Uncovering Forensic Evidence: A Path to Age Estimation through DNA Methylation.

Age estimation is a critical aspect of reconstructing a biological profile in forensic sciences. Diverse biochemical processes have been studied in their correlation with age, and the results have driven DNA methylation to the forefront as a promising biomarker. DNA methylation, an epigenetic modification, has been extensively studied in recent years for developing age estimation models in criminalistics and forensic anthropology. Epigenetic clocks, which analyze DNA sites undergoing hypermethylation or hypomethylation as individuals age, have paved the way for improved prediction models. A wide range of biomarkers and methods for DNA methylation analysis have been proposed, achieving different accuracies across samples and cell types. This review extensively explores literature from the past 5 years, showing scientific efforts toward the ultimate goal: applying age prediction models to assist in human identification.

Assessment of Blood and Semen Detection and DNA Collection from Swabs up to Three Months after Deposition on Five Different Cloth Materials.

Body fluid identification plays a crucial role in criminal investigations. Because of their presence in many cases, blood and semen are the most relevant body fluids in forensic sciences. Based on antigen-antibody reactions binding unique proteins for each body fluid, serological assays represent one of the most rapid and highly specific tests for blood and semen. Currently, few studies have assessed the factors affecting body fluid identification by applying these assays. This work aimed to study the effect of different fabrics from clothes and time since deposition on identification through immunochromatographic tests for blood and semen, DNA isolation, and STR profiling from these samples. Body fluids were deposited on black- and white-dyed denim and cotton fabrics, and on leather. Afterward, blood and semen were sampled at 1 day, 30 days, and 90 days after deposition and identified by using the SERATEC® HemDirect Hemoglobin Test and the PSA Semiquant and SERATEC® BLOOD CS and SEMEN CS tests, respectively. Laboratory and crime scene tests presented similar performances for the detection of blood and semen stains on every tested fabric. No differences were found on band intensities between timepoints for all fabrics. It was possible to recover and identify blood and semen samples up to three months after deposition and to obtain full STR profiles from all the tested fabrics. Both body fluid STR profiles showed differences in their quality between 1 and 90 days after deposition for all fabrics except for black cotton for semen samples. Future research will expand the results, assessing body fluid identification on other substrates and under different environmental conditions.

Making the Most of Lateral Flow Immunochromatographic Tests: An Efficient Protocol to Recover DNA.

Lateral flow immunochromatographic (LFI) tests are widely used in both biomedical and forensic sciences for different applications. In forensic sciences, their main use is to detect body fluids at crime scenes. However, there are situations in which the amount of potential biological evidence is so low that DNA extraction is favored with respect to the identification of body fluids. Here, an efficient and quick protocol is presented to integrate the detection of body fluids through LFI with DNA extraction from a sample swab and buffer, providing a complete characterization of the biological evidence. This protocol is a modification of a general DNA extraction silica-based kit, whose main application is for blood and tissues. Thus, it could be carried out in different settings (forensic labs, hospitals, other testing labs) without the necessity of buying a specific kit for swabs. The validation of this protocol is supported by the results presented here and previous publications from our group, obtaining DNA in good quantity and with good quality. This proves the potential application of the protocol in both forensic scenarios, to fully characterize biological evidence, and biomedical settings, to molecularly confirm the results of LFI tests.

Assessment of body fluid identification and DNA profiling after exposure to tropical weather conditions.

Despite current advances in body fluid identification, there are few studies evaluating the effect of environmental conditions. The present work assessed the detection of body fluids, blood, semen, and saliva, through lateral flow immunochromatographic (LFI) tests, exposed to tropical weather conditions over time, also evaluating the possibility of obtaining STR (short tandem repeat) profiles and identifying mitochondrial DNA (mtDNA) polymorphisms. Blood, semen, saliva samples, and mixtures of these fluids were deposited on polyester clothes and exposed to open-air tropical weather conditions for 1 month. The test versions from LFI (SERATEC®, Germany) Lab and crime scene (CS) used for the detection - one per each body fluid type - demonstrated that it is possible to identify body fluids and their mixtures up to 14 days after deposition. At 30 days, blood and semen were detected but not saliva. Full STR profiles were obtained from 14-day-old blood samples, and partial profiles were obtained from the remaining samples. It was possible to sequence mtDNA in the samples previously analyzed for STR profiling, and haplogroups could be assigned. In conclusion, this study demonstrated for the first time the possibility of body fluid identification and DNA profiling after exposure to tropical weather conditions for 1 month and also demonstrated the value of mtDNA analysis for compromised biological evidence.

