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An 86-year-old female was found unconscious the day after taking a prescribed tablet containing a combination of tramadol and acetaminophen. At admission to the hospital, marked hypoglycemia (blood glucose: 4 mg/dL) was confirmed, but serum insulin and C-peptide were within the normal range, which suggested that neither endogenous hyperinsulinemia nor exogenous insulin administration was responsible for the hypoglycemia. Despite resuscitation efforts, the woman subsequently died. At autopsy, there was renal disorder, but any pathological abnormalities that could have caused hypoglycemia were not observed. Blood tramadol and acetaminophen were in the therapeutic range. We speculate that the cause of fatal hypoglycemia was tramadol intake at the therapeutic dose. Older age and renal insufficiency are factors that could have potentially caused the fatal hypoglycemia in this case despite tramadol having been taken at a therapeutic dose. This is the first case report of fatal hypoglycemia following ingestion of a therapeutic dose of tramadol.

Forensic diagnosis of fatal hypothermia is considered difficult because no specific findings, such as molecular markers, have been identified. Therefore, determining the molecular mechanism in hypothermia and identifying novel molecular markers to assist in diagnosing fatal hypothermia are important. This study aimed to investigate microRNA (miRNA) and mRNA expression in iliopsoas muscle, which plays a role in homeostasis in mammals, to resolve the molecular mechanism in hypothermia. We generated rat models of mild, moderate, and severe hypothermia, then performed body temperature-dependent miRNA and mRNA expression analysis of the iliopsoas muscle using microarray and next-generation sequencing. Analysis showed that rno-miR-203a-3p expression was lower with decreasing body temperature, while Socs3 expression was significantly increased only by severe hypothermia. Luciferase reporter assays suggested that Socs3 expression is regulated by rno-miR-203a-3p. Socs3 and Mex3B small interfering RNA-mediated knockdown showed that suppressing Mex3B could induce the activation of Socs3, followed by a change in caspase 3/7 activity and adenosine triphosphate levels in iliopsoas muscle cells. These findings indicate that rno-miR-203a-3p and Mex3B are deactivated by a decrease in body temperature, whereby it contributes to suppressing apoptosis by accelerating Socs3. Accordingly, the rno-miR-203a-3p-Socs3-Casp3 or Mex3B-Socs3-Casp3 axis may be the part of the biological defense response to maintain homeostasis under extreme hypothermia.

We study a primordial black hole (PBH) formation scenario based on the Affleck-Dine (AD) mechanism and investigate two PBH mass regions: $M \sim 30 M_\odot$ motivated by the LIGO-Virgo observations of the binary black hole mergers and $M \gtrsim 10^4 M_\odot$ motivated by the observations of supermassive black holes at the center of galaxies. In the previous studies, it has been considered that the inhomogeneous AD baryogenesis generates regions with a large baryon asymmetry, some of which collapse into PBHs. In this paper, we show that this scenario is severely constrained due to the baryon asymmetry remaining outside PBHs, which would spoil the success of the big bang nucleosynthesis. Then, we propose an alternative scenario where the AD leptogenesis results in the inhomogeneous formation of Q-balls with lepton charges, which collapse into PBHs. As a result, we find that our scenario can explain the favorable PBH abundance without conflicting with the observational constraints., Comment: 25 pages, 8 figures

Based on the screening results of mass analyses using gas chromatography- mass spectrometry (GC-MS) and liquid chromatograph-tandem mass spectrometry (LC-MS/MS), we assessed the performance of Status DS10 (Status) and DRIVEN-FLOW® M8-Z (DF8), and compared the results with those of Triage DOA® (Triage) using 356 autopsy urine samples within one month of death. The sensitivity to benzodiazepines was 0.52 in Triage, 0.59 in Status, and 0.58 in DF8 with few false-positive cases. Triage detected triazolo-derivatives more easily than DF8. DF8 detected diazepam and nitro-benzodiazepines more easily than Status and Triage, with Status performing better than Triage. However, lorazepam detection with Status was difficult. There were 11 false-positive cases for amphetamines in Triage and 12 for Status-AMP at more than one week after death, but there were no false-positive in Status-MET and DF8. Tricyclic antidepressant (TCA) was detected in five cases by mass analysis, while there were 6 false-positive cases in Triage and 10 in both Status and DF8. In the TCA false-positive cases, tricyclic psychotics such as quetiapine, chlorpromazine, and carbamazepine existed. There were 23 true-positive and 6 false-positive cases for zolpidem in DF8 without false-negative cases. The accuracy of Status and DF8 for barbiturates or opiates was almost 1, but Triage was 0.98. There were no samples containing cocaine, THC, phencyclidine, or methadone. Based on the above, we conclude that Status and DF8 are comparable or slightly better than Triage, with fewer false-positive and fewer false-negative cases, except for TCA.

