Estimating the post-mortem interval (PMI) of human remains based on the histomorphology of dental pulp parameters is promising, but available evidence is scarce and sometimes contradictory without a scientific model. The aim of the study is to characterise the histomorphological changes of dental pulp associated with the decomposition of human remains by a qualitative and quantitative approach. The main aim is to establish a correlation based on post-mortem (PM) dental pulp histomorphology and the PMI, and whether pulp degradation could be an available medico-legal tool for PMI estimation beyond the first week after death (late PMI). The eligible sample consisted of 27 sound teeth from 16 healthy patients aged 16 to 72 years due to orthodontic or oral surgery treatment, to create PMI\'s simulating the death of the subject as the time elapsed from tooth avulsion. Data collected from patients (sex, date of birth, tooth position, date and hour of the avulsion, date and hour of pulp extraction) were anonymised in accordance with the requirements of Faculty of Dental Medicine of the University of Lisbon. The sample was divided into 9 groups of 3 teeth according to different PMI sets from T0 (baseline) up to 2 weeks (T0, 7, 12, 24, 36, 48, and 72 hours, 1 and 2 weeks). All the dental samples were stored at room temperature up to the time of pulp extraction and then prepared with haematoxylin and eosin stain. High-resolution microscopy was performed to obtain histological images. An operator performed the qualitative evaluation of blood vessels, collagen fibres, and the extra-cellular matrix (ECM) in PM pulps and measured the variation in cells/nuclei density by counting 6 different ROIs (Regions of Interest) for each pulp manually and automatically (quantitative analysis). Qualitative results showed that the degeneration of dental pulp appears 7 hours after death but histological changes in vessels, fibres, and ECM in PM dental pulp are characterised by high variability, consequently it is not possible to generalise the results for early PMIs. Quantitative measurements proved that cell count cannot be standardised due to the presence of superimposed layers of cells and nuclei fragmentation. Odontoblasts did not demonstrate evidence of cellular or nuclear lysis up to 14 PM suggesting their applicability in late PMIs. Future research will focus on late PMIs and different techniques of tooth preparation.

BACKGROUND: Accurate estimation of post-mortem interval (PMI) is crucial in forensic investigations. MicroRNAs (miRNAs or miRs) are small non-coding RNAs that remain relatively stable within the cell nucleus despite post-mortem changes.
OBJECTIVE: We assessed three target genes (miR-122, miR-133a, and miR-206) for PMI estimation using 72 healthy adult male BALB/c mice exposed to two different temperatures (4 and 21℃) at nine different time points over 10 days.
METHODS: Initially, the stability of the two reference genes (RNU6B and 5 srRNA) was evaluated using gene stability analysis tools (Delta Ct, Best Keeper, and Genorm) to select the optimal reference gene. RNU6B was found to be the most stable endogenous control. Subsequently, the expression patterns of miR-122, miR-133a, and miR-206 were analyzed within a 10-day PMI period using the heart, skeletal muscle, liver, and brain tissues.
RESULTS: At 4℃, miR-122 levels significantly decreased on days 8 and 10 in all tissues, with only the liver showing significant changes at 21℃. MiR-133a decreased over time in the heart, muscles, and brain, showing a dramatic decrease on days 8 and 10 in the heart and muscles at both temperatures. Although miR-206 levels decreased over time in muscles and liver at 4 ℃, these increased in the brain at 21 ℃, with no expression changes in other organs.
CONCLUSIONS: In summary, miR-122, miR-133a, and miR-206 are potential PMI markers in heart and skeletal muscle tissues.

Estimating the post-mortem interval (PMI) is an essential step in forensic investigations, particularly those involving homicides and unwitnessed deaths. However, traditional methods occasionally yield inconsistent estimates. Histological and molecular techniques are considered crucial in forensic pathology and are frequently employed to estimate the time interval of death. The gingiva is an oral mucosal tissue used to estimate PMI. This review aimed to examine the potential of histological methods to determine PMI using oral mucosal tissue, namely the gingiva, and to investigate changes that occur in oral mucosal tissue at different time intervals when compared with those in normal tissues. The oral mucosa comprises layers of stratified squamous epithelium and connective tissue. Similar to other body tissues, changes are known to occur in the gingiva after death, and these cellular and tissue changes should also be considered. Alterations in the gingiva include homogenisation, karyorrhexis, pyknosis, karyolysis, chromatin clumping, eosinophilia, collagen fibre degradation, and the loss of tissue architecture. Reviews collating the results of original trials have consistently reported how the oral mucosa is altered by autolysis and how such changes can be observed in histological tissue morphology after death. Histology is an acceptably accurate technique for estimating PMIs.

