Chemical investigation of biological trace evidence; toxicological screening of waste residues obtained from DNA extraction processes.

In a forensic scenario, if biological stains are found in very small quantities, these are usually collected for DNA analyses, considered essential for the forensic investigation and thus excluding possible investigations by other forensic disciplines as forensic toxicology. We developed an experimental study to evaluate the feasibility of analyzing DNA extraction residues obtained from DNA extraction procedures to perform toxicological analysis, with the aim to extract both genetic and toxicological information without affecting or compromising the genetic sample and/or DNA extraction. DNA extraction from four blood samples (fortified with 5 molecules of interest with a final concentrations of 1 µg/mL, 100 ng/mL, 10 ng/mL and 5 ng/mL, respectively) were analyzed with QIAGEN QIAmp® DNA Mini kit. Three waste residues collected from the DNA extraction were analyzed for the toxicological investigation via Solid-Phase Extraction and High-Performance Liquid Chromatography-Tandem Mass Spectrometry analyses (Thermo Scientific™ TSQ Fortis™ II Triple-Quadrupole Mass Spectrometer). The analytical investigation revealed that our analytes of interest were detected in two different residues of the DNA extraction procedure, allowing both genetic and toxicological analyses without affecting the DNA identification. At last, the experimental protocol was applied to a hypothetical case, with encouraging results and allowing the identification of our molecules of interest.

Proteomic analysis by mass spectrometry of postmortem muscle protein degradation for PMI estimation: A pilot study.

The determination of the postmortem interval is a topic of great forensic interest. The possibility of using new technologies has allowed the study of postmortem decay of biomolecules in the determination of PMI. Skeletal muscle proteins are promising candidates because skeletal muscle exhibits slower postmortem decay compared to other internal organs and nervous tissues, while its degradation is faster than cartilage and bone. In this pilot study, skeletal muscle tissue from pigs was degraded at two different controlled temperatures, 21 °C and 6 °C, and analysed at predefined times points: 0, 24, 48, 72, 96, and 120 h. The obtained samples were analysed by mass spectrometry proteomics approach for qualitative and quantitative evaluation of proteins and peptides. Immunoblotting validation was performed for the candidate proteins. The results obtained appeared significant and identified several proteins useful for possible postmortem interval estimation. Of these proteins, PDLIM7, TPM1, and ATP2A2 were validated by immunoblotting at a larger number of experimental points and at different temperatures. The results obtained are in agreement with those observed in similar works. In addition, the use of a mass spectrometry approach increased the number of protein species identified, providing a larger panel of proteins for PMI assessment.

Hepatitis E Virus RNA Detection from Hunted Wild Boars in Central Italy: an Epidemiological Investigation.

Every year, foodborne pathogens, including the hepatitis E virus (HEV), cause thousands of infections in different continents. Final consumers become infected through the ingestion of contaminated animal origin foodstuffs. Generally, in industrialized countries, HEV genotype 3 is involved in sporadic outbreaks. Infections have been described, in Europe and Japan as consequence of pork products and contaminated wild boar's primary or processed products (liver and muscle tissues) consumption. In Central Italy, hunting activities are largely practiced. In these small and rural communities, game meat and liver are ingested by hunters' families or at local and traditional restaurants. Therefore, these food chains can be considered critical HEV reservoirs. In this study, 506 liver and diaphragm tissues were collected from hunted wild boars in the Southern Marche region (Central Italy) and were screened for HEV RNA detection. From the 10.87% of liver and 2.76% of muscle samples, HEV3 subtype c was discovered. The observed prevalence values resulted in line with previous investigations performed in other Central Italian regions, but higher than Northern ones (3.7% and 1.9% from liver tissue). Therefore, the obtained epidemiological data highlighted the wide occurrence of HEV RNA circulation in a low-investigated area. Basing on results, a One-health approach was adopted due to the sanitary relevance of this Public Health concern.

Ultrasound Technology as Inactivation Method for Foodborne Pathogens: A Review.

An efficient microbiological decontamination protocol is required to guarantee safe food products for the final consumer to avoid foodborne illnesses. Ultrasound and non-thermal technology combinations represent innovative methods adopted by the food industry for food preservation and safety. Ultrasound power is commonly used with a frequency between 20 and 100 kHz to obtain an "exploit cavitation effect". Microbial inactivation via ultrasound derives from cell wall damage, the oxidation of intracellular amino acids and DNA changing material. As an inactivation method, it is evaluated alone and combined with other non-thermal technologies. The evidence shows that ultrasound is an important green technology that has a good decontamination effect and can improve the shelf-life of products. This review aims to describe the applicability of ultrasound in the food industry focusing on microbiological decontamination, reducing bacterial alterations caused by food spoilage strains and relative foodborne intoxication/infection.

