Molecular clocks in ancient proteins: Do they reflect the age at death even after millennia?

Age at death estimation in cases of human skeletal finds is an important task in forensic medicine as well as in anthropology. In forensic medicine, methods based on "molecular clocks" in dental tissues and bone play an increasing role. The question, whether these methods are applicable also in cases with post-depositional intervals far beyond the forensically relevant period, was investigated for two "protein clocks", the accumulation of D-aspartic acid (D-Asp) and the accumulation of pentosidine (Pen) in dentine. Eight teeth of skeletons from different burial sites in Austria and with post-depositional intervals between c. 1216 and c. 8775 years were analysed. The results of age at death estimation based on D-Asp and Pen in dentine were compared to that derived from a classical morphological examination. Age at death estimation based on D-Asp resulted consistently in false high values. This finding can be explained by a post-mortem accumulation of D-Asp that may be enhanced by protein degradation. In contrast, the Pen-based age estimates fitted well with the morphological age diagnoses. The described effect of post-mortem protein degradation is negligible in forensically relevant time horizons, but not for post-depositional intervals of thousands of years. That means that the "D-Asp clock" loses its functionality with increasing post-depositional intervals, whereas Pen seems to be very stable. The "Pen-clock" may have the potential to become an interesting supplement to the existing repertoire of methods even in cases with extremely long post-depositional intervals. Further investigations have to test this hypothesis.

Predominance of Anaerobic, Spore-Forming Bacteria in Metabolically Active Microbial Communities from Ancient Siberian Permafrost.

The prevalence of microbial life in permafrost up to several million years (Ma) old has been well documented. However, the long-term survivability, evolution, and metabolic activity of the entombed microbes over this time span remain underexplored. We integrated aspartic acid (Asp) racemization assays with metagenomic sequencing to characterize the microbial activity, phylogenetic diversity, and metabolic functions of indigenous microbial communities across a ∼0.01- to 1.1-Ma chronosequence of continuously frozen permafrost from northeastern Siberia. Although Asp in the older bulk sediments (0.8 to 1.1 Ma) underwent severe racemization relative to that in the youngest sediment (∼0.01 Ma), the much lower d-Asp/l-Asp ratio (0.05 to 0.14) in the separated cells from all samples suggested that indigenous microbial communities were viable and metabolically active in ancient permafrost up to 1.1 Ma. The microbial community in the youngest sediment was the most diverse and was dominated by the phyla Actinobacteria and Proteobacteria In contrast, microbial diversity decreased dramatically in the older sediments, and anaerobic, spore-forming bacteria within Firmicutes became overwhelmingly dominant. In addition to the enrichment of sporulation-related genes, functional genes involved in anaerobic metabolic pathways such as fermentation, sulfate reduction, and methanogenesis were more abundant in the older sediments. Taken together, the predominance of spore-forming bacteria and associated anaerobic metabolism in the older sediments suggest that a subset of the original indigenous microbial community entrapped in the permafrost survived burial over geological time.IMPORTANCE Understanding the long-term survivability and associated metabolic traits of microorganisms in ancient permafrost frozen millions of years ago provides a unique window into the burial and preservation processes experienced in general by subsurface microorganisms in sedimentary deposits because of permafrost's hydrological isolation and exceptional DNA preservation. We employed aspartic acid racemization modeling and metagenomics to determine which microbial communities were metabolically active in the 1.1-Ma permafrost from northeastern Siberia. The simultaneous sequencing of extracellular and intracellular genomic DNA provided insight into the metabolic potential distinguishing extinct from extant microorganisms under frozen conditions over this time interval. This in-depth metagenomic sequencing advances our understanding of the microbial diversity and metabolic functions of extant microbiomes from early Pleistocene permafrost. Therefore, these findings extend our knowledge of the survivability of microbes in permafrost from 33,000 years to 1.1 Ma.

Age estimation based on aspartic acid racemization in dentine: what about caries-affected teeth?

