Estimating bloodstain age in the short term based on DNA fragment length using nanopore sequencer.

We used a nanopore sequencer to quantify DNA fragments > 10,000 bp in size and then evaluated their relationship with short-term bloodstain age. Moreover, DNA degradation was investigated after bloodstains were wetted once with water. Bloodstain samples on cotton gauze were stored at room temperature and low humidity for up to 6 months. Bloodstains stored for 1 day were wetted with nuclease-free water, allowed to dry, and stored at room temperature and low humidity for up to 1 week. The proportion of fragments > 20,000 bp in dry bloodstains tended to decrease over time, particularly for fragments > 50,000 bp in size. This trend was modeled using a power approximation curve, with the highest R2 value (0.6475) noted for fragments > 50,000 bp in size; lower values were recorded for shorter fragments. The proportion of longer fragments was significantly reduced in bloodstains that were dried after being wetted once, and there was significant difference in fragments > 50,000 bp between dry conditions and once-wetted. This result suggests that even temporary exposure to water causes significant DNA fragmentation, but not extensive degradation. Thus, bloodstains that appear fresh but have a low proportion of long DNA fragments may have been wetted previously. Our results indicate that evaluating the proportion of long DNA fragments yields information on both bloodstain age and the environment in which they were stored.

Development of an Okinawa panel for biogeographic inference of Okinawans.

BACKGROUND: The Precision ID Ancestry Panel with 165 SNP markers was unable to differentiate between mainland Japanese and Okinawa Japanese or to distinguish either of them from other East Asian populations. AIM: An Okinawa panel was developed with the aim of further separating Okinawa Japanese individuals from mainland Japanese and other Asian groups. Seventy-five SNPs were selected using the most informative markers from the literature. Further, 22 SNPs were selected to separate Okinawa Japanese at minimum SNPs. SUBJECTS AND METHODS: Samples were collected from 48 unrelated individuals from mainland Japan and 46 unrelated residents of the Okinawa prefecture. Data were evaluated by STRUCTURE, principal component, and GenoGeographer analyses. RESULTS: The 22 SNP set had similar levels of differentiation in STRUCTURE and PCA analyses as the 75 SNP set. GenoGeographer analysis showed that, out of the 46 Okinawa Japanese individuals, the 75 SNP and 22 SNP sets correctly assigned the Okinawan population as the most likely population of origin for 32 and 31 individuals, respectively. CONCLUSION: Neither SNP set could completely differentiate between Okinawa Japanese and other Asian groups, however, these sets should be useful for crime investigation, when the sample, cost and time are limited.

Effect existence of aging on stutter ratio evaluated via Bayesian inference.

The stochastic behavior of the stutter ratio (SR) in capillary electrophoresis-based DNA typing is currently described and predicted using statistical models in forensic genetics. Clarifying this behavior can help obtain more objective and robust evidence to the court in terms of mixture interpretation. This study aimed to investigate the effect existence of aging on SR via a Bayesian framework. Nail scrapings and clippings were collected from 68 healthy individuals with informed consent. Samples were classified by age-class: young group (0-16 years; n = 36) and older-adult group (>61 years; n = 32). Then, they were compared in terms of their SRs for each simple repeat locus included in GlobalFiler Kit. Bayesian modeling was performed with lognormal distribution model, which implemented multiple linear regression, allele and age-class as explanatory variables. For all simple repeat loci, the median of the posterior distribution of the age-class parameter was a positive value. For CSF1PO and D7S820, the 95% credible interval of the posterior distribution did not include 0. Our data suggested that aging slightly increases the SR. These findings might help elucidate the stochastic behavior of SR.

Simplified detection of the species of origin of antler velvets using single-stranded tag hybridization chromatographic printed-array strip.

