Analysis of miRNA Expression Profiles in Traumatic Brain Injury (TBI) and Their Correlation with Survival and Severity of Injury.

Traumatic brain injury (TBI) is the leading cause of traumatic death worldwide and is a public health problem associated with high mortality and morbidity rates, with a significant socioeconomic burden. The diagnosis of brain injury may be difficult in some cases or may leave diagnostic doubts, especially in mild trauma with insignificant pathological brain changes or in cases where instrumental tests are negative. Therefore, in recent years, an important area of research has been directed towards the study of new biomarkers, such as micro-RNAs (miRNAs), which can assist clinicians in the diagnosis, staging, and prognostic evaluation of TBI, as well as forensic pathologists in the assessment of TBI and in the estimation of additional relevant data, such as survival time. The aim of this study is to investigate the expression profiles (down- and upregulation) of a panel of miRNAs in subjects deceased with TBI in order to assess, verify, and define the role played by non-coding RNA molecules in the different pathophysiological mechanisms of brain damage. This study also aims to correlate the detected expression profiles with survival time, defined as the time elapsed between the traumatic event and death, and with the severity of the trauma. This study was conducted on 40 cases of subjects deceased with TBI (study group) and 10 cases of subjects deceased suddenly from non-traumatic causes (control group). The study group was stratified according to the survival time and the severity of the trauma. The selection of miRNAs to be examined was based on a thorough literature review. Analyses were performed on formalin-fixed, paraffin-embedded (FFPE) brain tissue samples, with a first step of total RNA extraction and a second step of quantification of the selected miRNAs of interest. This study showed higher expression levels in cases compared to controls for miR-16, miR-21, miR-130a, and miR-155. In contrast, lower expression levels were found in cases compared to controls for miR-23a-3p. There were no statistically significant differences in the expression levels between cases and controls for miR-19a. In cases with short survival, the expression levels of miR-16-5p and miR-21-5p were significantly higher. In cases with long survival, miR-21-5p was significantly lower. The expression levels of miR-130a were significantly higher in TBI cases with short and middle survival. In relation to TBI severity, miR-16-5p and miR-21-5p expression levels were significantly higher in the critical-fatal TBI subgroup. Conclusions: This study provides evidence for the potential of the investigated miRNAs as predictive biomarkers to discriminate between TBI cases and controls. These miRNAs could improve the postmortem diagnosis of TBI and also offer the possibility to define the survival time and the severity of the trauma. The analysis of miRNAs could become a key tool in forensic investigations, providing more precise and detailed information on the nature and extent of TBI and helping to define the circumstances of death.

Molecular Autopsy in Asphyxia Deaths: Diagnostic Perspectives of miRNAs in the Evaluation of Hypoxia Response.

The forensic investigation of asphyxia deaths still poses a challenge due to the need to demonstrate vital exposure to hypoxic insult according to high levels of evidence. The pulmonary effects of hypoxia are complex and the understanding of the mechanisms underlying the acute pneumotoxicity induced by hypoxia is still incomplete. Redox imbalance has been suggested as the protagonist of the main acute changes in pulmonary function in the hypoxic context. The development of knowledge in biochemistry and molecular biology has allowed research in forensic pathology to identify some markers useful in immunohistochemical diagnostics of asphyxia deaths. Several studies have highlighted the diagnostic potential of markers belonging to the HIF-1α and NF-kB pathways. The central role of some highly specific microRNAs has recently been recognized in the complex molecular mechanisms involved in the hypoxia response; thus, several research activities are currently aimed at identifying miRNAs involved in the regulation of oxygen homeostasis (hypoxamiR). The aim of the manuscript is to identify, the miRNAs involved in the early stages of the cellular response to hypoxia, in order to characterize the possible implications in the forensic field of the determination of expression profiles. At present, more than 60 miRNAs involved in the hypoxia response with different expression profiles (upregulation and downregulation) have been identified. Despite the multiple and different effects on reprogramming following the hypoxic insult, the evaluation of the diagnostic implications of hypoxamiRs in the forensic field presupposes a specific treatment of the influences on HIF-1α regulation, cell cycle progression, DNA repair, and apoptosis.

