Inositol pyrophosphate profiling reveals regulatory roles of IP6K2-dependent enhanced IP7 metabolism in the enteric nervous system.

Inositol pyrophosphates regulate diverse physiological processes; to better understand their functional roles, assessing their tissue-specific distribution is important. Here, we profiled inositol pyrophosphate levels in mammalian organs using an originally designed liquid chromatography-mass spectrometry (LC-MS) protocol and discovered that the gastrointestinal tract (GIT) contained the highest levels of diphosphoinositol pentakisphosphate (IP7) and its precursor inositol hexakisphosphate (IP6). Although their absolute levels in the GIT are diet dependent, elevated IP7 metabolism still exists under dietary regimens devoid of exogenous IP7. Of the major GIT cells, enteric neurons selectively express the IP7-synthesizing enzyme IP6K2. We found that IP6K2-knockout mice exhibited significantly impaired IP7 metabolism in the various organs including the proximal GIT. In addition, our LC-MS analysis displayed that genetic ablation of IP6K2 significantly impaired IP7 metabolism in the gut and duodenal muscularis externa containing myenteric plexus. Whole transcriptome analysis of duodenal muscularis externa further suggested that IP6K2 inhibition significantly altered expression levels of the gene sets associated with mature neurons, neural progenitor/stem cells, and glial cells, as well as of certain genes modulating neuronal differentiation and functioning, implying critical roles of the IP6K2-IP7 axis in developmental and functional regulation of the enteric nervous system. These results collectively reveal an unexpected role of mammalian IP7-a highly active IP6K2-IP7 pathway is conducive to the enteric nervous system.

Proteomic profiling of sudden cardiac death with acquired cardiac hypertrophy.

BACKGROUND: Cardiac hypertrophy, which develops in middle-aged and older individuals as a consequence of hypertension and obesity, is an established risk factor for sudden cardiac death (SCD). However, it is sometimes difficult to differentiate SCD with acquired cardiac hypertrophy (SCH) from compensated cardiac hypertrophy (CCH), at autopsy. We aimed to elucidate the proteomic alteration in SCH, which can be a guideline for future postmortem diagnosis. METHODS: Cardiac tissues were sampled at autopsy. SCH group consisted of ischemic heart failure, hypertensive heart failure, and aortic stenosis. CCH group included cases of non-cardiac death with cardiac hypertrophy. The control group comprised cases of non-cardiac death without cardiac hypertrophy. All patients were aged > 40 years, and hypertrophic cardiomyopathy was not included in this study. We performed histological examination and shotgun proteomic analysis, followed by quantitative polymerase chain reaction analysis. RESULTS: Significant obesity and myocardial hypertrophy, and mild myocardial fibrosis were comparable in SCH and CCH cases compared to control cases. The proteomic profile of SCH cases was distinguishable from those of CCH and control cases, and many sarcomere proteins were increased in SCH cases. Especially, the protein and mRNA levels of MYH7 and MYL3 were significantly increased in SCH cases. CONCLUSION: This is the first report of cardiac proteomic analysis in SCH and CCH cases. The stepwise upregulation of sarcomere proteins may increase the risk for SCD in acquired cardiac hypertrophy before cardiac fibrosis progresses significantly. These findings can possibly aid in the postmortem diagnosis of SCH in middle-aged and older individuals.

Correlation of cytomegalovirus viral load between whole blood and plasma of congenital cytomegalovirus infection under valganciclovir treatment.

