Metabolomics investigation of post-mortem human pericardial fluid.

INTRODUCTION: Due to its peculiar anatomy and physiology, the pericardial fluid is a biological matrix of particular interest in the forensic field. Despite this, the available literature has mainly focused on post-mortem biochemistry and forensic toxicology, while to the best of authors' knowledge post-mortem metabolomics has never been applied. Similarly, estimation of the time since death or post-mortem interval based on pericardial fluids has still rarely been attempted. OBJECTIVES: We applied a metabolomic approach based on 1H nuclear magnetic resonance spectroscopy to ascertain the feasibility of monitoring post-mortem metabolite changes on human pericardial fluids with the aim of building a multivariate regression model for post-mortem interval estimation. METHODS: Pericardial fluid samples were collected in 24 consecutive judicial autopsies, in a time frame ranging from 16 to 170 h after death. The only exclusion criterion was the quantitative and/or qualitative alteration of the sample. Two different extraction protocols were applied for low molecular weight metabolites selection, namely ultrafiltration and liquid-liquid extraction. Our metabolomic approach was based on the use of 1H nuclear magnetic resonance and multivariate statistical data analysis. RESULTS: The pericardial fluid samples treated with the two experimental protocols did not show significant differences in the distribution of the metabolites detected. A post-mortem interval estimation model based on 18 pericardial fluid samples was validated with an independent set of 6 samples, giving a prediction error of 33-34 h depending on the experimental protocol used. By narrowing the window to post-mortem intervals below 100 h, the prediction power of the model was significantly improved with an error of 13-15 h depending on the extraction protocol. Choline, glycine, ethanolamine, and hypoxanthine were the most relevant metabolites in the prediction model. CONCLUSION: The present study, although preliminary, shows that PF samples collected from a real forensic scenario represent a biofluid of interest for post-mortem metabolomics, with particular regard to the estimation of the time since death.

PMI estimation through metabolomics and potassium analysis on animal vitreous humour.

INTRODUCTION: The estimation of post-mortem interval (PMI) remains a major challenge in forensic science. Most of the proposed approaches lack the reliability required to meet the rigorous forensic standards. OBJECTIVES: We applied 1H NMR metabolomics to estimate PMI on ovine vitreous humour comparing the results with the actual scientific gold standard, namely vitreous potassium concentrations. METHODS: Vitreous humour samples were collected in a time frame ranging from 6 to 86 h after death. Experiments were performed by using 1H NMR metabolomics and ion capillary analysis. Data were submitted to multivariate statistical data analysis. RESULTS: A multivariate calibration model was built to estimate PMI based on 47 vitreous humour samples. The model was validated with an independent test set of 24 samples, obtaining a prediction error on the entire range of 6.9 h for PMI < 24 h, 7.4 h for PMI between 24 and 48 h, and 10.3 h for PMI > 48 h. Time-related modifications of the 1H NMR vitreous metabolomic profile could predict PMI better than potassium up to 48 h after death, whilst a combination of the two is better than the single approach for higher PMI estimation. CONCLUSION: The present study, although in a proof-of-concept animal model, shows that vitreous metabolomics can be a powerful tool to predict PMI providing a more accurate estimation compared to the widely studied approach based on vitreous potassium concentrations.

Infant urinary metabolomic profile in a fatal acute methadone intoxication.

A case report suspicious for a Sudden Infant Death Syndrome is here described. Pathological findings were consistent with an acute respiratory failure while toxicological analysis revealed an elevated blood methadone concentration. Death was then ascribed to an acute methadone intoxication. In addition to the routinary approach, the urinary sample collected at autopsy was investigated with a 1H NMR metabolomic approach and the identified metabolomic profile was challenged with the urinary metabolomic profiles previously obtained from 10 newborns who experienced perinatal asphyxia and 16 healthy control newborns. Intriguingly, the urinary profile of the methadone intoxicated infant was very similar to those belonging to the perinatal asphyxia newborns, especially to those belonging to the newborns characterised by the worst outcome. The results offer several hints on a shared metabolic derangement between different mechanisms of asphyxia/hypoxia. To the best of the authors' knowledge, this is the first report of the use of a metabolomic approach in a pathological case, in which metabolomics offers useful additional information regarding the mechanism and the cause of death.