Cytokine storm and histopathological findings in 60 cases of COVID-19-related death: from viral load research to immunohistochemical quantification of major players IL-1ß, IL-6, IL-15 and TNF-α.

This study involves the histological analysis of samples taken during autopsies in cases of COVID-19 related death to evaluate the inflammatory cytokine response and the tissue localization of the virus in various organs. In all the selected cases, SARS-CoV-2 RT-PCR on swabs collected from the upper (nasopharynx and oropharynx) and/or the lower respiratory (trachea and primary bronchi) tracts were positive. Tissue localization of SARS-CoV-2 was detected using antibodies against the nucleoprotein and the spike protein. Overall, we tested the hypothesis that the overexpression of proinflammatory cytokines plays an important role in the development of COVID-19-associated pneumonia by estimating the expression of multiple cytokines (IL-1ß, IL-6, IL-10, IL-15, TNF-α, and MCP-1), inflammatory cells (CD4, CD8, CD20, and CD45), and fibrinogen. Immunohistochemical staining showed that endothelial cells expressed IL-1ß in lung samples obtained from the COVID-19 group (p < 0.001). Similarly, alveolar capillary endothelial cells showed strong and diffuse immunoreactivity for IL-6 and IL-15 in the COVID-19 group (p < 0.001). TNF-α showed a higher immunoreactivity in the COVID-19 group than in the control group (p < 0.001). CD8 + T cells where more numerous in the lung samples obtained from the COVID-19 group (p < 0.001). Current evidence suggests that a cytokine storm is the major cause of acute respiratory distress syndrome (ARDS) and multiple organ failure and is consistently linked with fatal outcomes.

Expanding frontiers of risk management: care safety in nursing home during COVID-19 pandemic.

BACKGROUND: Nursing homes provide long-term care and have residential-oriented hospitalizations characterized by medical, nursing and social-care treatments for a typically geriatric population. In the current emergency phase, the problem of infections in residential structures for the elderly is taking on considerable importance in relation to the significant prevalence rates of coronavirus disease 2019 (COVID-19). SAFETY IMPROVEMENT STRATEGIES: Prevention and control measures for severe acute respiratory syndrome coronavirus 2 infection in nursing homes should be planned before a possible outbreak of COVID-19 occurs and should be intensified during any exacerbation of the same. Each facility should identify a properly trained contact person-also external-for the prevention and control of infections, who can refer to a multidisciplinary support committee and who is in close contact with the local health authorities. The contact person should collaborate with professionals in order to prepare a prevention and intervention plan that considers national provisions and scientific evidence, the requirements for reporting patients with symptoms compatible with COVID-19 and the indications for the management of suspected, probable or confirmed cases of COVID-19. DISCUSSION: Adequate risk management in residential structures implies the establishment of a coordination committee with dedicated staff, the implementation of a surveillance program for the rapid recognition of the outbreaks, the identification of suitable premises and equipment, the application of universal precautions, the adaptation of care plans to reduce the possibility of contagion among residents and the protection of operators and staff training initiatives.

Widespread septic embolization in injection drug use mitro-aortic infective endocarditis as a remote cause of death.

Injection drug use-related infective endocarditis (IDU-IE) assumes peculiar epidemiological, pathogenetic, and prognostic characteristics that allow to consider it a distinct nosological entity, as well as a current problem of considerable social weight. Incidence is reasonably underestimated because diagnosis is often accidental in postmortem examination when drug-related death is suspected. In many cases, postmortem toxicological examinations are negative for acute drug abuse, and findings of infective endocarditis became relevant in the explanation of the mechanism of death. Extracardiac involvement of infective endocarditis is rarely reported as fatal. Fragmentation and embolization of bacterial vegetations can be associated with parenchymal infarcts, systemic spread of the infectious process by formation of an abscess. A case of septic shock as a consequence of the constant bacteremia determined by the continuous proliferation and release of bacteria into the circulation is presented in an injection drug user with left-sided endocarditis and widespread septic embolization. Authors reviewed forensic and medical literature and promote epidemiological value of medical and forensic autopsy. Extracardiac involvement of infective endocarditis may represent a remote and alternative cause of death in injection drug users, and an early diagnosis can be relevant for prognosis. Postmortem examination still represents a valuable opportunity of learning for clinicians and improving diagnostic accuracy with injection drug users. A call for changing of attitudes and practice toward autopsy is finally demanded.

