Animal Species and Blood Identification with Peptide Mass Fingerprinting.

As more families are acquiring pets and the opportunities for wild animals to appear in human neighborhoods are increasing, the number of cases and accidents involving animals is increasing. Hence, the need to identify animal species from blood left over at accident sites is increasing. Human hemoglobin is used as a marker for human blood. Although tandem mass spectrometry is the dominant methodology used in proteomics research, peptide mass fingerprinting, given its instant applicability, may be useful for screening animal species, as the amino acid sequences of hemoglobin from various animals differ. In this study, solutions that were easily purified─using hemoglobin reagents from humans, Japanese macaques, bears, cattle, goats, sheep, sika deer, pigs, wild boars, dogs, cats, and nutrias─were digested by trypsin, and subjected to database searched using Mascot. No candidate proteins were found in the blood of goats, sheep, sika deer, wild boars, pigs, or nutrias. However, bloodstains from all animal species except nutria (which is not registered in the database) yielded candidates, which were identified as the hemoglobin of origin or its relatives. This difference may be attributed to more contaminants being included in blood. Further narrowing was possible using the average mass obtained via infusion electrospray ionization mass spectrometry measurement of the undigested solution in Mascot results. Saliva, urine, semen, and sweat collected from humans were also examined and searched for mascots, but no hits were obtained. In conclusion, this method may be useful for estimating animal species and identifying blood in forensic science.

Dickkopf3 (Dkk3) is required for maintaining the integrity of secretory vesicles in the mouse adrenal medulla.

REIC (reduced expression in immortalized cells) has been identified as a gene whose expression was reduced in immortalized cultured cells. The REIC gene is identical to Dickkopf-3 (Dkk3), which encodes a secreted glycoprotein belonging to the Dkk family. Previously, we showed that Dkk3 protein is present in the mouse adrenal medulla. However, its role in this tissue has not been elucidated. To explore it, we performed electron microscopic (EM) studies and RNA-sequencing (RNA-seq) analysis on Dkk3-null adrenal glands. EM studies showed that the number of dense core secretory vesicles were significantly reduced and empty vesicles were increased in the medulla endocrine cells. Quantitative PCR (qPCR) analysis showed relative expression levels of chromogranin A (Chga) and neuropeptide Y (Npy) were slightly but significantly reduced in the Dkk3-null adrenal glands. From the result of RNA-seq analysis as a parallel study, we selected three of the downregulated genes, uncoupled protein-1 (Ucp1), growth arrest and DNA-damage-inducible 45 gamma (Gadd45g), and Junb with regard to the estimated expression levels. In situ hybridization confirmed that these genes were regionally expressed in the adrenal gland. However, expression levels of these three genes were not consistent as revealed by qPCR. Thus, Dkk3 maintains the integrity of secreting vesicles in mouse adrenal medulla by regulating the expression of Chga and Npy.

Unintentional Injury Deaths among Children: A Descriptive Study Using Medico-legal Documents in Okayama Prefecture, Japan (2001-2015).

According to the World Health Organization's World Report, approx. 950,000 children and young people < 18 years old die from an injury each year, and unintentional injury deaths account for a large portion of these cases. Here we used medico-legal documents to epidemiologically analyze the cases of unintentional injury deaths among children < 5 years old in Okayama Prefecture, Japan from 2001 to 2015. Age, sex, manner/cause of death, and various circumstances of the incident were investigated. There were 73 unintentional injury deaths during the study period. Drowning (n=29), suffocation (n=24), and transport accidents (n=13) were the major categories of unintentional injury deaths. Twenty-two cases (30.1%) were autopsied. Differences in the characteristics of the unintentional injury deaths by age were observed. Information which cannot be obtained from Vital Statistics was available from medico-legal documents, and detailed characteristics of unintentional injury deaths among children < 5 years old were elucidated. Investigating medico-legal information is one of the meaningful measures for the prevention of unintentional injury deaths among children in Japan.

Autopsy Case of Bilateral Optic Nerve Aplasia with Microphthalmia: Neural Retina Formation Is Required for the Coordinated Development of Ocular Tissues.

Human congenital anomalies provide information that contributes to the understanding of developmental mechanisms. Here we report bilateral optic nerve aplasia (ONA) with microphthalmia in the autopsy of the cadaver of a 70-year-old Japanese female. The gross anatomical inspection of the brain showed a cotton thread-like cord in the presumed location of the optic nerve tract or chiasm. Histologically, no neural retina, optic nerve bundle or retinal central vessels were formed in the eye globe, and the retinal pigment cells formed rosettes. The cornea, iris, and lens were also histologically abnormal. Immunohistochemically, no retinal cells expressed beta III tubulin, and Pax6- immunoreactive cells were present in the ciliary non-pigmented epithelial cells. This case of ONA could be attributed to the agenesis of retinal projection neurons as a sequel to the disruption of neural retina development. The neural retina formation would coordinate the proper development of ocular tissues.

