Proteomics in Forensic Sciences: Identification of the Nature of the Last Meal at Autopsy.

A long-term psychiatric 40 years-old male patient was found dead at 9:00 a.m. in the clinic where he lived. Death was caused by traumatic injuries, which the sanitary staff imputed to a fall. Nurses declared that the patient refused having breakfast, whereas at autopsy the stomach contained 350 g of whitish semifluid material. Using both shotgun and gel-based proteomics, we demonstrated that the chyme contained partly digested milk- and bread-derived proteins, eaten during a recent breakfast. The conflict between evidence and assertions of the attending sanitary staff prompted the Legal Authority to undertake detailed investigations to ascertain facts and possible responsibilities. The herein characterization provides insights in the in vivo mechanisms of gastric breakdown of food proteins in a real meal. ß-lactoglobulin was partially resistant to gastric digestion as confirmed by Western blot analysis, in contrast to caseins and wheat gluten proteins, which had been degraded by gastric fluids. In addition to a complex pattern of gastric proteins (e.g., mucin-5AC, pepsin A-3, pepsinogen C, gastric lipase, gastrokine-2, trefoil factors), chyme contained intact proteins and variably sized food-derived polypeptides arising from peptic and nonpeptic proteolytic cleavage as well as heterodimeric disulfide-cross-linked peptides. These findings suggest that the current analytical workflows offer only a partial picture of the real complexity of the human "digestome".

The effect of fluoride on the structure, function, and proteome of intestinal epithelia.

Fluoride exposure is widespread, with drinking water commonly containing natural and artificially added sources of the ion. Ingested fluoride undergoes absorption across the gastric and intestinal epithelia. Previous studies have reported adverse gastrointestinal effects with high levels of fluoride exposure. Here, we examined the effects of fluoride on the transepithelial ion transport and resistance of three intestinal epithelia. We used the Caco-2 cell line as a model of human intestinal epithelium, and rat and mouse colonic epithelia for purposes of comparison. Fluoride caused a concentration-dependent decline in forskolin-induced Cl- secretion and transepithelial resistance of Caco-2 cell monolayers, with an IC50 for fluoride of about 3 mM for both parameters. In the presence of 5 mM fluoride, transepithelial resistance fell exponentially with time, with a t1/2 of about 7 hours. Subsequent imaging by immunofluorescence and scanning electron microscopy showed structural abnormalities in Caco-2 cell monolayers exposed to fluoride. The Young's modulus of the epithelium was not affected by fluoride, although proteomic analysis revealed changes in expression of a number of proteins, particularly those involved in cell-cell adhesion. In line with its effects on Caco-2 cell monolayers, fluoride, at 5 mM, also had profound effects on Cl- secretion and transepithelial resistance of both rat and mouse colonic epithelia. Our results show that treatment with fluoride has major effects on the structure, function, and proteome of intestinal epithelia, but only at concentrations considerably higher than those likely to be encountered in vivo, when much lower fluoride doses are normally ingested on a chronic basis.

Postmortem Cholesterol Levels in Peripheral Nerve Tissue: Preliminar Considerations on Interindividual and Intraindividual Variation.

The study reports the evolution of the demyelinization process based on cholesterol ([CHOL]) levels quantified in median nerve samples and collected at different times-from death from both right and left wrists. The statistical data show that the phenomenon evolves differently in the right and left nerves. Such a difference can reasonably be attributed to a different multicenter evolution of the demyelinization. For data analysis, the enrolled subjects were grouped by similar postmortem intervals (PMIs), considering 3 intervals: PMI < 48 hours, 48 hours < PMI < 78 hours, and PMI > 78 hours. Data obtained from tissue dissected within 48 hours of death allowed for a PMI estimation according to the following equations: PMI = 0.000 + 0.7623 [CHOL]right (R = 0.581) for the right wrist and PMI = 0.000 + 0.8911 [CHOL]left (R = 0.794) for the left wrist.At present, this correlation cannot be considered to be definitive because of the limitation of the small size of the samples analyzed, because the differences in the sampling time and the interindividual and intraindividual variation may influence the demyelinization process.

On a defective Mitraclip® system: Considerations on the medical device regulation in Europe.