A technical report from the Italian SARS-CoV-2 outbreak. Postmortem sampling and autopsy investigation in cases of suspected or probable COVID-19.

SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is a coronavirus responsible for COVID-19 (coronavirus disease 2019) which resulted in a cluster of cases of pneumonia that originated in China around 31 December 2019 and has subsequently spread across the globe. Currently, COVID-19 represents a health emergency worldwide, leading, in severe cases, to pneumonia, severe acute respiratory syndrome, multiorgan dysfunction or failure, and death. In the context of limited scientific knowledge and evidence of SARS-CoV-2 infection, guidance is becoming increasingly necessary for pathologists who have to perform postmortem investigations on COVID-19 cases. The aim of the present report is to share a procedure applicable to cases of COVID-19-related death, particularly in cases of death without medical intervention and in the absence of an ascertained SARS-CoV-2 infection and/or COVID-19 diagnosis, therefore providing support for diagnostic activity in the present COVID-19 pandemic. For this purpose, a standard operating procedure for correct swab collection, autopsy investigation and tissue sampling is provided.

miR-1, miR-499 and miR-208 are sensitive markers to diagnose sudden death due to early acute myocardial infarction.

MicroRNAs (miRNAs) are strongly up-regulated under pathological stress and in a wide range of diseases. In recent years, miRNAs are under investigation for their potential use as biomarkers in cardiovascular diseases. We investigate whether specific cardio-miRNAs are overexpressed in heart samples from subjects deceased for acute myocardial infarction (AMI) or sudden cardiac death (SCD), and whether miRNA could help differentiate between them. Forty four cases of death due to cardiovascular disease were selected, respectively, 19 cases categorized as AMI and 25 as SCD. Eighteen cases of traumatic death without pathological cardiac involvement were selected as control. Immunohistochemical investigation was performed for CD15, IL-15, Cx43, MCP-1, tryptase, troponin C and troponin I. Reverse transcription and quantitative real-time PCR were performed for miR-1, miR-133, miR-208 and miR-499. In AMI group, stronger immunoreaction for the CD15, IL-15 and MCP-1 antibodies was detectable compared with SCD and control. Cx43 showed a negative reaction with respect to the other groups. Real-time PCR results showed a down-regulation of all miRNAs in the AMI group compared with SCD and control. The selected miRNAs presented high accuracy in discriminating SCD from AMI (miR-1 and miR-499) and AMI from control (miR-208) representing a potential aid for both clinicians and pathologists for differential diagnosis.

Postmortem computed tomography angiography (PMCTA) and traditional autopsy in cases of sudden cardiac death due to coronary artery disease: a systematic review and meta-analysis.

INTRODUCTION: Several studies have been performed to assess the efficacy of postmortem computed tomography angiography (PMCTA) in solving cases of sudden cardiac death, even in comparison with the traditional autopsy. However, the results were often inconsistent and inconclusive. Therefore, a global discussion on the subject through a systematic review of the literature and a meta-analysis is necessary. METHODS: A systematic search of PubMed was performed up to April 23, 2018. Studies exploring the role of PMCTA in cases of sudden cardiac death and the accuracy of this method in diagnosing the cause of death compared to traditional autopsy were included. RESULTS: The overall sensitivity and specificity of the seven included studies, using conventional autopsy as a reference standard, were 92% and 95%, respectively. The positive and negative likelihood ratios were, respectively, 20.76 (95% CI 1.16-370.2) and 0.08 (95% CI 0.03-0.17), showing that PMCTA represents a strong indicator of the posttest probability of disease. The diagnostic odds ratio and the area under the curve were, respectively, 261.54 (95% CI 1.87-5760.53) and 0.93 (95% CI 0.90-0.95), indicating a high diagnostic power of the test. CONCLUSION: PMCTA demonstrated a high accuracy in the diagnosis of parietal and luminal coronary changes but was less effective in detecting myocardial ischemia and necrosis. Therefore, the only radiological investigation is often insufficient to determine the cause of sudden death and the conventional autopsy remains the gold standard. However, PMCTA can improve the performance of the autopsy, serving as an aid and guide in the sampling phase for histopathological investigations.

