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Accuracy in the evaluation of death-induced tissue degradation for thanato-chronological purposes is strictly dependent on the condition of the biological source as well as on the precision of post-mortem interval (PMI) estimation. Thus, the optimization of tissue handling and identification of sensitive post-mortem biomarkers could help establish a timeline for post-mortem events. To this aim, we investigated the proteome changes in cortex samples of 6-week-old female SAMR1 mice over a post-mortem time course. After death, brain tissue was removed immediately (T0), and after 4, 8, 12, 24, and 32 h, four mice were used for each time period, and animals were maintained at 4 °C until brain removal. Dissected tissues were frozen at -80 °C until processed. Proteomic analysis, performed on samples related to early and late PMIs (<24 h and >24 h post-mortem, respectively) showed protein level changes as compared to T0 samples, with a remarkable increase in Calpain11 in the early PMI, as well as in Caspases 7 and 8 together with Gasdermin 3 in late PMI. These findings were confirmed by LIFT mass spectrometry technology and western blot analysis and, although requiring further investigation in other biological samples, suggest that these proteins could be considered as putative biomarkers of different PMIs.

Mast cells (MCs) are derived from hematopoietic progenitors, mature in vascularized tissues, and participate in innate and acquired immunity. Neuroinflammation is a highly debated topic in the biomedical literature; however, the impact of tumor necrosis factor (TNF) and IL-33 on MCs in the brain has not been widely addressed. MCs can be activated by IgE binding to FcεRI, as well as by different antigens. After activation, MCs mediate various immunological and inflammatory responses through TNF and IL-33. TNF has two receptors: TNFR1, a p55 molecule, and TNFR2, a p75 molecule. This cytokine is the only one of its kind to be stored in the granules of MCs and can also be generated by de novo synthesis via mRNA. In the central nervous system (CNS), TNF is produced almost exclusively by microglial cells, neurons, astrocytes, and, minimally, by endothelial cells. After its release into brain tissue, TNF rapidly induces the adhesion molecules endothelial leukocyte adhesion molecule 1 (ELAM-1), intercellular adhesion molecule 1 (ICAM-1), and vascular cell adhesion molecule 1 (VCAM-1) in endothelial cells. TNF causes the chemoattraction of neutrophils by inducing several molecules, including CXC chemokines (IL-8). Both MCs and microglial cells act as a primary barrier against foreign molecules in the CNS, producing pro-inflammatory cytokines such as IL-33. IL-33 belongs to the IL-1 family, is activated through the ST2L/IL1-RAcP receptor complex, and mediates both the innate and adaptive immune response. IL-33 is a nuclear transcription factor expressed in the brain, where it induces pro-inflammatory cytokines (TNF and IL-1) and chemokines (CCL2, CCL3, CCL5, and CXCL10). Therefore, MCs and microglia in the CNS are a source of pro-inflammatory cytokines, including TNF and IL-33, that mediate many brain diseases. The inhibition of TNF and IL-33 may represent a new therapeutic approach that could complement existing neuroinflammatory therapies.

Death is a multifaceted process wherein each individual cell and tissue has a metabolic homeostasis and a time of functional cessation defined by the dying process as well as by intrinsic and extrinsic factors. Decomposition is physiologically associated with the release of different types of volatile organic compounds (VOCs), and these form volaboloma mortis. The main purpose of this study was to record the volabolomic fingerprint produced by volatile molecules during the physiological decomposition process of human tissue and muscle cells. The volatile chemical signature has important implications for an open issue in forensics and pathology, namely the estimation of the postmortem interval (PMI), which decreases in accuracy with the passage of time. Volatile metabolites emitted from human tissues and muscle cells at 0, 24, 48, and 72 h were recorded in real time with an electronic nose sensor device. The key findings were the continuous sampling of VOCs emitted from tissues and cells. These showed a common behavior as time progressed; particularly, after 48 h the distributions became dispersed, and after 72 h they became more variable. Volabolomic fingerprinting associated with time progression relevant to the study of PMIs was reconstructed. Additionally, there may be broader applications, such as in dog training procedures for detecting human remains, and perhaps even for studying scavenger and insect attractants.