Age estimation based on aspartic acid racemization (AAR) in dentine is one of the most precise methods in adult age. Caries induces protein degradation and may have an impact on the kinetics of AAR in dentine. We systematically examined standardized prepared dentine samples from caries-affected teeth to clarify the question, if caries-affected teeth should not be used for age estimation based on AAR at all, or if the analysis of dentine samples from such teeth may be useful after removal of the caries-affected tissue according to clinical standards. Our results suggest that caries may lead to an extensive protein degradation even in macroscopically healthy-appearing dentine samples from caries-affected teeth and may significantly affect the precision of age estimation. To ensure the quality of age estimation based on AAR in forensic practice, we recommend using dentine samples from healthy teeth. If only caries-affected teeth are available, dentine samples from at least two teeth from the same individual should be analyzed as it seems unlikely that caries-induced protein degradation occurred with identical kinetics in two different teeth. In any case, results of the analysis of caries-affected teeth must be interpreted with caution.

Analysis of advanced glycation end products (AGEs) in dentine: useful for age estimation?

Ageing of the human organism results in the accumulation of modified molecules. Some of these molecular changes may be used for age estimation, as already shown for aspartic acid racemization (AAR). Another example for an accumulation of damaged molecules is advanced glycation end products (AGEs). We examined, (1) if the correlation between the concentration of AGEs (pentosidine) in root dentine and age is close enough to be used as basis for age estimation, and (2) if the combined analysis of AGEs and AAR in dentine may be a useful approach to rule out or to detect relevant effects of confounding factors in age estimation. We determined the pentosidine content of root dentine samples of 64 healthy teeth as well as in carious, "pink", diabetic and heated teeth, and in teeth after different storage times. In 23 teeth, the extent of aspartic acid racemization (AAR) was determined in parallel. We observed a close relationship between the concentration of pentosidine in dentine and chronological age (r = 0.94) in healthy teeth. The analysis of pentosidine in dentine can theoretically be used as a basis for age estimation in healthy teeth of non-diabetic individuals; diabetic individuals may exhibit very high pentosidine levels in dentine. This finding limits the application of this method, since information regarding the question if an unidentified person suffered from diabetes mellitus or not are missing in most cases. Moreover, the method is not suitable to identify or rule out the influence of confounding factors in age estimation based on AAR, since both methods are sensible to the most relevant confounding factors (caries, heat).

Aspartic acid racemization of root dentin used for dental age estimation in a Polish population sample.

Precise age determination of unidentified bodies and human remains is one of the essential tasks of forensic science. The aim of this study was to assess the applicability of using the enantiomeric composition of aspartic acid racemization in root and crown dentin for dental age estimation using a Polish population sample. Coronal and root dentin from four teeth groups from the mandible were studied using gas chromatography with mass spectrometry. The results demonstrated a very high correlation between the chronological age and enantiomeric composition in both of the dentin samples. Individual linear equations of root dentin with correlation coefficients between 0.96 and 0.98 and a standard estimation error of ±2.95-4.84 years validated the application of aspartic acid racemization as a significant practical contribution to everyday forensic medical practice. Discrepancies in methodological aspects and modifications that simplify the protocol are presented.

Age estimation based on aspartic acid racemization in human sclera.

Age estimation based on racemization of aspartic acid residues (AAR) in permanent proteins has been established in forensic medicine for years. While dentine is the tissue of choice for this molecular method of age estimation, teeth are not always available which leads to the need to identify other suitable tissues. We examined the suitability of total tissue samples of human sclera for the estimation of age at death. Sixty-five samples of scleral tissue were analyzed. The samples were hydrolyzed and after derivatization, the extent of aspartic acid racemization was determined by gas chromatography. The degree of AAR increased with age. In samples from younger individuals, the correlation of age and D-aspartic acid content was closer than in samples from older individuals. The age-dependent racemization in total tissue samples proves that permanent or at least long-living proteins are present in scleral tissue. The correlation of AAR in human sclera and age at death is close enough to serve as basis for age estimation. However, the precision of age estimation by this method is lower than that of age estimation based on the analysis of dentine which is due to molecular inhomogeneities of total tissue samples of sclera. Nevertheless, the approach may serve as a valuable alternative or addition in exceptional cases.