The use of medical devices is constantly growing and constitutes a valid aid to ailing people because of remarkable technological advances. The regulations on their circulation in Italy and Europe are inspired by the principle of free circulation: it is sufficient for a device to have the CE logo for it to be freely commercialized in all European countries. These regulations that favors commerce also expose the sick to the risk of harm from defective devices that have not been suitably checked prior to commercial release. This paper reports a case of a defective MitraClip® Delivery System, discusses other episodes of similar device malfunctioning, and analyzes the European legislation on medical devices. In the author's opinion, a careful "control" policy for devices, an adequate care in device manufacturing, an appropriate evaluation of pre- and post-marketing and suitable measures for the patient's safety are needed.

Environmental and occupational exposure to bisphenol A and endometriosis: urinary and peritoneal fluid concentration levels.

OBJECTIVES: The study aimed to give a first data set of bisphenol A (BPA) levels in the peritoneal fluid of patients suffering from endometriosis and to investigate the relationship between BPA exposure and endometriosis. METHODS: A questionnaire investigating the occupational context, life environment, and habits was administered to 68 patients suffering from endometriosis and 60 endometriosis-free subjects (control group). Urine and peritoneal fluids samples were collected and analysed by GC/MSMS for BPA dosage. RESULTS: Some of the investigated environmental/lifestyle risk factors (closeness to industries/activities at risk) were associated with an increase in endometriosis; smoking resulted as protective factor; others (use of food plastic boxes) did not seem to influence the onset of pathology. The association between the occupational exposure summarising all examined risk factors (working activity, personal protective equipment, seniority) and endometriosis was statistically significant (χ 2 = 5.252, p = 0.02). Contrasting results were obtained when specific activities were examined. Detectable urinary BPA levels were found in all analysed samples (patients: 1.17-12.68 pg/µl; mean ± SD, 5.31 ± 3.36 pg/µl; control group: 1.28-2.35 pg/µl; mean ± SD, 1.64 ± 0.49 pg/µl; median; 1.46 pg/µl), with a statistically significant difference between patients and controls, showing an association between BPA exposure and endometriosis. Only a few subjects from the control group supplied peritoneal fluid; hence, no comparison test with patients (range 0.39-1.46 pg/µl; mean ± SD, 0.67 ± 0.30 pg/µl; median, 0.58 pg/µl) was carried out. CONCLUSIONS: Results highlight the potential association between BPA exposure and endometriosis, as well as the current lack of knowledge regarding occupational exposure to BPA and the need of epidemiological studies focused on single activities/occupations, such as housewives, cleaners, students.

Non-receptor tyrosine kinases and the actin cytoskeleton in contractile vascular smooth muscle.

The contractility of vascular smooth muscle cells within the walls of arteries is regulated by mechanical stresses and vasoactive signals. Transduction of these diverse stimuli into a cellular response occurs through many different mechanisms, one being reorganisation of the actin cytoskeleton. In addition to a structural role in maintaining cellular architecture it is now clear that the actin cytoskeleton of contractile vascular smooth muscle cells is a dynamic structure reacting to changes in the cellular environment. Equally clear is that disrupting the cytoskeleton or interfering with its rearrangement, has profound effects on artery contractility. The actin cytoskeleton associates with dense plaques, also called focal adhesions, at the plasma membrane of smooth muscle cells. Vasoconstrictors and mechanical stress induce remodelling of the focal adhesions, concomitant with cytoskeletal reorganisation. Recent work has shown that non-receptor tyrosine kinases and tyrosine phosphorylation of focal adhesion proteins such as paxillin and Hic-5 are important for actin cytoskeleton and focal adhesion remodelling and contraction.

Pediatric poisoning management: How clinical practice can benefit from forensic approach.

Pediatric population represents the most vulnerable and at risk for unintentional poisoning, with children younger than 6 years old accounting for nearly half of poison exposures. Poisoning is a time-dependent emergency. The need to reach a scientific agreement on diagnostic protocol and treatment seems to be crucial to reduce morbidity and mortality. Starting from a buprenorphine pediatric intoxication case, this article highlights the limits and pitfalls of the traditional diagnostic approach. Diagnosis of drug intoxication was achieved after several days when an in-depth diagnostic investigation became necessary and complete forensic toxicological analyses were performed. Results evidenced an alarming lack of an unequivocal diagnostic protocol in case of suspect intoxication in structures not provided with a forensic toxicological service/unit. Collection of biological specimens according to forensic protocols at hospitalization plays a paramount role in the definitive diagnosis of intoxication. A diagnostic algorithm that focuses on medical history and biological specimen collection timing is herein proposed, in order to unify emergency approaches to the suspected poisoned child.