In this study, we developed a convenient and easy-to-use origin identification method for antler velvets based on a simple DNA extraction technique and single-stranded tag hybridization chromatographic printed-array strip (STH-PAS). The primer sets used to detect Cervus elaphus, Rangifer tarandus, and 12S rRNA did not engage in non-specific reactions such as primer dimer formation. In both the triplex and singleplex assays, the sensitivity was < 1 ng DNA. Moreover, Cervus elaphus DNA could be detected in OTC crude drug products. Although the detection sensitivity resulting from the simplified extraction was slightly lower than that obtained with extraction by conventional methods, the amount of DNA was sufficient even from a small sample. The choice of a triplex or singleplex assay will depend on the purpose of the test. For example, if it is important to determine whether the antler velvet is derived from Cervus elaphus or Rangifer tarandus, a triplex assay is appropriate. If it is necessary to explore whether antler velvet from Cervus elaphus is included in an OTC crude drug product, a singleplex assay using the Cervus elaphus primer set is informative. If it is necessary to explore whether powdered antler velvet includes counterfeit products (from Rangifer tarandus), a singleplex assay employing the Rangifer tarandus primer is appropriate. The singleplex assay detects minor components even at a 1,000:1 ratio. Our study thus demonstrated the utility of a method combining simple DNA extraction with STH-PAS for efficient identification of the origin of antler velvets.

A method for determining the origin of crude drugs derived from animals using MinION, a compact next-generation sequencer.

We evaluated whether MinION, an inexpensive portable sequencer, can be used to identify the origin of crude drugs derived from animals. Standard and nonstandard crude drugs with different species of origin were examined. In addition, standards mixed with nonstandard samples were used. As a target gene, cytochrome c oxidase I was amplified and sequenced. The fast mode results had a slightly lower match ratio than high-accuracy mode, but the animals of origin were correctly determined by BLAST for all samples. For antler velvet derived from Rangifer tarandus, even when the sequences were aligned based on Cervus elaphus, the animal of origin was determined correctly. Minor contents could be detected from mixtures of two animals, if the mixtures contained at least 19:1 mtDNA when the coverage allele-fraction threshold was 0.05. By contrast, in fast mode, two sequences could not be separated due to the low accuracy of the base-calling for each read. For fieldwork, the species of origin of crude drugs could be identified with only simple DNA extraction and library preparation. Therefore, MinION appears to be a convenient tool for identifying the origins of crude drugs derived from animals.

Highly sensitive sex determination method using the exon 1 region of the amelogenin gene.

Sex determination is a crucial factor in the identification of unidentified human remains. Sex determination by DNA analysis is particularly useful because it can be applied to samples for which morphological characteristics are unavailable. Because samples handled in forensic DNA typing are easily degraded by environmental factors and microorganisms, there is a need for a method that can accurately determine sex even in highly decayed samples. Previous studies mainly used sex differences in an intron of the amelogenin gene. However, this region is highly polymorphic, and there are cases where accurate sexing cannot be performed because of genetic mutations in the target region. Thus, for reliable sex determination, it is desirable to select loci with as few non-sexual polymorphisms as possible. In this study, we focused on the exon 1 region of the amelogenin gene, which has very little polymorphism other than sex differences. We developed a primer set for sex determination and compared it with the GlobalFiler™ PCR Amplification Kit (GF), which is widely used for forensic DNA typing. The results showed that the amount of DNA required for accurate sex determination was 25 pg for both methods, achieving equivalent sensitivity. Next, we compared the two methods using ancient human skeletons and found that the present method with its shorter amplicon was considerably superior to GF. The present method is simple, rapid, inexpensive, and suitable for analyzing highly degraded samples. Therefore, this method is expected to contribute to forensic sciences and physical anthropology.

Autopsy findings of post-COVID-19 vaccination deaths in Tokyo Metropolis, Japan, 2021.

BACKGROUND: COVID-19 vaccines have been used across Japan since 17 February 2021, and as of 17 April 2022, 1690 deaths potentially caused by vaccine-related adverse effects have been reported to the Ministry of Health, Labour and Welfare. However, the causal relationship between vaccination and death could not be fully evaluated because of a lack of sufficient information. METHODS: Autopsy cases in which deaths occurred within seven days after COVID-19 vaccination in Tokyo Metropolis and were handled by medical examiners were selected (n = 54). Age, sex, vaccine-related information, cause of death, and possible causal relationship between vaccination and death were examined. RESULTS: The mean age of the deceased individuals was 68.1 years, and the study sample consisted of 34 males (63.9%) and 20 females (37.0%). Thirty-seven and six individuals received Comirnaty and Spikevax, respectively (68.5% and 11.1% respectively). The manner of death included natural (n = 43), non-natural (n = 8), and undetermined (n = 3). The most frequent cause of death was ischemic heart disease (n = 16). Regarding causal relationships, 46 cases (85.2%) did not show a causal relationship to vaccination, except for myocarditis (n = 3), thrombosis-related death (n = 4), and others (n = 1). CONCLUSION: Although many cases of deaths after COVID-19 vaccination in this study showed no definite causal relationship between the vaccination and deaths, some cases showed possible adverse events such as myocarditis. Autopsies are essential for detecting vaccine-related deaths, and the Japanese death investigation system needs to be reinforced from this viewpoint.