Predictive risk factors of phenoconversion in idiopathic REM sleep behavior disorder: the Italian study "FARPRESTO".

Most patients with idiopathic REM sleep behavior disorder (iRBD) will develop an overt α-synucleinopathy over time, with a rate of phenoconversion of 73.5% after 12 years from diagnosis. Several markers of phenoconversion were identified; however, most studies investigated biomarkers separately, with retrospective study designs, in small cohorts or without standardized data collection methods. The risk FActoRs PREdictive of phenoconversion in idiopathic REM sleep behavior disorder: the Italian STudy (FARPRESTO) is a multicentric longitudinal retrospective and prospective study with a cohort of incident (prospective recruitment) and prevalent (retrospective recruitment) iRBD patients, whose primary aim is to stratify the risk of phenoconversion, through the systematic collection by means of electronic case report forms of different biomarkers. Secondary aims are to (1) describe the sociodemographic and clinical characteristics of patients with iRBD; (2) collect longitudinal data about the development of α-synucleinopathies; (3) monitor the impact of iRBD on quality of life and sleep quality; (4) assess the correlation between phenoconversion, cognitive performance, and loss of normal muscle atony during REM sleep; (5) identify RBD phenotypes through evaluating clinical, biological, neurophysiological, neuropsychological, and imaging biomarkers; and (6) validate vPSG criteria for RBD diagnosis. The FARPRESTO study will collect a large and harmonized dataset, assessing the role of different biomarkers providing a unique opportunity for a holistic, multidimensional, and personalized approach to iRBD, with several possible application and impact at different levels, from basic to clinical research, and from prevention to management. The FARPRESTO has been registered at clinicaltrials.gov (NCT05262543).

Looking for Post-Mortem Metabolomic Standardization: Waiting for Godot-The Importance of Post-Mortem Interval in Forensic Metabolomics.

A growing body of evidence suggests that the post-mortem interval exerts a strong effect on the metabolome, independently of the biological matrix or the cause of death. A sound and shared approach in standardization is mandatory.

Comparative use of aqueous humour 1H NMR metabolomics and potassium concentration for PMI estimation in an animal model.

Estimation of the post-mortem interval (PMI) remains a matter of concern in the forensic scenario. Traditional and novel approaches are not yet able to fully address this issue, which relies on complex biological phenomena triggered by death. For this purpose, eye compartments may be chosen for experimental studies because they are more resistant to post-mortem modifications. Vitreous humour, in particular, has been extensively investigated, with potassium concentration ([K+]) being the marker that is better correlated with PMI estimation. Recently, a 1H nuclear magnetic resonance (NMR) metabolomic approach based on aqueous humour (AH) from an animal model was proposed for PMI estimation, resulting in a robust and validated regression model. Here we studied the variation in [K+] in the same experimental setup. [K+] was determined through capillary ion analysis (CIA) and a regression analysis was performed. Moreover, it was investigated whether the PMI information related to potassium could improve the metabolome predictive power in estimating the PMI. Interestingly, we found that a part of the metabolomic profile is able to explain most of the information carried by potassium, suggesting that the rise in both potassium and metabolite concentrations relies on a similar biological mechanism. In the first 24-h PMI window, the AH metabolomic profile shows greater predictive power than [K+] behaviour, suggesting its potential use as an additional tool for estimating the time since death.

Lack of synergistic nephrotoxicity between vancomycin and piperacillin/tazobactam in a rat model and a confirmatory cellular model.