Evolution of post-mortem coronary imaging: from selective coronary arteriography to post-mortem CT-angiography and beyond.

Since the 1970s, remarkable efforts have been made in the post-mortem coronary study, especially by angiography, as an added tool to diagnose heart-related deaths. In more recent times, post-mortem CT (PMCT) and post-mortem CT-angiography (PMCTA) have become an established practice in numerous forensic units, because of the undeniable advantages these diagnostic instruments can offer: data acquisition times are increasingly fast, costs have become lower and, once acquired, data can be re-utilized and re-evaluated at any given time. This review aims to chart the history of post-mortem cardiac imaging, highlighting its evolution both in terms of methodology and technology as well as the contribution that forensic radiology has been able to offer to forensic pathology, not as an alternative to autopsy but as a guide and aid when performing one. Finally, the latest advances in the study of cardiac deaths are explored, namely by cardiac post-mortem MRI (PMMR), able to visualize all the various stages of a myocardial infarction, post-mortem MRI-angiography (PMMRA), useful in investigating coronary artery pathology and post-mortem cardiac micro-CT, able to provide near-histological levels of myocardial, coronary and valvular detail.

Immunohistochemical Evaluation of Aquaporin-4 and its Correlation with CD68, IBA-1, HIF-1α, GFAP, and CD15 Expressions in Fatal Traumatic Brain Injury.

Traumatic brain injury (TBI) is one of the leading causes of death and disability worldwide. Our understanding of its pathobiology has substantially increased. Following TBI, the following occur, edema formation, brain swelling, increased intracranial pressure, changes in cerebral blood flow, hypoxia, neuroinflammation, oxidative stress, excitotoxicity, and apoptosis. Experimental animal models have been developed. However, the difficulty in mimicking human TBI explains why few neuroprotective strategies, drawn up on the basis of experimental studies, have translated into improved therapeutic strategies for TBI patients. In this study, we retrospectively examined brain samples in 145 cases of death after different survival times following TBI, to investigate aquaporin-4 (AQP4) expression and correlation with hypoxia, and neuroinflammation in human TBI. Antibodies anti-glial fibrillary acid protein (GFAP), aquaporin-4 (AQP4), hypoxia induced factor-1α (HIF-1α), macrophage/phagocytic activation (CD68), ionized calcium-binding adapter molecule-1 (IBA-1), and neutrophils (CD15) were used. AQP4 showed a significant, progressive increase between the control group and groups 2 (one-day survival) and 3 (three-day survival). There were further increases in AQP4 immunopositivity in groups 4 (seven-day survival), 5 (14-dayssurvival), and 6 (30-day survival), suggesting an upregulation of AQP4 at 7 to 30 days compared to group 1. GFAP showed its highest expression in non-acute cases at the astrocytic level compared with the acute TBI group. Data emerging from the HIF-1α reaction showed a progressive, significant increase. Immunohistochemistry with IBA-1 revealed activated microglia starting three days after trauma and progressively increasing in the next 15 to 20 days after the initial trauma. CD68 expression demonstrated basal macrophage and phagocytic activation mostly around blood vessels. Starting from one to three days of survival after TBI, an increase in the number of CD68 cells was progressively observed; at 15 and 30 days of survival, CD68 showed the most abundant immunopositivity inside or around the areas of necrosis. These findings need to be developed further to gain insight into the mechanisms through which brain AQP4 is upregulated. This could be of the utmost clinicopathological importance.

Postmortem fertilization: New Italian government guidelines affirm the legitimacy of this procedure.