Child deaths with persistent neglected experiences from medico-legal documents in Japan.

BACKGROUND: Few studies have examined the actual conditions of fatal neglect in Japan. The aims of this study were to investigate persistent neglect among child fatalities using medico-legal documents, and to describe the characteristics of the socio-familial background and biological data. METHODS: The current study analyzed the documents of all postmortem external examinations and autopsies in children <2 years old carried out in one prefecture in Japan from 2006 to 2011. After examining 59 autopsy cases using modified the Maltreatment Classification System, we identified six children who experienced persistent neglect as study samples. RESULTS: Three children were found in unsanitary rooms and one was left alone inside a car. In two cases, age of mother at delivery was <21 years old. Three victims had 1 year older sibling. With regard to history of use of health services, three mothers missed some prenatal care visits, and two refused to receive neonatal home visits. With regard to biological data the average weight Z score of six children was -2.22 after being adjusted to average weight for age (in months) and sex. Three children had acute or chronic undernutrition. Three victims had thymic involution considered as being due to chronic child maltreatment. CONCLUSIONS: The present collaboration between public health and legal medicine has enabled investigation of the background and biological impact of experiences of persistent neglect. A multidisciplinary system of evaluating child death is needed to identify preventable factors in order to intervene in the case of neglected children in a timely manner.

Harderian Gland Development and Degeneration in the Fgf10-Deficient Heterozygous Mouse.

The mouse Harderian gland (HG) is a secretory gland that covers the posterior portion of the eyeball, opening at the base of the nictitating membrane. The HG serves to protect the eye surface from infection with its secretions. Mice open their eyelids at about 2 weeks of age, and the development of the HG primordium mechanically opens the eye by pushing the eyeball from its rear. Therefore, when HG formation is disturbed, the eye exhibits enophthalmos (the slit-eye phenotype), and a line of Fgf10+/- heterozygous loss-of-function mice exhibits slit-eye due to the HG atrophy. However, it has not been clarified how and when HGs degenerate and atrophy in Fgf10+/- mice. In this study, we observed the HGs in embryonic (E13.5 to E19), postnatal (P0.5 to P18) and 74-week-old Fgf10+/- mice. We found that more than half of the Fgf10+/- mice had markedly degenerated HGs, often unilaterally. The degenerated HG tissue had a melanized appearance and was replaced by connective tissue, which was observed by P10. The development of HGs was delayed or disrupted in the similar proportion of Fgf10+/- embryos, as revealed via histology and the loss of HG-marker expression. In situ hybridization showed Fgf10 expression was observed in the Harderian mesenchyme in wild-type as well as in the HG-lacking heterozygote at E19. These results show that the Fgf10 haploinsufficiency causes delayed or defective HG development, often unilaterally from the unexpectedly early neonatal period.

Successful Cardiac, Lung, and Kidney Transplantation from a Methanol-poisoned Donor.

Massive methanol exposure can lead to severe and detrimental effects that can result in death or brain death. As organs from patients with brain death after methanol ingestion are less likely to be recovered, these patients have been considered marginal donors. We present a case of successful multiple organ transplantation (heart, lungs, and kidneys) from a methanol-poisoned patient. Our experience illustrates that donor death from methanol intoxication does not preclude organ transplantation.

Involvement of a Basic Helix-Loop-Helix Gene BHLHE40 in Specification of Chicken Retinal Pigment Epithelium.

The first event of differentiation and morphogenesis in the optic vesicle (OV) is specification of the neural retina (NR) and retinal pigment epithelium (RPE), separating the inner and outer layers of the optic cup, respectively. Here, we focus on a basic helix-loop-helix gene, BHLHE40, which has been shown to be expressed by the developing RPE in mice and zebrafish. Firstly, we examined the expression pattern of BHLHE40 in the developing chicken eye primordia by in situ hybridization. Secondly, BHLHE40 overexpression was performed with in ovo electroporation and its effects on optic cup morphology and expression of NR and RPE marker genes were examined. Thirdly, we examined the expression pattern of BHLHE40 in LHX1-overexpressed optic cup. BHLHE40 expression emerged in a subset of cells of the OV at Hamburger and Hamilton stage 14 and became confined to the outer layer of the OV and the ciliary marginal zone of the retina by stage 17. BHLHE40 overexpression in the prospective NR resulted in ectopic induction of OTX2 and repression of VSX2. Conversely, BHLHE40 was repressed in the second NR after LHX1 overexpression. These results suggest that emergence of BHLHE40 expression in the OV is involved in initial RPE specification and that BHLHE40 plays a role in separation of the early OV domains by maintaining OTX2 expression and antagonizing an NR developmental program.