BACKGROUND: Vancomycin and piperacillin/tazobactam are reported in clinical studies to increase acute kidney injury (AKI). However, no clinical study has demonstrated synergistic toxicity, only that serum creatinine increases. OBJECTIVES: To clarify the potential for synergistic toxicity between vancomycin, piperacillin/tazobactam and vancomycin + piperacillin/tazobactam treatments by quantifying kidney injury in a translational rat model of AKI and using cell studies. METHODS: (i) Male Sprague-Dawley rats (n = 32) received saline, vancomycin 150 mg/kg/day intravenously, piperacillin/tazobactam 1400 mg/kg/day intraperitoneally or vancomycin + piperacillin/tazobactam for 3 days. Urinary biomarkers and histopathology were analysed. (ii) Cellular injury was assessed in NRK-52E cells using alamarBlue®. RESULTS: Urinary output increased from Day -1 to Day 1 with vancomycin but only after Day 2 for vancomycin + piperacillin/tazobactam-treated rats. Plasma creatinine was elevated from baseline with vancomycin by Day 2 and only by Day 4 for vancomycin + piperacillin/tazobactam. Urinary KIM-1 and clusterin were increased with vancomycin from Day 1 versus controls (P < 0.001) and only on Day 3 with vancomycin + piperacillin/tazobactam (P < 0.001, KIM-1; P < 0.05, clusterin). The histopathology injury score was elevated only in the vancomycin group when compared with piperacillin/tazobactam as a control (P = 0.04) and generally not so with vancomycin + piperacillin/tazobactam. In NRK-52E cells, vancomycin induced cell death with high doses (IC50 48.76 mg/mL) but piperacillin/tazobactam did not, and vancomycin + piperacillin/tazobactam was similar to vancomycin. CONCLUSIONS: All groups treated with vancomycin demonstrated AKI; however, vancomycin + piperacillin/tazobactam was not worse than vancomycin. Histopathology suggested that piperacillin/tazobactam did not worsen vancomycin-induced AKI and may even be protective.

Forensic NMR metabolomics: one more arrow in the quiver.

INTRODUCTION: NMR metabolomics is increasingly used in forensics, due to the possibility of investigating both endogenous metabolic profiles and exogenous molecules that may help to describe metabolic patterns and their modifications associated to specific conditions of forensic interest. OBJECTIVES: The aim of this work was to review the recent literature and depict the information provided by NMR metabolomics. Attention has been devoted to the identification of peculiar metabolic signatures and specific ante-mortem and post-mortem profiles or biomarkers related to different conditions of forensic concern, such as the identification of biological traces, the estimation of the time since death, and the exposure to drugs of abuse. RESULTS AND CONCLUSION: The results of the described studies highlight how forensics can benefit from NMR metabolomics by gaining additional information that may help to shed light in several forensic issues that still deserve to be further elucidated.

An evaluation of the objectivity and reproducibility of shear wave elastography in estimating the post-mortem interval: a tissue biomechanical perspective.

Cadaveric rigidity-also referred to as rigor mortis-is a valuable source of information for estimating the time of death, which is a fundamental and challenging task in forensic sciences. Despite its relevance, assessing the level of cadaveric rigidity still relies on qualitative and often subjective observations, and the development of a more quantitative approach is highly demanded. In this context, ultrasound shear wave elastography (US SWE) appears to be a particularly well-suited technique for grading cadaveric rigidity, as it allows non-invasive quantification of muscle stiffness in terms of Young's modulus (E), which is a widely used parameter in tissue biomechanics. In this pilot study, we measured, for the first time in the literature, changes in the mechanical response of muscular tissues from 0 to 60 h post-mortem (hpm) using SWE, with the aim of investigating its applicability to forensic practice. For this purpose, 26 corpses were included in the study, and the muscle mechanical response was measured at random times in the 0-60 hpm range. Despite the preliminary nature of this study, our data indicate a promising role of SWE in the quantitative determination of cadaveric rigidity, which is still currently based on qualitative and semiquantitative methods. A more in-depth study is required to confirm SWE applicability in this field in order to overcome some of the inherent limitations of the present work, such as the rather low number of cases and the non-systematic approach of the measurements.

Compassion fatigue, watching patients suffering and emotional display rules among hospice professionals: a daily diary study.

BACKGROUND: Hospice workers are required to regularly use emotional regulation strategies in an attempt to encourage and sustain terminally ill patients and families. Daily emotional regulation in reaction to constantly watching suffering patients may be intensified among those hospice professionals who have high levels of compassion fatigue. The main object of this study was to examine the relationship between daily exposition to seeing patient suffering and daily emotional work, and to assess whether compassion fatigue (secondary traumatic stress and burnout) buffers this relationship. METHODS: We used a diary research design for collecting daily fluctuations in seeing patients suffering and emotional work display. Participants filled in a general survey and daily survey over a period of eight consecutive workdays. A total of 39 hospice professionals from two Italian hospices participated in the study. RESULTS: Multilevel analyses demonstrated that daily fluctuations in seeing patients suffering was positively related to daily emotional work display after controlling for daily death of patients. Moreover, considering previous levels of compassion fatigue, a buffering effect of high burnout on seeing patients suffering - daily emotional work display relationship was found. CONCLUSIONS: A central finding of our study is that fluctuations in daily witness of patients suffering are positively related to daily use of positive emotional regulations. Further, our results show that burnout buffers this relationship such that hospice professionals with high burnout use more emotional display in days where they recurrently witness patients suffering.