Mast cells (MCs) are tissue cells that are derived from bone marrow stem cells that contribute to allergic reactions, inflammatory diseases, innate and adaptive immunity, autoimmunity, and mental disorders. MCs located near the meninges communicate with microglia through the production of mediators such as histamine and tryptase, but also through the secretion of IL-1, IL-6 and TNF, which can create pathological effects in the brain. Preformed chemical mediators of inflammation and tumor necrosis factor (TNF) are rapidly released from the granules of MCs, the only immune cells capable of storing the cytokine TNF, although it can also be produced later through mRNA. The role of MCs in nervous system diseases has been extensively studied and reported in the scientific literature; it is of great clinical interest. However, many of the published articles concern studies on animals (mainly rats or mice) and not on humans. MCs are known to interact with neuropeptides that mediate endothelial cell activation, resulting in central nervous system (CNS) inflammatory disorders. In the brain, MCs interact with neurons causing neuronal excitation with the production of neuropeptides and the release of inflammatory mediators such as cytokines and chemokines. This article explores the current understanding of MC activation by neuropeptide substance P (SP), corticotropin-releasing hormone (CRH), and neurotensin, and the role of pro-inflammatory cytokines, suggesting a therapeutic effect of the anti-inflammatory cytokines IL-37 and IL-38.

Nowadays, it is vital to have new, complete, and rapid methods to screen and follow pharmacotoxicological and forensic cases. In this context, an important role is undoubtedly played by liquid chromatography-tandem mass spectrometry (LC-MS/MS) thanks to its advanced features. This instrument configuration can offer comprehensive and complete analysis and is a very potent analytical tool in the hands of analysts for the correct identification and quantification of analytes. The present review paper discusses the applications of LC-MS/MS in pharmacotoxicological cases because it is impossible to ignore the importance of this powerful instrument for the rapid development of pharmacological and forensic advanced research in recent years. On one hand, pharmacology is fundamental for drug monitoring and helping people to find the so-called "personal therapy" or "personalized therapy". On the other hand, toxicological and forensic LC-MS/MS represents the most critical instrument configuration applied to the screening and research of drugs and illicit drugs, giving critical support to law enforcement. Often the two areas are stackable, and for this reason, many methods include analytes attributable to both fields of application. In this manuscript, drugs and illicit drugs were divided in separate sections, with particular attention paid in the first section to therapeutic drug monitoring (TDM) and clinical approaches with a focus on central nervous system (CNS). The second section is focused on the methods developed in recent years for the determination of illicit drugs, often in combination with CNS drugs. All references considered herein cover the last 3 years, except for some specific and peculiar applications for which some more dated but still recent articles have been considered.

We report the case of a 54-year-old man who died in a motorcycle accident due to loss of control of the vehicle on a viaduct. No other vehicles were apparently involved, except for a car hit by the motorcycle after it fell. A post-mortem CT scan (computed tomography scan) was performed showing complex head trauma with a subarachnoid hemorrhage and multiple skull and facial bone fractures. A forensic cinematic reconstruction performed by an engineer was needed to exclude other incident causes other than the loss of control. The multidisciplinary approach that included autopsy findings, a cinematic reconstruction, a helmet test and an examination played a key role in clarifying the dynamics of the accident, allowing us to explain how the death occurred despite the motorcyclist's helmet use. The cause of death was identified as a penetrating head trauma with cerebral material exposure, produced by the impact of the head against a fixed bolt in the guardrail base. Despite the use of the helmet, the impact force was enough to render the protection ineffective and allowed the bolt to penetrate through the helmet and the skull.