Partners in Postmortem Interval Estimation: X-ray Diffraction and Fourier Transform Spectroscopy.

The postmortem interval (PMI) is difficult to estimate in later stages of decomposition. There is therefore a need to develop reliable methodologies to estimate late PMI. This study aims to assess whether there is a correlation between changes in the mineral composition of human teeth and the estimation of PMI. X-ray diffraction (XRD) and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy techniques were performed to address this challenge. Forty healthy human teeth obtained from odontological clinics were stored at different times (0, 10, 25, 50 years; N = 10/group). XRD and ATR-FTIR parameters related to the structure and composition of teeth were studied. Our results showed that the crystallinity index, crystal size index, mineral-to-organic matrix ratio (M/M) and carbonate/phosphate ratio (C/P) had the strongest association with PMI. For larger PMIs, there was a significant increase in crystallinity, crystal size and M/M ratio, while the C/P ratio showed a specific decrease with increasing PMI. According to our results, the parameters of crystallinity, crystal size, M/M ratio and C/P ratio can be considered highly accurate in determining a PMI of 10 years of data; crystallinity and mineral maturity can be considered useful in determining a PMI of 25 years; and crystallinity and mineral maturity can be considered highly accurate in determining a PMI of 50 years. A particular XRD index was identified as the most suitable parameter to estimate PMI: crystallinity. The joint use of XRD and ATR-FTIR analyses could be a promising alternative for dating human teeth.

Estimation of sex based on postcranial elements in European American and Latin American populations.

This study assesses the use of postcranial elements for sex estimation taking population variability into account. European American and Latin American populations are independently analyzed. Nine measurements from postcranial elements were collected from 72 European American individuals, and 59 Latin American individuals. Statistical analyses were performed using the Statistical Package for Social Science (SPSS) version 15. Statistical analyses were conducted to corroborate that there were no intra- or interobserver errors. In both populations, significant differences were found on all measured traits between males and females, except Scapular Breadth in Latin Americans. According to Discriminant Function Analysis (DFA) in the European American population the Ulna Minimum Breadth of the Olecranon (UMBO) correctly classified 91.3% of individuals. When this parameter was combined with the Diameter of Humeral Head (HHD), the two correctly classified 98% of individuals. In Latin Americans, the UMBO correctly classified 82.4% of the individuals. When combined with HHD, the measurements correctly classified 79.4% individuals. UMBO is the most useful trait to correctly assign the sex of the remains in both populations. Including the HHD improved accuracy rates in the European American sample. These results are in agreement with previous studies, which named the humerus as one of the potentially useful bones to consider for sex estimation based on its accuracy. Thus, these two anatomical regions could be used alone or in combination with other methodologies for sex estimation, which is particularly important in situations of fragmentary remains and incomplete skeletons.

Evaluation of latent fingermark color contrast as aging parameter under different environmental conditions: A preliminary study.

This research expands previous studies in which color contrast between ridges and furrows of powder-enhanced latent fingermarks was explored as a possible aging parameter. The main goal is to test the sensitivity of the technique across a predetermined set of factors. In this case, experiment factors have included two donors who deposited sebaceous- and eccrine-rich fingermarks onto ceramic tile and polystyrene plastic. These were developed with either black carbon or titanium dioxide powder (TiO2 ) over eight time periods (0-72 days) and aged under three light conditions (direct light, shade, and darkness). The mean intensity (MI) and intensity amplitude (IA) metrics of color were collected from each image for statistical analyses. Results show that color contrast is affected significantly by substrate, secretion, and powder types, with an interaction effect between the substrate and powder type on both MI and IA metrics. The degree of light exposure did not have a noticeable impact on distinguishing aging patterns of fingermarks by neither powder methods. Different aging patterns were detected between sebaceous-rich and their eccrine-rich counterparts for all light conditions using regression analysis. All eccrine-rich fingermarks exhibited little (or minimal) change in IA over time, whereas sebaceous-rich samples showed varied patterns, from significant decreases to slight increases. These findings confirm and expand previous observations on the potential use of MI and IA as metrics to study latent fingermark degradation patterns that could eventually be used to estimate the age of a fingermark.

A Small Population Study on Friction Skin Ridges: Differences in Ridge Widths Between Latent and Inked Fingerprints.