Although posthumous reproduction (PHR) is viewed unfavorably by some, it may be a desirable option for subjects whose partners died before they could complete their family planning. With particular regard to posthumous embryo implantation, questions arise regarding the definition of "conception" when a couple undergoes in vitro fertilization while both are alive, but the embryo is implanted in a woman's womb after one parent has died. In accordance with Italian Law 40/2004, access to medically assisted reproduction is contingent upon the survival of both partners in a couple. The legislative prohibition remains in effect unless the application of the reproductive technique has already resulted in the formation of embryos, and implantation is permitted to uphold "the rights of all the subjects involved, including the conceived", as stated in Article 1 of Law 40/2004. Since the enactment of the legislation, a number of Italian courts have issued rulings on PHR on a case-by-case basis. Recent government guidelines in Italy have sought to balance these considerations, giving due weight to the will of the woman, the potential unborn child, and the previous consent of the donor partner.

A Hospital Medical Record Quality Scoring Tool (MeReQ): Development, Validation, and Results of a Pilot Study.

Hospital medical records are valuable and cost-effective documents for assessing the quality of healthcare provided to patients by a healthcare facility during hospitalization. However, there is a lack of internationally validated tools that measure the quality of the whole hospital medical record in terms of both form and content. In this study, we developed and validated a tool, named MeReQ (medical record quality) tool, which quantifies the quality of the hospital medical record and enables statistical modeling using the data obtained. The tool was applied to evaluate a sample of hospital individual patient medical records from a secondary referral hospital and to identify the departments that require quality improvement interventions and the effects of improvement actions already implemented.

Evaluation of Spontaneous Overtime Methemoglobin Formation in Post-Mortem Blood Samples from Real Cases in Critical Storage Conditions.

Nitrite/nitrate poisoning is an emerging problem, with an ongoing escalation of reported self-administration with suicidal intent in several countries. Nitrites toxicity mainly consists of their interaction with hemoglobin (Hb), causing its oxidization to methemoglobin (MetHb). In order to give support to the correct procedures for the analysis of these cases, this study aims to evaluate spontaneous sample degradation and consequent MetHb formation in the typical storage conditions of a forensic toxicology laboratory. Two different types of samples have been used in this study: the first stage of our study consisted of a retrospective analysis of blood samples obtained by judicial autopsies already stored in the toxicology laboratory, collected over four years (2018-2021), while the samples used for the second stage were appositely collected during judicial autopsies. The data obtained by the application of a derivative spectrophotometry method on these samples suggest that there seems not to be a maximum threshold for MetHb formation within which it is possible to state with a sufficient grade of certainty that the concentration of MetHb found is consistent with an ante-mortem formation and is not the result of an artifact due to sample degradation and storage conditions. On the other hand, the results suggest that MetHb formation depends on the time passed between sample collection and analysis, so that a tempestive sample processing, performed as soon as the samples are received in the laboratory, is crucial to obtain the maximum reliability and diagnostic values from the data when MetHb quantitation is necessary.

Recreational Cannabis Use: Regulatory Aspects, Public Opinion and Concerns for Public Health.

Cannabis represents the most used substance in the young population after alcohol and tobacco. The consumption of the substance undoubtedly represents an emerging public health issue, and the regulatory aspects are quite heterogeneous. Alternatives to drug prohibition, such as legalization or decriminalization, are a topic of growing debate among researchers, policy makers and the public. Limiting harmful use and combating illicit trafficking are the main objectives of legalization for recreational purposes, although liberalization raises critical issues that have not yet been resolved despite the experience gained in many countries. In the context of legalization, it is essential to allow the lawful production of cannabis, guarantee a supply of controlled quality cannabis, and ensure that the population understands the risks associated with the consumption of the substance. At the same time, it is essential to protect the safety and health of the population by limiting access to cannabis and preventing accidental exposure to fragile categories and children. Incomplete knowledge of law enforcement can create further complications. Incomplete knowledge of the implementation of the laws can create further complications; therefore adequate information must be favored. Scientific evidence helps to understand the potential harms and benefits of cannabis, but the impact of legalization - even in terms of reducing consumption - is not clearly documented. So, far more efforts need to be made in the future to broaden awareness of the general population on the recreational use of cannabis.

