Extremely low-frequency electromagnetic fields (ELF-MF) can modify the cell viability and regulatory processes of some cell types, including breast cancer cells. Breast cancer is a multifactorial disease where a role for ELF-MF cannot be excluded. ELF-MF may influence the biological properties of breast cells through molecular mechanisms and signaling pathways that are still unclear. This study analyzed the changes in the cell viability, cellular morphology, oxidative stress response and alteration of proteomic profile in breast cancer cells (MDA-MB-231) exposed to ELF-MF (50 Hz, 1 mT for 4 h). Non-tumorigenic human breast cells (MCF-10A) were used as control cells. Exposed MDA-MB-231 breast cancer cells increased their viability and live cell number and showed a higher density and length of filopodia compared with the unexposed cells. In addition, ELF-MF induced an increase of the mitochondrial ROS levels and an alteration of mitochondrial morphology. Proteomic data analysis showed that ELF-MF altered the expression of 328 proteins in MDA-MB-231 cells and of 242 proteins in MCF-10A cells. Gene Ontology term enrichment analysis demonstrated that in both cell lines ELF-MF exposure up-regulated the genes enriched in "focal adhesion" and "mitochondrion". The ELF-MF exposure decreased the adhesive properties of MDA-MB-231 cells and increased the migration and invasion cell abilities. At the same time, proteomic analysis, confirmed by Real Time PCR, revealed that transcription factors associated with cellular reprogramming were upregulated in MDA-MB-231 cells and downregulated in MCF-10A cells after ELF-MF exposure. MDA-MB-231 breast cancer cells exposed to 1 mT 50 Hz ELF-MF showed modifications in proteomic profile together with changes in cell viability, cellular morphology, oxidative stress response, adhesion, migration and invasion cell abilities. The main signaling pathways involved were relative to focal adhesion, mitochondrion and cellular reprogramming.

Tryptamine intoxications and fatalities are increasing, although these novel psychoactive substances (NPS) are not controlled in most countries. There are few data on the metabolic pathways and enzymes involved in tryptamine biotransformation. 4-acetoxy-N,N-diisopropyltryptamine (4-AcO-DiPT) is a synthetic tryptamine related to 4-hydroxy-N,N-diisopropyltryptamine (4-OH-DiPT), 4-acetyloxy-N,N-dipropyltryptamine (4-AcO-DPT), and 4-acetoxy-N,N-dimethyltryptamine (4-AcO-DMT). The aim of this study was to determine the best 4-AcO-DiPT metabolites to identify 4-AcO-DiPT consumption through human hepatocyte metabolism and high-resolution mass spectrometry. 4-AcO-DiPT metabolites were predicted in silico with GLORYx freeware to assist in metabolite identification. 4-AcO-DiPT was incubated with 10-donor-pooled human hepatocytes and sample analysis was performed with reversed-phase liquid chromatography coupled with high-resolution tandem mass spectrometry (LC-HRMS/MS) in positive- and negative-ion modes. Software-assisted LC-HRMS/MS raw data mining was performed. A total of 47 phase I and II metabolites were predicted, and six metabolites were identified after 3 h incubation following ester hydrolysis, O-glucuronidation, O-sulfation, N-oxidation, and N-dealkylation. All second-generation metabolites were derived from the only first-generation metabolite detected after ester hydrolysis (4-OH-DiPT). The metabolite with the second-most-intense signal was 4-OH-iPT-sulfate followed by 4-OH-DiPT-glucuronide, indicating that glucuronidation and sulfation are common in this tryptamine’s metabolic pathway. 4-OH-DiPT, 4-OH-iPT, and 4-OH-DiPT-N-oxide are suggested as optimal biomarkers to identify 4-AcO-DiPT consumption.

Forensic DNA phenotyping (FDP) provides the ability to predict the human external traits from unknown sample donors, directly from minute amounts of DNA found at the crime scene. We developed a MPS multiplex assay, with the aim of genotyping all 41 DNA markers included in the HIrisPlex-S system for simultaneous prediction of eye, hair and skin colours. Forensic samples such as blood, skeletal remains, touch DNA, saliva swab, artificially degraded samples together with individuals with known phenotypes and a set of 2800 M control DNA were sequenced on the Ion Torrent platform in order to evaluate the concordance testing results and the forensic suitability of the 41-plex MPS assay. The panel was evaluated by testing a different number of PCR cycles and the volume of reagents for library preparation. The study demonstrated that full and reliable profiles were obtained with 0.1–5 ng, even with high degraded DNA. The increment of the number of PCR cycles results in an improvement of correctly genotyping and phenotyping for samples with low amounts of degraded DNA but higher frequencies of artefacts were found. The high DNA degradation level did not influence the correct genotyping and phenotyping and the critical parameter affecting the result is the quantity of input DNA. Eye and hair colour was predicted in 92.60% of individuals and skin colour in 85.15% of individuals. The results suggest that this MPS assay is robust, highly sensitive and useful for human pigmentation prediction in the forensic genetic field.

