Epigenetic analyses in forensic medicine: future and challenges.

The possibility of using epigenetics in forensic investigation has gradually risen over the last few years. Epigenetic changes with their dynamic nature can either be inherited or accumulated throughout a lifetime and be reversible, prompting investigation of their use across various fields. In forensic sciences, multiple applications have been proposed, such as the discrimination of monozygotic twins, identifying the source of a biological trace left at a crime scene, age prediction, determination of body fluids and tissues, human behavior association, wound healing progression, and determination of the post-mortem interval (PMI). Despite all these applications, not all the studies considered the impact of PMI and post-sampling effects on the epigenetic modifications and the tissue-specificity of the epigenetic marks.This review aims to highlight the substantial forensic significance that epigenetics could support in various forensic investigations. First, basic concepts in epigenetics, describing the main epigenetic modifications and their functions, in particular, DNA methylation, histone modifications, and non-coding RNA, with a particular focus on forensic applications, were covered. For each epigenetic marker, post-mortem stability and tissue-specificity, factors that should be carefully considered in the study of epigenetic biomarkers in the forensic context, have been discussed. The advantages and limitations of using post-mortem tissues have been also addressed, proposing directions for these innovative strategies to analyze forensic specimens.

Artificial intelligence in healthcare: why not apply the medico-legal method starting with the Collingridge dilemma?

Technology has greatly influenced and radically changed human life, from communication to creativity and from productivity to entertainment. The authors, starting from considerations concerning the implementation of new technologies with a strong impact on people's everyday lives, take up Collingridge's dilemma and relate it to the application of AI in healthcare. Collingridge's dilemma is an ethical and epistemological problem concerning the relationship between technology and society which involves two approaches. The proactive approach and socio-technological experimentation taken into account in the dilemma are discussed, the former taking health technology assessment (HTA) processes as a reference and the latter the AI studies conducted so far. As a possible prevention of the critical issues raised, the use of the medico-legal method is proposed, which classically lies between the prevention of possible adverse events and the reconstruction of how these occurred.The authors believe that this methodology, adopted as a European guideline in the medico-legal field for the assessment of medical liability, can be adapted to AI applied to the healthcare scenario and used for the assessment of liability issues. The topic deserves further investigation and will certainly be taken into consideration as a possible key to future scenarios.

Expression of matrix metalloproteinase-9 (MMP-9) in human skin within 1 hour after injury through immunohistochemical staining: a pilot study.

Matrix metalloproteinase-9 (MMP-9) is involved in tissue remodeling and in skin wound healing. The present study focuses on the MMP-9 expression in epidermal wound healing within 1 h after injury, to test whether MMP-9 can be used to estimate the time of injury in forensic practice.A sample consisting of 5 individuals undergoing surgery was analyzed. With the consent of the patients, sections of skin were removed from the surgical wound at predefined time intervals. For each subject, 8 sections were taken, one for each time interval defined at 0 '- 1' - 3 '- 5' - 10 '- 15' - 30 '- 60' minutes. The specimens were immunostained with MMP-9, and the number of positively stained cells was examined.The number of positively stained cells showed an increasing trend as a function of time. Less than 30 positively stained cells were found in all cases within 3 min. At the post-infliction time of 5 min, the number of positively stained cells exceeded 30 in 3 out of 5 cases. The number of MMP-positive cells exceeded 40 in all cases in over 10 min.In the light of these results, the count of MMP-9 positive cells might be a useful marker in the wound-age estimation within 1 h in forensic setting. More research is required to collect more samples and to compare samples from the hyperacute phase with those from several days after injury.

Forensic species identification: practical guide for animal and plant DNA analysis.

The importance of non-human DNA in the forensic field has increased greatly in recent years, together with the type of applications. The molecular species identification of animal and botanical material may be crucial both for wildlife trafficking and crime scene investigation. However, especially for forensic botany, several challenges slow down the implementation of the discipline in the routine.Although the importance of molecular analysis of animal origin samples is widely recognized and the same value is acknowledged to the botanical counterpart, the latter does not find the same degree of application.The availability of molecular methods, especially useful in cases where the material is fragmented, scarce or spoiled preventing the morphological identification, is not well known. This work is intended to reaffirm the relevance of non-human forensic genetics (NHFG), highlighting differences, benefits and pitfalls of the current most common molecular analysis workflow for animal and botanical samples, giving a practical guide. A flowchart describing the analysis paths, divided in three major working areas (inspection and sampling, molecular analysis, data processing and interpretation), is provided. More real casework examples of the utility of non-human evidence in forensic investigations should be shared by the scientific community, especially for plants. Moreover, concrete efforts to encourage initiatives in order to promote quality and standardization in the NHFG field are also needed.

Current status of undergraduate teaching in forensic & legal medicine in Europe.