We had a forensic autopsy case in which drugs were detected in a cadaver that had been stored in a cold and wet condition for 5 years. The skin of the cadaver was hard, and the color was partly whitish or dark brown. Though the cadaver had transformed into adipocere in the wet and cold condition, QuEChERS extraction and LC-MS/MS revealed the presence of sulpiride and estazolam in the femoral muscle and bone marrow. The concentrations of sulpiride and estazolam in the femoral muscle were 10.6 ng/g and 39.9 ng/g, respectively. The result of a drug screening test led not only to the cause of death but also to the personal identification of the cadaver. The individual had a history of drug taking, which had been stored in his medical records at the hospital for a long time. The fact of taking sulpiride and estazolam at the same time was characteristic, and it was useful in identifying the cadaver in this case. The progress in analytical technology has made possible the detection of particle drugs from old or adipoceratous cadavers, but there have been no reports of particle drugs being detected in a cadaver that had been dead for 5 years and had transformed to adipocere, as in our present case. The analytical results by LC-MS/MS were certainly important for the diagnosis of the cause of death, and, moreover, they were useful for the purpose of personal identification.

We gave mice a 540 mg/kg dose of LD50 acephate, followed by an assessment of acephate, methamidophos (MP), and choline esterase (ChE) activity for up to 4 hours (hr) in order to investigate the time course of acephate intoxication. At 1 hr, the blood acephate and MP levels were 428 ± 90 µg/ml (mean ± SEM) and 4.2 ± 0.4 µg/ ml, respectively. The liver acephate levels were similar to those in the blood, but the liver MP levels were approximately 3.5 times that of the blood at 1 hr. The brain MP level tended to be higher than the blood MP at 1 hr. These levels decreased gradually over 4 hr, but the brain acephate and MP levels surpassed the blood levels significantly at 4 hr, and after 2 hr, respectively. Serum, liver, cerebrum, cerebellum, and brainstem cholinesterase activity (ChE) were inhibited at 1 hr, and remained inhibited in all but the cerebellum until the end of the experiment. The obtained data were applied to previously reported autopsy cases of acephate intake. Experimental data suggest that brain MP is involved in acute acephate-induced poisoning, even after a reduction in blood acephate. In autopsy cases with suspected acephate poisoning, the MP level in the brain should be considered in addition to the ChE activity to diagnose the cause of death.

A 25-year-old, emaciated man without medical treatment was found to have died suddenly at home by his mother. At autopsy, there were no injuries to his body, but significant circulatory insufficiency was observed. Electron microscopy revealed abnormal mitochondria in cells of the cardiac conduction system. The conduction system was filled with mitochondrial size abnormalities and mitochondrial cristae abnormalities. No notable abnormal findings were observed in other organs. Genetic examination of the blood revealed the mitochondrial pathogenetic variant m.3243AG. Epileptic seizures, diabetic ketoacidosis, and hyperosmolar hyperglycemic state were unlikely to be the cause of sudden death. The cause of death was diagnosed as arrhythmia possibly induced by the failure of the cardiac conduction system due to mitochondrial disease. This is a rare case of sudden death caused by an accumulation of abnormal mitochondria in the cardiac conduction system.

