Establishment of a random forest regression model to estimate the age of bloodstains based on temporal colorimetric analysis.

Bloodstain age estimation is important in forensic science. Although several studies have used spectroscopy to estimate bloodstain ages, this method has not yet been practically applied due to the need for expensive equipment and low reproducibility. Thus, we aimed to develop a bloodstain age estimation model that can be easily performed using a spectrophotometric colorimeter. First, bloodstains were prepared by placing blood obtained from five healthy volunteers on a plastic plate. The bloodstains were kept on conditions with various brightness and temperatures. Then, each bloodstain was dissolved in saline every 24 h to a final concentration of 1%, measured with a spectrophotometric colorimeter, and subjected to machine learning to generate a random forest regression (RFR) model, and finally, the prediction accuracy of the bloodstain age was verified. We also elucidated the mechanism of the color changes utilizing aminoguanidine, which is an inhibitor of Maillard reaction. Finally, we measured the time-dependent color changes of the blood fluids obtained from healthy volunteers and examined if the method could be potentially applied to estimate postmortem interval (PMI). Our results showed that the RFR model estimated the bloodstain age with no substantial assessment, and it was applicable to bloodstains, regardless of the brightness or temperature. The color changes were affected by the addition of aminoguanidine. Furthermore, the method could be applied to blood fluids, suggesting its potential usefulness for PMI estimation. Considering its feasibility, the present method could potentially be introduced to practical forensic sciences in the near future.

Establishing a Sequencing Method for the Whole Mitochondrial DNA of Domestic Dogs.

In human beings, whole mitochondrial DNA (mtDNA) sequencing has been widely used in many research fields, including medicine, forensics, and genetics. With respect to the domestic dog (Canis lupus familiaris), which is commonly recognized as being an additional member of the traditional human family structure, research studies on mtDNA should be developed to expand and improve our collective knowledge of dog medicine and welfare as it seems that there is still room for further development in these areas. Moreover, a simple and robust method for sequencing whole mtDNA that can be applied to various dog breeds has not yet been described in the literature. In the present study, we aim to establish such a method for the whole mtDNA sequencing of the domestic dog. In the experiments we conducted, oral mucosa DNA samples obtained from six Japanese domestic dogs were used as a template. We designed four primer pairs that could amplify approximately 5 kbp from each region of the mtDNA and validated several PCR conditions. Subsequently, the PCR amplicons were pooled and subjected to library preparation. The sequencing of the libraries was performed using next-generation sequencing (NGS), followed by bioinformatics analysis. Our results demonstrate that the proposed method can be used to perform highly accurate resequencing. We believe that this method may be useful for future research conducted to better understand dog medicine and welfare.

Usefulness of virtual reassembly of the skull and spine in cases of fragmentation due to high-energy trauma: A feasibility study.

With the widespread use of postmortem computed tomography (PMCT) beside forensic autopsies for investigation of causes of death, three-dimensional (3D) reconstruction and fusion imaging using PMCT data are now becoming common. In the present study, the applicability of virtual reassembly from PMCT data was investigated in three cases involving fragmentation of the skull or spine due to high-energy trauma, as in such cases it is sometimes difficult to obtain detailed information on fractures using macroscopic observation alone. In the first case, virtual reassembly of the skull provided more information about the fractures than conventional reconstruction with adhesive. In the second case, although the skull was severely fractured and could not be examined macroscopically, virtual reassembly allowed detailed visualization of the fractures. In the last case, virtual reassembly of the spine helped to clarify that the 6th-8th thoracic vertebrae had been run over by a vehicle at the scene. Thus, virtual reassembly was shown to be useful for assessment of injury patterns, and event reconstruction.

Postmortem genetic analysis of 17 sudden cardiac deaths identified nonsense and frameshift variants in two cases of arrhythmogenic cardiomyopathy.

