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.

The Role of Cytokines Produced via the NLRP3 Inflammasome in Mouse Macrophages Stimulated with Dental Calculus in Osteoclastogenesis.

Dental calculus (DC) is a common deposit in periodontitis patients. We have previously shown that DC contains both microbial components and calcium phosphate crystals that induce an osteoclastogenic cytokine IL-1ß via the NLRP3 inflammasome in macrophages. In this study, we examined the effects of cytokines produced by mouse macrophages stimulated with DC on osteoclastogenesis. The culture supernatants from wild-type (WT) mouse macrophages stimulated with DC accelerated osteoclastogenesis in RANKL-primed mouse bone marrow macrophages (BMMs), but inhibited osteoclastogenesis in RANKL-primed RAW-D cells. WT, but not NLRP3-deficient, mouse macrophages stimulated with DC produced IL-1ß and IL-18 in a dose-dependent manner, indicating the NLRP3 inflammasome-dependent production of IL-1ß and IL-18. Both WT and NLRP3-deficient mouse macrophages stimulated with DC produced IL-10, indicating the NLRP3 inflammasome-independent production of IL-10. Recombinant IL-1ß accelerated osteoclastogenesis in both RANKL-primed BMMs and RAW-D cells, whereas recombinant IL-18 and IL-10 inhibited osteoclastogenesis. These results indicate that DC induces osteoclastogenic IL-1ß in an NLRP3 inflammasome-dependent manner and anti-osteogenic IL-18 and IL-10 dependently and independently of the NLRP3 inflammasome, respectively. DC may promote alveolar bone resorption via IL-1ß induction in periodontitis patients, but suppress resorption via IL-18 and IL-10 induction in some circumstances.

Tracking the transition to agriculture in Southern Europe through ancient DNA analysis of dental calculus.

Archaeological dental calculus, or mineralized plaque, is a key tool to track the evolution of oral microbiota across time in response to processes that impacted our culture and biology, such as the rise of farming during the Neolithic. However, the extent to which the human oral flora changed from prehistory until present has remained elusive due to the scarcity of data on the microbiomes of prehistoric humans. Here, we present our reconstruction of oral microbiomes via shotgun metagenomics of dental calculus in 44 ancient foragers and farmers from two regions playing a pivotal role in the spread of farming across Europe-the Balkans and the Italian Peninsula. We show that the introduction of farming in Southern Europe did not alter significantly the oral microbiomes of local forager groups, and it was in particular associated with a higher abundance of the species Olsenella sp. oral taxon 807. The human oral environment in prehistory was dominated by a microbial species, Anaerolineaceae bacterium oral taxon 439, that diversified geographically. A Near Eastern lineage of this bacterial commensal dispersed with Neolithic farmers and replaced the variant present in the local foragers. Our findings also illustrate that major taxonomic shifts in human oral microbiome composition occurred after the Neolithic and that the functional profile of modern humans evolved in recent times to develop peculiar mechanisms of antibiotic resistance that were previously absent.

Effectiveness of decontamination protocols when analyzing ancient DNA preserved in dental calculus.

Ancient DNA analysis of human oral microbial communities within calcified dental plaque (calculus) has revealed key insights into human health, paleodemography, and cultural behaviors. However, contamination imposes a major concern for paleomicrobiological samples due to their low endogenous DNA content and exposure to environmental sources, calling into question some published results. Decontamination protocols (e.g. an ethylenediaminetetraacetic acid (EDTA) pre-digestion or ultraviolet radiation (UV) and 5% sodium hypochlorite immersion treatments) aim to minimize the exogenous content of the outer surface of ancient calculus samples prior to DNA extraction. While these protocols are widely used, no one has systematically compared them in ancient dental calculus. Here, we compare untreated dental calculus samples to samples from the same site treated with four previously published decontamination protocols: a UV only treatment; a 5% sodium hypochlorite immersion treatment; a pre-digestion in EDTA treatment; and a combined UV irradiation and 5% sodium hypochlorite immersion treatment. We examine their efficacy in ancient oral microbiota recovery by applying 16S rRNA gene amplicon and shotgun sequencing, identifying ancient oral microbiota, as well as soil and skin contaminant species. Overall, the EDTA pre-digestion and a combined UV irradiation and 5% sodium hypochlorite immersion treatment were both effective at reducing the proportion of environmental taxa and increasing oral taxa in comparison to untreated samples. This research highlights the importance of using decontamination procedures during ancient DNA analysis of dental calculus to reduce contaminant DNA.

The Postmedieval Latvian Oral Microbiome in the Context of Modern Dental Calculus and Modern Dental Plaque Microbial Profiles.