A new method for sex determination based on detection of SRY, STS and amelogenin gene regions with simultaneous amplification of their homologous sequences by a multiplex PCR.

We have developed a new method for sex determination based on simultaneous detection of the SRY (sex-determining region Y), STS (steroid sulfatase) and amelogenin (AMELX and AMELY) gene regions and their homologous sequences. The sex of 246 blood samples was correctly determined by this method. An AMELY-deleted male sample, which would have been erroneously considered female based solely on analysis of the amelogenin locus, was successfully identified as male by the present method. The detection limit of this method was 63 pg of genomic DNA, and the male DNA component could be detected from mixed samples having a male:female ratio as low as 1:10. This method was useful for degraded DNA and possessed the human specificity. Practical application to 35 autopsy cases is described.

Postmortem changes in myoglobin content in organs.

Postmortem changes in myoglobin concentrations in blood and organs were investigated using an enzyme immunoassay by animal experiments in combination with immunohistochemical staining of human cases. Blood myoglobin concentrations were found to increase drastically within a very short time after death. Those in striated muscle, however, did not change by day 14 postmortem. Myoglobin content in the liver and kidney increased slightly by day 5 postmortem, and more obviously by day 7 or later. However, almost no change was observed by day 5 in the kidney when the renal artery and vein had been ligated just after death. In the thyroid gland and the lung, the myoglobin content markedly increased by day 7 postmortem, with the logarithmical values rising nearly linearly as the time after death passed. In the thyroid gland, concentrations reached the level of the striated muscle. The mechanisms of postmortem myoglobin increase in organs are thought to be direct diffusion from the striated muscle and/or distribution through the blood. To estimate the postmortem interval, the determination of myoglobin content in the thyroid gland or the lung appears to be useful.

Quantitative analysis of DNA degradation in the dead body.

Postmortem degradation of DNA was quantitatively estimated. Brain, liver, kidney and muscle samples were obtained from sacrificed rats left at 20℃ or 4℃. The quantity of DNA was measured by real-time PCR using a primer set for a sequence in the Rsrc 1 gene. When the quantity of amplified DNA using 10ng Human Genomic DNA was defined as 100 RFU, the quantities in the brain, liver, kidney and skeletal muscle (each 2µg of dry weight) on the day of sacrifice were 253±11, 338±22, 556±14 and 531±12 Relative Fluorescence Units (RFU), respectively (mean±S.E., n＝5). The quantity of amplified DNA decreased to below 10 RFU in 1-3 weeks in the liver, kidney and skeletal muscle at 20℃, while that in the brain was more than 10 RFU for six weeks, demonstrating the usefulness of the brain as a sample for DNA analysis of decaying corpses. It was suggested that quantifying the amplified DNA in the brain at 20℃ and in the liver at 4℃ as well as the ratio of the quantity of amplified DNA in the liver to the brain at 4℃ might be useful for diagnosing time of death. This study provides the first quantitative analysis of the postmortem progress of DNA degradation in the corpse.

Fgf10-CRISPR mosaic mutants demonstrate the gene dose-related loss of the accessory lobe and decrease in the number of alveolar type 2 epithelial cells in mouse lung.