METODO, a prospective observational study to assess the efficacy and tolerability of methadone in heroin-addicted patients undergoing a methadone maintenance treatment: preliminary results at baseline evaluation.

METODO (methadone efficacy therapy optimization dosage on-going) is a prospective observational study to assess the efficacy and tolerability of methadone in 500 heroin-addicted patients taking a methadone maintenance treatment, enrolled through 2010 to 2011 in five Italian sites, observed over 2 years. The Opiate Dosage Adequacy Scale has been used for the evaluation of the "adequacy" of the methadone dosage and to stratify patients in adequate and not adequate groups. The treatment efficacy has been evaluated in correlation to the dosage adequacy during the visits. Moreover, patients have been evaluated according to the retention rate and duration of retention in treatment and a series of questionnaires.

The Identification of Drug Abuse.

Forensic toxicology has played a central role since its development in defining mechanisms of acute intoxication, often with a lethal outcome [...].

A novel test using dried blood spots for the chromatographic assay of methadone.

A novel test has been developed for the analysis of methadone in dried blood spot specimens from patients undergoing methadone maintenance treatment. An isocratic reversed-phase high-performance liquid chromatography method with coulometric detection has been optimized for the determination of methadone. The clean-up of dried blood spots was performed by means of an original microextraction by packed sorbent procedure after microwave-assisted extraction of the drug with a suitable solvent. Extraction yields were satisfactory, always being higher than 90.0 %. The calibration curve was linear over the 4-500 ng mL(-1) concentration range. The method had satisfactory sensitivity (limit of quantitation of 4 ng mL(-1)), precision (relative standard deviation less than 5.8 %), selectivity and accuracy (recovery greater than 87.0 %). It was successfully applied to dried blood spot samples collected from heroin-addicted patients undergoing methadone maintenance therapy at dosages between 40 and 240 mg day(-1). The statistical analysis (Bland-Altman plot) showed that the results were in good agreement with those found from the analysis of plasma samples obtained from the same patients. Thus, the method has proved to be suitable for the monitoring of methadone by means of dried blood spots.

Diquat Poisoning: Care Management and Medico-Legal Implications.

Acute chemical intoxication represents one of the major causes of Emergency Room admittance, and possible errors in diagnosis are extremely frequent, especially when patients present generic and non-specific symptoms. Diquat, a bipyridyl class of herbicides, exerts high intrinsic toxicity as a consequence of free oxygen radicals, leading to cellular death and organ dysfunctions. Following ingestion, with the major source of absorption for suicidal purposes, the chemical induces local irritating effects; systemic symptoms appear later, while specific symptoms can occur in the following 48 h. A smoker and hypertensive 50-year-old man arrives at the E.R., reporting that an episode of herbicide inhalation occurred few hours earlier. Physical examination evidenced alkalosis with hypoxemia, leucocytosis, mild hyperglycaemia and moderate increase in creatine kinase and myoglobin. Despite blood creatine kinase and myoglobin values that were higher than normal, he was prescribed with hydration and anti-pain therapy. During the night, the man left the hospital; he returned the next morning at 8:45 a.m., with cardiorespiratory arrest, medium fixed non-reactive mydriasis, diffused cyanosis of the skin and of the mucous membranes, as well as imperceptible pulse and peripheral pressure. Despite resuscitation attempts, the patient died at 9:30 a.m.; the body was immediately transferred to the morgue. Autopsy and toxicological analyses were carried out nine days later, evidencing paraquat ingestion for suicidal purposes. GC/MS analyses to verify the presence of diquat were performed on body fluids and gastric and colon contents; all specimens resulted positive, thus confirming the cause of death as herbicide ingestion (blood diquat concentration of 1.2 mg/L; more than twice the minimum to observe a systemic poisoning). The procedure followed for patient management resulted to be not in line with the provisions of both guidelines and good clinical practices. Staff did not perform clinical-diagnostical monitoring of the patient's condition or ask for more specific analyses (i.e., serum creatine phosphokinase monitoring). This misconduct led to a decrease in the patient's chances to survive.

