Plasma Concentrations of High Mobility Group Box 1 Proteins and Soluble Receptors for Advanced Glycation End-Products Are Relevant Biomarkers of Cognitive Impairment in Alcohol Use Disorder: A Pilot Study.

Alcohol use disorder (AUD) is a major component in the etiology of cognitive decline and dementia. Underlying mechanisms by which long-term alcohol abuse causes cognitive dysfunction include excessive oxidative stress and inflammation in the brain, activated by increased reactive oxygen/nitrogen species (ROS/RNS), advanced glycation end-products (AGEs) and high-mobility group box 1 protein (HMGB1). In a pilot study, we examine the potential clinical value of circulating biomarkers of oxidative stress including ROS/RNS, HMGB1, the soluble receptor for AGE (sRAGE), the brain biomarker of aging apolipoprotein D (ApoD), and the antioxidant regulator nuclear factor erythroid 2-related factor 2 (NRF2) as predictive indices for cognitive impairment (CI) in abstinent patients with AUD (n = 25) compared to patients with established Alzheimer's disease (AD, n = 26) and control subjects (n = 25). Plasma concentrations of sRAGE were evaluated with immunoblotting; ROS/RNS with a fluorometric kit; and HMGB1, ApoD, and NRF2 by ELISA. Abstinent AUD patients had higher sRAGE, ROS/RNS (p < 0.05), and ApoD (p < 0.01) concentrations, similar to those of AD patients, and lower NRF2 (p < 0.01) concentrations, compared to controls. These changes were remarkable in AUD patients with CI. HMGB1, and sRAGE correlated positively with duration of alcohol use (rho = 0.398, p = 0.022; rho = 0.404, p = 0.018), whereas sRAGE correlated negatively with periods of alcohol abstinence (rho = -0.340, p = 0.045). A predictive model including ROS/RNS, HMGB1, sRAGE, alcohol use duration, and alcohol abstinence periods was able to differentiate AUD patients with CI (92.3% of correct predictions, ROC-AUC= 0.90) from those without CI. In conclusion, we propose ROS/RNS, HMGB1, and sRAGE as stress biomarkers capable of predicting cognitive impairment in AUD patients.

Assessment of the efficiency of DNA isolation and profiling applying a temperature-driven method in human remains.

The identification of human remains is of utmost importance in a variety of scenarios. One of the primary identification methods is DNA. DNA extraction from human remains could be difficult, particularly in situations where the remains have been exposed to environmental conditions and other insults. Several studies tried to improve extraction by applying different approaches. ForensicGEM Universal (MicroGem) is a single-tube approach to DNA extraction and a temperature-driven method that could have some advantages with respect to previous techniques, among them, reducing the risk of contamination, not requiring specialized equipment, or several steps to perform. The aim of this study was to assess, for the first time, the efficiency of DNA extraction and quality of STR profiles applying the MicroGem protocol and modifications of this protocol from tooth samples in comparison with automatic extraction (AE). Our results indicated that AE and MicroGem performed similar, though with variability depending on the MicroGem modifications, increasing the DNA yield and STR profile quality when DNA is concentrated with Microcon. These findings demonstrated the efficiency of this methodology for DNA extraction from human remains while also providing a simple and quick technique suitable to apply in a variety of forensic scenarios.

A spit in time: identification of saliva stains and assessment of total DNA recovery up to 180 days after deposition.

The main aim of this work was to validate the detection of saliva samples from denim, cotton, and polyester fabrics aged up to six months, applying rapid immunochromatographic tests resulting in the analysis of nuclear and mitochondrial DNA recovered. A comparison was also carried out between two saliva detection tests, Laboratory and Crime Scene. 50 µl saliva samples (three per time and test) were deposited on denim, cotton, and polyester fabrics. After 1, 3, 7, 14, 21, 30, 60, 90, 150, and 180 days of storage at room temperature, the samples were recovered by swabbing and detected by SERATEC® Amylase (Laboratory) test and SERATEC® SALIVA CS (Crime Scene) test (SERATEC®, Göttingen, Germany). DNA was isolated from the swab extraction buffer applying a silica-based methodology, and quantified based on fluorescent and human-specific quantifications. Then, it was submitted to STR profiling and mtDNA sequencing. According to our results, saliva stains up to six months after deposition remain valid specimens. The intensity of the bands varied among fabric type and time. Total DNA was successfully recovered from all tested samples, though with the limitations of obtaining partial nuclear DNA profiles from the oldest samples. In contrast, complete characterization of mtDNA was achieved from all samples. Lab and CS tests performed similar on the detection of saliva, as well as, DNA yield and profiling. Future research will be able to expand these results, analyzing the stability of other body fluids and the sensitivity of rapid immunochromatographic tests to detect them.