Transthyretin (TTR) is a vertebrate-specific protein involved in thyroid hormone distribution in plasma, and its gene is thought to have emerged by gene duplication from the gene for the ancient TTR-related protein, 5-hydroxyisourate hydrolase, at some early stage of chordate evolution. We investigated the molecular and hormone-binding properties of the brown hagfish Paramyxine atami TTR. The amino acid sequence deduced from the cloned hagfish TTR cDNA shared 33-50% identities with those of other vertebrate TTRs but less than 24% identities with those of vertebrate and deuterostome invertebrate 5-hydroxyisourate hydrolases. Hagfish TTR, as well as lamprey and little skate TTRs, had an N-terminal histidine-rich segment, allowing purification by metal-affinity chromatography. The affinity of hagfish TTR for 3,3',5-triiodo-L-thyronine (T3) was 190 times higher than that for L-thyroxine, with a dissociation constant of 1.5-3.9nM at 4°C. The high-affinity binding sites were strongly sensitive to metal ions. Zn2+ and Cu2+ decreased the dissociation constant to one-order of magnitude, whereas a chelator, o-phenanthroline, increased it four times. The number of metal ions (mainly Zn2+ and Cu2+) was approximately 12/TTR (mol/mol). TTR was also a major T3-binding protein in adult hagfish sera and its serum concentration was approximately 8μM. These results suggest that metal ions and the acquisition of N-terminal histidine-rich segment may cooperatively contribute to the evolution toward an ancient TTR with high T3 binding activity from either 5-hydroxyisourate hydrolase after gene duplication.

A man and a woman were rescued from a room that had exploded. Many empty cassette gas cylinders were found in the room. The man and woman were hospitalized immediately for the treatment of burns. The woman died 6 days later, and the man died 31 days later without regaining consciousness. Carbonization and hardening of the frontal facial skin and parts of the left and right fingers were observed on the man's body. In both cases, systemic burns had led to progressive systemic edema and markedly suppressed circulation. Analytical samples for butanes obtained from their bodies at autopsy were stored at −20 °C for 14 and 25 days, respectively, before analysis. Normal butane and isobutane were quantified in the brain and subcutaneous adipose tissue of the woman. Only the isobutane was quantified in the adipose tissue of the man. The evidence suggests that the man lit a cigarette while breathing gas and the entire room exploded. Our results also suggest that butane can be detected in the adipose tissue of autopsy cases long after inhalation even under the present storage conditions, and isobutane may remain in adipose tissue longer than n-butane.

The efficacy of DRIVEN-FLOW® M7-II(DFM7II) for seven drug groups was compared with Triage DOA® (Triage) using 340 autopsy urine samples taken from bodies within 1 month of death based on mass screening analysis of GC/MS and LC-MS/MS. The sensitivity to benzodiazepines was 0.56 in Triage and 0.53 in DFM7II with few false positives, and their accuracy was 0.88. Triage detected triazolo diazepine derivatives more easily than DFM7II. DFM7II detected diazepam and nitro benzodiazepines more easily than Triage. There were nine amphetamine false-positive cases of more than 10 days after death in Triage, but these were absent in DFM7II during this period. The accuracy of amphetamines for Triage was 0.96 and for DFM7II was 1. Tricyclic antidepressant (TCA) was detected in five cases by mass analysis, while there were four false-positive cases using Triage and eight cases using DFM7II. In the TCA false-positive cases of both kits, tricyclic psychotics such as chlorpromazine, carbamazepine, and quetiapine were included as well as the drug poisoning cases. There were no samples containing cocaine or THC. The accuracy of DFM7II for opiate and barbiturates was 1, but those of Triage was less than 1. Based on the above, DFM7II is a more accurate kit with fewer false-positives for target drug groups, other than TCA, than Triage.

We had a forensic autopsy case that required additive pathological examination for the asbestos-related lung disease compensatory application afterwards. A man in his sixties with a history of occupational asbestos inhalation who had neither visited a hospital nor received a physical examination received forensic autopsy because of his death from unknown cause. An inmate said, "He developed cough and dyspnea, and died in the progression of the symptoms." The autopsy revealed widespread pleural plaques on both sides of the parietal pleura and multiple tumors in both sides of the lungs. The cause of death was diagnosed as lung cancer. Additional pathological examination was asked by his family to certify that he had suffered from asbestos-related lung disease in order to apply to the Asbestos-related Damage Relief Law. The Japanese criteria of the compensation law of asbestos-related lung cancer is the detection of more than 5,000 asbestos bodies per gram of dry lung tissue, while his number of asbestos bodies was 4,860. Asbestos bodies were reported to be accumulated in the distal lung parenchyma with no pathological changes. The present lung samples were collected from proximal section around the tumor, which might have made the number of asbestos bodies less than the criteria. Both the number of patients suffering from asbestos-related lung disease and the number of forensic autopsy cases have increased in Japan. Collecting lung samples from the appropriate lung section is essential and should be noted when the lung cancer is suspected at forensic autopsy in order to apply for asbestos-related lung disease compensation.