A 1H NMR metabolomic approach for the estimation of the time since death using aqueous humour: an animal model.

INTRODUCTION: The estimation of the time since death, or post-mortem interval (PMI), still remains a main conundrum in forensic science. Several approaches have been so far proposed from either a qualitative or a quantitative point of view, but they still lack reliability and robustness. Recently, metabolomics has shown to be a potential tool to investigate the time-related post-mortem metabolite modifications in animal models. OBJECTIVES: Here we propose, for the first time, the use of a 1H NMR metabolomic approach for the estimation of PMI from aqueous humour (AH) in an ovine model. METHODS: AH samples were collected at different times after death (from 118 to 1429 min). 1H NMR experiments were performed and spectral data analysed by multivariate statistical tools. RESULTS: A multivariate calibration model was built to estimate PMI on the basis of the metabolite content of the samples. The model was validated with an independent test set, obtaining a prediction error of 59 min for PMI < 500 min, 104 min for PMI from 500 to 1000 min, and 118 min for PMI > 1000 min. Moreover, the metabolomic approach suggested a picture of the mechanisms underlying the post-mortem biological modifications, highlighting the role played by taurine, choline, and succinate. CONCLUSION: The time-related modifications of the 1H NMR AH metabolomic profile seem to be encouraging in addressing the issue of a reproducible and robust model to be employed for the estimation of the time since death.

Urinary 1H NMR metabolomics profile of Italian citizens exposed to background levels of arsenic: a (pre)cautionary tale.

Objectives: Arsenic is a toxic metal ubiquitous in the environment and in daily life items. Long-term arsenic exposure is associated with severe adverse health effects involving various target organs. It would be useful to investigate the existence of metabolic alterations associated with lifestyle and/or with the environment. For this purpose, we studied the correlation between urinary arsenic levels and urinary proton nuclear magnetic resonance spectroscopy (1H NMR) metabolomics profiles in a non-occupationally nor environmentally arsenic exposed general population.Methods: Urine samples were collected from 86 healthy subjects. Total and non-alimentary urinary arsenic (U-naAs) levels, namely the sum of arsenite, arsenate, monomethylarsonate and dimethylarsinate, were measured and 1H NMR analysis was performed. Orthogonal Projection to Latent Structures was applied to explore the correlation between the metabolomics profiles and U-naAs levels.Results: Despite the extremely low U-naAs levels (mean value = 6.13 ± 3.17 µg/g creatinine) of our studied population a urinary metabolomics profile related to arsenic was identified.Conclusion: The identified profile could represent a fingerprint of early arsenic biological effect and could be used in further studies as an indicator of susceptibility, also in subjects exposed to a low arsenic dose, with implications in occupational health, toxicology, and public health.

A novel method for post-mortem interval estimation based on tissue nano-mechanics.

Forensic estimation of post-mortem interval relies on different methods, most of which, however, have practical limitations or provide insufficient results, still lacking a gold standard method. In order to better understand the phenomenon of rigor mortis and its applicability to the post-mortem interval estimation, we decided to use atomic force microscopy, a tool often employed to measure mechanical properties of adherent cells. Thus, we surgically removed skeletal muscle samples of three forensic cases from 0 to 120 h post-mortem and quantitatively evaluate two parameters: the Young's modulus (E), which gives information about the sample stiffness, and the hysteresis (H), which estimates the contribution of viscous forces. Despite being a preliminary study, the obtained results show that the temporal behavior of E well correlates with the expected evolution of rigor mortis between 0 and 48 h post-mortem, and then monotonically decreases over time. Unfortunately, it is strongly affected by inter-individual variability. However, we found that H provides measurable data along a time-dependent curve back to the starting point, and these data measured on different subjects collapse onto a single master curve, getting rid of the inter-individual variability. Although a larger sampling should be performed to improve the result reliability, this finding is strongly suggestive that the evaluation of rigor mortis should involve the measure of the nanoscale dissipative behavior of muscular tissues.

