Susceptibility to recombinant SARS-CoV-2 spike protein entry in the lungs of high-fat diet-induced obese mice.

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Obesity is a major risk factor for the development of COVID-19. Angiotensin-converting enzyme 2 (ACE2) is an essential receptor for cell entry of SARS-CoV-2. The receptor-binding domain of the S1 subunit (S1-RBD protein) in the SARS-CoV-2 spike glycoprotein binds to ACE2 on host cells, through which the virus enters several organs, including the lungs. Considering these findings, recombinant ACE2 might be utilized as a decoy protein to attenuate SARS-CoV-2 infection. Here, we examined whether obesity increases ACE2 expression in the lungs and whether recombinant ACE2 administration diminishes the entry of S1-RBD protein into lung cells. We observed that high-fat diet-induced obesity promoted ACE2 expression in the lungs by increasing serum levels of LPS derived from the intestine. S1-RBD protein entered the lungs specifically through ACE2 expressed in host lungs and that the administration of recombinant ACE2 attenuated this entry. We conclude that obesity makes hosts susceptible to recombinant SARS-CoV-2 spike proteins due to elevated ACE2 expression in lungs, and this model of administering S1-RBD protein can be applied to new COVID-19 treatments.

Double suicide in Japan in the post-war reconstruction period, with reference to contemporary Japan.

After Japan's post-war reconstruction, in the early 1950s, cases of double suicide, in which two people (particularly young lovers) leave a suicide note and die together, were a relatively frequent occurrence. During the three-year period between 1954 and 1956, 5466 suicides were recorded in the special wards of Tokyo, including 79 cases of double suicides, accounting for 158 deaths. In these double suicide cases (2.89% of all deaths by suicide), the evidence revealed that 65.8% involved lovers and 29.1% involved married couples. By contrast, contemporary data indicate a large drop in suicide pacts between lovers to 15.9% and an increase between spouses to 48.8%. Conceivably, the relatively high double-suicide rate after post-war reconstruction reflected difficulties for the younger generation in reconciling 'marriage based primarily on love' and the traditional family system, specifically marriage problems and stress caused by rapidly changing post-war values. One notable difference between victims of double suicide in 1954-1956 and the contemporary period is the younger average age of the former. Another important shift was found in the most common causes of death among victims of double suicide: in 1954-1956 these were poisoning by cyanide or hypnotic drugs, compared to carbon monoxide poisoning and hanging in modern times. We discuss similarities and differences concerning double suicides in relation to social and economic conditions in Japan in the 1950s and today.

Assessing age-related heart changes by comparing cases of sudden death during bathing and control cases in Japan.

We analyzed 400 deaths that occurred in the bathtub during a 10-year period in the central area of Kanagawa prefecture in Japan. There were 72 (18%) medico-legal autopsy cases. The average age at death was 76.4 ± 11.9 years. Drowning (n = 21, 70.8%) was the most common cause of death in the 72 autopsy cases. The study examined the bodies of 40 cases within a postmortem interval of 3 days. The mean age of the 40 cases of sudden death during bathing was 68.6 ± 12.5 years. Results revealed cardiac hypertrophy in 12 cases (30%), lipofuscin deposition in 39 cases (97.5%), basophilic degeneration in 12 cases (30%), anisocytosis of the nucleus of myocardial cells in 18 cases (45%), perivascular fibrosis in 17 cases (42.5%), amyloid deposits in 1 case, and aortic valve calcification in 1 case. The hearts of control subjects who had lived to 20-99 years were also examined; the frequency of each change was higher in people older than 70 years. There was no statistically significant difference in age-related cardio-pathological changes between cases of sudden death during bathing in people in their 70s and controls in their 70s. It can be concluded that this age-related histopathological index is not related to sudden death during bathing. A large number of elderly people, including those without heart disease, have died during bathing. Preventive measures against sudden death during bathing are strongly recommended, e.g., elderly people should not be left totally unsupervised while they bathe.

Rare primary peritoneal mucinous adenocarcinoma in a 69-year-old man.

Primary peritoneal mucinous adenocarcinoma is rare in men. The low-grade tumor consisted of mucin-producing columnar cells with minimal nuclear atypia. Relationship to pseudomyxoma peritonei and disseminated peritoneal adenomucinosis is discussed.

Individual identification using the RapidHIT™ ID system for forensic samples.