BACKGROUND: Congenital cytomegalovirus (CMV) infection (cCMV) can cause sensorineural hearing loss and neurodevelopmental disabilities in children. Oral valganciclovir (VGCV) therapy has been reported to improve long-term audiological and neurodevelopmental outcomes in patients with cCMV. The levels of CMV DNA in whole blood have been monitored in previous studies. However, quantitative methods using whole blood have not been standardized. Recently, the plasma viral load has been standardized and widely used in CMV-associated diseases. METHODS: CMV viral loads in whole blood and plasma were serially measured in 24 patients with a confirmatory diagnosis of cCMV during oral VGCV therapy using an in-house real-time PCR assay. Plasma samples were assayed using the Cobas 6800 system (Roche Diagnostics) in addition to an in-house assay. RESULTS: Plasma CMV viral loads were remarkably decreased at the end of therapy compared to before therapy. A significant correlation of CMV levels between whole blood and plasma was observed (Spearman's ρ = 0.566). The levels of CMV DNA before therapy were significantly correlated with the period of decreasing the viral loads to below the detection limit, not only in whole blood (Spearman's ρ = 0.901) but also in plasma (Spearman, ρ = 0.804). Finally, CMV viral loads between the in-house assay and commercially available standardized assay in 75 plasma samples with positive PCR results for CMV were compared; a significant correlation was observed between the results of both assays. CONCLUSIONS: There was a significant correlation between the two assays (Spearman, ρ = 0.882), suggesting that CMV plasma viral loads measured by the standardized assay are widely used to monitor the levels of CMV DNA in patients with cCMV during oral VGCV therapy.

Gastric aspiration in sudden unexpected infant death of Prader-Willi syndrome: immunohistochemical detection of feeding components.

Prader-Willi syndrome (PWS) in infants is characterized by hypotonia and poor sucking with feeding difficulties. Two autopsy cases of sudden unexpected death during sleep after tube feeding are described herein. For one, gastric aspiration caused by the possible milk regurgitation was suspected. Immunohistochemical examination of lung sections was performed using three antibodies to human α-lactalbumin, human gross cystic disease fluid protein 15, and cow whey ß-lactoglobulin. Five cases of sudden unexpected infant death occurring earlier than at 6 months old were selected as controls. Marked immune-staining for infant formula in one PWS subject was evident within terminal bronchioles and alveoli with granular and amorphous features. However, no positive staining was apparent in the other subject, who exhibited contrasting features in milk distribution. Among control cases, one showed mild staining in the bronchiole, but the others did not. The antibody to ß-lactoglobulin reacted specifically with formula, with no nonspecific background. Gastric contents in the airway can be a difficult issue because of the consequent terminal gasping. However, because of an episode of antemortem symptoms of potential regurgitation, and from findings at autopsy such as petechiae, we inferred that fatal regurgitation occurred in this PWS infant after tube feeding. Several clinical reports have described milk aspiration, but this pathological report is the first related to aspiration in PWS during tube feeding.

An instance of homicide by electrocution with hand-made electrode plates.

Homicide by electrocution is rare in forensics, and the identification of the cause of death can be quite difficult when the electric device is removed from the scene. We present an instance where the police were unsure of homicide in the initial investigation. The offender used hand-made electrode plates for electrocution, which produced unique electric marks different from those produced by common electric devices such as electric wires. To the best of our knowledge, this is the first report of homicide by electrocution with electrode plates. We believe that the macroscopic and microscopic findings in this instance are quite valuable for forensic practitioners.

Y chromosome analysis for common surnames in the Japanese male population.

For many years of Japan's long history, Japanese surnames have been handed down patrilineally. This study investigated relations between major surnames and Y chromosomal polymorphism among the Japanese male population. To analyze genetic phylogeny in namesakes, the Y-single nucleotide polymorphism (SNP) plus Y-short tandem repeat (STR) approach was employed. A haplogroup based on SNPs and haplotypes at 17 STR loci were typed in 567 unrelated volunteers recruited in Kanagawa, Japan. Samples covered 27 common surnames such as Satoh and Suzuki, each name having 10-55 bearers. Significant difference was found for SNP haplogroup compositions and a multidimensional scaling plot using STR haplotypes in several surname groups. By contrast, these common surnames displayed wide diversity with phylogenetic networks, suggesting that no genetic drift event has occurred in their history. In all, 22 descent clusters were found, as judgcriteria ed by ad hoc of groups within five mutational steps in the 15 STR loci with the same haplogroup. The times of the most recent common ancestor ranged from 279 to over 2577 years. According to the approximate millennium span of Japanese surname history, descent criteria are expected to be reasonable for grouping within four step-neighbors. High heterogeneity of common surnames resembles that observed for England and Spain, but not for Ireland. Our results highlight that common Japanese surnames consist of descent clusters and many singletons, reflecting a mixture of long-term bearers and short-term bearers among the population. The genetic study of this population revealed characteristic features of Japanese surnames.