Thyroid hormones (THs) play a critical role in amphibian metamorphosis, during which the TH receptor (TR) gene, thrb, is upregulated in a tissue-specific manner. The Xenopus laevis thrb gene has 3 TH response elements (TREs) in the 5' flanking regulatory region and 1 TRE in the exon b region, around which CpG sites are highly distributed. To clarify whether exposure to 3,3',5-triiodothyronine (T3) affects histone and RNA polymerase II (RNAPII) modifications and the level of DNA methylation in the 5' regulatory region, we conducted reverse transcription-quantitative polymerase chain reaction, bisulfite sequencing and chromatin immunoprecipitation assay using X. laevis cultured cells and premetamorphic tadpoles treated with or without 2 nM T3. Exposure to T3 increased the amount of the thrb transcript, in parallel with enhanced histone H4 acetylation and RNAPII recruitment, and probably phosphorylation of RNAPII at serine 5, in the 5' regulatory and exon b regions. However, the 5' regulatory region remained hypermethylated even with exposure to T3, and there was no significant difference in the methylation status between DNAs from T3-untreated and -treated cultured cells or tadpole tissues. Our results demonstrate that exposure to T3 induced euchromatin-associated epigenetic marks by enhancing histone acetylation and RNAPII recruitment, but not by decreasing the level of DNA methylation, in the 5' regulatory region of the X. laevis thrb gene.

Transthyretin (TTR) diverged from an ancestral 5-hydroxyisourate hydrolase (HIUHase) by gene duplication at some early stage of chordate evolution. To clarify how TTR had participated in the thyroid system as an extracellular thyroid hormone (TH) binding protein, TH binding properties of recombinant little skate Leucoraja erinacea TTR was investigated. At the amino acid level, skate TTR showed 37-46% identities with the other vertebrate TTRs. Because the skate TTR had a unique histidine-rich segment in the N-terminal region, it could be purified by Ni-affinity chromatography. The skate TTR was a 46-kDa homotetramer of 14.5kDa subunits, and had one order of magnitude higher affinity for 3,3',5-triiodo-l-thyronine (T3) and some halogenated phenols than for l-thyroxine. However, the skate TTR had no HIUHase activity. Ethylenediaminetetraacetic acid (EDTA) treatment inhibited [(125)I]T3 binding activity whereas the addition of Zn(2+) to the EDTA-treated TTR recovered [(125)I]T3 binding activity in a Zn(2+) concentration-dependent manner. Scatchard analysis revealed the presence of two classes of binding site for T3, with dissociation constants of 0.24 and 17nM. However, the high-affinity sites were completely abolished with 1mM EDTA, whereas the remaining low-affinity sites decreased binding capacity. The number of zinc per TTR was quantified to be 4.5-6.3. Our results suggest that skate TTR has tight Zn(2+)-binding sites, which are essential for T3 binding to at least the high-affinity sites. Zn(2+) binding to the N-terminal histidine-rich segment may play an important role in acquisition or reinforcement of TH binding ability during early evolution of TTR.

Transthyretin (TTR) is a plasma thyroid hormone (TH) binder that emerged from an ancient hydroxyisourate hydrolase by gene duplication. To know how an ancient TTR had high affinity for THs, molecular and TH binding properties of lamprey TTRs were investigated. In adult serum, the lipoprotein LAL was a major T3 binder with low affinity. Lamprey TTRs had an N-terminal histidine-rich segment, and had two classes of binding sites for 3,3′,5-triiodo-L-thyronine (T3): a high-affinity and a low-affinity site. Mutant TTRΔ3-11, lacking the N-terminal histidine-rich segment, lost the high-affinity T3 binding site. [125I]T3 binding to wild type TTR and mutant TTRΔ3-11, was differentially modulated by Zn2+. Zn2+ contents of wild type TTR were 7–10/TTR (mol/mol). Our results demonstrate that lamprey TTR is a Zn2+-dependent T3 binder. The N-terminal histidine-rich segment may be essential for neo-functionalization (i.e., high-affinity T3 binding activity) of an ancient TTR after gene duplication.