The RapidHIT™ ID system produces GlobalFiler™ analysis results after a short operating time. This device is effective because it automatically extracts DNA from oral mucosal cells or from blood stains and saliva collected at a crime scene, with subsequent polymerase chain reaction performed to produce a DNA profile. Two types of dedicated cartridges are available for RapidHIT™ ID: the RapidHIT™ ID ACE GlobalFiler Express sample cartridge for oral cells and other samples and the RapidINTEL™ sample cartridge for minute samples, such as blood stains. Previously validated specimens include oral mucosa cells and blood stains left at crime scenes. There have been no reports of blood and nail clipping samples collected from the postmortem bodies at the time of death. This report summarizes the results of using the RapidHIT™ ID system by collecting a variety of actual forensic samples from postmortem bodies at different stages of decomposition, which were subsequently analyzed using these cartridges.

Emergence and Characterization of Acute Coronary Syndrome in Adults After Confirmed or Missed History of Kawasaki Disease in Japan: A Japanese Nationwide Survey.

Background: Acute coronary syndrome (ACS), which is emerging in adults long after confirmed (followed-up or lost-to-follow), or missed Kawasaki disease (KD), is poorly characterized. Methods and Results: A Japanese retrospective nationwide hospital-based questionnaire survey of ACS during 2000-09 was conducted to characterize such patients. Among a total of 67 patients (median age 35, male 76%) recruited, low conventional coronary risks (≤1/6) was noted in 75%, a diagnosis of ST-elevation and myocardial infarction or cardiac arrest in 66%, medication before ACS in 22% (warfarin in 4%), and no prior history of acute myocardial infarction in 94%. One-month mortality was 19%. KD diagnosis was made in 32 during acute illness (Group A), in which 17 were lost to follow, and retrospectively in the other 35 from coronary imaging at ACS (Group B). Group A developed ACS at lower coronary risks (≤2/5 in 87 vs. 65% in group B, p = 0.043) at a younger age (26.5 vs. 40 yo, p < 0.001). In group A, followed-up patients developed ACS under medication before ACS (87 vs. 0% in lost-to-follow patients, p < 0.001) for giant aneurysm in culprit lesions (69 vs. 29%, p = 0.030). One-month mortality was comparable between groups A and B, and between patients followed-up and lost-to-follow in group A. The culprit lesion in group A was characterized by the association of an aneurysm ≥6 mm in acute KD (100%), lack of significant stenosis (61%) or giant aneurysm (50%) in the long-term (median interval 16 y), and the presence of intravascular ultrasound-derived calcification at ACS (86%). Conclusions: The present retrospective nationwide questionnaire survey demonstrated nationwide emergence of initial ACS in young adults at low coronary risks, who are followed-up or lost-to-follow after confirmed KD and initial coronary aneurysms ≥6 mm.

Sudden Infant Death After Vaccination: Survey of Forensic Autopsy Files.

Sudden infant deaths might be attributable to adverse reaction to vaccination, but separating them from coincidental occurrences is difficult. This study retrospectively investigated vaccination-related details and postmortem findings for 57 cases of sudden death in children 2 years or younger. Data were extracted from autopsy files at the Department of Forensic Medicine, Tokai University School of Medicine. Vaccination histories were available in 50 cases based on the maternity passbook. Of the 32 cases in which any vaccines were administered, 7 infants (21.9%) had received immunization within 7 days of death. The most frequent vaccine cited as the last immunization before death was Haemophilus influenzae B. Although a temporal association of vaccines with sudden death was present for two 3-month-old and one 14-month-old infants in whom death occurred within 3 days of receiving the H. influenzae type b and other vaccinations, a definitive relationship between the vaccine and death could not be identified. Histopathological examinations revealed pneumonia and upper respiratory infection as contributing to death in their cases. Moreover, all 3 cases showed hemophagocytosis in the spleen and lymph nodes, which are similar features to hemophagocytic lymphohistiocytosis. Judgment of the disorders as truly related to vaccination is difficult, but suspicious cases do exist. Forensic pathologists must devote more attention to vaccination in sudden infant death cases.

Extensive gastric necrosis secondary to acute gastric dilatation: A case report.

We report a case of sudden death in a patient who developed extensive gastric necrosis secondary to acute gastric dilatation. A 36-year-old man with mental retardation (but without difficulties in activities of daily living), developed an illness after a meal out with friends, necessitating 3 hospital visits. He returned home after receiving drug therapy; however, his condition deteriorated, and he was transferred to our hospital via ambulance. Whole-body computed tomography performed upon admission revealed gastric dilatation. A stomach tube was inserted, and 2000 mL of gastric aspirate was obtained. The patient died approximately 5 h later despite receiving treatment. Autopsy revealed 1000 mL of gastric contents and extensive gastric necrosis. He was diagnosed with extensive gastric necrosis secondary to acute gastric dilatation.