Cannabidiol (CBD) exhibits anti-inflammatory, anxiolytic, antiseizure, and neuroprotective proprieties without addictive or psychotropic side effects, as opposed to Δ9-tetrahydrocannabinol (THC). While recreational cannabis contains higher THC and lower CBD concentrations, medical cannabis contains THC and CBD in different ratios, along with minor phytocannabinoids, terpenes, flavonoids and other chemicals. A volumetric absorptive microsampling (VAMS) method combined with ultra-high-performance liquid chromatography coupled with mass spectrometry in tandem for quantification of CBD, THC and their respective metabolites: cannabidiol-7-oic acid (7-COOH-CBD); 7-hydroxy-cannabidiol (7-OH-CBD); 6-alpha-hydroxy-cannabidiol (6-α-OH-CBD); and 6-beta-hydroxycannabidiol (6-β-OH-CBD); 11- Hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC) and 11-Nor-9-carboxy-Δ9-tetrahydrocannabinol (THCCOOH). After overnight enzymatic glucuronide hydrolysis at 37°C, samples underwent acidic along with basic liquid-liquid extraction with hexane: ethyl acetate (9:1, v/v). Chromatographic separation was carried out on a C18 column, with the mass spectrometer operated in multiple reaction monitoring mode and negative electrospray ionization. Seven patients with intractable epilepsy were dosed with various CBD-containing formulations and blood collected just before their daily morning administration. The method was validated following international guidelines in toxicology. Linear ranges were (ng/ml) 0.5–25 THC, 11-OH-THC, THCCOOH, 6-α-OH-CBD and 6-β-OH-CBD; 10–500 CBD and 7-OH-CBD; and 20–5000 7-COOH-CBD. 7-COOH-CBD was present in the highest concentrations, followed by 7-OH-CBD and CBD. This analytical method is useful for investigating CBD, THC and their major metabolites in epilepsy patients treated with CBD preparations employing a minimally invasive microsampling technique requiring only 30 µL blood.

Background4-Hydroxy-N,N-methylpropyltryptamine (4-OH-MPT) is a psychedelic tryptamine whose use is regulated in several countries. Due to unspecific effects, consumption can be ascertained only through toxicological analyses. However, the trace amounts of tryptamines are usually challenging to detect in biological samples. 4-OH-MPT metabolism was characterized to identify optimal metabolite markers of intake in clinical/forensic toxicology.Research design and methods4-OH-MPT was incubated with 10-donor-pooled human hepatocytes to simulate in vivo conditions; samples were analyzed by liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS/MS), and data were processed with Compound Discoverer from Thermo Scientific. LC-HRMS/MS and data mining were supported by in silico metabolite predictions (GLORYx).ResultsThree phase I and four phase II metabolites were identified, including N-oxidation and N-demethylation at the alkylamine chain, and O-glucuronidation and sulfation at the hydroxylindole core.Conclusions4-OH-MPT metabolic fate was consistent with the human metabolism of tryptamine analogues: we suggest 4-OH-MPT-N-oxide and 4-hydroxy-N,N-propyltryptamine (4-OH-PT) as metabolite biomarkers of 4-OH-MPT consumption after glucuronide/sulfate hydrolysis in biological samples to improve detection of 4-OH-MPT and phase I metabolites; 4-OH-MPT-glucuronide is suggested as an additional biomarker when hydrolysis is not performed. Further research on the metabolism of structural analogues is necessary to evaluate the specificity of 4-OH-MPT metabolite biomarkers.