Flunitrazepam (FNZ) is a short-intermediate acting benzodiazepine derivative, and easily reduced to 7-amino FNZ. We autopsied the buried cadaver of homicide victim two years after the strangulation following ingestion of FNZ. The whole body of the cadaver had become moderately decomposed and adipocere without any obvious dehiscence. There was a fracture of the lamina thyroid cartilage. Gas chromatography/mass spectrometry (GC/MS) was performed on bone marrow sample and the stomach contents. Only 7-amino FNZ could be detected in the stomach contents. The detection of 7-amino FNZ brought important information about the ante-mortem circumstances. To our knowledge, such detection of FNZ product in a cadaver after a long period of time has not been reported previously.

BACKGROUND The severity of renal dysfunction correlates with fatal outcome after hemorrhagic shock. However, the precise mechanism for increasing renal dysfunction in response to the degree and the progression of hemorrhagic shock has not been clearly demonstrated. In this study, we examined the role of p38 mitogen-activated protein kinase (MAPK) activation on the progression of renal dysfunction by studying the differential severity of bleeding. METHODS Hemorrhagic shock was studied by quantitatively grading four groups of hemorrhaging rats: not hemorrhaged (Sham group); hemorrhaged up to 16.7% of their total body blood volume (16.7% group); hemorrhaged up to 25% (25% group); and hemorrhaged up to 33% (33% group). Mean arterial blood pressure and renal blood flow were recorded up to 5 hours after the bleeding. Kidneys were excised for assays of p38 MAPK and mRNA of the proinflammatory cytokines, such as tumor necrosis factor-α and interleukin-1β, and for histopathological study. The levels the cytokines and creatinine were measured in the renal venous blood. RESULTS As the amount of bleeding increased, the initial activation of p38 MAPK and the expression of renal cytokines were progressively enhanced. The severity of renal dysfunction, manifested by serum creatinine concentration, histologic damage score, and neutrophil accumulation in the kidney, was well correlated with the degree of initial p38 MAPK activation. CONCLUSIONS The increase of initial p38 MAPK activation after hemorrhagic shock quantitatively enhanced the ensuing renal dysfunction in response to the degree and the progression of hemorrhagic shock.

An experimental DNA extraction kit (new kit) was recently developed to extract DNA from degraded skeletal remains without the need for powdering the samples. We compared the utility of the new kit with the conventional phenol/chloroform method using real-time quantitative PCR and multiplex STR analysis. The new kit yielded large amounts of DNA from a compact bone fragment compared with the conventional phenol/chloroform method. We were able to extract sufficient DNA for STR analysis from 75% (3 of 4) and 60% (3 of 5) of the un-powdered tooth and bone samples, respectively, using the new kit. We were able to perform mini-STR analysis of the remaining samples using DNA extracted with the new kit. Furthermore, we successfully performed mitochondrial DNA sequencing of every sample. The new kit simplifies the DNA extraction procedure as it does not require powdering samples. Decreasing the number of procedural steps in DNA extraction will be beneficial in controlling DNA contamination in laboratories. Our results suggest that the new kit may be used for the simple, simultaneous extraction of DNA from multiple samples.

Information regarding the ancestral and geographical origins of biological evidence samples may be useful for crime investigators as they narrow down the possible donors of the sample. A method for simultaneous analysis of seven biallelic markers (M130, M131, M57, M125, M175, M122 and M134) was developed for forensic application. M57, M125 and M131 are included to identify haplogroups inferred as having originated in the Japanese archipelago. Our method employs allele-specific PCR and fragment analysis using fluorescently labeled primers and capillary electrophoresis. This method can be used to assign a haplogroup from both of degraded male DNA samples and DNA samples containing a mixture of female and male DNA by designing PCR primers that generate small amplicons and are highly specific for targets on the Y chromosome. A total of 1346 samples from Japanese males collected from the four major islands and Okinawa island were classified into seven Y binary haplogroups i.e., C-M130, C-M131, D-M57, D-M125, O-M175, O-M122 and O-M134, and a "no-mutation detected" group and their frequencies were 0.0617, 0.0565, 0.1441, 0.182, 0.3418, 0.11, 0.0847 and 0.0193, respectively. Samples of "no-mutation detected" were further analyzed by direct sequencing for identification of the major haplogroup to which they belong. Along with the haplogroup data, we report haplotype data for the 16 Y-STR markers included in the AmpFlSTR ® Yfiler™ PCR amplification kit (Applied Biosystems). These data will be useful in the prediction of haplogroups based on Y-STR haplotypes.