Violence against Women and Stress-Related Disorders: Seeking for Associated Epigenetic Signatures, a Pilot Study.

BACKGROUND: Violence against women is a relevant health and social problem with negative consequences on women's health. The interaction between genome and environmental factors, such as violence, represents one of the major challenges in molecular medicine. The Epigenetics for WomEn (EpiWE) project is a multidisciplinary pilot study that intends to investigate the epigenetic signatures associated with intimate partner and sexual violence-induced stress-related disorders. MATERIALS AND METHODS: In 2020, 62 women exposed to violence (13 women suffering from sexual violence and 49 from Intimate Partner Violence, IPV) and 50 women with no history of violence were recruited at the Service for Sexual and Domestic Violence. All women aged 18-65 were monitored for their physical and psychological conditions. Blood samples were collected, and DNAs were extracted and underwent the epigenetic analysis of 10 stress-related genes. RESULTS: PTSD prevalence in victims was assessed at 8.1%. Quantitative methylation evaluation of the ten selected trauma/stress-related genes revealed the differential iper-methylation of brain-derived neurotrophic factor, dopamine receptor D2 and insulin-like growth factor 2 genes. These genes are among those related to brain plasticity, learning, and memory pathways. CONCLUSIONS: The association of early detection of posttraumatic distress and epigenetic marker identification could represent a new avenue for addressing women survivors toward resilience. This innovative approach in gender-based violence studies could identify new molecular pathways associated with the long-term effects of violence and implement innovative protocols of precision medicine.

Trichinella spp. detection in hunted wild boar (Sus scrofa) diaphragm biopsies in Central Italy.

Trichinellosis is a globally diffused foodborne parasitic disease caused by nematodes of the Trichinella complex. During evolution, guided by ecological interactions, natural selection, and biochemical "intelligence", these parasites developed admirable strategies to infect the host's organism. One of the most fascinating is represented by the nurse cell formation in muscular tissue (e.g., diaphragm, skeletal muscle, extrinsic ocular muscles, etc.). This strategy allowed the parasite to adapt and conquer the wider host species spectrum, including ungulates and humans. Consumption of undercooked meat from infected wild ungulates constitutes the most important source of infection for the human species. In this study, we show the prevalence of Trichinella spp. in hunted wild boars (Sus scrofa) in Central Italy. During the hunting season 2021/2022 in the province of Rieti, 554 wild boar diaphragm biopsies were collected for Trichinella spp. screening, in accordance with Regulation EU 1375/2015. An artificial digestion method was used for the detection of Trichinella spp. larval forms. The results revealed a positivity of 0.18% (1/554), and molecular biology identification demonstrated the presence of Trichinella britovi in the positive sample. This species is the most diffused in wild ungulate populations in Central Italy and the most frequently isolated in human patients with trichinellosis from this area, showing a close epidemiological relation between Homo sapiens and Sus scrofa for Trichinella spp. diffusion in an ecosystem. Epidemiological surveillance, in receptive animal species destined for human consumption and at any One Health level, represents the main "winning" strategy in the control of this worldwide, widespread foodborne parasitic disease.

Intradiaphragmatic abscesses in a wild boar (Sus scrofa): Inspective implications based on anatomopathological evidences.

The intradiaphragmatic localization of an abscess is rarely described in humans and in other animal domestic and wild species, and can be caused by penetrative traumas (i.e., firearm injuries). Here we describe two intradiaphragmatic abscesses in a hunted adult male wild boar (Sus scrofa) pluck, associated with adhesion phenomena with the contiguous anatomical structures (pleural, phrenic, and glissonian serosas) and observed during the post mortem inspection, in accordance with the Reg. EU 627/2019. One of these lesions also presented a phreno-abdominal fistula. We found in cytopathological evaluation of the neoformations' content the presence of spheroidal bacterial soma, characterized by linearly concatenated "Streptococcus-like" aggregation pattern. Furthermore, microbiological assays revealed a polymicrobial pattern characterized by the presence of telluric microorganisms, some of which have a marked pyogenic action (Streptococcus suis type I, Sphingomonas paucimobilis, Carnobacterium divergens, and Lactobacillus sakei). Our results and collected evidence demonstrate the pathogenetic hypothesis of bacterial contamination secondary to penetrative trauma caused by a not-mortal projectile's wound, defining the inspective behaviour according to the cogent legislation. These lesions, in the reason of their potential relation to toxemia, bacteremia and septicemia phenomena, represent a sanitary risk that impose, from a normative point of view, the total condemnation of the carcass. These inspective implications, originating from the observation and interdisciplinary description of the anatomopathological and microbiological features of the lesions, are directly projected on the normative scenario, providing useful elements to guarantee the health of the consumer.

