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.

Bioprotective role of platelet-derived microvesicles in hypothermia: Insight into the differential characteristics of peripheral and splenic platelets.

BACKGROUND: Most platelets are present in peripheral blood, but some are stored in the spleen. Because the tissue environments of peripheral blood vessels and the spleen are quite distinct, the properties of platelets present in each may also differ. However, no studies have addressed this difference. We previously reported that hypothermia activates splenic platelets, but not peripheral blood platelets, whose biological significance remains unknown. In this study, we focused on platelet-derived microvesicles (PDMVs) and analyzed their biological significance connected to intrasplenic platelet activation during hypothermia. METHODS: C57Bl/6 mice were placed in an environment of -20 °C, and their rectal temperature was decreased to 15 °C to model hypothermia. Platelets and skeletal muscle tissue were collected and analyzed for their interactions. RESULTS: Transcriptomic changes between splenic and peripheral platelets were greater in hypothermic mice than in normal mice. Electron microscopy and real-time RT-PCR analysis revealed that platelets activated in the spleen by hypothermia internalized transcripts, encoding tissue repairing proteins, into PDMVs and released them into the plasma. Plasma microvesicles from hypothermic mice promoted wound healing in the mouse myoblast cell line C2C12. Skeletal muscles in hypothermic mice were damaged but recovered within 24 h after rewarming. However, splenectomy delayed recovery from skeletal muscle injury after the mice were rewarmed. CONCLUSIONS: These results indicate that PDMVs released from activated platelets in the spleen play an important role in the repair of skeletal muscle damaged by hypothermia.

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.

Karnofsky performance status and visual analogue scale scores are simple indicators for quality of life in long-term AYA survivors who received allogeneic hematopoietic stem cells transplantation in childhood.

The purpose of this study was to investigate Karnofsky performance status (KPS) scores and visual analogue scale (VAS) scores to explain which domains in the standardized self-reported quality of life (QOL) are instrumental for long-term hematopoietic stem cell transplantation (HSCT) survivors. We conducted a nationwide cross-sectional questionnaire study on 221 survivors with allogeneic-HSCT in 28 pediatric centers. Patient-reported QOL was assessed at a single time point using the 36-item Short-Form Survey (SF-36), the Functional Assessment of Cancer Therapy-Bone Marrow Transplant (FACT-BMT), and VAS scores. KPS scores were significantly correlated with both physical and role component summary scores of the SF-36, while the VAS provided by the patient (VASpt) was significantly correlated with the mental component summary score of the SF-36 and many subscales of the FACT-BMT. The VAS provided by the participants' attending physician (VASdoc) was correlated well with KPS scores. A VASpt score more than 40% lower than KPS scores suggested mental health problems. In conclusion, KPS scores might be considered as an indicator for physical and role/social components and VASpt score as an indicator for mental components and HSCT-specific QOL.

Identification of cell cycle-associated and -unassociated regulators for expression of a hepatocellular carcinoma oncogene cyclin-dependent kinase inhibitor 3.

Human cyclin-dependent kinase inhibitor 3 (CDKN3) is a known oncogene in hepatocellular carcinoma (HCC) and its expression is promoted during tumor development. CDKN3 serves as a cell cycle regulator and its dysregulation is considered to be a causal factor for tumor progression. However, the molecular basis of the regulation of CDKN3 expression remains largely elusive. Using in silico approach, we identified CDKN3SE, a super enhancer (SE), and enhancer RNA (eRNA) candidates transcribed from this SE. Among the eRNA candidates, the expression of CDKN3eRNA was detected in the human HCC model cell line HepG2, and was found to facilitate the expression of CDKN3 without affecting the cell proliferation rate. In silico screening revealed two DNA-binding transcription factors, upstream stimulatory factor (USF) 1 and 2, involved in the regulation of CDKN3eRNA expression on CDKN3SE. A knock-down of USF1/USF2 expression in the HepG2 cells did not affect CDKN3eRNA expression, while the expression of CDKN3 was down-regulated. In a USF2 dominant negative HepG2 cell line generated by genome editing, a drastically altered cell shape and lowered cell proliferation rate were found; however, the expression of CDKN3eRNA appeared unaffected. Thus, the present study illustrated two regulators for CDKN3 expression: USF2, as a cell cycle-associated protein regulator, and CDKN3eRNA, as a cell cycle-unassociated RNA regulator.

A long non-coding RNA as a direct vitamin D target transcribed from the antisense strand of the human HSD17B2 locus.

Vitamin D (VD) exerts a wide variety of actions via gene regulation mediated by the nuclear vitamin D receptor (VDR) under physiological and pathological settings. However, the known target genes of VDR appear unlikely to account for all VD actions. We used in silico and transcriptomic approaches in human cell lines to search for non-coding RNAs transcriptionally regulated by VD directly. Four long non-coding RNAs (lncRNAs), but no microRNAs (miRNAs), were found, supported by the presence of consensus VDR-binding motifs in the coding regions. One of these lncRNAs (AS-HSD17ß2) is transcribed from the antisense strand of the HSD17ß2 locus, which is also a direct VD target. AS-HSD17ß2 attenuated HSD17ß2 expression. Thus, AS-HSD17ß2 represents a direct lncRNA target of VD.