MicroRNA profiling of gastric content from breast-fed and formula-fed infants to estimate last feeding: a pilot study.

Recently, we were consulted about a challenging case, where an infant died by poisoning and the drug-dependent mother insisted that she unintentionally gave the toxic drug through breast milk. Accordingly, we investigated the utility of immunoblotting and microRNA (miRNA) profiling of the infant's gastric content (GC) to differentiate between breast-feeding and formula-feeding. As a pilot study, we sampled the GC from breast-fed (GCB) and formula-fed (GCF) infants, as well as gastric juice (GJ) from fasted adults at autopsy. Breast milk (BM) samples were collected from volunteers within 1 year post-delivery. By immunoblotting, lactoferrin and gross cystic disease fluid protein (GCDEP) were clearly detected in BM, but could not be detected in GCB. Similarly, ß-lactoglobulin was detected in formula milk, but could not be detected in GCF. Meanwhile, miRNA sequencing revealed that the miRNA expression profile of GCB was closer to BM than GCF and GJ. Especially, miR-151a and miR-186 were more abundant in BM and GCB than in GCF and GJ. Our study is the first to elucidate the human GJ miRNA profile and demonstrate the possibility that miR-151a and miR-186 in GC may be the biomarker of breast-feeding.

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.

A descriptive study of solitary death in Yokohama City.

BACKGROUND: The solitary death rate in Japan is expected to continue increasing because of its growing super-aged society and the rapid growth of home care in the country. To accurately determine the actual status of solitary deaths, we used a novel analysis method of combining vital statistics and ambulatory care information in Yokohama City. METHODS: Data of persons who died at home in 2013 were obtained from death certificate notifications. We also obtained the emergency transportation records that matched the cases of these death certificate notifications. Then, we gathered information regarding age, gender, marital status, and cause of death for the matched cases. RESULTS: There were 1890 "suspected unnatural deaths," in which most solitary deaths could be included, among all citizens who died at home (n = 4847). We were able to match 1503 of these cases with emergency transportation records. These 1503 cases were divided into two groups, "solitary death" (n = 349) and "un-solitary death" (n = 1154) according to the postmortem interval until finding (PMI-f). Pearson's χ2 tests conducted for the two groups revealed that there were significant differences regarding the proportion of persons who were elderly, unmarried, male, and had a hepatic disease and senility. A logistic regression analysis also showed that an increased likelihood of a prolonged PMI-f was associated with males and an unmarried status with hepatic diseases. CONCLUSIONS: Unmarried, male sex, and liver diseases are independent risks for solitary death in Yokohama City.

The importance of the rate of pure "attended deaths at home" for objective outcome indicator for assessing the prevalence of home care in Japan.

BACKGROUND: No study has yet been performed on the importance of the rate of pure "attended deaths at home," excluding examined deaths subjected to a postmortem examination. Therefore, in the present study, we investigated actual state of pure "attended deaths at home," in order to provide reference data for the future development of end-of-life care at home. METHODS: We performed a detailed survey in Yokohama City according to the type of death, age, and underlying cause of death in cases of home deaths, based on the detailed version of the Vital Statistics Survey Death Forms. Then, we divided deaths occurring in each municipality in Kanagawa Prefecture into two categories: "examined deaths" or "attended deaths," which were also stratified by the place of death, based on the Vital Statistics, and data on number of death cases subjected to postmortem examination from the Kanagawa Prefectural Police Headquarters. RESULTS: In 2013, the survey in Yokohama City showed large differences in age distribution and cause of death between examined and attended deaths. In 2014, home deaths accounted for 15.7% of all deaths in the prefecture, whereas the overall proportion of attended deaths at home was 6.9%. CONCLUSIONS: We should utilize the rate of pure "attended deaths at home" for objective outcome indicator.