Sudden death, or unexpected natural death of a healthy individual, is a serious problem in all nations. Sudden cardiac death (SCD) mainly due to ischemic heart diseases is the top cause of sudden death. However, there are pathophysiological conditions, referred to as sudden arrhythmic death syndrome, in which no apparent lesion can be identified even after complete conventional or ordinary autopsy. While postmortem genetic analyses have accumulated evidence about underlying genetic abnormality in such cases, the precise relationships between genetic background and the phenotype have been largely elusive. In this study, a retrospective investigation of 17 autopsy cases in which lethal arrhythmia was suspected to be the cause of death was carried out. Genetic analysis focusing on 72 genes reported to be associated with cardiac dysfunctions was performed, in combination with detailed histopathological and postmortem imaging examination, and a family study. As a result, in two cases of suspected arrhythmogenic cardiomyopathy (ACM), we found a nonsense variant in PKP2 and frameshift variant in TRPM4 gene. In contrast, the other 15 cases showed no morphological changes in the heart despite the presence of a frameshift variant and several missense variants, leaving the clinical significance of these variants obscure. The findings of the present study suggest that nonsense and frameshift variants could be involved in the morphological abnormality in cases of SCD due to ACM, while missense variants alone rarely contribute to massive structural changes in the heart.

Acute subdural hematoma caused by rupture of a mycotic aneurysm due to meningitis associated with infectious endocarditis: comparison of autopsy findings with postmortem computed tomography.

Forensic pathologists often encounter cases of acute subdural hematoma (SDH) due to trauma, whereas those attributable to endogenous causes are rare. Here, we report a case of the latter type in a 42-year-old man who was found dead at home after several months of fever and malaise. Postmortem computed tomography (PMCT) and autopsy were undertaken to clarify the cause of death. PMCT images revealed a fatal SDH and a localized hyper-density area in the right parietal lobe; macroscopic and microscopic examinations revealed SDH due to rupture of a mycotic aneurysm (MA) associated with meningitis. The PMCT images also indicated thickening and calcification of the mitral valve, while autopsy demonstrated infective endocarditis (IE). In addition, PMCT demonstrated a low-density area in the spleen, which was shown to be a splenic abscess at autopsy. PMCT also demonstrated tooth cavities. Based on the findings of autopsy, the cause of death was considered to be SDH due to rupture of the MA resulting from meningitis with IE and splenic abscess. Although PMCT was unable to clarify the significance of any individual feature, a retrospective review of the PMCT images might have suggested IE, bacteremia, or ruptured MA leading to SDH. This case suggests that, instead of interpreting individual features demonstrated on PMCT images, integrated interpretation of overall PMCT findings might provide clues for identifying causes of death, despite the fact that PMCT lacks diagnostic accuracy for infectious diseases such as IE and meningitis.

Emergence of an erythroid cell-specific regulatory region in ABO intron 1 attributable to A- or B-antigen expression on erythrocytes in Hominoidea.

A- and B-antigens are present on red blood cells (RBCs) as well as other cells and secretions in Hominoidea including humans and apes such as chimpanzees and gibbons, whereas expression of these antigens on RBCs is subtle in monkeys such as Japanese macaques. Previous studies have indicated that H-antigen expression has not completely developed on RBCs in monkeys. Such antigen expression requires the presence of H-antigen and A- or B-transferase expression in cells of erythroid lineage, although whether or not ABO gene regulation is associated with the difference of A- or B-antigen expression between Hominoidea and monkeys has not been examined. Since it has been suggested that ABO expression on human erythrocytes is dependent upon an erythroid cell-specific regulatory region or the + 5.8-kb site in intron 1, we compared the sequences of ABO intron 1 among non-human primates, and demonstrated the presence of sites orthologous to the + 5.8-kb site in chimpanzees and gibbons, and their absence in Japanese macaques. In addition, luciferase assays revealed that the former orthologues enhanced promoter activity, whereas the corresponding site in the latter did not. These results suggested that the A- or B-antigens on RBCs might be ascribed to emergence of the + 5.8-kb site or the corresponding regions in ABO through genetic evolution.

Hypothermic death resulting from extreme freezing with characteristic postmortem computed tomography findings: A case report and review of the literature.

We report a case of hypothermic death that resulted from extreme freezing, with characteristic postmortem computed tomography (PMCT) findings. A 75-year-old man died in a deeply frozen state. In PMCT, there was a lack of increase in the bilateral lung-field attenuation. Urinary retention, with a hypodense area of frozen urine, was observed in the bladder. Changes that appeared to involve the crystallization of serum in frozen blood were observed in the aorta. Based on the scene and his circumstances, it was speculated that he died of hypothermia. Present case and our review revealed that although PMCT findings from hypothermic death that resulted from deep freezing are very rare, the characteristic PMCT findings may help determine the cause of death.

Post-traumatic cerebral infarction caused by thrombus in the middle cerebral artery.