The Perfect Match: Assessment of Sample Collection Efficiency for Immunological and Molecular Findings in Different Types of Fabrics.

Body fluid identification at crime scenes can be crucial in retrieving the appropriate evidence that leads to the perpetrator and, in some cases, the victim. For this purpose, immunochromatographic tests are simple, fast and suitable for crime scenes. The potential sample is retrieved with a swab, normally a cotton swab, moistened in a specific buffer. Nonetheless, there are other swab types available, which have been proven to be efficient for DNA isolation and analysis. The aim of this study is to evaluate the efficiency of different swab types for body fluid identification as well as DNA isolation and characterization. Fifty microliters of human saliva were deposited in three different types of fabric (denim, cotton, and polyester). After 24 h at room temperature, samples were recovered by applying three different swab types, and the tests were performed. Subsequently, total DNA was recovered from the sample buffer. Cotton swabs performed worse in denim and cotton fabrics in both immunochromatography tests and DNA yield. No differences were observed for polyester. In contrast, and except for two replicates, it was possible to obtain a full DNA profile per fabric and swab type, and to identify the mtDNA haplogroup. In this paper, the impact of swab types on body fluid identification through the application of immunochromatographic tests is analyzed for the first time. This work corroborates previous research related to the influence of swab types in nuclear DNA isolation and characterization.

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.

Identifying Methylation Patterns in Dental Pulp Aging: Application to Age-at-Death Estimation in Forensic Anthropology.

Age-at-death estimation constitutes one of the key parameters for identification of human remains in forensic investigations. However, for applications in forensic anthropology, many current methods are not sufficiently accurate for adult individuals, leading to chronological age estimates erring by ±10 years. Based on recent trends in aging studies, DNA methylation has great potential as a solution to this problem. However, there are only a few studies that have been published utilizing DNA methylation to determine age from human remains. The aim of the present study was to expand the range of this work by analyzing DNA methylation in dental pulp from adult individuals. Healthy erupted third molars were extracted from individuals aged 22-70. DNA from pulp was isolated and bisulfite converted. Pyrosequencing was the chosen technique to assess DNA methylation. As noted in previous studies, we found that ELOVL2 and FHL2 CpGs played a role in age estimation. In addition, three new markers were evaluated-NPTX2, KLF14, and SCGN. A set of CpGs from these five loci was used in four different multivariate regression models, providing a Mean Absolute Error (MAE) between predicted and chronological age of 1.5-2.13 years. The findings from this research can improve age estimation, increasing the accuracy of identification in forensic anthropology.

"The big sleep: Elucidating the sequence of events in the first hours of death to determine the postmortem interval".

Recent developments on postmortem interval estimation (PMI) take an advantage of the autolysis process, pointing out to the analysis of the expression of apoptosis and autophagy genes towards this purpose. Oxidative stress plays a role in this signaling as a regulatory mechanism and/or as a consequence of cell death. Additionally, melatonin has been implicated on apoptosis and autophagy signaling, making melatonin a suitable target for PMI determination. The aim of this study was to investigate the early PMI through the analysis of the expression of autophagy genes as well as oxidative stress and melatonin receptor. Our results demonstrated a rapidly increased on the expression of autophagy genes according to the expected sequence of events, then a marked decrease in this expression, matched with the switch to the apoptosis signaling. These results revealed potential candidates to analyze the PMI in the first hours of death, helping to estimate the time-since-death.

Latent Fingermark Aging Patterns (Part IV): Ridge Width as One Indicator of Degradation.

This fourth article of the series is taking an in-depth analysis at the visible aging of latent fingermarks regarding changes in ridge widths over time. The objective is to quantify and statistically describe significant ridge size variations under controlled indoor conditions. The effect of three environmental variables are examined: type of secretion (sebaceous- and eccrine-rich) and type of substrate (glass and polystyrene) when aged in three light conditions (direct natural light, shade, and dark). Prior to width measurements, fresh and aged fingermarks were powdered with titanium dioxide (TiO2 ) and sequentially photographed at predetermined times over 6 months. Three independent observers measured the ridges from thirty predetermined locations using strategically placed intersecting lines on the print. Results indicate that fingermarks deposited on glass are more resilient to degradation compared with those deposited on plastic. The presence of direct natural light plays a negligible role on degradation compared to secretion and substrate types.

When forensic odontology met biochemistry: Multidisciplinary approach in forensic human identification.