Background Preliminary data suggested that fat embolism could explain the importance of visceral obesity as a critical determinant of coronavirus disease-2019 (COVID-19). Methods We performed a comprehensive histomorphologic analysis of autoptic visceral adipose tissue (VAT), lungs and livers of 19 subjects with COVID-19 (COVID-19+), and 23 people without COVID-19 (controls). Human adipocytes (hMADS) infected with SARS-CoV-2 were also studied. Results Although there were no between-group differences in body-mass-index and adipocytes size, a higher prevalence of CD68+ macrophages among COVID-19+ VAT was detected (p = 0.005) and accompanied by crown-like structures presence, signs of adipocytes stress and death. Consistently, human adipocytes were successfully infected by SARS-CoV-2 in vitro and displayed lower cell viability. Being VAT inflammation associated with lipids spill-over from dead adipocytes, we studied lipids distribution by ORO. Lipids were observed within lungs and livers interstitial spaces, macrophages, endothelial cells, and vessels lumen, features suggestive of fat embolism syndrome, more prevalent among COVID-19+ (p < 0.001). Notably, signs of fat embolism were more prevalent among people with obesity (p = 0.03) independently of COVID-19 diagnosis, suggesting that such condition may be an obesity complication exacerbated by SARS-CoV-2 infection. Importantly, all infected subjects' lungs presented lipids-rich (ORO+) hyaline membranes, formations associated with COVID-19-related pneumonia, present only in one control patient with non-COVID-19-related pneumonia. Importantly, transition aspects between embolic fat and hyaline membranes were also observed. Conclusions This study confirms the lung fat embolism in COVID-19+ patients and describes for the first time novel COVID-19-related features possibly underlying the unfavorable prognosis in people with COVID-19 and obesity.

The growing use of massively parallel sequencing (MPS) for the whole mitochondrial genome analysis in forensic laboratories, requires the establishment of efficient workflows and interpretation procedures, to support the feasibility of the technology and the reliability of the data. In the case of reference samples, such as blood and buccal swabs, the generation of mtDNA profiles by using MPS is relatively simple. Conversely, many forensic casework samples still pose challenges for the MPS, data interpretation and reporting of mtDNA. This is especially true for the analysis of shed hairs, which are one of the most common evidence types and which are among the most limited in terms of DNA quantity and quality. Due to these limitations, every step involved in the analysis become essentials and should be performed in order to obtain the best performance, optimizing the outcomes and minimizing the errors. In light of this, we present a study focusing on the extraction, quantification and MPS of the whole mtDNA in hair shafts, with the aims of set-up and validate a methodological pipeline to obtain the best sequencing results. The overall performance of the MPS panel, mainly in terms of total coverage, amplicons coverage and different primer pools efficiency, was evaluated also in relation to the different hair fragments, the mtDNA copy number used for libraries preparation and its degradation state.

The rising use of designer benzodiazepines (DBZD) is a cat-and-mouse game between organized crime and law enforcement. Non-prohibited benzodiazepines are introduced onto the global drug market and scheduled as rapidly as possible by international authorities. In response, DBZD are continuously modified to avoid legal sanctions and drug seizures and generally to increase the abuse potential of the DBZD. This results in an unpredictable fluctuation between the appearance and disappearance of DBZD in the illicit market. Thirty-one DBZD were considered for review after consulting the international early warning database, but only 3-hydroxyphenazepam, adinazolam, clonazolam, etizolam, deschloroetizolam, diclazepam, flualprazolam, flubromazepam, flubromazolam, meclonazepam, phenazepam and pyrazolam had sufficient data to contribute to this scoping review. A total of 49 reports describing 1 drug offense, 2 self-administration studies, 3 outpatient department admissions, 44 emergency department (ED) admissions, 63 driving under the influence of drugs (DUID) and 141 deaths reported between 2008 and 2021 are included in this study. Etizolam, flualprazolam flubromazolam and phenazepam were implicated in the majority of adverse-events, drug offenses and deaths. However, due to a general lack of knowledge of DBZD pharmacokinetics and toxicity, and due to a lack of validated analytical methods, total cases are much likely higher. Between 2019 and April 2020, DBZD were identified in 48% and 83% of postmortem and DUID cases reported to the UNODC, respectively, with flualprazolam, flubromazolam and etizolam as the most frequently detected substances. DBZD toxicology, public health risks and adverse events are reported.