Recent advantages in paleomicrobiology have provided an opportunity to investigate the composition of ancient microbial ecologies. Here, using metagenome analysis, we investigated the microbial profiles of historic dental calculus retrieved from archaeological human remains from postmedieval Latvia dated 16-17th century AD and examined the associations of oral taxa and microbial diversity with specific characteristics. We evaluated the preservation of human oral microbiome patterns in historic samples and compared the microbial composition of historic dental calculus, modern human dental plaque, modern human dental calculus samples and burial soil microbiota. Overall, the results showed that the majority of microbial DNA in historic dental calculus originated from the oral microbiome with little impact of the burial environment. Good preservation of ancient DNA in historical dental calculus samples has provided reliable insight into the composition of the oral microbiome of postmedieval Latvian individuals. The relative stability of the classifiable oral microbiome composition was observed. Significant differences between the microbiome profiles of dental calculus and dental plaque samples were identified, suggesting microbial adaptation to a specific human body environment.

Ancient DNA Reconstructs the Genetic Legacies of Precontact Puerto Rico Communities.

Indigenous peoples have occupied the island of Puerto Rico since at least 3000 BC. Due to the demographic shifts that occurred after European contact, the origin(s) of these ancient populations, and their genetic relationship to present-day islanders, are unclear. We use ancient DNA to characterize the population history and genetic legacies of precontact Indigenous communities from Puerto Rico. Bone, tooth, and dental calculus samples were collected from 124 individuals from three precontact archaeological sites: Tibes, Punta Candelero, and Paso del Indio. Despite poor DNA preservation, we used target enrichment and high-throughput sequencing to obtain complete mitochondrial genomes (mtDNA) from 45 individuals and autosomal genotypes from two individuals. We found a high proportion of Native American mtDNA haplogroups A2 and C1 in the precontact Puerto Rico sample (40% and 44%, respectively). This distribution, as well as the haplotypes represented, supports a primarily Amazonian South American origin for these populations and mirrors the Native American mtDNA diversity patterns found in present-day islanders. Three mtDNA haplotypes from precontact Puerto Rico persist among Puerto Ricans and other Caribbean islanders, indicating that present-day populations are reservoirs of precontact mtDNA diversity. Lastly, we find similarity in autosomal ancestry patterns between precontact individuals from Puerto Rico and the Bahamas, suggesting a shared component of Indigenous Caribbean ancestry with close affinity to South American populations. Our findings contribute to a more complete reconstruction of precontact Caribbean population history and explore the role of Indigenous peoples in shaping the biocultural diversity of present-day Puerto Ricans and other Caribbean islanders.

Differential preservation of endogenous human and microbial DNA in dental calculus and dentin.

Dental calculus (calcified dental plaque) is prevalent in archaeological skeletal collections and is a rich source of oral microbiome and host-derived ancient biomolecules. Recently, it has been proposed that dental calculus may provide a more robust environment for DNA preservation than other skeletal remains, but this has not been systematically tested. In this study, shotgun-sequenced data from paired dental calculus and dentin samples from 48 globally distributed individuals are compared using a metagenomic approach. Overall, we find DNA from dental calculus is consistently more abundant and less contaminated than DNA from dentin. The majority of DNA in dental calculus is microbial and originates from the oral microbiome; however, a small but consistent proportion of DNA (mean 0.08 ± 0.08%, range 0.007-0.47%) derives from the host genome. Host DNA content within dentin is variable (mean 13.70 ± 18.62%, range 0.003-70.14%), and for a subset of dentin samples (15.21%), oral bacteria contribute > 20% of total DNA. Human DNA in dental calculus is highly fragmented, and is consistently shorter than both microbial DNA in dental calculus and human DNA in paired dentin samples. Finally, we find that microbial DNA fragmentation patterns are associated with guanine-cytosine (GC) content, but not aspects of cellular structure.

Patients with dental calculus have increased saliva and gingival crevicular fluid fetuin-A levels but no association with fetuin-A polymorphisms.

Fetuin-A is a potent inhibitor of calcium-phosphate precipitation and of the calcification process, therefore it can also be related with dental calculus. Thus, we aimed to investigate a possible relationship between fetuin-A gene polymorphism and the presence of dental calculus. A possible relationship between serum, saliva and gingival crevicular fluid (GCF) levels of fetuin-A was also investigated. Fetuin-A c.742C > T and c.766C > G polymorphisms were investigated in 103 patients with or without dental calculus. Additionally, serum, saliva and GCF fetuin-A levels of patients were compared according to dental calculus presence. A significant difference was not observed in the distribution of the fetuin-A c.742C > T and c.766C > G polymorphisms between patients with or without dental calculus. Saliva and GCF fetuin-A concentrations of patients with dental calculus were statistically higher than those without dental calculus (P=0.001, P=0.036 respectively). According to our results, fetuin-A c.742C > T and c.766C > G polymorphisms were not associated with presence of dental calculus. However, higher GCF and saliva fetuin-A levels were detected in patients with dental calculus than in patients without dental calculus, which may result from an adaptive mechanism to inhibit mineral precipitation and eventually calculus formation.