A woman in her 80s was found unconscious after being hit by a car while crossing a road. After admission to hospitals, computed tomography (CT) scans revealed traumatic brain injury (TBI), and the patient was treated symptomatically. However, despite improvement of TBI in CT images, she died unexpectedly. Postmortem CT demonstrated cerebral infarction in the territory of the right middle cerebral artery (MCA). Histopathological examination revealed lumen-obstructing thrombosis and intimal injury upstream of the thrombosis in the right MCA. These findings suggested that the intimal injury in the MCA had led to thrombus formation, and thromboembolism in the region distal to the injury leading to post-traumatic cerebral infarction (PTCI). Both postmortem CT and autopsy were able to reveal the final condition of the deceased, which had not been fully anticipated by the clinicians who had treated her after the accident. The longitudinal antemortem to postmortem course revealed by multiple CT images and the histopathological examination provided crucial clues to the pathogenesis of PTCI in this case.

Development of a detector tube for screening tadalafil and its analogues in adulterated sexual enhancement products.

Sexual enhancement products adulterated with phosphodiesterase 5 inhibitors (PDE-5i) pose a serious public health concern. Tadalafil and its analogues (Tds) are PDE-5i frequently detected as adulterants. In this study, a Td detector tube for the rapid detection of Tds was developed based on the color change reaction between sulfuric acid and Tds. The specificity of this test method was evaluated using 13 Tds, all of which elicited positive results. Additionally, 30 commonly found adulterants in dietary supplements, 11 active pharmaceutical ingredients of psychotropic drugs and 18 food ingredients were tested and obtained no false-positive results, except levomepromazine. The test tube accurately detected the presence or absence of Tds in 54 commercially available products. The visual detection limit was 2-50 and 5-20 µg/ml for Tds and tadalafil-spiked samples with matrix, respectively. The applicability of the developed detector tube to a semiquantitative test using digital image analyses were investigated using red, green, and blue color values. The results of the recovery test suggested that the tube test was affected by the dark-colored matrix. The results of semiquantitative analyses of tadalafil for five marketed products were consistent with the liquid chromatographic quantification results, except for the blue value. The detector tube developed in this study can facilitate with the rapid screening of Tds in adulterated sexual enhancement products.

Usefulness of a tissue optical clearing technique for forensic autopsy.

Forensic pathologists are required to investigate lethal trauma or disease at autopsy. In addition to massive contusions of various organs, a number of small features with potentially fatal implications also need to be sought. Since such lesions may need microscopic examinations for detailed evaluation, it is important to select suitable anatomic locations for tissue sampling. For practical screening of small lesions, we have developed a tissue optical clearing (TOC) technique for forensic autopsy. The technique involves clearing with a non-toxic organic solvent, ethyl cinnamate, which renders excised organs transparent, while hemorrhages or blood-containing vessels remain opaque. Using this technique, tiny hemorrhages in the spinal cord were able to be identified by gross examination, allowing proper selection of locations for tissue sampling. Subsequent histopathological evaluation was successfully performed with no apparent artifacts related with the TOC procedure. In addition, a combination of TOC and targeted CT angiography allowed feasible examination of the arterial occlusive lesion in the superior mesenteric artery, and when combined with micro-CT scanning it was useful for evaluating the lumen of the coronary artery with stent implantation. The results obtained so far indicated that TOC could complement routine forensic autopsy procedures when detailed evaluation of small lesions is required.

Reduction of blood group A antigen on erythrocytes in a patient with myelodysplastic syndrome harboring somatic mutations in RUNX1 and GATA2.

BACKGROUND: Reduction of blood group ABO antigens on red blood cells (RBCs) is well known in patients with leukemias, and this reduction of ABO expression is strongly associated with DNA methylation of the ABO promoter. Previously, we reported a two-nucleotide deletion in RUNX1 encoding an abnormally elongated protein lacking the trans-activation domain in a patient with myelodysplastic syndrome (MDS) showing A-antigen loss on RBCs. This prompted us to investigate the underlying mechanism responsible for A-antigen reduction on RBCs in another patient with MDS. STUDY DESIGN AND METHODS: Screening of somatic mutations was carried out using a targeted sequencing panel with genomic DNA from peripheral blood mononuclear cells from the patient and eleven MDS controls without A- or B-antigen loss. DNA methylation of the ABO promoter was examined by bisulfite genomic sequencing. Transient transfection assays were performed for functional evaluation of mutations. RESULTS: Screening of somatic mutations showed missense mutations in RUNX1 and GATA2 in the patient, while no mutation was found in exons of those genes in the controls. There was no significant difference in ABO promoter methylation between the patient and the controls. Transient transfection experiments into COS-7 and K562 cells suggested that the amino acid substitutions encoded by those mutations reduced or lost the trans-activation potential of the ABO expression. CONCLUSION: Considering the discrepancy between the variant frequencies of these mutations and the ratios of the RBCs with A-antigens loss, the antigen reduction might be associated with these somatic mutations and hypermethylation of the ABO promoter.