Introduction Defining extreme temperatures as the cause of death remains challenging. It is mostly based on circumstantial, macroscopic and microscopic features. Methods We retrospectively compared groups of cases of fatal hypothermia, fatal hyperthermia and non-extreme temperature-related deaths. We analysed specific histological findings, focusing on samples from the liver, pancreas and kidney. Results Between 1 January 2013 and 31 December 2016, 15 autopsies were performed for deaths related to extreme temperatures. They included 11 cases of fatal hypothermia (group A), four cases of fatal hyperthermia (group B) and eight controls (group C). Perinuclear hepatocyte vacuolisation was observed in seven cases of hypothermia, one case of hyperthermia and four controls. Pancreatic cytoarchitecture was well preserved in two cases of hypothermia, one case of hyperthermia and two controls. No particular microscopic feature was found in pancreatic samples. Renal epithelial tubular cell vacuolisation was observed in seven cases of hypothermia and one case of hyperthermia, while it was absent in all controls. Chromogranin A (CgA) was markedly positive in the pancreatic tissue of five cases of fatal hypothermia and one control, and mildly positive in one case of fatal hyperthermia. No significant p-values were observed for any comparisons ( p > 0.05), except when hypothermia cases group were compared to the control group for the Armanni–Ebstein phenomenon test ( p = 0.0078). Conclusions Although our study did not find a specific microscopic marker, hepatocyte vacuolisation, the Armanni–Ebstein phenomenon and pancreatic CgA positivity, taken together, may be useful tools to confirm hypo- and hyperthermia-related deaths, in addition to circumstantial and macroscopic findings.

OBJECTIVE The aim of this review was to explore recent pieces of evidence focused on the use of miRNAs for PMI estimation both in humans and animal experiments, with particular interest on the best miRNAs to use as reference/target markers in different tissues or biological fluids. MiRNAs are innovative biomarkers used in clinical and research field; they appear very attractive, being introduced in forensic research scenarios even for PMI estimation. MATERIALS AND METHODS Data from PubMed and Scopus were analyzed from January 2013 to August 2020. Based on inclusion/exclusion criteria, high-quality articles have been selected to become the subject of this review. RESULTS A total of 737 papers were found but, after titles/abstracts screening for inclusion criteria and a full-text careful selection, 33 papers were deeply studied. After the exclusion of 19 papers, 15 articles remained. Eight papers dealt with animals (mice/rats), two both with animals and humans (for method validation previously built), while 5 exclusively with humans. Myocardium (6/15) and brain (6/15) were the most studied tissues, respectively in mice/rats and humans. PMI considered was up to 7.5 days in mouse studies and less than 3 days in human models. CONCLUSIONS Because of their significant stability in both early and long PMI, miRNAs are the cleverest reference markers to be used. Temperature and environmental conditions influence mostly mRNA, while miRNAs are less susceptible to them. The best miRNA to choose depends on its tissue specificity, i.e., miR-9 and miR-125 in brain or miR-1 and miR-133 in skeletal muscle/heart.

Mycobacterium chimaera is a non-tuberculous mycobacterium, member of the Mycobacterium avium complex (MAC), which has become a global public health concern due to infection following cardiac surgery performed with contaminated heater-cooler units. M. chimaera infection is characterized by a long latency, non-specific signs and symptoms and high mortality rates. Thus, the diagnosis is still challenging both for forensic pathologists and for clinicians. Clinical manifestations of M. chimaera infection include endocarditis, hepatitis, nephritis, encephalitis and chorioretinitis. A constant histopathologic finding is the presence of non-caseating granulomas, with multinucleated giant cells and histiocytes. Hereby, we present two cases of fatal disseminated M. chimaera infection following aortic valve surgery reporting clinical history and post-mortem findings. Further, we provide a brief overview of the literature with a special focus on histopathological characteristics of M. chimaera infection. The aim of this article is to provide a complete synopsis of histopathological characteristics useful for forensic pathologists.

BACKGROUND:Synthetic cathinones (SCs) are designer analogs of the natural active principle of khat. Since their appearance on the black market in 2003, their popularity has increased annually, and they have become the most seized class of new psychoactive substances reported to the UNODC Early Warning Advisory system. The constant introduction of newly synthesized molecules makes this issue difficult to monitor. The authors reviewed the most recent SC-related fatalities worldwide to highlight new trends of consumption, reporting acute pharmacological and toxicological symptoms, scene investigations, analytical methods, and reported SC concentrations in diverse biological matrices.METHODS:A literature search was performed using scientific databases such as PubMed, Scopus, Science Direct, Web of Science, and Research Gate to identify relevant scientific publications from 2017 to 2020. In addition, a search was conducted through the EU EWS.RESULTS:From 2017 to 2020, 31 different SCs were identified in 75 reported fatal intoxications in the literature, alone or in combination with other substances. The most abused SCs were N-ethylpentylone, N-ethylhexedrone, and 4-chloromethcathinone. The EU EWS included less detail on 72 additional SC-related fatalities from 2017 to 2020.CONCLUSIONS:New SCs continuously replace older natural and synthetic stimulant drugs, making determining the cause of death difficult. Analytical methods and high-performance mass spectrometry instruments are essential to detect the low concentrations of these potent new SCs. Little data are available on the pharmacology of these new drugs; the evaluation of toxicological antemortem and postmortem findings provides critical data on the drug's pharmacology and toxicology and for the interpretation of new SC cases.