Morphological changes in the width of latent fingermark ridges occur naturally over time. This could be used to examine the aging process of latents and eventually estimate time of deposition. In a crime context, it is common practice to compare a questioned (aged) fingermark with a database of known (inked) prints. Therefore, in the absence of fresh fingermarks for aging purposes, it is of interest to reveal correlations between these two categories of fingerprints with regard to the widths of their ridges. The present study explores correlations of ridge widths between flat and rolled inked prints with latent fingermarks visualized with carbon black (CB) and titanium dioxide (TiO2 )-based powders among a small population of ten donors. Results revealed consistent differences between the ridge widths of latent and inked prints as well as flat and rolled impressions. Latent fingermarks visualized with CB and TiO2 powders showed ridges with comparable widths.

Frailty, Cognitive Decline, Neurodegenerative Diseases and Nutrition Interventions.

Currently the human population is aging faster. This leads to higher dependency rates and the transformation of health and social care to adapt to this aged population. Among the changes developed by this population is frailty. It is defined as a clinically detectable syndrome, related to the aging of multiple physiological systems, which prompts a situation of vulnerability. The etiology of frailty seems to be multifactorial and its pathophysiology is influenced by the interaction of numerous factors. Morley et al. propose four main mechanisms triggering the frailty: atherosclerosis, sarcopenia, cognitive deterioration and malnutrition, with their respective metabolic alterations. Malnutrition is associated with cognitive impairment or functional loss, but it is also known that an inadequate nutritional status predisposes to cognitive frailty. Additionally, nutritional factors that may influence vascular risk factors will potentially have an effect on dementia decline among patients with cognitive frailty. This review aims to describe the nutritional factors that have been researched so far which may lead to the development of frailty, and especially cognitive decline.

Setting the light conditions for measuring root transparency for age-at-death estimation methods.

Age-at-death estimation is one of the main goals in forensic identification, being an essential parameter to determine the biological profile, narrowing the possibility of identification in cases involving missing persons and unidentified bodies. The study of dental tissues has been long considered as a proper tool for age estimation with several age estimation methods based on them. Dental age estimation methods can be divided into three categories: tooth formation and development, post-formation changes, and histological changes. While tooth formation and growth changes are important for fetal and infant consideration, when the end of dental and skeletal growth is achieved, post-formation or biochemical changes can be applied. Lamendin et al. in J Forensic Sci 37:1373-1379, (1992) developed an adult age estimation method based on root transparency and periodontal recession. The regression formula demonstrated its accuracy of use for 40 to 70-year-old individuals. Later on, Prince and Ubelaker in J Forensic Sci 47(1):107-116, (2002) evaluated the effects of ancestry and sex and incorporated root height into the equation, developing four new regression formulas for males and females of African and European ancestry. Even though root transparency is a key element in the method, the conditions for measuring this element have not been established. The aim of the present study is to set the light conditions measured in lumens that offer greater accuracy when applying the Lamendin et al. method modified by Prince and Ubelaker. The results must be also taken into account in the application of other age estimation methodologies using root transparency to estimate age-at-death.

Ridge Width Correlations between Inked Prints and Powdered Latent Fingerprints.

A methodology to estimate the time of latent fingerprint deposition would be of great value to law enforcement and courts. It has been observed that ridge topography changes as latent prints age, including the widths of ridges that could be measured as a function of time. Crime suspects are commonly identified using fingerprint databases that contain reference inked tenprints (flat and rolled impressions). These can be of interest in aging studies as they provide baseline information relating to the original (nonaged) ridges' widths. In practice, the age of latent fingerprints could be estimated following a comparison process between the evidentiary aged print and the corresponding reference inked print. The present article explores possible correlations between inked and fresh latent fingerprints deposited on different substrates and visualized with TiO2 . The results indicate that the ridge width of flat inked prints is most similar to fresh latent fingerprints, and these should be used as the comparison standard for future aging studies.

Latent Fingermark Aging Patterns (Part III): Discontinuity Index as One Indicator of Degradation.

This article is the third in a series of reports exploring quantifiable visual parameters of the aging process of latent fingermarks. On this occasion, research is focused on the occurrence of ridge discontinuities (i.e. breakages) as a function of time. Experiment variables included type of secretion (eccrine and sebaceous), substrate (glass and plastic), and exposure to natural light (dark, shade, and direct light) over a 6 months period. Fingermarks were sequentially visualized with titanium dioxide powder, photographed, and the number of naturally occurring ridge discontinuities subsequently evaluated. A semi-quantitative value, named Discontinuity Index, was used to better characterize this aging parameter. Results indicated that ridges of sebaceous depositions on glass were generally less affected by the environmental conditions compared with those on plastic surface. In addition, aging in darkness was not always the best condition for preservation, and the direct exposure to light seemed not to affect the degradation under certain conditions.

Relationship Between Mitochondrial DNA Mutations and Aging. Estimation of Age-at-death.