Identification of 5F-Cumyl-PINACA, a Synthetic Cannabinoid, in the Herbal Material Used for Recreational Purposes in the Province of Trieste: Public Health Implications.

BACKGROUND: In recent years, the phenomenon of the production and trade of synthetic cannabinoids has grown, becoming a public health issue worldwide. The recent access- to the ED of the hospital of Trieste- of people who complained of episodes of hallucinations, sensation of poisoning, tachycardia, and air hunger following the inhalation of "Che Sballo platinum", have highlighted the need to perform further analysis on the contents of the packet sold as an air freshener, produced in Koper (Slovenia). OBJECTIVE: This paper wants to be an alert about the possible consequences on health due to the spreading of "Che Sballo platinum" in the province of Trieste. METHODS: The package contents were analyzed by a multi-target screening method of MRM-IDAEPI experiment. The result was then confirmed, and quantification was achieved via LC-ESI-MS/MS analysis in MRM mode using QTrap 6500 + Sinergy hydro column 100 x 2 mm 1.9 um transitions MRM1 368.3 → 250.0; MRM2 368.3 → 233.0. RESULTS: The initial screening tested negative for THC and showed positive results for 5F-Cumyl- PINACA. Quantitation result reported dose by the package of 8.5 mg of the compound. Formal notification was sent to the Italian Health Authorities (Notification No. 2021110205). CONCLUSION: Consumption of 5F-Cumyl-PINACA results in much more potent effects than marijuana. Lack of information about the actual concentration of the substance on the packaging does not allow drug users to have an adequate dosage, with possible toxic consequences on health. Further investigations must be done to discover the true extent of the phenomenon.

Mental health law: a comparison of compulsory hospital admission in Italy and the UK.

In Europe, the mental health law legal framework has had several changes throughout the years to achieve and develop new reforms, better mental health care, and protect the human rights of patients. The UK national data shows rising detention rates and the disproportionate use of the legal framework among people from black and minority ethnic groups. At the national level, compulsory admissions are lower in Italy; it also shows that it has increased in the last few years in both countries. The lack of ethnic national data, especially in Italy, limited the ability to understand compulsory admission, discrimination, and stigma in mental health. The present study aims to compare the legal framework of mental health law and compulsory hospital admission in Italy and the UK. A review of each country's latest amendments to mental health law and the number of compulsory hospital admissions was conducted to understand the impact of changes in mental health care.

Cannabis and Driving: Developing Guidelines for Safety Policies.

INTRODUCTION: The dynamism in the regulatory frameworks concerning the consumption of cannabinoids has placed their effects on cognitive and psychomotor skills at the center of the scientific debate. In consideration of the potential repercussions on public safety, particular attention has been focused on the impairment of driving skills, opening up the need to specifically regulate driving under the effects of cannabinoids. PHARMACOKINETICS: Both native cannabinoids and metabolites show a long positivity at low concentrations in the biological fluids, especially in the case of chronic consumption. Qualitative positivity to cannabinoids does not itself prove the presence of detrimental effects, which require the presence of active substances at relevant concentrations. Driving Skill Impairment: Multiple studies highlight a tetrahydrocannabinol (THC) concentration- based alteration of driving skills mainly affecting automatisms, whereas skills subjected to cognitive control are preserved up to higher dosages. The evidence relating to associations with other substances, chronic consumption and other cannabinoids, on the other hand, is still burdened by a high degree of uncertainty. Regulation Policies: Different models can be adopted in the regulation of driving under the effects of cannabinoids: sanctions can be applied in case of qualitative positivity, cannabinoids concentration above a defined threshold, or in presence of a demonstrated state of cognitive alteration. CONCLUSION: "Per se limit" with a quantitative THC cut-off between 3.5 and 5 ng/ml can currently be considered the most balanced choice. Finally, the analysis carried out allowed to identify pitfalls in both scientific and legislative fields for the improvement of safety policies.