Diagnostic significance of the histopathology of bone marrow macrophages in forensic autopsies.

Forensic pathologists often encounter autopsies that require an assessment of antemortem general conditions (e.g., infection, metabolic disorders). To establish evaluation clues for such cases, we quantitatively examined macrophages and the general pathology of bone marrow in samples from 180 forensic autopsy cases of decedents with various conditions. Hematoxylin-eosin staining, Berlin blue staining, and immunostainings for CD163, CD138, and CD61 were performed. We determined the numbers per field (density) of total macrophages, swollen macrophages, macrophages with hemophagocytosis, and hemosiderin-laden macrophages. Each density was standardized by identifying its ratio to the total number of macrophages. The decedents' background data (cause of death, other pathological findings, postmortem interval, antemortem symptoms, and presence of resuscitation) were extracted. No correlations were found between the postmortem interval and the other decedent data, indicating that these data are not affected by postmortem changes. In the group in which inflammatory disease was the cause of death, there were significant elevations in the ratio of the swollen macrophage density to total macrophages. Significantly higher ratios of the density of swollen and hemophagocytic macrophages were observed in the group in which conditions with a prolonged agonal period were the cause of death. The group with a return of spontaneous circulation to resuscitation showed a significantly higher ratio of macrophage density with hemophagocytosis. This study provides the first statistical analysis focused on bone marrow histopathology in forensic autopsies. The results will be useful for elucidating causes of death and agonal-period conditions.

A sudden infant death associated with atrial septal aneurysm and abnormality of the connecting site between the inferior vena cava and right atrium.

An atrial septal aneurysm (ASA) is a rare cardiac anomaly characterized by varicose bulgingofthe atrial septum (oval fossa) into the left or right atrium. Pathogenesis and clinical significance of ASA are controversial. We report an autopsy case of a huge undiagnosed ASA with abnormality of the connecting site between the inferior vena cava and the right atrial ostium in a 2-month-oldJapanesefemale who died suddenly and unexpectedly. She was born at 36 weeks 4 days (body weight 3,110 g). No abnormality was detected during pregnancy or delivery. The postnatal growth was normal with no cardiac problem detected at the 1-month checkup. The ASA bulged off in a mass to the left atrium (width, 0.8 cm; excursion ratio, 53%), reaching close to the inflow site of the right pulmonary vein, with dilation of the pulmonary vein. The connecting site between the inferior vena cava and the right atrium was atypically located 1.6 cm away from the atrioventricular groove. Although most cases of ASA in an infant resolve physiologically as the infant grows, the infant in the present case is thought to have had an exceptional pathological ASA, possibly causing supraventricular arrhythmia. The abnormality of the connecting site between the inferior vena cava and the right atrium might have affected the development and continuation of the ASA.

Estimating individual mtDNA haplotypes in mixed DNA samples by combining MinION and MiSeq.

We tried to estimate individual mtDNA haplotypes in mixed DNA samples by combining MinION and MiSeq. The BAM files produced by MiSeq were viewed using Integrative Genomics Viewer (IGV) to verify mixed bases. By sorting the reads according to base type for each mixed base, partial haplotypes were determined. Then, the BAM files produced by MinKNOW were viewed using IGV. To determine haplotypes with IGV, only mixed bases determined by MiSeq were used as target bases. By sorting the reads according to base type for each target base, each contributor's haplotype was estimated. In mixed samples from two contributors, even a haplotype with a minor contribution of 5% could be distinguished from the haplotype of the major contributor. In mixed samples of three contributors (mixture ratios of 1:1:1 and 4:2:1), each haplotype could also be distinguished. Sequences of C-stretches were determined very inaccurately in the MinION analysis. Although the analysis method was simple, each haplotype was correctly detected in all mixed samples with two or three contributors in various mixture ratios by combining MinION and MiSeq. This should be useful for identifying contributors to mixed samples.