The role of collective affective commitment in the relationship between work-family conflict and emotional exhaustion among nurses: a multilevel modeling approach.

BACKGROUND: Work-family conflict (WFC) is a crucial problem in nursing because of the demanding conditions of the job, such as strenuous shifts, physical and emotional workload, and intense patient involvement. Using a multilevel approach, this study investigated the moderating role of collective affective commitment as a protective resource in the relationship between WFC and emotional exhaustion. METHODS: The sample included 647 nurses from 66 working units in 4 Italian hospitals. A self-administrated questionnaire was administered to nurses. To analyze data, hierarchical linear modeling was used to examine cross-level relationships between variables. RESULTS: The results indicated that emotional exhaustion increased with augmenting of WFC and that this relationship was stronger when collective affective commitment was low and weaker when it was high. CONCLUSIONS: The study thus suggests that collective affective commitment may be considered a protective resource for nurses. Moreover, the results show that high work-family conflict should not represent a serious problem when nurses have high affective commitment. Interventions at both individual and group level are discussed in order to mitigate WFC, promoting collective affective commitment and thus reducing emotional exhaustion.

Could an Innovative Training Program Including Contact Sports and Counseling Help Young People With Traits of Psychopathy and A History of School Dropout?

BACKGROUND: The aim was to assess the effects of a training program inclusive of contact sports and counseling on school dropout, quality of life (QoL) and psychopathologic symptoms in the youth with a history of school dropout and psychopathic personality traits. METHODS: The Experimental Group (EG) consisted of 32 subjects (male 90.6%; age 19.6±4.3 years); the Control Group (CG) consisted of an equal number matched for gender and age with the same psychological features. At the beginning of the experimental Training Program (T0), both cohorts were assessed by a diagnostic psychiatric interview (SCID ANTAS), the Short Form Health Survey (SF-12) to evaluate QoL, the Psychopathy Checklist - Revised (PCL-R) for the assessment of psychopathic traits, the Self Reporting Questionnaire (SRQ) to measure general psychopathology. At the end of the program (T1), the coorths were evaluated by SF-12 and SRQ. RESULTS: Twenty-seven subjects in the EG (84.4%) completed the course and underwent the evaluation at T1. The SF-12 score significantly increased from T0 to T1 in both groups, albeit this was more evident in the EG than in the CG, owing to an interaction between time and group. SRQ score significantly decreased in the EG from T0 to T1, while in the CG it did not, although the interaction between time and group was not significant. CONCLUSION: The experimental training program was effective in improving QoL and countering school dropout in young citizens with psychopathic traits. Further studies are needed to clarify if such results are due to a relationship between the practical tasks approach including contact sports and an improvement in mentalization processes.

Optical coherence tomography in forensic sciences: a review of the literature.

Optical coherence tomography (OCT) is an interferometric imaging technique that has revolutionized clinical ophthalmology since the first half of the 1990's. Despite this approach being successfully employed in ophthalmology and having great potential in forensic cases, its use in different forensic fields appears to be quite limited. In this review we reviewed the scientific literature regarding the application of OCT in forensic science and legal medicine from 1995 to 2019. Our research showed the usefulness of this approach for the study of coronary injuries, postmortem ocular changes, forensic entomology, and several other applications of specific forensic interest (the study of blood stains, fingerprints, and hair bulbs for personal identification, as well as the study of materials found in the crime scene for comparation, or anti-fraud investigation). The creation of specific 'ad hoc' devices and a better knowledge of this type of technology by pathologists will be a fundamental step to continue to develop the use of OCT forensic fields.

Ocular surface and respiratory tract damages from occupational, sub-chronic exposure to fluorspar: case report and other considerations.