Antagonistic action of a synthetic androgen ligand mediated by chromatin remodeling in a human prostate cancer cell line.

The clinical use of androgen receptor (AR) antagonists has been successful in treating prostate cancer patients, inducing remission of androgen-dependent tumors. However, a couple of years after treatment, prostate tumors transition into an androgen-independent state with altered gene expression profiles, but the molecular basis is not understood. Since the AR antagonists trigger this transition, we assessed whether AR antagonists induce chromatin reorganization in an androgen-dependent prostate cancer cell line (LNCaP). Treatment of LNCaP cells with two clinically used AR antagonists (bicalutamide [Bic] and enzalutamide [Enz]) expectedly resulted in antagonistic effects on cell proliferation, AR transactivation, and dihydrotestosterone (DHT)-induced expression of AR target genes. Thus, the antagonists expectedly acted to antagonize the transactivation function of AR activated by androgen binding. By ChIP-qPCR assay, AR bound to Bic, but not Enz, was recruited to an endogenous consensus AR-binding site within the kallikrein-related peptidase 3 gene promoter after treatment with Bic, similar to the effect of DHT. By ATAC-seq analysis of the cells after long-term treatment for 5 days, Bic and dihydrotestosterone DHT induced different chromatin reorganization patterns and gene expression profiles, suggesting that Bic exhibited a distinct action from that by DHT. Thus, these results suggest that the action of a known AR antagonist is mediated by chromatin reorganization in a prostate cancer cell line.

A pilot study of game-based learning programs for childhood cancer survivors.

BACKGROUND: Childhood cancer survivors lacking awareness on their potential risks of late effects often fail to seek adequate follow-up care. Patient education matching their preference is of great importance to improve their adherence to survivorship care. In this study, we developed two age-dependent game-based learning programs, which enable continuous approaches for childhood cancer survivors along their intellectual maturation. Then, we assessed the effectiveness of the programs. METHODS: Childhood cancer survivors over 10 years of age who regularly visited a long-term follow-up clinic were enrolled in this study. They were requested to play either of two different types of game tools, one for school children and another for adolescents and young adults, for one month at home. To evaluate the educational effects of the programs, they were examined for health management awareness, self-esteem, and knowledge on cancer-related late effects before and after the intervention with age-based questionnaires and knowledge tests. RESULTS: Among 83 participants, 49 (59.0%) completed the assessments over the period of 12 months. The health management awareness and knowledge levels increased significantly at 1-month after the intervention as compared to the baseline in both school children and adolescents/young adults (for health management awareness, p = 0.011 in elementary school children; p = 0.007 in junior high school children; p < 0.001 in adolescents/young adults; for knowledge levels, p < 0.001 in school children; p < 0.001 in adolescents/young adults). The effect was maintained for 12 months in school children while it decreased in adolescents and young adults with time. Self-esteem significantly increased at 1-month (p = 0.002 in school children; p = 0.020 in adolescents/young adults) and was maintained for 12 months in both age groups. CONCLUSION: The game-based learning programs enhanced health locus of control and self-esteem in childhood cancer survivors. The game-based learning programs could be applied effectively to survivorship care as a new modality of patient education. TRIAL REGISTRATION: This study was retrospectively registered in UMIN-CTR ( UMIN000043603 ) on March 12, 2021.

Gemtuzumab Ozogamicin Followed by Unrelated Cord Blood Transplantation With Reduced-intensity Conditioning for a Child With Refractory Acute Promyelocytic Leukemia.

There is no established treatment for patients with acute promyelocytic leukemia (APL) refractory to targeted therapies with all-trans retinoic acid (ATRA) and/or arsenic trioxide (ATO). We report here a case of an 8-month-old girl with APL who failed standard ATRA-combined chemotherapy. Although molecular remission was achieved after introducing ATRA/ATO combination therapy, molecular relapse occurred during the ATO consolidation courses. Subsequent molecular remission was rapidly achieved after administering 2 doses of gemtuzumab ozogamicin. She was successfully treated with unrelated cord blood transplantation using reduced-intensity conditioning. Gemtuzumab ozogamicin might be a preferable choice for patients with APL refractory to standard therapy.

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.

Skeletal and gene-regulatory functions of nuclear sex steroid hormone receptors.