Unexpected death in a young child associated with anomalous aortic origin of the left main coronary artery without physical exertion: A case of an anomalous coronary artery with highly abundant elastic fibers.

Sudden death due to anomalous aortic origin of a coronary artery is far less common among young children in the absence of exercise stress. This report describes the case of a 2-year-old boy with a lower respiratory tract infection who suffered sudden cardiac arrest in his bed at home. The autopsy revealed that the left coronary artery (LCA) originated from the right sinus of Valsalva with an acute angle takeoff and traveled between the aorta and the pulmonary trunk (an interarterial course). Upon histological examination, the LCA, before reaching its major branches, was located adjacent to the outside of the aortic wall without an intramural passage, and the arterial wall was composed almost exclusively of elastic fibers without media containing smooth muscle cells throughout the entire length of the abnormal running. Screening tests for respiratory virus infection detected enterovirus in the lung tissue. In association with an acute angle takeoff and interarterial course, the wall structure with highly abundant elastic fibers that are more flexible tissues among blood vessel components might suggest their vulnerability to compression during the great vessels' systolic expansion in the sympathetic activation induced by the viral infection, leading to fatal myocardial ischemia without physical exertion.

Estimating included animal species in mixed crude drugs derived from animals using massively parallel sequencing.

We developed a method that can detect each animal species of origin for crude drugs derived from multiple animal species based on massively parallel sequencing analysis of mitochondrial genes. The crude drugs derived from animals investigated in this study were Cervi Parvum Cornu and Trogopterorum feces, which are derived from a mix of different animal species, two chopped cicada sloughs, and two commercial Kampo drugs. The mitochondrial 12S rRNA, 16S rRNA, and cytochrome oxidase subunit I gene regions were amplified and sequenced using MiSeq. The ratios of haplotype to total number of sequences reads were calculated after sequence extraction and trimming. Haplotypes that exceeded the threshold were defined as positive haplotypes, which were compared with all available sequences using BLAST. In the Cervi Parvum Cornu and Trogopterorum feces samples, the haplotype ratios corresponded roughly to the mixture ratios, although there was a slight difference from mixture ratios depending on the gene examined. This method could also roughly estimate the compositions of chopped cicada sloughs and Kampo drugs. This analysis, whereby the sequences of several genes are elucidated, is better for identifying the included animal species. This method should be useful for quality control of crude drugs and Kampo drugs.

Dissecting lesions in a culprit artery of a hemorrhagic focus in the basal ganglia: Histopathological analysis by serial sectioning.

In a hypertensive hemorrhagic focus of the basal ganglia, the culprit arteries have been reported to be associated with dissecting lesions, whose topographical relationship to the rupture sites remains to be clarified. Herein we describe multiple dissecting lesions in the culprit artery of hypertensive hemorrhage of the basal ganglia. A 1.0 × 0.8 × 0.8 cm-sized bleeding globe was confirmed at a left lenticulostriate artery and histologically analyzed by serial sectioning. Three independent dissecting lesions were identified in the culprit artery. They were situated near the bifurcations, ranging from 240 to 3200 µm in length. The dissections mainly occurred between the intima and media with disruption of the internal elastic lamina (IEL), forming a fresh thrombus within the false lumen. Two rupture sites causing the cerebral hematoma were confirmed away from the dissecting lesions. One was situated close but not adjacent to the longest dissecting lesion; the other, measuring approximately 150 µm in diameter, was adjacent to the bifurcation of an artery. The histopathological findings suggest that the dissecting lesion resulted from medial detachment following IEL disruption in the process of arterial rupture of the culprit artery. We conclude that this was a secondary manifestation during the rupture rather than a cause of the arterial rupture.

Modeling the minus two base pair stutter ratio of the D1S1656 locus: A sequence-based mixture distribution model.