Characteristic postmortem computed tomography findings of ingestion of benzine.

There are some reports investigating the cause of death by examining the contents of the stomach and duodenum using postmortem computed tomography, but most of these have been based on radiopaque contents. Here, we report a case of suicide after ingesting a large amount of benzine. Although the gastric contents were radiolucent, the characteristic postmortem computed tomography imaging findings helped to determine the cause of death.

Investigation of the applicability of virtual gastroscopy based on postmortem computed tomography to detect changes in the stomach, along with reports of three rare cases.

Postmortem computed tomography is now being used more commonly for routine forensic investigation. The use of 3D reconstruction techniques including virtual gastroscopy is effective and also improves the speed of interpretation, recognition, and description of specific clinical conditions. However, it has been unclear whether postmortem virtual endoscopy could be applicable for medicolegal autopsy or whether it could complement pathological examination at autopsy. Here, we investigated the applicability of postmortem virtual gastroscopy by reviewing 295 medicolegal autopsy cases seen at our institution, and found four cases in which the technique had been able to demonstrate features corresponding to changes that were evident at autopsy. Thus,postmortem virtual gastroscopy would have only rarely been effective forvisualizing any change in the stomach in such cases. In addition, we describe in detail three of those cases in which virtual gastroscopy had been able to visualize changes in the stomach, including a gastric ulcer, a polyp, and the presence of foamy fluid, which were all verified at autopsy. In those cases, virtual gastroscopy was useful for understanding features in the stomach of the deceased, which were revealed by axial images of the abdomen, to forensic pathologists who were not familiar with PMCT 2D images. Taken together, our findings suggest that postmortem virtual gastroscopy might help facilitate clear, straightforward sharing of information about PMCT images of complex anatomical structures among radiologists and forensic pathologists, as well as non-medical professionals with a limited knowledge of anatomy and physiology.

A cell-specific regulatory region of the human ABO blood group gene regulates the neighborhood gene encoding odorant binding protein 2B.

The human ABO blood group system is of great importance in blood transfusion and organ transplantation. ABO transcription is known to be regulated by a constitutive promoter in a CpG island and regions for regulation of cell-specific expression such as the downstream + 22.6-kb site for epithelial cells and a site in intron 1 for erythroid cells. Here we investigated whether the + 22.6-kb site might play a role in transcriptional regulation of the gene encoding odorant binding protein 2B (OBP2B), which is located on the centromere side 43.4 kb from the + 22.6-kb site. In the gastric cancer cell line KATOIII, quantitative PCR analysis demonstrated significantly reduced amounts of OBP2B and ABO transcripts in mutant cells with biallelic deletions of the site created using the CRISPR/Cas9 system, relative to those in the wild-type cells, and Western blotting demonstrated a corresponding reduction of OBP2B protein in the mutant cells. Moreover, single-molecule fluorescence in situ hybridization assays indicated that the amounts of both transcripts were correlated in individual cells. These findings suggest that OBP2B could be co-regulated by the + 22.6-kb site of ABO.

Single molecule in situ hybridization reveals distinct localizations of schizophrenia risk-related transcripts SNX19 and AS3MT in human brain.

