviors during pregnancy among pregnant women in a northeastern province of Thailand. METHOD: A total of 405 pregnant women who attended antenatal care clinics at one of the government hospitals in the province were invited to participate in this cross-sectional study. Dentists in the hospitals measured pregnant women's gingivitis and dental calculus status using mouth mirrors and explorers. A structured questionnaire was used to obtain variables of interest. Linear regression analysis, Beta and 95% confidence interval (CI) were applied. RESULTS: The majority were 20-24 years old (33.6%). Most of the participants had received upper secondary education (37.6%). Majority had gingivitis (88.1%) and dental calculus (88.6%). The findings revealed that age (Beta = -0.129, 95%CI = -0.269, -0.016), educational level (Beta = 0.118, 95% CI = 0.110, 0.183), and oral health literacy (Beta = 0.283, 95% CI = 0.156, 0.319) were statistically significant factors associated with oral healthcare behaviors. CONCLUSION: Younger pregnant women had better oral healthcare behaviors than older pregnant women and pregnant women had better oral healthcare behaviors due to higher educational levels and oral health literacy. Oral health promotion should be improved through oral health literacy, and interventions should be added to improve oral care skills particularly in older pregnant women as they are at a greater risk for poor oral healthcare behaviors.

Analysis of starch grains trapped in human dental calculus in Áspero, Peru during the Initial Formative Period (3000-1800 BCE).

The objective of this research is to identify the plants consumed and to determine their dietary importance in Áspero, an urban center on the coast of the Supe Valley, Peru. Consequently, starch grains trapped in the human dental calculus of nine individuals were recovered, while the results from one individual from the Sacred City of Caral, located in the interior of Supe Valley, are presented. Eight species of food plants were identified, among them C3 plants: sweet potato, squash, potato, chili pepper, algarrobo, manioc and bean and C4 plant: maize. Previous isotopic analysis indicates that C3 plants formed the foundation of the diet at Áspero and Caral. Our results indicate a high ubiquity of C3 plants like sweet potato (100%) and squash (90%) suggesting, with caution, that these taxa were an important C3 source in the menu. Maize, C4 plant, showed a similar ubiquity (100%) to sweet potato and squash, however, previous isotopic analysis indicate that maize was a marginal food in Áspero and Caral. These results support that the absence and abundance of starch grains cannot be employed to directly infer the frequency of intake of C3 and C4 plants within a small population, as suggested by previous studies.

Efficacy of two diode lasers in the removal of calculus from the root surface: An in vitro study.

INTRODUCTION: Scaling and root planning (SRP) is still the gold standard of nonsurgical periodontal therapy, and it has been accompanied by several supportive therapies in recent years. One of the most studied methods is the diode laser, thanks to its thermal and bactericidal properties. Our trial intended to verify whether it could influence the chemical bond between calculus and the root surface. OBJECTIVES: The aim of the study was to assess the efficacy of the diode laser prior the mechanical removal of calculus in an in vitro application. The reduction in time and the number of strokes required to clean the untreated root surfaces were evaluated as primary outcomes. The pressure was considered as a secondary outcome. METHODS: A total of 75 extracted human teeth with subgingival calculus were assigned equally among three treatment groups (n = 25) according to the size of the occupied areas, which were classified by evaluating the pixel numbers. The groups were assigned to either no pretreatment application (A), Laser Diode Fox III (Sweden & Martina) (B) or Wiser Laser Evolution (Doctor Smile) (C). The weight for instrumentation was calibrated for an After Five curette (Hu-Friedy, Chicago). A new set of tools was used for each group, and the curettes were sharpened after each use with the Sidekick sharpener (Hu-Friedy, Chicago). RESULTS: A Kruskal-Wallis test was used to assess the significance for each considered parameter. The results were statistically significant for each parameter for the two groups where the laser was used compared to the control group. CONCLUSIONS: Despite the limitations of an in vitro study, data showed that the diode laser had an overall positive effect on root debridement, facilitating SRP in terms of stroke count, time, and pressure.

Ultra-high performance liquid chromatography high-resolution mass spectrometry for metabolomic analysis of dental calculus from Duke Alessandro Farnese and Maria D'Aviz.

Dental calculus is a valuable resource for the reconstruction of dietary habits and oral microbiome of past populations. In 2020 the remains of Duke Alessandro Farnese and his wife Maria D'Aviz were exhumed to get novel insights into the causes of death. This study aimed to investigate the dental calculus metabolome of the noble couple by untargeted metabolomics. The pulverized samples were decalcified in a water-formic acid mixture, extracted using methanol/acetonitrile and analyzed by ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS) using a reversed-phase separation followed by electrospray ionization and full scan in positive and negative ion mode. Waters Synapt-G2-Si High-Definition hybrid quadrupole time-of-flight mass spectrometer was used. Significant features were then identified using MSE acquisition mode, recording information on exact mass precursor and fragment ions within the same run. This approach, together with data pre-treatment and multivariate statistical analysis allowed for the identification of compounds able to differentiate between the investigated samples. More than 200 metabolites were identified, being fatty acids, alcohols, aldehydes, phosphatidylcholines, phosphatidylglycerols, ceramides and phosphatidylserines the most abundant classes. Metabolites deriving from food, bacteria and fungi were also determined, providing information on the habits and oral health status of the couple.