Medico-Legal Issues in Cremation: Comparative Analysis of International Legislation.

Cremation has seen a constant increase in popularity all around the world. Because of its extensively destructive nature, however, a series of medico-legal issues arise concerning identification, forensic autopsy, external examination, histological, toxicological and genetic exams to be performed not in the immediacy of death. The aim of this study is to compare the international legislation on cremation, seeking the response of various countries to their medico-legal issues. Several affinities but also some differences were found. Similarities include the need for a certificate by a medical examiner excluding any medico-legal issues and non-natural causes of death and the expression of consent to cremation given by the deceased when still alive otherwise by relatives. Significant differences were found in German law, which provides for a second medical examination prior to cremation and in Italian law providing for the medical examiner to collect biological samples from the body and store them for a minimum of ten years for any future purpose of justice. The Italian approach could give a plausible solution to the medico-legal issues raised by cremation with the imperative premise, however, we need to look deeply into its privacy and consent implications, cost-benefits rate, sample collection and storage protocol.

Screening of several drugs of abuse in Italian workplace drug testing: performance comparisons of on-site screening tests and a fluorescence polarization immunoassay-based device.

According to the Italian laws, some categories of workers entrusted with duties possibly constituting a threat to security, physical safety, and health of third parties have to be screened to exclude the use/abuse of the following drugs of abuse: opiates, cocaine, cannabinoids, amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine, methadone, and buprenorphine. Toxicological tests can be performed with urinary on-site rapid screening devices, provided that sensitivities up to specified cutoffs are ensured. The present study reports performances, in terms of sensitivity, specificity, and accuracy, of an automatic on-site test and of an FPIA-based device, using gas chromatography/mass spectrometry (GC/MS) as a reference methodology. Three levels of concentration were tested, corresponding to the cutoff and to 2 and 3 times the limits, respectively. In terms of sensitivities, neither the on-site nor the benchtop instrumentations gave positive results, since values of zero percentage were obtained for concentrations up to 2-fold the limits. Even if good results were obtained in terms of specificity and accuracy by both devices, none of them seem to be adequate for the current application to the toxicological screening at workplaces. In fact, a rapid screening device can be used for drug tests provided that it ensures sensitivity at the prescribed cutoffs. Data showed that such is completely rejected and a more sensitive instrumentation should be preferred.

Forensic Toxicological and Medico-Legal Evaluation in a Case of Incongruous Drug Administration in Terminal Cancer Patients.

BACKGROUND: In most cases, palliative care is prescribed to adults diagnosed with cancer. The definition of the most suitable therapy for an effective sedation in terminal cancer patients still represents one of the most challenging goals in medical practice. Due to their poor health, the correct dosing of drugs used for deep palliative sedation in terminal cancer patients, often already on polypharmacological therapy, can be extremely complicated, also considering possible drug-to-drug interactions that could lead to an increased risk of overdose and/or incongruous administration with fatal outcomes. The case of a terminal cancer patient is presented, focusing on the "adequacy" of administered therapy. MATERIALS AND METHODS: A young male, affected by Ewing sarcoma, attending a palliative care at his own home, died soon after midazolam administration. Toxicological and histological analyses were performed on body fluids and organ fragments. RESULTS AND DISCUSSION: Morphological reliefs evidenced a neoplastic mass, composed of lobulated tissue with a lardy, pinkish-gray consistency, extending from the pleural surface to the lung parenchyma, also present at the sacrum region (S1-S5), at the anterior mediastinum level, occupying the entire left pleural cavity, and infiltrating the ipsilateral lung. Metastatic lesions diffused to rachis and lumbar structures. The brain presented edema and congestion. Toxicological analyses evidenced blood midazolam concentrations in the range of 0.931-1.690 µg/mL, while morphine was between 0.266 and 0.909 µg/mL. Death was attributed to cardiorespiratory depression because of a synergic action between morphine and midazolam. The pharmacological interaction between midazolam and morphine is discussed considering the clinical situation of the patient. The opportunity to proceed with midazolam administration is discussed starting from guidelines recommendation. Finally, professional liability outlines are highlighted.