Combined effects of multiple linked loci on pairwise sibling tests.

The advanced multiplex STR system, PowerPlex Fusion, includes four linked locus pairs. The conventional Identifiler system has one pair of linked loci. Therefore, sibling tests conducted using the advanced system might be more affected by linkage than those conducted using the conventional system. This study simulated single and combined effects of the four linked locus pairs on pairwise sibling tests. Simulated genotypes of 100,000 pairs of full siblings and nonrelatives were constructed according to allele frequencies of the Japanese population. The single linkage effect was evaluated for simulated genotype data by calculating both the likelihood ratio accounting for the linkage between two loci and the likelihood ratio ignoring the linkage. The combined effect was obtained by multiplication of the respective single effects. Furthermore, we investigated the possibility that ignoring the linkage affects subject classification by introducing a scale of the likelihood ratio into sibling tests. The single effect in the Identifiler analysis was 0.645-1.746 times if the linkage was ignored. Overestimations and underestimations were predictable from the identical-by-state status at two linked loci. The combined effect in the PowerPlex Fusion analysis was 0.217-7.390 times. Ignoring the linkage rarely caused a false conclusive or inconclusive result, even from PowerPlex Fusion analysis. Application of the advanced system improved sibling tests considerably. The additional examined loci were more beneficial than the adverse effect of the linkage derived from the four linked locus pairs.

Forensic Characteristics of Body Abandonment by Housemates in Japan.

INTRODUCTION:  In Japan, many cases occur wherein housemates fail to report dead bodies found in their homes. However, only individual cases are reported through press and court records, and analysis including unreported cases has not been conducted. In this study, we evaluated cases handled by our Forensic Science Department in which housemates did not immediately report a dead body found in their home. We analyzed the overall picture and forensic characteristics of such cases, stratifying whether the abandoners were estimated hikikomori. METHODS:  Of the 1,179 legal autopsy cases handled by the Department of Forensic Medicine of Tokai University from January 1, 2017, to July 1, 2023, we evaluated 45 cases in which housemates did not immediately report dead bodies. The characteristics analyzed were body age, cause of death, autopsy findings, duration from the body's discovery by the abandoner to the police report, the reason for the lack of report in the first body discovery by the abandoner, and the reason for the report. In this study, the criteria for estimating whether a hikikomori abandoned the body were (1) the police provided the information that the person was a hikikomori or (2) the person met the following four criteria: 20-64 years old, unemployed, not in school, and living with parents. RESULTS:  Positive significant differences were found in the body's decomposition and the time from the body's discovery to the report to the police when the abandoner was suspected to be a hikikomori for more than one, four, or eight days. No significant differences were found in the cause of death. Regarding the reported characteristics, when the abandoner was an estimated hikikomori, positive and significant differences were found for recognizing the body and did not report immediately due to shock. Conversely, negative and significant differences were found for the person who reported as the abandoner. CONCLUSION:  This is the first study that reports on body abandonment by housemates and elaborates on its complications to forensic doctors. The incidence rate of abandonment is higher than expected. This study suggests that hikikomori are more likely to hide the bodies for longer, which hinders the death cause investigation.

A case of a motor vehicle collision suspected as associated with development of angiosarcoma.

Trauma has been suspected as a factor leading to development of angiosarcoma, a malignant soft-tissue tumor. We conducted a forensic autopsy to investigate a putative relation between a motor vehicle collision and the driver's later death from angiosarcoma. A vehicle operated by a man in his 60 s collided with an oncoming vehicle at a curve. The victim noticed no injury at the scene. However, 45 days later, he was transferred to an emergency room with dyspnea and bloody sputum. After diagnosis of angiosarcoma, he died of respiratory failure 132 days later. The bereaved family speculated about a relation between the collision and angiosarcoma onset. At autopsy, tumor cells of the scalp had metastasized to the lung, pleura, liver, and spleen. Histopathological examinations revealed characteristic features of angiosarcoma with positive immune-staining for CD31, CD34, and factor VIII. When a person dies some time after a collision, it is designated as a delayed death. In such cases, the relevance of trauma to the person's death is often an issue of concern. Because the interval between trauma and angiosarcoma development was short, only 45 days, the angiosarcoma might be coincidental. Therefore, we rejected the relation. Forensic experts sometimes need to investigate such inquiries.