Genome-wide association studies have identified single nucleotide polymorphisms (SNPs) associated with schizophrenia risk. Integration of RNA-sequencing data from postmortem human brains with these risk SNPs identified transcripts associated with increased schizophrenia susceptibility, including a class of exon 9-spliced isoforms of Sorting nexin-19 (SNX19d9) and an isoform of Arsenic methyltransferase (AS3MT) splicing out exons 2 and 3 (AS3MTd2d3). However, the biological function of these transcript variants is unclear. Defining the cell types where these risk transcripts are dominantly expressed is an important step to understand function, in prioritizing specific cell types and/or neural pathways in subsequent studies. To identify the cell type-specific localization of SNX19 and AS3MT in the human dorsolateral prefrontal cortex (DLPFC), we used single-molecule in situ hybridization techniques combined with automated quantification and machine learning approaches to analyze 10 postmortem brains of neurotypical individuals. These analyses revealed that both pan-SNX19 and pan-AS3MT were more highly expressed in neurons than non-neurons in layers II/III and VI of DLPFC. Furthermore, pan-SNX19 was preferentially expressed in glutamatergic neurons, while pan-AS3MT was preferentially expressed in GABAergic neurons. Finally, we utilized duplex BaseScope technology, to delineate the localization of SNX19d9 and AS3MTd2d3 splice variants, revealing consistent trends in spatial gene expression among pan-transcripts and schizophrenia risk-related transcript variants. These findings demonstrate that schizophrenia risk transcripts have distinct localization patterns in the healthy human brains, and suggest that SNX19 transcripts might disrupt the normal function of glutamatergic neurons, while AS3MT may lead to disturbances in the GABAergic system in the pathophysiology of schizophrenia.

Histone deacetylase inhibitors suppress ACE2 and ABO simultaneously, suggesting a preventive potential against COVID-19.

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide as a pandemic throughout 2020. Since the virus uses angiotensin-converting enzyme 2 (ACE2) as a receptor for cellular entry, increment of ACE2 would lead to an increased risk of SARS-CoV-2 infection. At the same time, an association of the ABO blood group system with COVID-19 has also been highlighted: there is increasing evidence to suggest that non-O individuals are at higher risk of severe COVID-19 than O individuals. These findings imply that simultaneous suppression of ACE2 and ABO would be a promising approach for prevention or treatment of COVID-19. Notably, we have previously clarified that histone deacetylase inhibitors (HDACIs) are able to suppress ABO expression in vitro. Against this background, we further evaluated the effect of HDACIs on cultured epithelial cell lines, and found that HDACIs suppress both ACE2 and ABO expression simultaneously. Furthermore, the amount of ACE2 protein was shown to be decreased by one of the clinically-used HDACIs, panobinostat, which has been reported to reduce B-antigens on cell surfaces. On the basis of these findings, we conclude that panobinostat could have the potential to serve as a preventive drug against COVID-19.

Superimposed CT imaging using fusion function to visualize the relationship between the knife and the wound path in a stabbing victim.

With the increasing use of postmortem computed tomography (PMCT) in medicolegal autopsies, three-dimensional (3D) models of injured areas can now be generated from multislice computed tomography images. However, since PMCT has low sensitivity for detecting injuries in solid organs in the absence of contrast administration, it has been difficult to demonstrate the tracks of stab wounds leading to solid organ injury using 3D reconstruction. Here, we report one homicide case with two stab wounds. On the skin surface, the stab wounds were located on the neck and anterior chest wall. A medicolegal autopsy revealed that one stab wound in the neck had penetrated the wall of the right pleural cavity and the upper portion of the right lung whereas the other stab wound in the anterior chest wall had penetrated the right diaphragm and the heart. To illustrate the tracks of the stab wounds, superimposed CT images of the body, the excised organ, and a knife model were constructed to obtain a 3D model. This allowed clear and concise visualization of the complex relationship of the knife to the heart incision and the stab wound on the chest surface.

dotdotdot: an automated approach to quantify multiplex single molecule fluorescent in situ hybridization (smFISH) images in complex tissues.

Multiplex single-molecule fluorescent in situ hybridization (smFISH) is a powerful method for validating RNA sequencing and emerging spatial transcriptomic data, but quantification remains a computational challenge. We present a framework for generating and analyzing smFISH data in complex tissues while overcoming autofluorescence and increasing multiplexing capacity. We developed dotdotdot (https://github.com/LieberInstitute/dotdotdot) as a corresponding software package to quantify RNA transcripts in single nuclei and perform differential expression analysis. We first demonstrate robustness of our platform in single mouse neurons by quantifying differential expression of activity-regulated genes. We then quantify spatial gene expression in human dorsolateral prefrontal cortex (DLPFC) using spectral imaging and dotdotdot to mask lipofuscin autofluorescence. We lastly apply machine learning to predict cell types and perform downstream cell type-specific expression analysis. In summary, we provide experimental workflows, imaging acquisition and analytic strategies for quantification and biological interpretation of smFISH data in complex tissues.