Study of the fragmentation pattern of ketamine-heptafluorobutyramide by gas chromatography/electron ionization mass spectrometry.

Ketamine is an anaesthetic compound used in human and veterinary medicine with hallucinogen properties that have resulted in its increased illicit use by teenagers at rave parties. Although several gas chromatography/mass spectrometry (GC/MS) methods have been reported for the quantification of the drug both in urine and in hair, its electron ionization (EI) fragmentation after derivatization with different reagents has been not yet fully investigated. The present work reports the study of the fragmentation of ketamine, derivatized with heptafluorobutyric anhydride (HFBA-Ket), using gas chromatography/electron ionization mass spectrometry (GC/EI-MS). The complete characterization of the fragmentation pattern represented an intriguing exercise and required tandem mass spectrometry (MS(n)) experiments, high-resolution accurate mass measurements and the use of deuterated d(4)-ketamine to corroborate the proposed structures and to characterize the fragment ions carrying the unchanged aromatic moiety. Extensive fragmentation was observed, mainly located at the cyclohexanone ring followed by rearrangement of the fragment ions, as confirmed by the mass spectra obtained from the deuterated molecule. The GC/EI-MS analysis of HFBA-Ket will represent a useful tool in forensic science since high-throughput analyses are enabled, preserving both the GC stationary phase and the cleanliness of the mass spectrometer ion optics.

Simultaneous determination of disulfiram and bupropion in human plasma of alcohol and nicotine abusers.

An isocratic high-performance liquid-chromatographic method has been developed for the simultaneous determination of disulfiram and bupropion in human plasma samples. Analyses were carried out on a C(8) reversed-phase column using a mobile phase composed of 50% acetonitrile and 50% aqueous phosphate buffer, containing triethylamine. Diode-array detection was used, operating at a wavelength of 250 nm. For the clean-up of plasma samples, a solid phase extraction procedure, based on C(2) cartridges, was implemented. Extraction yields of the analytes were satisfactory, being always higher than 84%. The calibration curve was linear over the 5-500 ng mL(-1) plasma concentration range for both disulfiram and bupropion. The method showed a high sensitivity (limit of detection of 1.5 ng mL(-1)) and satisfactory precision, selectivity and accuracy. The application to human plasma samples obtained from some alcohol and nicotine abusers also gave good results.

Biological monitoring of nurses exposed to doxorubicin and epirubicin by a validated liquid chromatography/fluorescence detection method.

OBJECTIVES: Occupational exposure to antineoplastic drugs can represent a potential health risk for hospital staff. Assessing exposure is the first step in providing a safe work environment; the present study aimed to perform a biological monitoring (BM) of nurses exposed to doxorubicin and epirubicin. In order to assure data accuracy and reproducibility, the high-performance liquid chromatography with fluorescence detection method was validated. METHODS: Validation experiments were carried out according to the Food and Drug Administration guidelines. A detailed questionnaire about workplace practices and work organization was administered to 56 nurses of oncology department of two hospitals (A and B) located in southern Italy. End-shift urine samples were collected. Amounts of drugs handled were registered. RESULTS: The quantification and detection limits were 1.1 and 0.6 pg microl(-1) (doxorubicin) and 2.0 and 1.2 pg microl(-1) (epirubicin); moreover, the analytical method fulfilled all guidelines requirements. Questionnaire information evidenced that vertical laminar flow hoods were present in both hospitals, surfaces were cleaned with inappropriate detergents, no antispilling devices were adopted, and gloves were not changed during the work shift. A lower percentage of positive samples was found in the hospital where higher amounts of anthracyclines were handled (3.4% in A and 14.8% in B), suggesting individual incorrect working/cleaning practices in hospital A and overall hygienic standards to be improved in hospital B, where 'critical practices' were carried out. CONCLUSIONS: Results showed the crucial role of adopting effective safety precautions and handling practices to reduce exposure. Environmental and BM should be performed to discriminate between incorrect personal working modalities and general hygienic standards.

Deprescription in elderly patients with type 2 diabetes mellitus.