Forensic sciences play a vital role in the criminal justice system by providing insights into the identity of victims and suspects, causes of death, and other crucial pieces of evidence. In this research paper, we will explore the utility of forensic sciences, its techniques and applications, and the critical role of the forensic pathologist in analyzing human remains. For this purpose, we have analyzed a series of human remains and cadavers found in a state of decomposition, illustrating the medico-legal investigations carried out. Specifically, 50 cases from Calabrian experience are reported from 2003 to 2023 in different contexts of both judicial and archaeological interest and discovered by chance. In any case, anthropological, odontological, genetic, entomological, and forensic radiological investigations were carried out with the supervision of the forensic pathologist in all cases. The varied composition of the sample made it possible to understand the methods and the various specialists involved in such diversified cases. Furthermore, a review of the scientific literature on the topic of human remains was carried out. In particular, by delving into these topics, we aim to provide a comprehensive understanding of forensic anthropology and forensic sciences and their significance in the criminal justice system.

Forensic microbiology is a relatively new area of forensic sciences. It considers the potential of microorganisms to be used in criminal investigations. As most studies involve the role of bacteria in fields like post-mortem interval estimation, personal identification or geolocation, the data on the role of fungi is comparatively scarce. Forensic mycology involves the application of fungi and their structures in forensic cases. The aim of this review is the evaluation of the current state of knowledge on fungi associated with human cadavers and their possible role in estimating the time since death. In accordance with the available reports, we focused on the relation between microscopic fungi isolated from human corpses and the cadaver condition e.g., the stage of decomposition. We also emphasised the contrast between the reported methodologies and attempted to standardise research methods in forensic mycology from sample collection to its storage, mycological analysis and identification of the obtained fungal cultures. Moreover, the potential usage of microscopic fungi in criminal cases was discussed based on various case reports.

The family Sarcophagidae is very diverse in Brazil. Due to their living habits, they are the subject of many medical, veterinary, sanitary, and entomological studies. However, Sarcophagidae species are still poorly studied in forensic entomology, although they are frequently reported in carcasses and even human corpses. Thus, this study aims to identify and compare the developmental stages and intrapuparial morphological characteristics of Peckia (Euboettcheria) collusor to serve as an auxiliary tool in forensic entomology. The pupae collected after zero hour at 27 °C and 32 °C were sacrificed every three hours until the first 24 h and then every six hours until the emergence of the first adults, using 30 pupae each time, totaling 1560 for 27 °C and 1290 for 32 °C. The intrapuparial development time of this fly species under laboratory-controlled conditions was 288 h at 27 °C and 228 h at 32 °C. The 2820 pupae were analyzed according to temperature and classified into eight possible stages. This contributed to the selection of 16 key morphological characteristics to identify the age of the pupae. The identified intrapupal morphological characteristics have great potential to help researchers, experts, technical assistants, and forensic entomologists estimate the minimum post-mortem interval (minPMI) of cadavers.