Hepatitis E Virus Detection in Hunted Wild Boar Liver and Muscle Tissues in Central Italy.

In different European countries, including Italy, hepatitis E virus (HEV) has been recognized as an emerging public health concern. Humans are infected through the orofecal route by the ingestion of contaminated uncooked or undercooked animal-origin foodstuffs. Wild boars (Sus scrofa) have gained a crucial role as viral reservoirs. HEV-3 is the most frequently identified genotype from hunted wild boar liver and muscle tissues. The Marche region, more specifically Ascoli Piceno province, is characterized by a rooted hunting tradition and related product consumption. In this research study, 312 liver and 296 muscle specimens were screened using biomolecular assays, and HEV RNA was detected from 5.45% and 1.35% of liver and muscle samples, respectively. Phylogenetic analysis revealed that positive animals were infected by genotype 3 subtype c. Based on the environmental pathogen characteristics, HEV has also evolved to guarantee its survival in a wild environment. Therefore, wild boars and ruminants have a key role in its persistence. Epidemiological data regarding HEV circulation have resulted as necessary, and biomolecular analysis represents an important means of monitoring and establishing preventive measures. A multidisciplinary approach could provide a wide perspective regarding HEV and infectious implications on human, animal, and environmental health.

Hepatitis E virus detection in hunted wild boar (Sus scrofa) livers in Central Italy.

Hepatitis E virus (HEV) is a zoonotic pathogen, responsible for numerous cases of infection in humans. Transmission occurs through the orofecal route, and ingestion of contaminated foods represents an important risk factor for final consumer's health. Wild animal species, in particular wild boar (Sus scrofa), are the main virus reservoirs; liver is the target organ, from which, through the hematic diffusion, HEV reaches different tissues and organs, as muscular one. The hygienic-sanitary critical issues connected with game meat food chain in general, and particularly wild boar, with special regards to any geographical area where this animal species can be directly in contact with humans, domestic ones (i.e., domestic pig), and other wild reservoirs (i.e., wild ruminants), finds favorable environmental conditions, have induced us to conduce the present scientific investigation. During the hunting season 2019/2020, a total of 156 wild boar livers were collected from provided plucks at slaughterhouse in Ascoli Piceno. Nested RT-PCR was used for the viral RNA detection. Results demonstrated a positivity of 5.12% (8/156), and the circulation in the screened area of genotype 3 subtype c, which is frequently identified in Central Italy. HEV sanitary relevance and the emerging role of any food chains in its transmission impose further detailed studies. The molecular screening of hunted wild boars' livers can provide important information about virus's circulation in wild animal populations in a specific area.

Telemedicine Applications in the Era of COVID-19: Telesurgery Issues.

Telemedicine allows for the effective delivery of health care to patients at a distance through the application of information technology to the field of medicine. This is optimal during the COVID-19 pandemic to reduce interpersonal contact to mitigate contagion. Among the possible Telemedicine applications, there is Telesurgery, which involves more and more surgical specialties thanks to the numerous benefits in quality and cost containment. In the growing field of Telesurgery, its technical and legal implications must be considered. In this study, a traditional review of the scientific literature was carried out to identify the most relevant issues of interest in Telesurgery. The problematic legal aspects identified are mainly related to the difference in legislation between different geographical areas, which is critical in the case of malpractice. In addition, there is the possibility of a malicious hacker attack on the transmitted data stream either to steal sensitive data or to harm the patient. Finally, there are inherent difficulties with the technology used, such as latency issues in data transmission. All these critical issues are currently not adequately addressed by current legislation. Therefore, one can only hope for a legislative action to allow Telesurgery to be used safely.

Histomorphological aspects of cadaveric skin and its possible use in forensic genetics.