Age estimation in humans through the analysis of aspartic acid racemization from teeth: A scoping review of methods, outcomes, and open research questions.

Teeth are considered the most resistant structures in the human body. In forensic odontology, teeth are useful for human identification, especially when dental age estimation is necessary. Despite numerous studies, there is no consensus regarding the best methods for dental age estimation. The analysis of aspartic acid racemization, however, has shown promising results. This scoping review aimed to present a descriptive synthesis of the current literature regarding dental age estimation through aspartic acid racemization. Four electronic databases were screened: PubMed, Scielo, Web of Science, and Scopus. Cross-sectional studies published before April 2021 were selected. From 206 articles found, 26 met the eligibility criteria. Several experimental protocols and laboratory settings were detected, but the different protocols did not seem to significantly reduce error rates in dental age estimation. The analysis of aspartic acid racemization in human dental tissues produced accurate and potentially reliable results for age estimation. Aspartic acid racemization stands out especially in the adulthood - age category in which other methods struggle to deliver proper performances. Studies with larger samples, independent testing, and standardized laboratory procedures are necessary. Equator-like reporting guidelines are encouraged to enable future systematic reviews and meta-analyses.

Application of Aspartic Acid Racemization for Age Estimation in a Spanish Sample.

Correct age-at-death estimation in adult individuals is one of the challenges of forensic investigation. Forensic anthropology macroscopic techniques are non-invasive methods for this purpose. However, several methods need to be applied to accurately estimate age, and the difference between chronological and predictive age may still be around ±10 years. New research trends are focused on the inherent process of aging, which produces changes in tissues and organs at different biochemical levels. One of the oldest and most studied approaches in this field is aspartic acid racemization. The accuracy of this technique in age estimation has been widely demonstrated. However, only a few studies have assessed its accuracy in different populations. The aim of this research was to assess the accuracy of aspartic acid racemization in a Spanish sample and its applicability to forensic cases. Dentin from fifteen third molars from two Spanish populations (ages 19-70 years old) was isolated and D and L forms of aspartic acid were detected through GC/MS, according to a previous published protocol. D/L ratios were calculated and after the application of a regression analysis, a formula for age estimation was developed. The results were similar to previous studies, obtaining an R = 0.91 between racemization ratios and age and a mean absolute error (MAE) between chronological and predictive age of 5 years. These results were ratified by leave-one-out cross-validation, as well as the application of the formula to five teeth of a known age. Despite these promising results, this technique is not exempt from drawbacks; thus, further studies are required to apply this methodology to forensic cases and to combine it with forensic anthropology findings.

Aspartic acid racemization and repair in the survival and recovery of hyperthermophiles after prolonged starvation at high temperature.

Long-term survivability is well-known for microorganisms in nutrient-depleted environments, but the damage accrued by proteins and the associated repair processes during the starvation and recovery phase of microbial life still remain enigmatic. We focused on aspartic acid (Asp) racemization and repair in the survival of Pyrococcus furiosus and Thermococcus litoralis under starvation conditions at high temperature. Despite the dramatic decrease of viability over time, 0.002% of P. furiosus cells (2.1×103 cells/mL) and 0.23% of T. litoralis cells (2.3×105 cells/mL) remained viable after 25 and 50 days, respectively. The D/L Asp ratio in the starved cells was approximately half of those from the autoclaved cells, suggesting that the starving cells were capable of partially repairing racemized Asp. Transcriptomic analyses of the recovered cells of T. litoralis indicated that the gene encoding Protein-L-isoaspartate (D-aspartate) O-methyltransferase (PIMT) might be involved in the repair of damaged proteins by converting D-Asp back to L-Asp during the resuscitation of starved cells. Collectively, our results provided evidence that Asp underwent racemization in the surviving hyperthermophilic cells under starved conditions and PIMT played a critical role in the repair of abnormal aspartyl residues during the initial recovery of starved, yet still viable, cells.

Analysis of 14C, 13C and Aspartic Acid Racemization in Teeth and Bones to Facilitate Identification of Unknown Human Remains: Outcomes of Practical Casework.