Unexpected sudden death on arrival in a healthy middle-aged man associated with COVID-19-related diffuse cardiac injury: A case report.

Coronavirus disease 2019 (COVID-19) is an emerging respiratory infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). COVID-19 has been reported to lead to acute cardiac injury, but previous research indicated that the mechanism is different from that of other viruses and remains poorly understood. Herein, we describe a case of COVID-19-associated sudden death, in a healthy 47-year-old man after developing diffuse cardiac necrosis. Two days before death, the patient developed general malaise without respiratory symptoms. The patient's fatigue worsened with time, and he ultimately developed cardiac arrest in an ambulance; however, resuscitation was unsuccessful. Antigen testing performed at the hospital revealed that the patient was positive for SARS-CoV-2 virus. At autopsy, contraction band necrosis was observed insularly in all areas of the myocardium. CD42b immunohistochemical staining indicated platelet aggregation in the microvessels around the cardiac necrosis area, suggesting COVID-19 can be fatal for healthy people by microcirculatory disturbance due to diffuse cardiac injury arising from platelet activiation. This unique mechanism can be a novel therapeutic target of COVID-19-related cardiac injury.

Construction of 13-locus X-STR multiplex PCR system that reinforces Argus X-12 kit discrimination capacity: Application to the Malay population.

Closely linked groups of markers on the X chromosome are very useful for testing complex kinship relationships involving X-STR transmission. The Argus X-12 kit, a unique commercially available kit, can obtain haplotypes of 4 linkage groups (LGs) consisting of 3 markers. Although many population data have been reported for forensic purposes, differences in discrimination ability exist between LG1 and LG2, 3, and 4 in East Asian populations, and the data of this kit would become more useful if the discrimination ability of the latter groups were increased. Therefore, for matches found using this kit for some linkage group data, then to increase the identification ability, we additionally introduced 13 X-STR loci and established a method allowing comparison using data from 25 loci. The 13X-STRs add two locus data to each of LG2, 3, and 4, and also add two closely linked group (CLG) data between LG2 and 3 and LG3 and 4 in one multiplex PCR. Assessment of this method for a Malay population for which data by Argus X-12 had already been reported showed that the frequencies of distinct haplotypes in LG2, 3, and 4 were increased by 33.0-42.6 %, and frequencies of unique haplotypes increased by 45.4-59.2 %. The respective haplotype diversity values of the additional 3-locus and 4-locus CLGs were 0.9838 and 0.9939, which helps to improve discriminatory power and to predict recombination locations on the X chromosome. Although we have been testing these loci with Japanese subjects, this system would also be useful for the Malay population.

Layer-specific proteomic profiling of human normal heart.

BACKGROUND: The organized functioning of the anisotropic myocardial layers-including the inner longitudinal, middle circular, and outer longitudinal layers-is essential for stable systemic circulation. However, the proteomic profile of each myocardial layer has not been studied yet. Here, we aimed to elucidate the layer-specific proteomic profile of human cardiac tissue using microscopic sampling. METHODS: Normal hearts were obtained from five autopsy cases, and cardiomyocytes were microdissected separately from the three myocardial layers of the left ventricle. Histological analysis and shotgun proteomic profiling were performed, followed by immunohistochemical analysis. RESULTS: Histologically, no significant changes were observed among the three layers regarding cardiomyocyte diameter and myocardial fibrosis. Totally 1220 proteins-comprising 9404 peptides-were identified from 15 samples, of which the expression levels of 92 proteins were significantly altered among the layers. Gene ontology enrichment analysis revealed that the proteins specifically elevated in the inner and outer layers mostly belonged to the actin filament-binding protein group. In particular, MYH1 was highly expressed in cardiomyocytes in the outer layer, and CTNNA3 was highly expressed at the intercalated disc in the inner layer. CONCLUSIONS: This is the first report on layer-specific proteomic profiling of human normal hearts. Anisotropic profiles of actin filament-binding proteins in myocardial layers may contribute to the anisotropic contractile and conductive abilities of the heart. Knowledge of the layer-specific proteome profiles of a human heart in the normal state can aid in further research on cardiac pathology, such as the prognosis and treatment of focal myocardial infarction.