The European Council of Legal Medicine (ECLM) is the body established in 1992 to represent practitioners forensic & legal medicine and is composed of delegates of the countries of the European Union (EU) and from other countries which form part of Europe to a current total of 34 member countries. The aims of this study were to determine the current status of undergraduate forensic & legal medicine teaching in the curriculum of medical studies in ECLM countries and to use the results of this study to determine whether it would be appropriate to develop new guidelines and standards for harmonising the content of undergraduate forensic medicine training across ECLM member countries. A detailed questionnaire was sent to all individuals or organisations listed on the ECLM contact database. Responses were received from 21 of 33 countries on the database. These responses showed considerable emphasis on undergraduate teaching of forensic medicine in all countries with the exception of Belgium and the United Kingdom. There was great general consistency in the subjects taught. The data from this survey provide a baseline which should assist in developing a strategy to harmonise forensic & legal medicine undergraduate training in member countries of the ECLM. The ECLM is now in a good position to establish a pan-European working group to coordinate a consensus document identifying an appropriate and modern core undergraduate forensic medicine curriculum that can be presented to the medical education authorities in each country, and which can be adapted for local requirements, based on available personnel, the forensic medicine structure in the country, and most importantly, the needs of the local population.

Postmortem biochemistry of GFAP, NSE and S100B in cerebrospinal fluid and in vitreous humor for estimation of postmortem interval: a pilot study.

Postmortem interval (PMI) is a challenging issue in forensic practice. Although postmortem biomarkers of traumatic brain injury (TBI) are recognised as an emerging resource for PMI estimation, their role remains controversial. This study aims to evaluate postmortem concentrations of three TBI biomarkers (GFAP, NSE and S100B) in two matrices (cerebrospinal fluid and vitreous humor), in order to find out if these markers could be adopted in PMI estimation. Thirty-five deceased individuals with known PMI who underwent forensic autopsy at the University of Parma were examined. Matrices were collected during autopsy, then biomarker concentrations were determined through the enzyme-linked immunosorbent assay. Statistical significance of the data in relation to PMI was studied. The correlation of biomarkers with PMI, examined with samples divided into six groups according to the number of days elapsed since death, was not statistically significant, although S100B in cerebrospinal fluid showed an increasing trend in cases from 1 to 5 days of PMI. Comparison between cases with 1 day of PMI and those with 2 or more days of PMI showed a statistically significant correlation for GFAP and NSE in cerebrospinal fluid. GFAP and NSE in cerebrospinal fluid represent appropriate biomarkers in PMI estimation to distinguish cases with one day of PMI from those with two or more days of PMI. The current study was limited by the scarcity of the cohort and the narrow spectrum of cases. Further research is needed to confirm these observations.

Applications of forensic anthropology methodology: accuracy of virtual face reproductions performed on the Tenchini collection.

Facial reproduction validation uses the methodology typical of forensic anthropology to confirm the accuracy of three-dimensional reproductions of faces. Achieving high accuracy in virtual facial reproductions is still under study. In the present paper, the Tenchini collection, which contains wax reproductions of prisoners' faces and their skulls, was used. By creating facial reproductions on skulls from this collection, the result was compared with the real face of the person to whom the skull belongs. The three-dimensional volume of each examined skull of Tenchini collection was acquired via CT scan and photogrammetry. Subsequently, the virtual reproduction of each skull was processed using three-dimensional graphics software (ZBrush, Pixologic®). The morphological comparison parameters used in the field of personal identification by forensic anthropology were used. The blind procedure required the operator to see the mask subsequent to the facial reproduction phase, so that the facial reproduction cannot exploit the information that knowledge of the mask would have produced. The parameters used in the study of facial reproductions have shown discrepancies between the characteristics of the masks and the facial reproductions, partly expected because due to the lack of soft tissue on the skull. However, a high degree of accuracy in the facial reproductions performed due to the applied methodology was documented. The present study allowed us to observe how these parameters can be useful to study the accuracy of facial reproduction and identify what difficulties are encountered in producing a result close to the real appearance.

Father figure: Study of the age progression process from old pictures and its value in forensic sciences.

The application of age progression to the search for missing persons demonstrates its usefulness in being able to reconstruct the aged face based on the current age of a person who has been missing for many years. Age progression takes into account facial physiognomic elements and their changes over time. Based on these premises, the present study aims to investigate the evolution of somatic features, from childhood to adulthood, of one of the authors' father using photos at different ages of him. Sixteen photos were collected depicting an individual of Italian origin between the age of 1 and 72. The photos were processed in Adobe© Photoshop software to graphically highlight the shape development of individual physiognomic elements. The shape of the face, eyebrows, eyes, nose, mouth, and cheeks were analysed in detail using a morphological approach. The ageing processes that occurred in this case were discussed on the basis of the literature. The present study demonstrates the in-depth analysis of the dynamics of facial growth in a single case. Nonetheless, aging is different for each individual, so further studies are necessary to observe facial development on as many subjects as possible.

The forensic imaging technique of age progression used within missing people cases: The Italian Missing Children association realizing age progression for Missing Child Kenya.