The identification of unknown human remains represents an important task in forensic casework. If there are no clues as to the identity of the remains, then the age, sex, and origin are the most important factors to limit the search for a matching person. Here, we present the outcome of application of so-called bomb pulse radiocarbon (14C derived from above-ground nuclear bomb tests during 1955-1963) analysis to birthdate human remains. In nine identified cases, 14C analysis of tooth crowns provided an estimate of the true date of birth with an average absolute error of 1.2 ± 0.8 years. Analysis of 14C in tooth roots also showed a good precision with an average absolute error of 2.3 ± 2.5 years. Levels of 14C in bones can determine whether a subject has lived after 1955 or not, but more precise carbon turnover data for bones would be needed to calculate date of birth and date of death. Aspartic acid racemization analysis was performed on samples from four cases; in one of these, the year of birth could be predicted with good precision, whereas the other three cases are still unidentified. The stable isotope 13C was analyzed in tooth crowns to estimate provenance. Levels of 13C indicative of Scandinavian provenance were found in known Scandinavian subjects. Teeth from four Polish subjects all showed higher 13C levels than the average for Scandinavian subjects.

Preparation of dentin standard samples for age estimation based on increased aspartic acid racemization rate by heating.

Age estimation in adults based on aspartic acid racemization (AAR) provides fewer errors and higher precision than that based on bone morphology for the identification of cadavers. The technique has been established in some labs as a routine method. However, as the essential requisites for the technique, a wide age range of teeth of the same type as the target tooth must be collected for calibration for each examination. We investigated whether dentin standard samples could be prepared by increasing the AAR rate via heat. Powdered dentin was prepared from a maxillary first premolar (13 years) and heated for 0-72 h at 110 °C. The extent of AAR increased significantly with heating time and the correlation was strong (r = 0.913; p < 0.01). Similar results were found for a mandibular canine (24 years, r = 0.948; p < 0.01) and a maxillary third molar (20 years, r = 0.944; p < 0.01). We attempted to estimate the age of four maxillary first premolars of persons aged 25-58 years by using the heated samples (18 years, 12 h to 7 days). The differences between the actual and estimated ages were within ±5 years. The stability of the AAR rates in the powdered dentin during storage at 22-25 °C, 4 °C, and -30 °C was examined after 1 year and no significant changes had occurred. We were able to prepare dentin standard samples and created a calibration curve. This is a pilot study that needs to be validated before it can be used in forensic practice.

Age estimation of living Indian individuals based on aspartic acid racemization from tooth biopsy specimen.

BACKGROUND: Age estimation in living individuals is imperative to amicably settle civil and criminal disputes. A biochemical method based on amino acid racemization was evaluated for age estimation of living Indian individuals. DESIGN: Caries-free maxillary/mandibular premolar teeth (n = 90) were collected from participants with age proof documents and divided into predefined nine age groups. MATERIALS AND METHODS: Dentine biopsy from the labial aspect of the tooth crown was taken with an indigenously developed microtrephine. The samples were processed and subjected to gas chromatography. Dextrorotatory:levorotatory ratios were calculated, and a regression equation was formulated. RESULTS: Across all age groups, an error of 0 ± 4 years between protein racemization age and chronological age was observed. CONCLUSION: Aspartic acid racemization from dentine biopsy samples could be a viable and accurate technique for age estimation of living individuals who have attained a state of skeletal maturity.

Datación mediante racemización del ácido aspártico en dentina humana / Dating by aspartic acid racemization in human dentine

La determinación de la edad al momento de la muerte sobre la base de la recemización del ácido aspártico (AAR) en la dentina humana se ha aplicado con éxito en odontología forense desde hace varios años. El uso de este procedimiento en la dentina proporciona una solución sencilla y rentable, cuyo método puede lograr una presición de más menos 3 años. Actualmente esta determinación no se encuentra estandarizada, inconsistencia que se debe resolver si se pretende aplicar con éxito la técnica para la determinación de la edad en casos de interés forense. Se pretende aplicar con éxito la técnica para la determinación de la edad en casos de interés forense...