CRISPR/Cas9-mediated gene editing often generates founder generation (F0) mice that exhibit somatic mosaicism in the targeted gene(s). It has been known that Fibroblast growth factor 10 (Fgf10)-null mice exhibit limbless and lungless phenotypes, while intermediate limb phenotypes (variable defective limbs) are observed in the Fgf10-CRISPR F0 mice. However, how the lung phenotype in the Fgf10-mosaic mutants is related to the limb phenotype and genotype has not been investigated. In this study, we examined variable lung phenotypes in the Fgf10-targeted F0 mice to determine if the lung phenotype was correlated with percentage of functional Fgf10 genotypes. Firstly, according to a previous report, Fgf10-CRISPR F0 embryos on embryonic day 16.5 (E16.5) were classified into three types: type I, no limb; type II, limb defect; and type III, normal limbs. Cartilage and bone staining showed that limb truncations were observed in the girdle, (type I), stylopodial, or zeugopodial region (type II). Deep sequencing of the Fgf10-mutant genomes revealed that the mean proportion of codons that encode putative functional FGF10 was 8.3 ± 6.2% in type I, 25.3 ± 2.7% in type II, and 54.3 ± 9.5% in type III (mean ± standard error of the mean) mutants at E16.5. Histological studies showed that almost all lung lobes were absent in type I embryos. The accessory lung lobe was often absent in type II embryos with other lobes dysplastic. All lung lobes formed in type III embryos. The number of terminal tubules was significantly lower in type I and II embryos, but unchanged in type III embryos. To identify alveolar type 2 epithelial (AECII) cells, known to be reduced in the Fgf10-heterozygous mutant, immunostaining using anti-surfactant protein C (SPC) antibody was performed: In the E18.5 lungs, the number of AECII was correlated to the percentage of functional Fgf10 genotypes. These data suggest the Fgf10 gene dose-related loss of the accessory lobe and decrease in the number of alveolar type 2 epithelial cells in mouse lung. Since dysfunction of AECII cells has been implicated in the pathogenesis of parenchymal lung diseases, the Fgf10-CRISPR F0 mouse would present an ideal experimental system to explore it.

Factors affecting the choice of suicide method in Okayama: a database analysis from a forensic perspective.

The annual number of suicides in Japan increased sharply in 1998, and since that time it has consistently exceeded 30,000 per year. In this study, we analyze a database of personal and background characteristics of 824 cases (605 men, 219 women) who completed suicide in Okayama Prefecture in 2002 and 2003. The data were obtained with cooperation from the police. Using the methodologies in a previous European study as a model, we classified the suicide methods into 8 categories. To examine the generational and regional differences in the choice of methods, we stratified the sample into 4 age groups (< or =24, 25 approximately 44, 45 approximately 64, and > or =65) and 2 regional groups (Okayama/Kurashiki vs. other areas). Our results on gender differences in 7 of the suicide methods were mostly similar to the European data. However, our data showed a remarkably higher proportionate male-to-female mortality ratio for poisoning by other substances (ICD-10, X65-X69 codes) (1.83, 1.15-2.92). In terms of generational differences in the choice of suicide methods, the Mantel-Haenszel test of homogeneity was significant for most of the categories in our study, suggesting an impact of age on how people commit suicide. There were no remarkable regional differences in our sample. An epidemic curve for suicides via carbon monoxide poisoning using charcoal briquets revealed a trend of time clustering not observed in the other 6 means. The database constructed and used in this study contains richer information than conventional death statistics and is expected to provide helpful knowledge and insights for future epidemiological studies.

Cyanide concentrations in blood and tissues of fire victims.

Cyanide poisoning has been regarded to contribute the fatal outcome in fire victims. The toxicity of inhaled hydrogen cyanide (HCN) at the cellular level was evaluated considering the impact of methemoglobin (MetHb) produced by fire gases. Cyanide (CN) concentrations and total hemoglobin contents were measured in right heart blood (RHB) and seven organs/tissues (basal ganglia, brain stem, heart, lung, liver, kidney and psoas muscle) collected from 20 fire fatalities. MetHb and carboxyhemoglobin saturations were also measured in RHB. The amount of CN probably bound to the cytochrome c oxidase of the tissue cells (CCO-CN) was extrapolated from CN and hemoglobin contents in RHB and organs/tissues, MetHb saturation in RHB and binding capacity of MetHb for CN. CN concentrations in RHB showed a wide range with the highest concentration of 8.927 µg/mL. The lung contained the largest CN content among organs/tissues with the mean concentration of 2.219 µg/g, then the heart (0.259 µg/g) and it was lower than 0.100 µg/g in others. Exceedingly large amount of CN in the lung could be explained by high hemoglobin content, being the port of entry of HCN and postmortem diffusion of fire gases. CCO-CN was theoretically present in about 20% of organ/tissue samples, most commonly in the basal ganglia (10 samples, with the mean of 0.059 µg/g) followed by heart (eight samples, with the mean of 0.109 µg/g). No CCO-CN was found in liver and kidney. HCN might have the effect on brain and heart.

Localization of the ultraviolet-sensor Opn5m and its effect on myopia-related gene expression in the late-embryonic chick eye.