The age progression technique allows to update identification photos of individuals who have been missing for years. This method is based on the study of the facial variation and on the dynamics of facial growth. Knowledge about changes in the distribution of facial fat and the tone of the cartilage allows realistic predictions of the facial appearance after many years. Beside these elements, age progression is also supported by similar characters between family members. This is why photos of the missing and family members are used to create an age progression, making the new identikit accurate. Non-profit associations are crucial for the dissemination of information on the problem of missing persons. In Italy, the Italian Missing Children association provides for free an age progression service with the support of forensic anthropology. With the activation of a protocol, consisting of guidelines, and a form for the request for age progression by family members, the Italian Missing Children has started a collaboration with Missing Child Kenya. It allowed to disseminate up-to-date photos of the presumed current appearance of the missing children in Kenya. Thus, in the present study some cases are reported, in which age progression technique was applied. The photos provided by Italian Missing Children were shown in video documentaries made by Missing Child Kenya, allowing to continue the dissemination of age progression photo during the Covid quarantines and to keep the population active in the search for the missing.

Medically assisted suicide in Italy: Recent legal developments on a controversial topic.

On 16th June 2022, the first case of lawful 'medically assisted suicide' took place on Italian soil. This event is a result of decade-long debates on informed consent and end-of-life care stimulated by medical jurisprudence. The authors first retrace the crucial moments that allowed this to happen and underline the problems still to be solved. The cases of DJ Fabo, Davide Trentin, Mario and Fabio Ridolfi are discussed, signalling how they influenced the path implemented by Italian jurisprudence.

HIF-1α expression by immunohistochemistry and mRNA-210 levels by real time polymerase chain reaction in post-mortem cardiac tissues: A pilot study.

INTRODUCTION: The postmortem diagnosis of acute myocardial ischemia (AMI) represents a challenging issue in forensic practice. Immunohistochemical studies and gene expression studies are becoming a promising field of research in forensic pathology. The present study aims to evaluate HIF-1α expression through immunohistochemistry (IHC), and mRNA-210 level using real-time polymerase chain reaction (RT-PCR), in order to define if HIF-1α and mRNA-210 in post-mortem myocardium could be adopted in the diagnosis of AMI. MATERIALS AND METHODS: Thirty-five deceased individuals, who underwent forensic autopsy at the Legal Medicine Service of the University of Parma, between 2010 and 2018, were investigated. The cohort was divided into two groups according to the cause of death (sudden deaths caused by AMI vs control cases). Cardiac specimens were collected during autopsy, then samples were processed for morphological evaluation using haematoxylin-eosin staining, for IHC, and for RT-PCR. HIF-1α expression and mRNA-210 levels were investigated. RESULTS: Statistical evaluation demonstrated statistically significant differences in terms of number of IHC positive vessels, leukocytes, and cardiomyocytes between the two groups. Moreover, in the majority of cases, immunostaining positivity was observed only in myocardial and subendocardial samples. With reference to mRNA-210, the difference between the two groups proved to be statistically significant. CONCLUSIONS: The present study indicates that HIF-1α and mRNA-210 in post-mortem cardiac specimens could represent appropriate biomarkers in the diagnosis of AMI. The current study was primarily limited by the scarcity of the cohort, so further research is required to confirm these preliminary observations.

Taxonomic and metabolic development of the human gut microbiome across life stages: a worldwide metagenomic investigation.

The human gut microbiota is a dynamic community of microorganisms that undergo variable changes over the entire life span. To thoroughly investigate the possible fluctuations of the microbiota throughout human life, we performed a pooled analysis of healthy fecal samples across different age groups covering the entire human life span. Our study integrated data from 79 publicly available studies and new stool samples from an Italian cohort, i.e., the Parma Microbiota project, resulting in 6,653 samples processed through the shotgun metagenomic approach. This approach has allowed species-level taxonomic reconstruction of the gut microbiota and investigation of its metabolic potential across the human life span. From a taxonomic point of view, our findings confirmed and detailed at species-level accuracy that the microbial richness of the gut microbiota gradually increases in the first stage of life, becoming relatively stable during adolescence. Moreover, the analysis identified the potential core microbiota representative of distinct age groups, revealing age-related bacterial patterns and the continuous rearrangement of the microbiota in terms of relative abundances across the life span rather than the acquisition and loss of taxa. Furthermore, the shotgun approach provided insights into the functional contribution of the human gut microbiome. The metagenomic analysis revealed functional age-related differences, particularly in carbohydrate and fiber metabolism, suggesting a co-evolution of the microbiome assembly with diet. Additionally, we identified correlations between vitamin synthesis, such as thiamine and niacin, and early life, suggesting a potential role of the microbiome in human physiology, in particular in the functions of the host's nervous and immune systems. IMPORTANCE: In this study, we provided comprehensive insights into the dynamic nature of the human gut microbiota across the human life span. In detail, we analyzed a large data set based on a shotgun metagenomic approach, combining public data sets and new samples from the Parma Microbiota project and obtaining a detailed overview of the possible relationship between gut microbiota development and aging. Our findings confirmed the main stages in microbial richness development and revealed specific core microbiota associated with different age stages. Moreover, the shotgun metagenomic approach allowed the disentangling of the functional changes in the microbiome across the human life span, particularly in diet-related metabolism, which is probably correlated to bacterial co-evolution with dietary habits. Notably, our study also uncovered positive correlations with vitamin synthesis in early life, suggesting a possible impact of the microbiota on human physiology.