The skin is rarely considered as good biological material for successful DNA typing when a corpse is found in a leathery, mummified or partially skeletonised state, as bones and teeth are the gold standard in these cases. This study evaluates the histomorphological aspects of nuclear chromatin (Lillie's staining) in leathery and mummified skin samples as an indicator for possible successful DNA typing. Chromatin was found in samples that underwent mummification or partial skeletonisation but not in samples in a wet type of post-mortem transformation, such as saponification or leathery transformation. As a preliminary result, a positive detection of DNA profiles was only observed in 1-year-old mummified or partially skeletonised samples. These findings suggest that specific areas of skin, even from severely deteriorated cadavers, can show nuclear chromatin and DNA. These preliminary results raise the potential use of skin samples as an alternative source of DNA in highly degraded corpses.

Evaluation of critical aspects in clinical and forensic management of sexual violence: A multicentre Ge.F.I. project.

Violence against women is a violation of human rights, crossing all cultures, classes, levels of education, earnings, ethnic and age groups. We conducted a retrospective study to review forensic records of sexual assault examinations carried out in different Italian health facilities and to correlate these findings with the results of the forensic DNA analyses. The goal was to determine which factors could have affected the obtained results, to identify the fundamental aspects to search for while examining a sexual assault victim in order to gather useful evidence to identify the offender and reconstruct the dynamics of the fact. We analysed 102 cases that occurred between 2006 and 2017, coming from ten participating laboratories. Despite a relatively limited number of cases, this study shows that the ability to ascertain the presence of male biological material in the samples collected is not a problem for forensic laboratories and seems to be influenced by other factors, such as how much time elapsed between the event and the sampling, the availability of the aggressor's biological material on the victim and the identification of biological fluids/stains. Therefore, the need for health structures to adopt specific protocols has been highlighted. It is necessary for health structures to define specific pathways and adopt homogeneous procedures or operational protocols, and it is essential to provide adequate training for health personnel. The results of the study could be useful in drafting and revising protocols/guidelines implemented in Italian hospital. Issues related to the limited number of analyses requested by Italian Authorities are also discussed.

A Pilot Study on the Diagnosis of Fatal Electrocution by the Detection of Myocardial Microhemorrhages.

In electrocutions, death may be caused by alterations in the heart conduction system provoking ventricular fibrillation. This study aims to identify histological cardiac markers of high- and low-voltage electrocution. Two groups of decedents were evaluated: group A included 14 fatalities caused by high- or low-voltage electrocution and group B (control) included 14 fatalities due to other traumatic or disease causes. Myocardial sampling with microscopic examination was performed on all the hearts using the hematoxylin and eosin and Masson's trichrome stains to investigate morphological characteristics that could indicate the damage caused by high- and low-voltage electrocutions. Interstitial myocardial hemorrhagic infiltration was the only differentiating finding, which was shown only in high-voltage electrocution. This pathological finding has not been previously reported, and it may be specific to high-voltage electrocution deaths. Further studies are warranted.

Loss of alpha-smooth muscle actin expression associated with chronic intestinal pseudo-obstruction in a young Miniature Bull Terrier.

BACKGROUND: Chronic intestinal pseudo-obstruction (CIPO) is a rare clinical syndrome in veterinary medicine characterized by severe intestinal dysmotility without evidence of mechanical occlusion of the intestinal lumen. The exact pathogenesis of CIPO is unknown. CASE PRESENTATION: A 1-year-old male Miniature Bull Terrier dog was presented with a history of chronic weight loss, regurgitation, lethargy, vomiting and diarrhea. The dog was submitted for exploratory laparotomy. A full thickness intestinal biopsy was taken and a CIPO was suspected. The clinical condition deteriorated and the dog was euthanized. At gross examination the small intestine was severely dilated. Histologically severe fibrosis of the submucosa and severe atrophy of the tunica muscularis were present in small intestine and colon. Immunohistochemical examination with a panel of antibodies for gastro-intestinal neuromuscular disease-associated antigens revealed a severely reduced expression of alpha-smooth muscle actin in the tunica muscularis. CONCLUSIONS: This case report describes the gross, histological and immunohistochemical findings of CIPO affecting a 1-year-old Miniature Bull Terrier; on the basis of these findings a myopathic form of CIPO is hypothesized in this case.

A particularly complex case of matricide by means of plastic bag suffocation: Case report.