Matrix-Gla Protein rs4236 [A/G] gene polymorphism and serum and GCF levels of MGP in patients with subgingival dental calculus.

AIM: Matrix-Gla Protein (MGP) is one of the major Gla-containing protein associated with calcification process. It also has a high affinity for Ca2+ and hydroxyapatite. In this study we aimed to evaluate the MGP rs4236 [A/G] gene polymorphism in association with subgingival dental calculus. Also a possible relationship between MGP gene polymorphism and serum and GCF levels of MGP were examined. MATERIAL AND METHODS: MGP rs4236 [A/G] gene polymorphism was investigated in 110 patients with or without subgingival dental calculus, using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) techniques. Additionally, serum and GCF levels of MGP of the patients were compared according to subgingival dental calculus. RESULTS: Comparison of patients with and without subgingival dental calculus showed no statistically significant difference in MGP rs4236 [A/G] gene polymorphism (p=0.368). MGP concentrations in GCF of patients with subgingival dental calculus were statistically higher than those without subgingival dental calculus (p=0.032). However, a significant association was not observed between the genotypes of AA, AG and GG of the MGP rs4236 gene and the serum and GCF concentrations of MGP in subjects. CONCLUSION: In this study, it was found that MGP rs4236 [A/G] gene polymorphism was not to be associated with subgingival dental calculus. Also, that GCF MGP levels were detected higher in patients with subgingival dental calculus than those without subgingival dental calculus independently of polymorphism, may be the effect of adaptive mechanism to inhibit calculus formation.

Successful enrichment and recovery of whole mitochondrial genomes from ancient human dental calculus.

OBJECTIVES: Archaeological dental calculus is a rich source of host-associated biomolecules. Importantly, however, dental calculus is more accurately described as a calcified microbial biofilm than a host tissue. As such, concerns regarding destructive analysis of human remains may not apply as strongly to dental calculus, opening the possibility of obtaining human health and ancestry information from dental calculus in cases where destructive analysis of conventional skeletal remains is not permitted. Here we investigate the preservation of human mitochondrial DNA (mtDNA) in archaeological dental calculus and its potential for full mitochondrial genome (mitogenome) reconstruction in maternal lineage ancestry analysis. MATERIALS AND METHODS: Extracted DNA from six individuals at the 700-year-old Norris Farms #36 cemetery in Illinois was enriched for mtDNA using in-solution capture techniques, followed by Illumina high-throughput sequencing. RESULTS: Full mitogenomes (7-34×) were successfully reconstructed from dental calculus for all six individuals, including three individuals who had previously tested negative for DNA preservation in bone using conventional PCR techniques. Mitochondrial haplogroup assignments were consistent with previously published findings, and additional comparative analysis of paired dental calculus and dentine from two individuals yielded equivalent haplotype results. All dental calculus samples exhibited damage patterns consistent with ancient DNA, and mitochondrial sequences were estimated to be 92-100% endogenous. DNA polymerase choice was found to impact error rates in downstream sequence analysis, but these effects can be mitigated by greater sequencing depth. DISCUSSION: Dental calculus is a viable alternative source of human DNA that can be used to reconstruct full mitogenomes from archaeological remains. Am J Phys Anthropol 160:220-228, 2016. © 2016 The Authors American Journal of Physical Anthropology Published by Wiley Periodicals, Inc.

Patients with dental calculus have increased saliva and gingival crevicular fluid fetuin-A levels but no association with fetuin-A polymorphisms

ABSTRACT: Fetuin-A is a potent inhibitor of calcium-phosphate precipitation and of the calcification process, therefore it can also be related with dental calculus. Thus, we aimed to investigate a possible relationship between fetuin-A gene polymorphism and the presence of dental calculus. A possible relationship between serum, saliva and gingival crevicular fluid (GCF) levels of fetuin-A was also investigated. Fetuin-A c.742C > T and c.766C > G polymorphisms were investigated in 103 patients with or without dental calculus. Additionally, serum, saliva and GCF fetuin-A levels of patients were compared according to dental calculus presence. A significant difference was not observed in the distribution of the fetuin-A c.742C > T and c.766C > G polymorphisms between patients with or without dental calculus. Saliva and GCF fetuin-A concentrations of patients with dental calculus were statistically higher than those without dental calculus (P=0.001, P=0.036 respectively). According to our results, fetuin-A c.742C > T and c.766C > G polymorphisms were not associated with presence of dental calculus. However, higher GCF and saliva fetuin-A levels were detected in patients with dental calculus than in patients without dental calculus, which may result from an adaptive mechanism to inhibit mineral precipitation and eventually calculus formation.