OBJECTIVES: To report the ocular surface and respiratory tract damages due to a sub-chronic (3 months), occupational exposure to fluorspar dust in case of inconsistent use of personal protective equipment. METHODS: A 50-year-old man was referred to our clinic with bilateral conjunctival injection, ocular foreign body sensation and symptoms of ocular discomfort. He reported having 3 days before an urgent hospital admission for dyspnoea with odynophagia, hyposmia, nausea, vomiting, headache and asthenia. Otorhinolaryngological examination revealed a severe hypertrophic rhinopharyngitis and a significant decrease in overall sensitivity to olfactory stimuli. General anamnesis was negative for previous diseases or medication use. RESULTS: Ocular examination revealed a diffuse injection of the ocular surface with some areas of conjunctival and limbal ischaemia, a best-corrected visual acuity of 20/25 in both eyes, an intraocular pressure of 23 and 21 mmHg, respectively, in the right and in the left eyes, and a normal fundus oculi. Assessment of the tear film and ocular surface showed the presence of dry eye disease. Based on medical history and clinical tests, initial diagnostic hypothesis was ocular surface burn due to chemical exposure, i.e. to fluorspar. Therefore, anterior segment fluorescein angiography was performed to confirm the extension of conjunctival and limbal ischaemia. Thus, ocular ischaemia was detected in approximately 10 clock hours of limbus and in 50% of conjunctiva. CONCLUSIONS: The present work remarks the importance of using protective equipment for preventing ocular and respiratory tract damages in workers occupationally exposed to the 'acid-grade fluorite'.

Morphological analysis of corneal findings modifications after death: A preliminary OCT study on an animal model.

The aim of this work was to describe, for the first time, the morphological modifications, in a three-dimensional mode, of the central cornea at different intervals since death. The study design involved the analysis of 30 eyes (15 heads) of female, adult sheep (>2 years) sacrificed at a local slaughterhouse. The eyes, after animal decapitation, were examined in situ, without enucleation. Ocular globes were stored at well-known temperature (within a range of 12-22 °C) and humidity (within a range of 50-60%). The instrumental analysis was executed using a portable spectral-domain OCT (SD-OCT) system (iVue SD-OCT, Optovue Inc, Fremont, CA) calibrated to the corneal mode. OCT imaging was performed at different time-points since death. Pachymetric map, morphological and ultrastructural analysis (epithelium, stroma, and endothelium), were performed for each time-point. After an initial thinning of tissues and an enhancement of epithelial reflectivity, stromal thickness increased from the 2nd up to the 6th hour. Subsequently, a new trend incorneal thinning was observed in association with the appearance ofone or more demarcation lines between the anterior andposterior stroma. After the 12th hour, a recurrence of corneal swelling was detected in association with thedelamination of stromal tissue. Since the 24th hour, the epithelium disappeared in 50% of cases and the anterior chamberdepth progressively decreased. At the 48th hour, various ocular structures showed the onset of putrefaction processes, such as theappearance of hyper-reflective dots in anterior chamber, iridocorneal contact, and the massive vacuolization of theposterior stroma until the total delamination. The portable OCT system is a useful approach for in situ postmortem corneal examination, and it may be potentially applied for the selection of donor cornea in transplantology and for the determination of post-mortem intervals in forensic medicine.

Firing a loaded gun during sleep in an elderly man with a "perfect storm" of risk factors including severe obstructive sleep apnea.

PURPOSE: We report a case of firing a loaded gun during sleep in a geriatric patient with undiagnosed major sleep disorders and multiple risk factors for sleep violence. Polysomnographic findings, diagnostic challenges, and forensic implications in this unprecedented geriatric case are discussed. METHODS: A 75-year-old employed man, married for 32 years, presented to a sleep center reporting to having fired a shot in his bedroom during sleep while his wife was away, without memory of hearing the gunshot. The day before the event, the patient had a normal life, apart from serious worries about recent nearby burglaries that prompted his sleeping with a loaded gun placed behind his bed. The patient underwent a sleep medicine workup, including nocturnal video polysomnography (vPSG). RESULTS: The patient and his wife were unaware of any sleep problems. Upon careful questioning, only mild daytime sleepiness and rare episodes of minor abnormal motor behavior were reported. At vPSG, sleep structure was markedly disrupted with only one clear sleep cycle with REM sleep that had preserved REM-atonia; severe obstructive sleep apnea and moderately severe periodic limb movement activity were documented. Brief abnormal movements from REM sleep without apparent precipitant were recorded. CPAP therapy was effective. CONCLUSIONS: In this case, there was a "perfect storm" of sleep and psychological risk factors that converged to strongly promote precipitous arousals with sleep-related violence in a patient with documented sleep motor dyscontrol. Primary care physicians, including geriatric specialists, should question patients and their spouses about any symptoms of sleep disorders.