When human remains are found, the priority of the investigation is to ascertain the identity of the deceased. A positive identification is a key factor in providing closure for the family of the deceased; it is also required to issue the death certificate and therefore, to settle legal affairs. Moreover, it is difficult for any forensic investigation involving human remains to be solved without the determination of an identity. Therefore, personal identification is necessary for social, legal and forensic reasons. In the last thirty years forensic odontology has experienced an important transformation, from primarily involving occasional dental identification into a broader role, contributing to the determination of the biological profile. In the same way, "DNA fingerprinting" has evolved not only in terms of improving its technology, but also in its application beyond the "classical": helping with the estimation of sex, age and ancestry. As these two forensic disciplines have developed independently, their pathways have crossed several times through human identification operations, especially the ones that require a multidisciplinary approach. Thus, the aim of this review is to describe the contributions of both forensic odontology and molecular biology/biochemistry to human identification, demonstrating how a multidisciplinary approach can lead to a better and more efficient identification.

Evaluation of macroscopic changes and the efficiency of DNA profiling from burnt teeth.

Identification of human remains subjected to incineration is extremely challenging. Our study evaluates the macroscopic changes and efficiency of DNA profiling in burnt teeth under controlled temperature and time conditions. 28 teeth were exposed to temperatures between 100 and 700°C for a duration of 1-15min. Two non-burnt teeth were used as control. Macroscopic changes were evaluated and recorded. DNA was extracted using a silica-based methodology. Efficiency of DNA profiling was assessed through Quantitative PCR for STRs. Burnt teeth reached chalky white appearance at 400°C 5min and fractures were observed from 300°C 10min. Amplification of STRs was very low from 300°C and 1 or 5min. In contrast, the housekeeping gene, GAPDH, was amplified in all combinations of temperatures and times. Although it is possible to amplify the housekeeping gene at high temperature, DNA profiling is difficult to obtain, probably due to small size of these regions making them more prone to degradation.

Human mitochondrial DNA and nuclear DNA isolation from food bite marks.

OBJECTIVE: Bite mark analysis is used for comparison between bite marks on a bitten object and the suspects' teeth. However, if it is not possible to obtain a correct match, it is important to recover salivary DNA. Previous studies have tried to isolate human nuclear DNA from bitten foods but were not completely successful. In the present work, we studied the efficiency of human nuclear and mitochondrial DNA isolation from bite marks in cheese, a donut and an apple. DESIGN: Using a double swab technique and silica-based DNA extraction kit, nuclear and mitochondrial DNA were isolated. Human housekeeping genes were amplified to analyse the efficiency of nuclear DNA profiling. mtDNA was sequencing and haplogroup assign. RESULTS: Although cheese and apple samples showed the highest concentration of DNA, the purity of DNA on the apple was low. Moreover, apple samples failed to amplify the two human housekeeping genes, GAPDH and RPL22. In contrast, cheese samples have high purity and amplification efficiency. Donut samples showed an intermediate value and low amplification efficiency. In spite of these results, isolation and characterization/sequencing of human mitochondrial DNA was completely successful in the three samples, which pointed out the possibility of identification through this type of DNA. CONCLUSIONS: This research indicated that it is possible to recover and isolate human nuclear and mitochondrial DNA from bitten foods, although the quantity and purity of nuclear DNA depends on the type of food. That is of significance important in forensic sciences for the correct identification of a suspect.

Cell death proteins as markers of early postmortem interval.

Estimation of time since death is one of the challenges in forensic science. There are many approaches to estimate the postmortem interval, including both physical and thanatochemistry methods. Decomposition is triggered by a process called autolysis, which induces destructive changes in the cell leading to cell death. Based on the process of cell death signaling, this study analyzed the early postmortem interval (2-8 h since death) using the study of the mRNA expression of Fas Ligand (FasL) and phosphatase and tensin homologue deleted on chromosome 10 (PTEN) by Quantitative-PCR. Results of the study indicate a time-dependent increase in the mRNA levels of both proteins up until 6 h after death. Using a regression analysis in these first 6 h, a positive linear correlation was found between the mRNA expression of these proteins and the time since death. Since PTEN and FasL are implicated in signaling pathways, both proteins are potential candidates to analyze the time since death in time intervals of 6 h or less. Further research is needed to find additional cell death markers and expand the time period for time since death estimation.

Applications of physiological bases of ageing to forensic sciences. Estimation of age-at-death.

Age-at-death estimation is one of the main challenges in forensic sciences since it contributes to the identification of individuals. There are many anthropological techniques to estimate the age at death in children and adults. However, in adults this methodology is less accurate and requires population specific references. For that reason, new methodologies have been developed. Biochemical methods are based on the natural process of ageing, which induces different biochemical changes that lead to alterations in cells and tissues. In this review, we describe different attempts to estimate the age in adults based on these changes. Chemical approaches imply modifications in molecules or accumulation of some products. Molecular biology approaches analyze the modifications in DNA and chromosomes. Although the most accurate technique appears to be aspartic acid racemization, it is important to take into account the other techniques because the forensic context and the human remains available will determine the possibility to apply one or another methodology.