The significance of biomechanical analyses for forensic time since death estimations has recently been demonstrated. Previous biomechanical analyses successfully discriminated post-mortem brain tissue from tissue with a post-mortem interval of at least one day when held at 20 °C. However, the practical utility of such analyses beyond day one at 20 °C was limited. This study investigates the storage, loss, and complex shear modulus of various brain regions in sheep stored at 4 °C in 24-hour intervals over four days post-mortem using rheometry tests. The aim is to identify the critical biomechanical tissue property values to predict post-mortem time and assess the temperature sensitivity of the rheometry method by comparing results to recent findings at 20 °C. Thirty sheep brains were examined, including the frontal lobe, parietal lobe, anterior and posterior deep brain, superior colliculi, pons, medulla, and cerebellum. Rheometry tests were conducted, and receiver operator characteristic analyses were employed to establish cut-off values. At 4 °C storage, all investigated biomechanical properties of the examined brain regions remained stable for at least one day post-mortem. Using cerebellar samples stored at 4 °C, a post-mortem interval of at least two days could be determined with excellent diagnostic ability. Complex shear modulus values below 1435 Pa or storage modulus values below 1313 Pa allowed prediction of two or more days post-mortem. Comparisons between 4 °C and 20 °C revealed brain region-specific results. For instance, the complex shear moduli of the anterior deep brain at 4 °C were significantly higher on all individual testing days when compared to 20 °C. In contrast, the combined medulla and pons samples were similar on each day. Rheometry testing of brain tissue consistently stored at 4 °C since death proved valuable for forensic time since death estimations starting from two days after death.

Succession patterns of carrion insects on large mammal\'s carrion has been widely studied, notably to estimate the post-mortem interval in forensic investigations as accurately as possible. However, little attention has been paid to the carrion insects living inside these bones once a carcass is skeletonized. One very recent study documented flies emerging from pig carcasses, and only scarce authors reported the presence of other carrion insects taking advantage of the bone marrow. We, thus, aimed to (1) estimate the frequency of inner-bone space colonization by carrion insects, with particular attention to bone-skipper flies; (2) identify the insects living inside the carrion bones; and (3) determine whether or not carrion insects found within the bones can successfully exit the bones and complete their development. We extensively sampled 185 large mammals\' bones collected from twelve vulture feeding stations and four isolated carcasses in southwest France and northern Spain. Sampled bones were opened, and the insects found inside were identified. For two bones, foramen, i.e., the holes providing a natural entrance and exit to the bone\'s inner cavity, was monitored with a camera to assess the insect\'s putative exit. We describe the entomofauna, i.e., the set of insect species, living within the bones, and illustrate insects\' ability to exit the bones for their subsequent development and maturity. These results are discussed in the framework of carrion insect conservation and forensic entomology perspectives.

Blow flies (Diptera: Calliphoridae) are generally early colonisers of fresh cadavers, enabling the estimation of a minimum post-mortem interval (minPMI) based on an accurate aging of the oldest immature stages associated with a cadaver. In blow flies, the pupal stage and the subsequent development of the adult take place inside a protective case, the puparium, formed from the hardened and darkened cuticle of the third instar larva. Because the puparium is an opaque structure that shows virtually no external changes, qualitative analyses of the internal tissues can be very informative for determining reliable age-specific morphological markers. Those analyses can be performed using either non-invasive but expensive and not widely accessible techniques, or traditional histological methods, which are invasive as they require the serial sectioning of the sample. Histological methods are often readily available for forensic researchers and practitioners; however, the histological study of blow fly intra-puparial stages has traditionally been hampered by the poor paraffin infiltration of tissues due to the abundance of fat bodies, resulting in usually fragmented sections and the subsequent loss of relevant information. We present here an effective method for the preparation of histological sections of blow fly intra-puparial stages, maximising the paraffin infiltration while enabling the production of clean and entire sections that allow for the use of reliable age-specific morphological markers, thus improving the accuracy of minPMI estimations when access to more costly techniques is not feasible.

Estimation of the post-mortem interval (PMI) is mainly based on the state of body cooling, post-mortem lividity (livor mortis) and post-mortem muscle stiffness (rigor mortis). However, the time span of development of these post-mortem phenomena are influenced by a variety of factors concerning the body of the deceased and the environment in which the body is found. Subsequently, this leads to a substantial spread in upper and lower limits of PMI based on determination of the state of these phenomena. Moreover, interpretation of post-mortem phenomena like lividity, rigor and interpretation of the correction factor for Henssge\'s nomogram is subjective. For this reason, PMI estimations are often broad, possibly too broad to be helpful for answering questions which are relevant for the criminal investigation. Therefore, combining the outcome of different methods for estimating the PMI, the so-called compound method, is recommended. Supravital muscle reaction by mechanical stimulation of skeletal muscle is a less known aspect of the compound method. Here we present a series of cases series in which supravital muscle reaction contributed to a more precise estimation of the PMI.