The aim of this study was the reconstruction of SARS-CoV-2 evolutionary dynamics in time and space in Italy and Europe between February and June 2020. The cluster analysis showed that pure Italian clusters were observed mainly after the lockdown and distancing measures were adopted. Lineage B and B.1 spread between late January and early February 2020, from China to Veneto and Lombardy, respectively. Lineage B.1.1 most probably evolved within Italy and spread from central to south Italian regions, and to European countries. The lineage B.1.1.1 entered Italy only in the second half of March and remained localized in Piedmont until June 2020. In conclusion, the reconstructed ancestral scenario suggests a central role of China and Italy in the widespread diffusion of the D614G variant in Europe in the early phase of the pandemic and more dispersed exchanges involving several European countries from the second half of March 2020.

Purpose: The decomposition process of human bodies in marine environment is not well understood, and it is influenced by external variables related to the geographical area where the body is submerged. We report the application of two decomposition scores, the Heaton's score and the van Daalen's score, on a casuistry of human bodies recovered from the Northern Adriatic Sea. The aims of this study are to verify whether the marine environment of a Mediterranean climate area may affect the applicability of both scores and to develop a prediction model that can be applied on bodies recovered in salt water. Methods: A retrospective study was performed on 61 human bodies recovered between 2005 and 2019 from coastal water of the Northern Adriatic Sea nearby the Italian regions Emilia-Romagna and Marche. For each of the 61 cases included, the Total Aquatic Decomposition Score (TADS) was calculated with the Heaton's score and the Van Daalen's score. The prediction model was assessed through multiple regression analyses, and the determination coefficients (r(2)) between TADS and PMSI (expressed in days) and between TADS and Accumulate Degrees Days (ADD) were studied. The prediction model was applied to the entire case sample, to bodies recovered during the warm season and to bodies recovered during the cold season. Results: All bodies were recovered floating, and a very poor scavenging activity was observed. The regression analyses showed a strong correlation between the TADS and the total case sample using both scores and both independent variables (PMSI and ADD). The determination coefficients were greater than 0.95 also when considering the total case sample. Discussion: The proposed prediction models are not significantly influenced by seasonality, contrarily to what observed on bodies recovered in fresh water in the same climate area. However, the ADD model, which also consider the water temperature, should be preferred for higher decomposition stages. This study helps increase the accuracy of PMSI estimation in bodies recovered from a marine environment of the Northern Adriatic Sea. (C) 2020 Elsevier B.V. All rights reserved.

From two COVID-19-related deaths, samples of lung, heart and kidney were collected and processed for Transmission and Scanning Electron Microscopy (TEM and SEM) with the aim of identifying the virus. Virions of SARS-CoV-2 were found in all tissues by TEM and SEM, corroborating the hypothesis that the virus enters the cells of different organs. This is the first report identifying SARS-CoV-2 in different human tissues by TEM and SEM.

OPINION article Front. Psychiatry, 23 January 2020 | https://doi.org/10.3389/fpsyt.2019.01006

No analytical assay is currently available for the simultaneous determination of CBD major metabolites in serum or urine samples of individuals treated with medical cannabis or CBD-based pharmaceuticals. We developed and validated a method using ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) for quantifying cannabidiol (CBD) and its metabolites, cannabidiol-7-oic acid (7-COOH-CBD), 7- hydroxycannabidiol (7-OH-CBD), 6-alpha-hydroxycannabidiol (6-α-OH-CBD) and 6-beta-hydroxycannabidiol (6-β-OH-CBD) in serum and urine samples of an individual treated with medical cannabis. The ionization was performed by electrospray in negative mode to reach the sensitivity required to detect trace amounts, with limits of quantification ranging from 0.05 to 0.1 ng/mL. The method is accurate (average inter/intra-day error, <15%), precise (inter/intra-day imprecision, <15%) and fast (8 min run time) and it is an essential tool to investigate CBD pharmacokinetics and pharmacodynamics in individuals treated with medical cannabis or with CBD-based medical preparations.