The main aim of this study was to investigate the post-mortem proteolytic degradation process of selected tissue antigens and correlate it to the post-mortem interval. During the autopsy of 12 cadavers (time interval ranging 1 day-2 years after death) samples of skin, liver, kidney, and spleen were collected. All samples were formalin-fixed and paraffin-embedded. Four µm paraffin sections were used for hematoxylin-eosin staining and immunohistochemical analysis (Ki67, Vimentin, Pan cytokeratin, and CD20). Data reported here show that immunohistochemical reactivity preservation was related to the characteristics of the tissues. In particular, the most resistant tissue was the skin, where the autolysis phenomena were not appreciable before 5 days. On the contrary, the liver and the spleen underwent early autolysis, while the kidney displayed an early autolysis of the tubules and a late one of the glomeruli. As concerns specific antigens, immunoreactivity was lost earliest for nuclear antigens as compared to cytoplasmic ones. In conclusion, our results demonstrate that immunohistochemical detection of specific antigens may be useful in estimating the post-mortem interval, especially when we need to know whether the post-mortem interval is a few days or more than 7-10 days.

Human postmortem skeletal muscles are a unique source of satellite cells for skeletal muscle regenerative studies. Presomite and somite satellite cells obtained by postmortem muscles have been established as populations of human skeletal muscle precursor cells able to proliferate and differentiate in vitro . It is extremely interesting to have access to a large amount of postmortem human skeletal muscle precursor cells, especially from craniofacial as well as limb skeletal muscles in order to evaluate their potential application not only for the fundamental understanding of muscle physiology and diseases but also for drug testing in a challenging 3D-shaping muscles like skeletal muscle microphysiological systems., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Pietrangelo, Demontis, Santangelo, Pini, Bonelli, Rosato, Roberti, Locatelli, Tartaglia, Marramiero, Verratti, Bondi, Fulle, D’Aloja and D’Ovidio.)

Background: Since the beginning of the worldwide spread of severe acute respiratory syndrome coronavirus 2 to date, important knowledge has been obtained about the virus behavior in living subjects and on inanimate surfaces; however, there is still a lack of data on virus persistency on dead bodies and the risk of contagion from cadavers., Case Presentation: The present case shows the persistency of the severe acute respiratory syndrome coronavirus 2 viral genome in nasopharyngeal swabs performed on a drowned Caucasian man, aged 41 years old, who was completely asymptomatic when he was alive, up to 41 days after death. Specific real-time reverse transcriptase-polymerase chain reaction (TaqMan 2019-nCoV Assay Kit v2; Thermo Fisher Scientific, Italy and Realquality RQ-SARS-CoV-2, AB Analytical) was used to evaluate the swabs., Conclusions: This data reflect the importance of postmortem swabs in all autopsy cases, and not only in potential severe acute respiratory syndrome coronavirus 2-related death, and also highlight the necessity to evaluate virus positivity a long time after the moment of death, even if a low initial viral load was assessed., (© 2022. The Author(s).)

Therapeutic drug monitoring (TDM) of antiseizure medications (ASMs) represents a valuable tool to establish an appropriate patient therapy, to collect important information about drugs' interactions and to evaluate patient's metabolic capabilities. In recent years, a new volumetric absorptive microsampling technique using VAMS ® technology and Mitra ® devices, consisting of a sampling technique for the collection of fixed-volume capillary blood, was developed. These new devices provide a new home-sampling technique for whole blood that has been spread out to simplify sample collection from finger-pricks. This review is aimed to compare published articles concerning the application of VAMS ® in epilepsy and to identify the strengths and improvement points for the TDM of antiseizure medications. VAMS ® allowed a minimally invasive blood sampling even in the absence of trained personnel. Good stability data have indicated that storage and delivery can be facilitated only for specific ASMs. Trueness and precision parameters have been evaluated, and the hematocrit (HCT) effect was minimized.