AIMS: The Primary aim is to verify physicians' adherence to the 2016 Italian diabetes guidelines therapeutic targets, and their habits on deprescription in elderly persons with Type 2 Diabetes Mellitus (T2DM). Secondary aims are the assessment of the potential impact of the targets' changes in 2018 Italian guidelines, and the outcomes of deprescription in the management of T2DM. METHODS: Observational retrospective cohort study, enrolling persons with T2DM, aged > 75 years, who attended a visit throughout 2017, and a second visit 6 months later in our outpatient clinic. RESULTS: Of the 387 patients included, 336 (87, 8%) were on target, according to 2016 guidelines. Deprescription was advisable in 62% of patients on target. Among those, 22% were deprescribed. In patients undergoing deprescription, during the following 6 months, no severe hypoglycemia occurred (versus 5 cases in the prior 6 months). Glycated Hemoglobin (HbA1c) increased (p < 0.05) from 47.0 [41.7-51.0] to 53.0 [45.4-59.5] mmol/mol). Applying to the sample the 2018 Italian Guidelines targets, 57.2% would have been on target, 18.5% above, and 24.3% below (needing deprescription). CONCLUSION: In our study, a minority of suitable patients received deprescription. Deprescription led to a significant reduction in severe hypoglycemia rate, whereas HbA1c remained on target in the majority of cases.

Mass spectrometry-based proteomics for the forensic identification of vomit traces.

Proteomics was exploited to assess the nature of possible traces of vomit found on the scene of an alleged sexual assault. In the case in point, a woman reported to the police to be raped five days before by a cousin of hers in his car. The woman declared she had vomited in the car before fainting definitely, due to alcohol or possible drugs covertly slipped in her drinks. The suspect confirmed the sexual intercourse, but he claimed consensual sex while the woman was fully conscious. To establish consent and hence subsistence of the crime, the Magistrate requested toxicological analyses on items sampled from the car and from woman's boots. Negative results obtained from toxicological analyses could not exclude the actual assumption of psychoactive substances by the alleged victim, due to sample aging. On the contrary, proteomic analysis disclosed a pattern of 249 gene products including signature endogenous and food-derived proteins along with a multitude of peptide digests, clearly indicative of vomit, thereby supporting the victim's report in the case under examination. Proteomics also provided detailed information about the nature of meal, which might contribute to frame the crime scene in similar cases. SIGNIFICANCE: The identification of traces of vomit supported the report of the victim's report according to which she vomited before definitely losing consciousness, so providing key contribution to establish consent for the sexual intercourse. This is the first time that proteomics is used to identify traces of vomit for forensic purposes. In spite of the scantiness of the biological specimen available, proteomics was successful to define a panel of characteristic endogenous proteins as well as to identify partly digested food-proteins arising from a complex meal. Proteomics is increasingly used as a forensic technique, well complementing the existing tools. In general, assessing traces of vomit in biological specimens and characterizing the nature of food ingested at the molecular level could afford probative elements to frame a crime scene.

Association between migraine and headache attributed to stroke: a case-control study.

BACKGROUND: Several studies were carried out to investigate the occurrence of headache attributed to acute stroke in patients with a lifetime history of migraine. METHODS: In a case-control series of 96 acute stroke patients with a lifetime history of migraine (M+) and 96 stroke patients without (M-), ischemic stroke patients only, without secondary infarction, were selected. The headache attributed to acute ischemic stroke was then analyzed. RESULTS: (M+) patients complained of headache more often than (M-) patients (P < .0001), mainly in the 24 hours before stroke onset (P < .0001). Migraine-like features of headache were recognized in a greater proportion of cases in the (M+) patient group with ischemic stroke (P < .018). A preferential brainstem location of ischemic stroke in (M+) patients emerged compared with (M-) patients (P = .014). DISCUSSION: The high prevalence of headache attributed to stroke in (M+) patients, in a relevant proportion of cases presenting as a sentinel headache, suggests that cerebral ischemia lowers the threshold for head pain more easily in these "susceptible" patients. The most frequent involvement of the brainstem in (M+) patients with ischemic infarction concurs with recent reports that emphasized a greater headache frequency when cerebral infarctions are localized in this structure or deep brain gray matter.