Fulminant group A streptococcal infection without gangrene in the extremities: Analysis of five autopsy cases.

Five autopsy cases of fulminant group A streptococcal infection without gangrene in the extremities are presented. Clinical course of the fulminant illness was short (2-4 days). One pathological autopsy case was aged (86-years-old), and hemorrhagic cystitis was observed. The other four forensic autopsy cases were young (24-38 years-old) with the mean age of 32, and the primary infective lesions were located in the postpartum endometrium, tonsil and bronchus (2 cases). Systemic coccal dissemination with poor neutrophilic reaction was seen in two of five cases. Bilateral renal cortical necrosis was noted in three cases (including two with bacterial embolism). Hemophagocytosis, probably resulting from hypercytokinemia, was characteristic in three cases without bacterial embolism. Gram-positive cocci colonizing the hemorrhagic and necrotizing lesions were consistently immunoreactive for streptococcal antigens and Strep A (a carbohydrate antigen on group A streptococci). Neutrophilic reaction was mild in the primary infected foci. Clinicians should note that fulminant streptococcal infection (streptococcal toxic shock syndrome) in young and immunocompetent patients may not be associated with gangrene in the extremities. Autopsy prosecutors (diagnostic and forensic pathologists) must recognize the difficulty in making an appropriate autopsy diagnosis, particularly when bacterial embolism is not associated.

Cytokine Elevation in Sudden Death With Respiratory Syncytial Virus: A Case Report of 2 Children.

Respiratory syncytial virus (RSV) is the most common viral cause of bronchiolitis and pneumonia in young children worldwide. Premature birth, bronchopulmonary dysplasia, congenital heart disease, and Down syndrome are risk factors for high mortality and prolonged morbidity after RSV infection. Conversely, many previously healthy, full-term children are also admitted to the hospital because of RSV, and some of them experience severe sequelae or die due to the virus. Various complications of RSV infection have been reported, such as encephalopathy, encephalitis, and cardiomyopathy. However, the pathogenesis of serious cases in children without an underlying disease has not been elucidated. In this report, we present 2 RSV-related deaths of children who were born at full-term and developed normally up to the age of 19 months. Their cardiopulmonary arrests occurred within half a day after the onset of symptoms, such as cough and high fever. Many postmortem examinations were performed to investigate their unexpected deaths. Histopathological examinations revealed extensive bronchiolitis and mild pneumonia accompanying airway obstruction. Immunostaining revealed the presence of the virus mainly in bronchial epithelia, but not in alveoli. Complete brain edema was prominent, and encephalopathy was developing. Blood tests revealed that the IL-6 level was elevated more than >200-fold above normal, despite a normal C-reactive protein level. Because IL-6 may reflect the severity of bronchial epithelial damage and contribute to brain edema, an extreme elevation of IL-6 may predict the risk for sudden death in children with RSV infection.

Trend of homicide-suicide in Kanagawa Prefecture (Japan): Comparison with western countries.

We examined 169 deceased persons and 76 homicide-suicide cases reported in Japan's Kanagawa Prefecture from 1999 to 2011. The relationships between homicide-suicide perpetrators and homicide victims; the numbers of victims; their age, sex, causes and places of death; motivation; and the presence or absence of a suicide note were extracted and examined. The relationship between homicide-suicide perpetrators and homicide victims was examined based on findings from the following: 24 married couples (31%), 22 parents and children aged ≥18 years (29%), 19 parents and children aged ≤17 years (25%), seven families (9%), two couples (3%) and two miscellaneous relationships (3%). The perpetrators comprised 39 men and 40 women, with a mean age of 51.6 years. The victims comprised 39 men and 51 women, with a mean age of 35.4 years. In our study, approximately half of the perpetrators were female, which differed greatly from the reports from Western countries, where most perpetrators were male. Homicide-suicides among married couples accounted for a higher proportion of overall homicide-suicide deaths in Western countries. In Japan, homicide-suicide occurred more frequently with parents and children. Cases in which a mother committed suicide after having killed her young children accounted for a high proportion of these deaths. Because these events occur so frequently in Japan, we recommend making particular efforts to reduce homicide-suicides among mothers and children.

Evaluation of Y chromosomal SNP haplogrouping in the HID-Ion AmpliSeq™ Identity Panel.