Currently, forensic research is multidisciplinary with new methods and parameters useful to define the cause and time of death as well as survival/agony times. The identification of biochemical markers able to estimate agonal period has been studied by many forensic researchers. It is known that the estimation of agonal time in different types of death is not always easy, hence our interest in literature's data. The studies analyzed in this review confirm the important role of thanatobiochemistry for the estimation of survival times. Regardless of the death cause, the survival/agony time between the primary event and death influences markers concentrations in biological samples (e.g., blood, urine, cerebrospinal fluid). Different biomarkers can be used for qualitative evaluations in deaths with short and long agony (e.g., C-reactive protein, ferritin, GFAP, etc.). Instead, the quantitative interpretation showed limits due to the lack of reference cut-offs. Thanatobiochemistry is a useful tool to confirm what emerged from autopsies findings (macroscopic and histological analysis), but further studies are desirable to confirm the evidence emerging from our review of the literature.

Notwithstanding life-threatening haemorrhagic complications, endobronchial ultrasound-guided transbronchial needle aspiration represents a cornerstone in the evaluation of NSCLC patients. Due to its low invasiveness and satisfactory tolerability even in high-risk patients, it is usually scheduled in one-day surgery hospital stay. Moreover, EBUS-TBNA offers a viable alternative to other conventional endoscopic procedures such as mediastinoscopy with the addiction to gain access also to hilar nodes. We report an unexpected and rare event of post-bronchoscopy fatal endobronchial haemorrhage in a 67-year-old female patient with a right S1 solitary pulmonary nodule and concomitant multiple bilateral lymphadenopaties. According to clinical staging the patient was unfit for upfront surgery and endoscopic procedure for tissue diagnosis was scheduled. The immediate postoperative period was uneventful as no intraoperative injuries were reported. Twenty-four hours later, the patient claimed respiratory distress and, after admission to the Emergency Department, a massive and uncontrolled bleeding coming from the lower respiratory tract was diagnosed leading to a death. Although one-day surgery EBUS-TBNA is the standard of care in patients undergoing endoscopic exploration, current guidelines seem to be murky about proper case stratification in order to plan an exhaustive observation time especially in high-risk patients., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/med-20-33). The authors have no conflicts of interest to declare., (2021 Mediastinum. All rights reserved.)

A new liquid chromatography-tandem mass spectrometry (LC-MS/MS) confirmation method for the direct analysis of 17 drugs starting from 200μL of diluted oral fluid (OF), in a single chromatographic run, was developed and validated. Cocaine, benzoylecgonine (BEG), cocaethylene, Δ-9-tetrahydrocannabinol (Δ-9-THC), buprenorphine, 6-acetylmorphine (6AM), morphine, codeine, methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine (MDMA), methylenedioxyamphetamine (MDA), 3,4-Methylenedioxy-N-ethylamphetamine (MDE), ketamine, N-methyl-1-(1,3-benzodioxol-5-yl)-2-butanamine (MBDB) were determined in a chromatographic run of 12min only with no sample pre-treatment, after the addition of 15 different internal standards (ISs). The method met all requirements in terms of linearity, accuracy (precision and trueness), recovery, and stability requested by FDA guidelines. Carry-over and interferences were negligible, as well as the matrix effects. LLOQs are below the limits defined by European guidelines and Italian national laws. The original oral fluid collections are stable at least six months at -20°C and one week at +4°C., Competing Interests: Declaration of Competing Interest Elisa Bassotti is employee at Eureka Lab Division. Paolo Dossetto is employee at Sciex. The other authors declare that does not exist any economic interest or any conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Ethanol (ethylic alcohol) represents the most commonly used drug worldwide and is often involved in clinical and forensic toxicology. Based on several reports, excessive alcohol consumption is the main contributing factor in traffic accidents, drownings, suicides, and other crimes. For these reasons, it becomes essential to analyze the alcohol concentration during autopsy. Although blood is usually used for alcohol analysis in post-mortem cases, it could suffer alterations, putrefaction, and microbial contaminations. As an alternative to whole blood, vitreous humor has been successfully used in medico-legal studies. In this work, post-mortem specimens were analyzed for ethanol determination. The analysis of blood and vitreous humor were carried-out using gas chromatography-flame ionized detector (GC-FID) with a total run time of 6 min. The method was validated in terms of limit of detection, limit of quantification, dynamic range, sensibility, recovery, precision and trueness. A linear regression analysis indicated a coefficient of determination (R 2 ) of 0.9981. The study confirmed no statistically differences between alcohol concentration in blood and vitreous humor, leading vitreous humor as an excellent matrix that could be used as an alternative to whole blood in toxicological analysis in cases where blood is not available.