The wide variety of sex hormone actions underlie bone growth and health, and their actions mediate gene regulation by the cognate nuclear receptors. Nuclear androgen and estrogen receptors (AR, and ERα/ERß) are hormone-dependent and DNA binding- transcription regulatory factors, and gene regulation by sex hormones often accompany with chromatin remodeling under aid of a number of co-regulators. As sex hormone biosynthesis is under highly regulated systemic and local regulations, the skeletal actions of sex hormones could be inferred from only the phenotypic abnormalities in skeleton in mouse genetic models deficient of nuclear receptors selectively in specific types of bone cells as well as at specific cell differentiation stages. Anabolic androgen actions and anti-bone resorptive estrogen actions are discussed here from the phenotypic abnormalities in such model mice. Though rapid gene regulation by sex hormones may not require chromatin reorganization, dynamic chromatin reconfiguration looks to facilitate profound and long-term hormonal actions. In this review, we focus the recent findings in gene regulation at a chromatin level, particularly of the function of enhancer RNAs transcribed from strong enhancers, and in the role of liquid-liquid phase separation state in transcription initiation through chromatin reconfiguration.

Impact of chronic GVHD on QOL assessed by visual analogue scale in pediatric HSCT survivors and differences between raters: a cross-sectional observational study in Japan.

A nationwide cross-sectional survey was conducted in long-term survivors of allogeneic hematopoietic stem cell transplantation (HSCT) in childhood to investigate the effect of chronic graft-versus-host disease (cGVHD) on quality of life (QOL) and differences in QOL assessments between raters. QOL was evaluated by a visual analogue scale (VAS). Assessments were compared between the survivor, guardian, and attending pediatrician for those aged 15 years or younger, and between the survivor and attending pediatrician for those aged 16 years or older. For cGVHD, severity scores were obtained by organ and their association with the VAS score was analyzed. The average pediatrician-rated VAS score was higher than that of other raters for both patient age groups (< 15 years and > 16 years). By organ, involvement of the skin, digestive organs, and joints in GVHD affected the VAS scores. A high joint score was associated with a low VAS score, and conversely, a high lung score was associated with a low pediatrician-rated VAS score. Our results indicate that differences between raters must be considered when evaluating QOL of HSCT survivors, because patients appeared to experience grater inconvenience and difficulties due to joint GVHD than their pediatricians perceived.

Changes of cancer diagnosis disclosure to children in Japan in the last 20 years.

BACKGROUND: The practice of cancer diagnosis disclosure to children has been changed with the times. The regulations of clinical trials in the 2000s might change the practice in Japan. However, the perspective of this topic among children and adults has not been investigated in detail. METHODS: We studied changes in the practice of information sharing with children with cancer at pediatric cancer centers and the perspective of cancer diagnosis disclosure to children among school children, their parents and pediatric oncologists in the last 20 years by comparing the results of questionnaire surveys conducted in 1998, 2008 and 2018. RESULTS: This study revealed that the performing rate has increased with the times, but the institutions actively performing for children aged 7-9 years were 36.4% even in the 2018 survey. More than 70% of children wished diagnosis disclosure if they suffer from cancer in the series of surveys, while the ratio of parents who tell cancer diagnosis to their children hovered at 34.5 to 53.7% (p < 0.001 in all surveys). The ratio of pediatric oncologists having the policy to perform diagnosis disclosure proactively increased from 9.3 to 60.0%, while that of parents having the same policy stayed at 5.3% even in 2018. CONCLUSIONS: The performing rate of information sharing with children with cancer was significantly changed in the last 20 years. The opinion gaps were observed between parents and children and between parents and pediatric oncologists.

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.

Profiling of Androgen-Dependent Enhancer RNAs Expression in Human Prostate Tumors: Search for Malignancy Transition Markers.

INTRODUCTION: Although the ability of androgens to promote prostate cancer development has been known for decades, the molecular mechanisms of androgen receptor (AR) signaling in the tumorigenesis remain unclear. Enhancer RNAs (eRNAs) transcribed from strong enhancers, or super-enhancers (SEs), have recently emerged as a novel class of regulatory non-coding RNAs (ncRNAs) that facilitate transcription, including that of androgen target genes, through chromatin looping to position enhancers proximate to the promoters. The aim of this study was to assess androgen-dependent transcription in prostate tumors of eRNAs (designated as KLK3eRNAs) from the SE of the KLK3 gene encoding the prostate-specific antigen (PSA) protein, a clinical marker of prostate carcinogenesis. MATERIALS AND METHODS: The androgen-induced KLK3eRNAs were identified in the LNCaP human prostate cancer cell line. The expressions of these KLK3eRNAs together with KLK3 and AR mRNA transcripts were assessed by qRT-PCR in prostate tumor samples from five prostate cancer patients. RESULTS: Androgen-induced KLK3eRNAs have been identified in the LNCaP cells, and their expression was further analyzed in tumors of prostate cancer patients. Transcripts of the tested KLK3eRNAs have been detected in all clinical samples, but their expression patterns differed between individual tumor specimens. We found a statistically significant correlation between the levels of the KLK3 and AR mRNAs with those of the previously reported KLK3eRNAs, while such correlation was not observed for novel KLK3eRNAs described in our recent report. CONCLUSION: Presented data suggest that prostate tumor development may associate with epigenetic reorganization in the KLK3 genomic regulatory elements reflected by changes of the KLK3eRNA expression. Our findings support a potential of eRNAs profiling to be used as diagnostic marker.