Clinical Practice Guidelines for the Management of Congenital Cytomegalovirus Infection in Japan 2023: Executive Summary.

Congenital cytomegalovirus (cCMV) infection is the most common congenital infection in developed countries. Although a standard therapy has not yet been established, evidence for the management of cCMV infection has been accumulating. The first edition of the "Clinical Practice Guidelines for the Management of Congenital Cytomegalovirus Infection" was published in Japan in 2023. This summary outlines the clinical questions (CQs) in the guidelines, with reference to the Japanese Medical Information Distribution Service Manual. Overall, 20 CQs with statements regarding prenatal risk assessment, prevention and management at diagnosis (CQs 1-1-1-3), diagnosis (CQs 2-1-2-6), treatment (CQs 3-1-3-7) and follow-up requirements (CQs 4-1-4-4) have been discussed. For each statement, the levels of recommendation, evidence and consensus rates were determined. These guidelines will assist in the management of patients with cCMV infection.

The effectiveness and limitations of triphenyltetrazolium chloride to detect acute myocardial infarction at forensic autopsy.

Triphenyltetrazolium chloride (TTC) is one of the most conventional stains to detect infarcted area of the heart in animal experiments. However, its availability and limitations have not been thoroughly discussed in the forensic field. Here, authors stained human hearts with TTC soon after the harvest. Photographs of the samples were analyzed using image analysis software, which evaluated the occupying ratio of the stained area on the surface of each slice. The results showed that the stainability of TTC declines with the length of the postmortem interval (PMI). Specimens reacted well to TTC within 1.5 days after death and then decreased the stainability logarithmically with PMI (y = - 0.294 In (x) + 1.0441; x = PMI, y = TTC-stained area / total myocardial area, R = 0.5673). Samples with old myocardial infarction produced clear TTC contrast; normal tissue is vivid red, and fibrotic myocardium is white discoloration. In acute myocardial infarction cases where death occurred within 9 hours after the attack, however, the detection of infarcted area was very difficult even when PMI was less than 1.5 days. In summary, the TTC method may be useful within 1.5 days after death, but short suffering period before death disturbs its staining efficiency.

Case Report: Intraventricular Cerliponase Alfa Treatment in a Patient with Advanced Neuronal Ceroid Lipofuscinosis Type 2.

Neuronal ceroid lipofuscinosis type 2 (CLN2) is an autosomal recessive lysosomal disease caused by decreased activity of the enzyme tripeptidyl peptidase 1 (TPP1) due to pathogenic variants in the TPP1 gene. Cerliponase alfa, a recombinant proenzyme form of TPP1, has shown efficacy in preventing motor and language function decline in early-stage CLN2. However, the safety and effects of this therapy in advanced-stage CLN2 are unclear. We herein report a case of intraventricular cerliponase alfa treatment for over a year in a patient with advanced-stage CLN2. The results suggest the safety and potential efficacy of treatment at an advanced stage of CLN2.

Pituitary abscess presenting a very rapid progression: report of a fatal case.

Pituitary abscess is a rare disease presenting with nonspecific clinical symptoms, and diagnosis is often difficult. This disease is potentially life-threatening, but most cases have a chronic and indolent course. We report a case of a 60-year-old man with a pituitary abscess associated with pituitary adenoma who died 5 days after the onset of clinical symptoms without a definitive diagnosis. Postmortem computed tomography and autopsy findings revealed a sellar mass with cystic change and extension toward the optic chiasm. Histopathology of the lesion demonstrated an abscess with suppurative meningitis and encephalitis. The disturbance of the cardiac autonomic nervous system because of hypothalamus involvement was suggested as the cause of rapid progression and death. This case provides useful information for clinicians to avoid a lethal outcome.