BACKGROUND We present a report of a rare cardiac malformation case as well as a review of the literature. In addition, the diagnostic features are discussed. CASE REPORT The case of a female newborn who died on her third day of life was studied at the Institute of Legal Medicine, University of Chieti-Pescara (Italy). The investigations around her death revealed a cardiac congenital malformation, seen as a rare variant of a common arterial trunk, in which the aorta was fused with the right branch of the pulmonary artery. The ascending aorta showed hypoplasia, while the coronary arteries were free of any pathological findings. The atrial septum showed a closed foramen ovale and the ventricular septum did not show any defect. Only an isolated right ventricular hypertrophy and dilation with no other cardiac abnormalities was found. The cause of death was acute respiratory failure on the third day of extrauterine life when the ductus Botalli closed. The karyotype analysis performed in this case was normal, and the fluorescent in situ hybridization analysis did not show the 22q11.2 microdeletion suggestive of the DiGeorge syndrome. CONCLUSIONS These findings underline the value of 3-dimensional/4-dimensional ultrasound imaging when added to a fetal cardiology screening program, and the need for improvements in postnatal screening routines by using pulse oximetry in order to discover isolated vascular defects before circulatory collapse occurs, as well as to reduce the medico-legal disputes in cases of missed diagnosis. We found the relevant literature search lacked a description of this congenital malformation, which supports our deeper perinatal investigation.

Intragastric balloon (IGB) therapy is a widely used technique to counter obesity, and it is known to be safe and effective. Although there can be a high rate of side effects following IGB therapy, most are self-healing and they are mainly accommodative in nature. Few cases of Wernicke's syndrome under IGB therapy have been described in the literature, and to the best of our knowledge, none have been fatal. We present here a case of a 51-year-old woman who underwent IGB therapy over 8 months. Late diagnosed Wernicke's syndrome that first appeared as lower limb neuropathy progressively immobilized the patient, until she required bed rest. Finally, a major complication of pneumonia caused her death. Wernicke's syndrome has been mainly related to bariatric surgery techniques, but it must also be linked to IGB therapy (and also to other restrictive weight-loss interventions). As the use of IGB therapy spreads, the literature must alert physicians to this complication. Wernicke's syndrome is a severe but reversible condition when diagnosed and treated early.

Surgical extraction of teeth due to dental pathologies is a relatively common procedure in modern man. The healing of the wound that results occurs in gradual and sequential stages, such that the analysis of this repair process can be very useful in forensic investigations on human remains. The following study reports on a particular case where the remodeling of a tooth socket allowed an estimation of the time that had elapsed from the day of the surgical extraction of the tooth to the time of death. The corpse was that of a woman of 34 years. It was in an advanced state of decomposition, as it was largely skeletonized. Macroscopic, radiographic, and histological examinations of the oral cavity showed the initial stages of alveolar bone remodeling of the first left mandibular molar, which was characterized by: (i) a small reduction in the vertical height of the vestibular surface with respect to the theoretical original position of the tooth; (ii) resorption of the intra-alveolar septum and lamina dura; and (iii) formation of new immature bone, which covered the entire inner surface of the socket. This study established that the subject died 13-42days after the tooth extraction. Knowing the date of the dental extraction provided by the police investigation, it was possible to provide an estimate of the date of death., (Copyright © 2017 Elsevier B.V. All rights reserved.)