A total of 3046 males of Chinese, Malay, Thai, Japanese, and Indian population affinity were previously typed for the Y STR loci DYS19, DYS385 (counted as two loci), DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS456, DYS458, DYS635, DYS448, and Y GATA H4 using the AmpFlSTR Yfiler kit. These samples were assessed for population genetic parameters that impact forensic statistical calculations. All population samples were highly polymorphic for the 16 Y STR markers with the marker DYS385 being the most polymorphic, because it is comprised of two loci. Most (2677 out of a total of 2806 distinct haplotypes) of the 16 marker haplotypes observed in the sample populations were represented only once in the data set. Haplotype diversities were greater than 99.57% for the Chinese, Malay, Thai, Japanese, and Indian sample populations. For the Y STR markers, population substructure correction was considered when calculating the rarity of a Y STR profile. An F(ST) value, rather than a R(ST) value, is more appropriate under a forensic model. Because the F(ST) values are very small within the Asian populations, the estimate of the rarity of a haplotype comprised of 10-16 markers does not need substructure correction. However haplotypes with fewer markers may require F(ST) corrections when calculating the rarity of the profile.

A 49-year-old male captain fell and unfortunately died in a hold tank where he had entered to rescue his fainting co-worker on the disposing waste fluid left there. An autopsy revealed that the captain died from drowning in the waste fluid. In order to clarify the cause of their falling in the tank, the gas in the hold tank was analyzed. The concentration of oxygen was 18.86 to 19.31%, carbon dioxide was 7.28 to 9.07% and the other gases, including hydrogen sulfide, were assessed to be under the normal level. It was concluded that the intoxication of carbon dioxide generated from the waste fluid fermentation was the cause of this fatal accident through loss of consciousness. It is necessary to recognize that carbon dioxide is a dangerous and deleterious gas in circumstances where the gas can be produced.

An 80-year-old woman was intubated with a spiral endotracheal tube via a tracheal stoma during an arytenoidectomy. After being connected to the ventilator, the victim quickly became cyanosed, showed a decrease in blood pressure, and fell into cardiopulmonary arrest. Despite continuous resuscitation, the victim died. Necropsy found a tracheal perforation located 2.0 cm distal from the tracheal stoma that led to the right pleural space through the mediastinal space. We concluded that the tip of spiral endotracheal tube passed through the membranous part of the trachea into the pleural space and caused a hemopneumothorax followed by blood aspiration and death. Tracheal perforation is a rare, but life-threatening complication following a tracheostomal intubation. The inappropriate use of a tracheal tube stylet, guiding catheter, dilating forceps, and oversize tracheal tube have been demonstrated to cause airway injuries. However, fatal incorrect intubation with a spiral endotracheal tube via a tracheal stoma that resulted in death has not been reported previously.

Short tandem repeat studies are powerful tools for parentage analysis and for identification of missing persons, victims of murder, and victims of mass fatalities when reference samples are unavailable. The primer in the Identifiler® kit failed to amplify an allele at the D19S433 locus, producing a silent (“null”) allele. The causal mutation is a base change (G>A) 32 nucleotides downstream from the 3′ end of the AAGG repeats. The silent alleles are problematical in parentage analysis because when transmitted, they can cause a parent–child inconsistency that is unrelated to Mendelian genetics. The inconsistency is sometimes termed an “apparent opposite homozygosity” and it produces false evidence of nonparentage. Alternative primers were designed to amplify the D19S433 locus alleles and they detect the silent allele. Frequencies of the (no longer) silent allele were determined to be 0.0114 in 176 people from Shizuoka (Honshu) and 0.0128 in 156 people from Okinawa.