Recent studies show that exposure to ultraviolet (UV) light suppresses ocular elongation, which causes myopia development. However, the specific mechanisms of this process have not been elucidated. A UV-sensor, Opsin 5 (Opn5) mRNA was shown to be present in extraretinal tissues. To test the possibility that UV-signals mediated by Opn5 would have a direct effect on the outer connective tissues of the eye, we first examined the expression patterns of a mammalian type Opn5 (Opn5m) in the late-embryonic chicken eye. Quantitative PCR showed Opn5m mRNA expression in the cornea and sclera. The anti-Opn5m antibody stained a small subset of cells in the corneal stroma and fibrous sclera. We next assessed the effect of UV-A (375 nm) irradiation on the chicken fibroblast cell line DF-1 overexpressing chicken Opn5m. UV-A irradiation for 30 min significantly increased the expression of Early growth response 1 (Egr1), known as an immediate early responsive gene, and of Matrix metalloproteinase 2 (Mmp2) in the presence of retinal chromophore 11-cis-retinal. In contrast, expression of Transforming growth factor beta 2 and Tissue inhibitor of metalloproteinase 2 was not significantly altered. These results indicate that UV-A absorption by Opn5m can upregulate the expression levels of Egr1 and Mmp2 in non-neuronal, fibroblasts. Taken together with the presence of Opn5m in the cornea and sclera, it is suggested that UV-A signaling mediated by Opn5 in the extraretinal ocular tissues could influence directly the outer connective tissues of the chicken late-embryonic eye.

Postmortem increase of flecainide level in cardiac blood.

We encountered a case of fatal congestive heart failure that occurred under the influence of flecainide. In this case, an extreme postmortem increase in the flecainide level was identified in cardiac blood. The patient had been administered 400 mg/day of flecainide for seven days before death. Antemortem plasma obtained 13 h before death showed a flecainide concentration of 2.5 mg/L and a pH of 7.4. In comparison, centrifuged supernatants of postmortem right and left cardiac blood contained flecainide concentrations of 13.8 and 44.2 mg/L, respectively, with pH of 5.5 in both samples. This increase in blood flecainide concentration was attributed to postmortem redistribution, as about 18 h had passed between the last intake of flecainide and death.

Distribution of orally ingested hydrochloric acid in the thoracoabdominal cavity after death.

The authors encountered a case of hydrochloric acid (HCl) poisoning, thought to be caused by oral ingestion of concentrated HCl. Coagulation of the surface of the tongue and the mucosa of the pharynx, esophagus, and stomach were observed at forensic autopsy. An overabundance of Cl- was found in the gastric contents, corresponding to 8.19 mL of concentrated HCl. This was suggested to be a lethal oral dose of concentrated HCl, and the cause of death was determined to be HCl poisoning. Measuring the pH and concentrations of various ions in body fluids and contents of the alimentary tract enabled postmortem diffusion of HCl to be determined.

Comparison of German and Japanese general practitioners' awareness of suicide and attitudes toward patients with suicidal ideation.

The authors designed a questionnaire to investigate the differences in German and Japanese general practitioners? (GP) awareness of suicide and attitudes toward patients with suicidal ideation in their respective societies. The purpose of this study was to obtain insights leading to a better means of suicide prevention in primary care in Japan. The background for conducting the study was declining suicides in the past 20 years and the lower suicide rate in Germany compared with the present situation in Japan, where the number of suicides has in recent years continued to exceed 30,000, resulting in a suicide rate approximately 2 times higher than that in Germany. The questionnaire was randomly mailed to GPs in Okayama-Prefecture (western Japan) and Hamburg-State (northern Germany) and was collected in the same way. The patterns of answers were compared between the 2 countries, and the differences were statistically analyzed. Japanese GPs seem to have a lower will to prevent suicide in daily practice compared to German GPs and a great lack of knowledge about treatment of suicidal patients. These observations suggest that improving GPs? interest in the problem of suicide and providing training programs for the treatment of patients with suicidal intentions might be a means of achieving better suicide prevention in Japan.

Measurement of Postmortem 1,5-anhydroglucitol in Vitreous Humor for Forensic Diagnosis.

In forensic diagnosis, postmortem blood glucose is known to be susceptible to change after death. However, the 1,5-anhydroglucitol (1,5-AG) concentrations in plasma and cerebrospinal fluid (CSF) reflect the mean blood glucose level for a short period of time. In this study, we compared the postmortem 1,5-AG concentrations in vitreous humor and CSF in 47 subjects to evaluate the utility of this concentration in the vitreous humor for forensic diagnosis. The postmortem 1,5-AG concentrations in vitreous humor (mean±SD: 20.2 ± 8.7 µg/mL) and CSF (16.8 ± 8.7 µg/mL) did not differ significantly and showed a strong correlation (r(2) = 0.87, p < 0.01). These results suggest that the vitreous humor 1,5-AG concentration provides useful information on the antemortem blood glucose level, in addition to the HbA1c value and the CSF 1,5-AG concentration.