The Y chromosomal haplogroup determined from single nucleotide polymorphism (SNP) combinations is a valuable genetic marker to study ancestral male lineage and ethical distribution. Next-generation sequencing has been developed for widely diverse genetics fields. For this study, we demonstrate 34 Y-SNP typing employing the Ion PGM™ system to perform haplogrouping. DNA libraries were constructed using the HID-Ion AmpliSeq™ Identity Panel. Emulsion PCR was performed, then DNA sequences were analyzed on the Ion 314 and 316 Chip Kit v2. Some difficulties became apparent during the analytic processes. No-call was reported at rs2032599 and M479 in six samples, in which the least coverage was observed at M479. A minor misreading occurred at rs2032631 and M479. A real time PCR experiment using other pairs of oligonucleotide primers showed that these events might result from the flanking sequence. Finally, Y haplogroup was determined completely for 81 unrelated males including Japanese (n=59) and Malay (n=22) subjects. The allelic divergence differed between the two populations. In comparison with the conventional Sanger method, next-generation sequencing provides a comprehensive SNP analysis with convenient procedures, but further system improvement is necessary.

Self-inflicted Firearm Discharge from Heating Using a Gas Burner.

A male in his 70s was found lying dead in the living room of his house. A gunshot entrance wound was observed in the left orbit, with a lead slug and wadding left in the skull, which exhibited fatal cranio-cerebral trauma. A cartridge had been discharged from a handmade launcher, or zip gun, that had been fixed to a spare gun barrel on a pipe chair, by heating the launcher from the side using a gas burner. The deceased had owned guns for hunting in the past and had returned the license, but he had retained a spare barrel and live cartridges at home. In this unique case of suicide, a zip gun was discharged by heating with a gas burner.

Effects of using the GlobalFiler™ multiplex system on parent-child analyses of cases with single locus inconsistency.

Parent-child analyses sometimes reveal inconsistency of shared alleles at only one locus. This is conventionally called "single locus exclusion", which results from mutational events and the presence of null alleles. Here, in parent-child analyses of the Japanese population, we detected exclusions by using the GlobalFiler™ system comprising 21 short tandem repeat loci. One- or two-step mutations resulting from strand slippage causing gain or loss were observed in seven of 221 parent-child transmissions. The incidences of single locus inconsistency of alleles were 5.88×10(-2) and 8.40×10(-3) for paternal and maternal relationships, respectively. With calculation using a set of 15 loci in the Identifiler® multiplex system, the combined likelihood ratio (CLR) values were limited to less than 100 in all five cases accompanied by single inconsistency. The addition of six loci recovered the CLR values to over 10,000 in three cases. Application of this advanced system may increase the detected occurrence of mutational events, but it should be beneficial for inference in parent-child analyses, particularly in cases accompanied by genetic inconsistency.

Allelic Imbalance of mRNA Associated with α2-HS Glycoprotein (Fetuin-A) Polymorphism.

Alpha 2-HS glycoprotein (AHSG), also designated as fetuin-A, exhibits polymorphism in population genetics consisting of two major alleles of AHSG(∗) 1 and AHSG(∗) 2. The serum level in the AHSG(∗) 1 homozygote is significantly higher than that of the AHSG(∗) 2 homozygote. This study examined the molecular mechanism for the cis-regulatory expression. To quantitate allele-specific mRNA in intra-assays of the heterozygote, RT-PCR method employing primers that were incorporated to the two closely located SNPs was developed. The respective magnitudes of AHSG(∗) 1 to AHSG(∗) 2 in the liver tissues and hepatic culture cells of PLC/PRF/5 were determined quantitatively as 2.5-fold and 6.2-fold. The mRNA expressional difference of two major alleles was observed, which is consistent with that in the serum level. The culture cells carried heterozygous genotypes in rs4917 and rs4918, but homozygous one in rs2248690. It was unlikely that the imbalance was derived from the SNP located in the promotor site. Furthermore, to investigate the effect of mRNA degradation, RNA synthesis in the cell culture was inhibited potently by the addition of actinomycin-D. No marked change was apparent between the two alleles. The results indicated that the cis-regulatory expressional difference is expected to occur at the level of transcription or splicing of mRNA.

MicroRNA Stability in Postmortem FFPE Tissues: Quantitative Analysis Using Autoptic Samples from Acute Myocardial Infarction Patients.