A total of 1079 Japanese males were typed for the following 16 Y chromosomal short tandem repeat (Y-STR) markers: DYS456, DYS389I, DYS390, DYS389II, DYS458, DYS19, DYS385, DYS393, DYS391, DYS439, DYS635, DYS392, Y GATA H4, DYS437, DYS438 and DYS448 using an AmpFlSTR® Yfiler™ PCR Amplification kit (Applied Biosystems). A total of 950 haplotypes for the 16 Y-STR markers were detected and, of these, 886 haplotypes were unique. The most frequent haplotype was found in 22 Japanese males. The haplotype diversity was 0.9992, indicating a high potential for differentiating between male individuals. There were 10 haplotypes with no allele detected at the DYS448 marker. Thus, the presence of such atypical haplotypes should be noted, when DNA typing results obtained from degraded DNA samples and/or DNA mixture samples from more than one male individual are being interpreted.

It has generally been accepted that cardiovascular failure can be induced by thermal injury (Reynolds et al. 1995; Murphy et al. 1998; Carlson & Horton 2006). Previous studies (Horton et al. 1995; Horton 1996; Maass et al. 2002) demonstrated that post-burn myocyte apoptosis occurred in the ventricular myocardium and was temporally correlated with the development of cardiac depression. Comstock et al. (1998) found apoptosis in lipopolysaccharide (LPS)-challenged cardiac myocytes due to the production of tumour necrosis factor-α (TNF-α) and its release from myocytes. It is well known that cardiac myocytes themselves produce TNF-α by the stimulation of bacterial LPS and that the local myocardial TNF-α seems to be a potential source of the TNF-α affecting myocardial function (Tanaka et al. 1994; Hickson-Bick et al. 2006; Shang et al. 2006). The existence of endogenous TNF-α in the cardiovascular system has been reported by several workers (Tanaka et al. 1994; Ballard-Croft et al. 2001; Mukherjee et al. 2003). However, the process in which this mediator induces myocardial apoptosis has not been clearly established, because even the basic question of which cells in the cardiovascular system synthesizes TNF-α during burn injury has not yet been determined. A previous study by Horton et al. (2004) suggested that the burn-related loss of gastrointestinal barrier function and translocation of microbial products were upstream mediators of post-burn myocardial inflammatory signalling and dysfunction. Recent studies (Ballard-Croft et al. 2001; Kyoi et al. 2006; Li et al. 2006) have shown that p38 mitogen-activated protein kinase (MAPK) activation is one aspect of the signalling cascade that culminates in post-burn secretion of TNF-α and contributes to post-burn myocardial apoptosis. We have previously demonstrated that bacterial LPS enhances the activation of the renal p38MAPK after burn injury and post-burn renal failure can be more easily observed in infant rats than in adult rats possibly because of the immature intestinal barrier against bacterial LPS (Kita et al. 2004). Thus, the present study was designed to show post-burn TNF-α localization in the cardiovascular system and the time course of the above-mentioned candidates in post-burn injury by using an infant rat model with a specific inhibitor of p38MAPK in order to estimate the valid cause of heart failure following burn shock.

Cardiac dysfunction is a well-known complication of hemorrhagic shock as a consequence of local inflammatory response. Several studies have indicated that p38 mitogen-activated protein kinase (MAPK) is a key mediator in organ dysfunction that is associated with the inflammatory state through the activation of proinflammatory cytokines such as TNF-alpha and IL-1beta. Whether the same applies to cardiac dysfunction after hemorrhagic shock has not been clearly determined. Therefore, in this study, the role of p38 MAPK on cardiac dysfunction after hemorrhagic shock was studied up to 5 h after a hemorrhage using FR167653, a specific inhibitor of p38 MAPK phosphorylation. The p38 MAPK phosphorylation, the cardiac mRNA expressions of TNF-alpha and IL-1beta, and intracardiac serum concentrations of each cytokine and creatine phosphokinase-MB isozyme increased after a hemorrhage. Activated neutrophil accumulation in the heart, histological inflammation-related injuries, and frequent ventricular arrhythmia were observed in the late phase after hemorrhagic shock. FR167653 inhibited these hemorrhagic changes except the induction of the primary hypotensive state. These results demonstrate that p38 MAPK phosphorylation in hemorrhagic shock plays an important role in the cardiac expression of the proinflammatory cytokines and in the development of cardiac dysfunction relative to the inflammatory responses.