A rare case of homicide with plastic bag suffocation is presented in which forensic genetic investigations were carried out on the inner surface of a plastic bag placed over the head of an elderly woman, bedridden after a stroke. The results obtained suggested that she had been murdered and hinted at the perpetrator of the crime. In fact, it emerged that biological traces left by the victim matched those of her principal caregiver, her psychotic daughter, who later confessed to the crime. The old woman also had a son affected by a serious illness, whose genetic profile was found on the same bag. In a later interview, he stated that his sister had tried to kill him too, thus confirming the genetic findings and allowing the investigators to hypothesise that the daughter had used the same means to try to kill him. Based on these results, the usefulness of forensic genetic testing when investigating complex plastic bag suffocation deaths is highlighted. This peculiar case deserves interest, since no murder of this kind has ever been reported in the Italian forensic literature.

Overshoot during phenotypic switching of cancer cell populations.

The dynamics of tumor cell populations is hotly debated: do populations derive hierarchically from a subpopulation of cancer stem cells (CSCs), or are stochastic transitions that mutate differentiated cancer cells to CSCs important? Here we argue that regulation must also be important. We sort human melanoma cells using three distinct cancer stem cell (CSC) markers - CXCR6, CD271 and ABCG2 - and observe that the fraction of non-CSC-marked cells first overshoots to a higher level and then returns to the level of unsorted cells. This clearly indicates that the CSC population is homeostatically regulated. Combining experimental measurements with theoretical modeling and numerical simulations, we show that the population dynamics of cancer cells is associated with a complex miRNA network regulating the Wnt and PI3K pathways. Hence phenotypic switching is not stochastic, but is tightly regulated by the balance between positive and negative cells in the population. Reducing the fraction of CSCs below a threshold triggers massive phenotypic switching, suggesting that a therapeutic strategy based on CSC eradication is unlikely to succeed.

The molecular characterization of a depurinated trial DNA sample can be a model to understand the reliability of the results in forensic genetics.

The role of DNA damage in PCR processivity/fidelity is a relevant topic in molecular investigation of aged/forensic samples. In order to reproduce one of the most common lesions occurring in postmortem tissues, a new protocol based on aqueous hydrolysis of the DNA was developed in vitro. Twenty-five forensic laboratories were then provided with 3.0 µg of a trial sample (TS) exhibiting, in mean, the loss of 1 base of 20, and a molecular weight below 300 bp. Each participating laboratory could freely choose any combination of methods, leading to the quantification and to the definition of the STR profile of the TS, through the documentation of each step of the analytical approaches selected. The results of the TS quantification by qPCR showed significant differences in the amount of DNA recorded by the participating laboratories using different commercial kits. These data show that only DNA quantification "relative" to the used kit (probe) is possible, being the "absolute" amount of DNA inversely related to the length of the target region (r(2) = 0.891). In addition, our results indicate that the absence of a shared stable and certified reference quantitative standard is also likely involved. STR profiling was carried out selecting five different commercial kits and amplifying the TS for a total number of 212 multiplex PCRs, thus representing an interesting overview of the different analytical protocols used by the participating laboratories. Nine laboratories decided to characterize the TS using a single kit, with a number of amplifications varying from 2 to 12, obtaining only partial STR profiles. Most of the participants determined partial or full profiles using a combination of two or more kits, and a number of amplifications varying from 2 to 27. The performance of each laboratory was described in terms of number of correctly characterized loci, dropped-out markers, unreliable genotypes, and incorrect results. The incidence of unreliable and incorrect genotypes was found to be higher for participants carrying out a limited number of amplifications, insufficient to define the correct genotypes from damaged DNA samples such as the TS. Finally, from a dataset containing about 4500 amplicons, the frequency of PCR artifacts (allele dropout, allele drop-in, and allelic imbalance) was calculated for each kit showing that the new chemistry of the kits is not able to overcome the concern of template-related factors. The results of this collaborative exercise emphasize the advantages of using a standardized degraded DNA sample in the definition of which analytical parameters are critical for the outcome of the STR profiles.

World War One Italian and Austrian soldier identification project: DNA results of the first case.

We report the results of an attempt to identify the supposed remains of a famous World War I (WWI) Italian soldier who was killed in battle along the Italian front in 1915. Thanks to the availability of offspring from both paternal and maternal lineage Y-STRs and mtDNA were analysed and both showed a clear exclusion scenario: the remains did not belong to the supposed war hero. This is the first effort of identification of the remains of soldiers who perished during World War I within a multidisciplinary project aimed at the retrieval of historical and cultural aspects linked to WWI, and the systematic study of the remains of soldiers and ultimately their identification. This last step involves both Italian and Austrian laboratories.