In this study, we propose a stutter ratio for a minus two base pair stutter (-2bpSR) model of the D1S1656 locus in capillary electrophoresis (CE)-based short tandem repeat (STR) typing. DNA from a total of 108 Japanese individuals was analyzed via massively parallel sequencing to investigate the length of the longest uninterrupted stretch of two base repeat motif (2bpLUS value) within repetitive structures involving the flanking region. Additionally, -2bpSR data was collected using the GlobalFiler Kit on a 3500xL Genetic Analyzer. As a result of sequencing analysis, all alleles were classified into two types by their 2bpLUS values. The -2bpSR differed significantly between the types. Then, we modeled the -2bpSR with a mixture log-normal distribution using the classification of alleles based on the 2bpLUS values. Furthermore, probabilities of the sequence type within each repeat number in the mixture log-normal distribution model were estimated using logistic regression for each of the five major detected populations. This study is expected to enable interpretation of STR typing while considering minus two base pair stutter at the D1S1656 locus.

Bloodstain examination and DNA typing from hand-washed bloodstains on clothes.

We investigated whether bloodstain examination and DNA typing can be performed on washed bloodstains on clothes. Blood was dropped onto T-shirts made from 100% cotton or 100% polyester. After drying, the T-shirts were hand-washed with handwashing soap, dishwashing detergent, laundry detergent, soap, or just water until the bloodstains could not be seen. After drying the T-shirts, DNA and RNA were extracted simultaneously from the bloodstained areas using commercial kits. RNA was reverse-transcribed to DNA, and then the detection of the mRNAs for HBB, ACTB, and 18S rRNA was examined. DNA was quantified via real-time PCR, and then STR typing was performed with a commercial kit. The luminol and leucomalachite green tests were used as preliminary bloodstain tests, and an immuno-chromatography kit was used to identify human bloodstains. DNA could be extracted from all washed bloodstains, but more DNA was extracted from cotton T-shirts than from polyester T-shirts. STR typing was successful for all bloodstains without issues such as PCR inhibition. In the human bloodstain identification test using mRNA, almost all bloodstains produced a Ct value for HBB and all bloodstains produced a Ct value for 18S rRNA, whereas few bloodstains produced a Ct value for ACTB. All bloodstains reacted positively to luminol, but some were negative for leucomalachite green. Most of the bloodstains did not react positively in the human bloodstain identification test using the immuno-chromatography kit. The results suggest that human bloodstain identification and DNA typing can still be performed after clothes with bloodstains are washed.

The origin identification method for crude drugs derived from arthropods and annelids using molecular biological techniques.

We evaluated whether the origins of crude drugs derived from arthropods and annelids could be identified using molecular biological techniques. DNA was extracted from 20 crude drugs prepared from different animals using a commercial kit with added phenol treatment. The target regions used to identify origin were the mitochondrial 16S ribosomal RNA (rRNA), 12S rRNA, and cytochrome oxidase subunit I (COI) gene regions. Extracted DNA was amplified by polymerase chain reaction, and then sequenced by the Sanger method. The aligned sequences were compared with all available sequences using BLAST to estimate the origins of the crude drugs. The origin of crude drugs used in this study could be estimated using this method. The COI region was the best for identifying origin among three regions examined, based on the success rate of PCR amplification and analysis. Moreover, the 12S rRNA region was also useful for origin identification, with the exception of the earthworm. However, the origin of some crude drugs could not be strictly identified due to matches to various species in all three regions. One likely cause was that the species of origin of a crude drug has not been registered in DNA databases. We found that even the same crude drug from the same pharmaceutical company had different origins by production lot or import source country. Therefore, this method is useful not only for DNA-based origin identification but also quality control of production lots.

Estimation of the number of contributors to mixed samples of DNA by mitochondrial DNA analyses using massively parallel sequencing.