MicroRNAs (miRNAs) are very short (18-24 nucleotides) nucleic acids that are expressed in a number of biological tissues and have been shown to be more resistant to extreme temperatures and pH compared to longer RNA molecules, like mRNAs. As miRNAs contribute to diverse biological process and respond to various kinds of cellular stress, their utility as diagnostic biomarkers and/or therapeutic targets has recently been explored. Here, we have evaluated the usefulness of miRNA quantification during postmortem examination of cardiac tissue from acute myocardial infarction (AMI) patients. Cardiac tissue was collected within one week of the patient's death and either frozen (19 samples) or fixed in formalin for up to three years (36 samples). RNA integrity was evaluated with an electropherogram, and it appears that longer RNAs are fragmented after death in the long-term fixed samples. Quantitative PCR was also performed for seven miRNAs and three other small RNAs in order to determine the appropriate controls for our postmortem analysis. Our data indicate that miR-191 and miR-26b are more suitable than the other types of small RNA molecules as they are stably detected after death and long-term fixation. Further, we also applied our quantitation method, using these endogenous controls, to evaluate the expression of three previously identified miRNA biomarkers, miR-1, miR-208b, and miR-499a, in formalin-fixed tissues from AMI patients. Although miR-1 and miR-208b decreased (1.4-fold) and increased (1.2-fold), respectively, in the AMI samples compared to the controls, the significance of these changes was limited by our sample size. In contrast, the relative level of miR-499a was significantly decreased in the AMI samples (2.1-fold). This study highlights the stability of miRNAs after death and long-term fixation, validating their use as reliable biomarkers for AMI during postmortem examination.

Self-inflicted fatal shotgun wound from a homemade weapon.

A case of suicide, a male shot in the abdomen using a homemade weapon, is described herein. Postmortem examination revealed bleeding into the thoracic and abdominal cavities, a collapsed liver and other organs with numerous pellets. The contact-shot wound was not noticed during the initial investigation at the scene, but it was elucidated during autopsy. A simple launcher had been assembled from pipes of a clothes rack frame made for home use. The deceased had once owned a shotgun, but his firearm license had been relinquished 5 years prior. A shotgun shell that he had kept was used. Presumably, the injuries from the homemade weapon were not noticed initially because of strict gun control rules in Japan.

"Dead in hot bathtub" phenomenon: accidental drowning or natural disease?

Sudden death in a hot bathtub occurs frequently in Japan, particularly among elderly people. This retrospective report describes the epidemiologic circumstances and physical findings at autopsy. In total, 268 victims were found unconscious or dead during tub bathing. After postmortem examination, the manner of death was judged as natural cause in 191 (71.2%) and accidental drowning in 63 (23.5%) cases. Mean age (SD) was 72.1 (15.2) years with no significant difference between males and females. A seasonal difference was evident: the winter displayed the highest frequency. Drowning water inhalation, which was confirmed in 72% of victims, was absent in the others. The most common observations on postmortem examination were cardiac ischemic changes and cardiomegaly. Water inhalation signs were evident in a significantly fewer victims exhibiting these factors. In contrast, inhalational findings were observed more frequently in victims with other backgrounds such as alcohol intake, mobility disturbance, and history of epilepsy. Annual mortality in Japan from accidental drowning in persons aged older than 75 years is 33 deaths per 100,000 population. However, this number may be considerably underestimated as pathologists tend to regard lack of water inhalation as indicating a natural cause of death. Confusion in diagnosis remains consequent to the accidental and natural aspects of "dead in hot bathtub" phenomenon.

Evaluation of the allele-sharing approach, known as the IBS method, in kinship analysis.

To infer relatedness from genetic data based on short tandem repeats, the exact method, in which shared allele frequencies are applied to relevant equations, has been conventionally used. An alternative approach is the IBS method that is based on the number of shared alleles between individuals. In the present study, the performance of the IBS method in pairwise kinship analysis was compared with the exact method using simulated data of 10,000 genotype pairs for 15 loci in the ABI Identifiler system. The likelihood ratio in allele-sharing of zero, one and two was calculated from joint probabilities based on allele frequencies of the Japanese population. Whereas the IBS method generally produced lower values of combined indices, smaller deviations of the distributions were evident. The threshold for identification of full siblings relative to non-relatives was comparable with that of the exact method, indicating that both inference powers were almost identical. The likelihood ratio in the IBS method depends on the heterozygosity at a locus, and heterozygosities of the 15 loci were consistent across various population groups, particularly in East Asians. The convenience of fixed LR values in the IBS method is beneficial for cases with uncertain allele frequencies and rare alleles.