Some studies have pointed to the relationship between mitochondrial DNA (mtDNA) mutations and age in different tissues, which are potentially interesting in aging research and in forensic identification because they could help to improve the estimation of age-at-death. The present study aims to evaluate the mutations in mtDNA from dentin and pulp and their relation with age. Healthy erupted third molars were extracted from individuals from two Spanish populations, aged 20-70. When analyzing the amplification of hypervariable region 2 of the mtDNA by real-time polymerase chain reaction, a negative strong linear correlation was found between the mtDNA amplification and age in dentin from both populations. In contrast, a significant correlation between mtDNA amplification and age in pulp was not discovered, probably due to the majority of the mitochondria are placed in dentin. A difference in mtDNA damage between these two populations was also detected, indicating the role of ancestry as a component. The findings from this research enrich the current studies related to aging and mitochondrial damage and provide a new quantitative tool for estimating the age-at-death that, in combination with traditional age markers, could improve identification accuracy in forensic cases.

Improved age determination of blood and teeth samples using a selected set of DNA methylation markers.

Age estimation from DNA methylation markers has seen an exponential growth of interest, not in the least from forensic scientists. The current published assays, however, can still be improved by lowering the number of markers in the assay and by providing more accurate models to predict chronological age. From the published literature we selected 4 age-associated genes (ASPA, PDE4C, ELOVL2, and EDARADD) and determined CpG methylation levels from 206 blood samples of both deceased and living individuals (age range: 0-91 years). This data was subsequently used to compare prediction accuracy with both linear and non-linear regression models. A quadratic regression model in which the methylation levels of ELOVL2 were squared showed the highest accuracy with a Mean Absolute Deviation (MAD) between chronological age and predicted age of 3.75 years and an adjusted R(2) of 0.95. No difference in accuracy was observed for samples obtained either from living and deceased individuals or between the 2 genders. In addition, 29 teeth from different individuals (age range: 19-70 years) were analyzed using the same set of markers resulting in a MAD of 4.86 years and an adjusted R(2) of 0.74. Cross validation of the results obtained from blood samples demonstrated the robustness and reproducibility of the assay. In conclusion, the set of 4 CpG DNA methylation markers is capable of producing highly accurate age predictions for blood samples from deceased and living individuals.

mtDNA Mutations and Their Role in Aging, Diseases and Forensic Sciences.

Mitochondria are independent organelles with their own DNA. As a primary function, mitochondria produce the energy for the cell through Oxidative Phosphorylation (OXPHOS) in the Electron Transport Chain (ETC). One of the toxic products of this process is Reactive Oxygen Species (ROS), which can induce oxidative damage in macromolecules like lipids, proteins and DNA. Mitochondrial DNA (mtDNA) is less protected and has fewer reparation mechanisms than nuclear DNA (nDNA), and as such is more exposed to oxidative, mutation-inducing damage. This review analyzes the causes and consequences of mtDNA mutations and their relationship with the aging process. Neurodegenerative diseases, related with the aging, are consequences of mtDNA mutations resulting in a decrease in mitochondrial function. Also described are "mitochondrial diseases", pathologies produced by mtDNA mutations and whose symptoms are related with mitochondrial dysfunction. Finally, mtDNA haplogroups are defined in this review; these groups are important for determination of geographical origin of an individual. Additionally, different haplogroups exhibit variably longevity and risk of certain diseases. mtDNA mutations in aging and haplogroups are of special interest to forensic science research. Therefore this review will help to clarify the key role of mtDNA mutations in these processes and support further research in this area.

Sex determination from dentin and pulp in a medicolegal context.

BACKGROUND: The techniques used to determine the sex of skeletons are limited. The authors conducted a study to analyze the accuracy of sex identification from dentin and pulp via DNA isolation. METHODS: The authors extracted DNA from the dentin and pulp of 14 teeth by using a silica-based methodology. They used the amelogenin gene to determine the sex via polymerase chain reaction. ß-actin, a housekeeping gene, was used as a control gene. The authors checked the results in agarose gel and semiquantified them by using gel analysis software. RESULTS: The DNA yield depended on the type of tooth and was lowest in the smallest teeth (that is, incisors). In all cases, the authors were able to identify the sex, as well as the control gene, which suggests the potential to identify other genes, such as short tandem repeats. CONCLUSIONS: It is possible to correctly identify a person's sex from dentin and pulp; in instances in which one dental material is not available, the other material can be used with the same efficiency. Practical Implications. The results of this study are applicable to forensic dentistry, particularly in situations in which there is commingling of remains and fragmentary remains, and there may be only one tooth with which to identify a person's sex.