We evaluated whether the number of contributors to mixed DNA samples can be estimated by analyzing the D-loop of mitochondrial DNA using massively parallel sequencing. The A- (positions 16,209-16,400) and B- (positions 30-284) amplicons in hypervariable regions 1 and 2, respectively, were sequenced using MiSeq with 2 × 251 cycles. Sequence extraction and trimming were performed using CLC Genomics Workbench 11 and the number of observed haplotypes was counted for each amplicon type using Microsoft Excel. The haplotype ratios were calculated by dividing the number of counted reads of the corresponding haplotype by the total number of sequence reads. Haplotypes that were over the threshold (5%) were defined as positive haplotypes. The number of larger positive haplotypes in either of the two amplicon types was defined as the number of contributors. Samples were collected from seven individuals. Seventeen mixed samples were prepared by mixing DNA from two to five contributors at various ratios. The number of contributors was correctly estimated from almost all of the mixed samples containing equal amounts of DNA from two to five people. In mixed samples of two or three people, the minor components were detected down to a ratio of 20:1 or 8:2:1. However, heteroplasmy, base deletions, and sharing of the same haplotypes caused incorrect estimations of the number of contributors. Although this method still has room for improvement, it may be useful for estimating the number of contributors in a mixed sample, as it does not rely on forensic mathematics.

Anthracene-Attached Persistent Tricyclic Aromatic Hydrocarbon Radicals.

Anthracene-attached tricyclic aromatic hydrocarbon radicals having different central polygons, Ant-5, Ant-6, and Ant-7, were synthesized to evaluate the role of an anthracene substituent group in the stability and reactivity of tricyclic aromatic hydrocarbon radicals. The bulky anthryl group effectively protects a carbon atom with high spin density, resulting in high persistence of the radicals. On the other hand, the combination of the anthryl group and the tricyclic aromatic scaffold makes the molecular structure drastically change from a twisted form to a folded form and an unpaired electron moves into the anthryl moiety, eventually affording a tail-to-tail σ-dimer.

Establishment of widely applicable DNA extraction methods to identify the origins of crude drugs derived from animals using molecular techniques.

We established widely applicable DNA extraction methods to identify the origins of crude drugs derived from animals. Twenty-one samples including 17 kinds of crude drug derived from animals were examined. DNA was extracted from most of the crude drugs by adjustment of the QIAamp® DNA Mini Kit. DNA extraction was performed successfully using phenol to remove impurities after applying a proteinase treatment. DNA extraction was performed successfully by decalcification treatment using ethylenediaminetetraacetic acid (EDTA), before applying the proteinase treatment for crude drugs having high calcium content, such as those from oyster shell and cuttlefish bone. DNA could not be extracted from sea-ear shell using the EDTA decalcification treatment, but was extracted successfully using a TBONE EX KIT. The mitochondrial 16S ribosomal RNA (rRNA) gene region was amplified, and Basic Local Alignment Search Tool (BLAST) analysis was performed after sequencing. Polymerase chain reaction (PCR) products of approximately 600 bp in length were obtained from all samples except donkey glue, one of the two seahorses, and longgu. Drug origins were determined in all samples by sequence analysis based on the BLAST results, and match rates were >97 %. Moreover, 16 samples had a match rate >99 %. Our DNA extraction methods were widely applicable to evaluation of many crude drugs derived from animals, and proved very useful for identifying the origins of such drugs.

Fatal Intracranial Aneurysms and Dissections Causing Subarachnoid Hemorrhage: An Epidemiological and Pathological Analysis of 607 Legal Autopsy Cases.

BACKGROUND: There are no detailed reports, in terms of epidemiology and pathology, on intracranial aneurysms and on dissections that were found in unexpected fatal subarachnoid hemorrhage (SAH) cases. In this report we analyzed, based on large-sized medicolegal autopsy cases, the detailed epidemiology and pathological aspects of both lesions. METHODS: We analyzed 607 autopsy cases of unexpected fatal SAHs including 496 aneurysms and 111 dissections. RESULTS: The following results were obtained: (1) Patients who died of dissections were younger than those who died of aneurysms; (2) symptom prevalence rates of aneurysms were 31.9%, appearing to be lower than those in previous studies; (3) a significantly higher prevalence of clinical symptoms was found in patients with dissections (60.5%) than patients with aneurysms; (4) hypertensive cardiomegaly was indicated in more than 80%, while no obvious difference in incidence in hypertensive cardiomegaly was noted between aneurysms and dissections; (5) aneurysms were found to occur much more frequently in the anterior communicating artery (31.9%) and vertebral arteries (7.5%), while dissections were found much more commonly in vertebral arteries (93.7%); and (6) the size of aneurysms was much smaller in general than that previously regarded as a risk factor of rupturing. CONCLUSIONS: These data might help in the prompt intervention in SAH and also in the prevention of lethal SAH in clinical settings.