Sequence variation in hypervariable regions HV1 and HV2 in the control region of mitochondrial DNA (mtDNA) was studied in 100 unrelated Japanese individuals living in Miyazaki Prefecture (in southern Japan) by PCR amplification and direct sequencing. PCR and sequencing primer sets designed for the C-stretch region at around position 16189 in HV1 and position 310 in HV2 were used. Sequence comparison revealed the existence of 90 different haplotypes in 120 variable positions, and of these 82, were unique, 6 were observed twice and 2 were observed three times. Genetic diversity was estimated at 0.999 and the probability of two randomly selected sequences matching (Random Match Probability, RMP) was 1.24%. The C-stretch region in HV1 was observed in 35% of individuals. The average number of nucleotide differences was 9.61 for HV1 and HV2. The majority of sequence variations were substitutions, particularly transitions from thymidine to cytosine (42.6%). Sequence heteroplasmy was not found in this study. Based on the observed polymorphic sites in HV1 and HV2, haplogroups D4 (D4a), M7a, M7b, N9a were the most commonly observed clusters. The resulting data were compared with some existing Japanese mtDNA databases. The present data contribute to expansion of the Japanese mtDNA database, particularly the database for Miyazaki Prefecture. These results show that an mtDNA database is very useful in forensic examination for identification of individuals.

This report aimed to present the postmortem finding of toluene in a homicide victim buried under the ground for six years. The bones of the skull and limbs were exposed, and the remainder of the subcutaneous tissues, brain and heart had formed into adipocere. There were numerous fractures in the skull and the anterior side of the ribs. A cardiac contusion extending into the cavity of the right ventricle was also observed. No other obvious injuries were identified on the body. The concentration of toluene in the bone marrow within the head of the humerus was 58.4 μg/g. The cause of death was suspected as heart rupture, possibly from a forceful impact or compression of the anterior chest under toluene intoxication. This report presents a rare case where toluene intake by a human was disclosed by autopsy even after several years of death.

— The p38 mitogen-activated protein kinase (MAPK) pathway is a proinflammatory signal transduction pathway for the production of cytokines and cellular response to stress, such as bacterial LPS or ischemia. We examined the effects of FR167653, a specific inhibitor of p38 MAPK, to explore the relationship between intestinal barrier damage and remote renal dysfunction. Immunohistochemical data showed the accumulation of neutrophils in the intestine after burn, and a horseradish peroxidase (HRP) tracer experiment showed burn-induced intestinal barrier damage. Our quantitative bacterial culture data demonstrated that viable bacteria reached the remote organs after burn and prevented the invading viable bacteria from using FR167653. Western blotting identified increased phosphorylation of p38 MAPK in the kidney after burn, and it may also have shown the possibility that endotoxin associated with the bacterial translocation enhances the activation of the p38 MAPK pathway. We blocked the intestinal barrier damage using FR167653, which resulted in reduced neutrophils in the intestine. FR167653 also prevented the increased phosphorylation of p38 MAPK in the kidney, which resulted in reduced neutrophils in the glomerulus and the reduction of tumor necrosis factor (TNF)-α and interleukin (IL)-1β mRNA in the kidneys, and, finally, prevented burn-induced renal failure. This study provides evidence for the hypothesis that the p38 MAPK pathway controls inflammatory mediators and not only improves intestinal function but also reduces remote renal failure after burn. We identified the pathophysiologic role of the p38 MAPK pathway in the development of renal failure after burn.

The transmission of a C16,291C/T heteroplasmy in the HV1 region of human mitochondrial DNA (mtDNA) was examined in buccal cells from 13 maternally-related individuals across three generations and in additional tissues (hair, blood, or finger nails) from three members of this family. The ratio of C:T at nucleotide position (np) 16,291 showed wide intra- and intergenerational variation as well as tissue variation within individuals. Our results demonstrate that one or two sequence differences between samples in the mtDNA does not warrant an exclusion. To avoid false exclusions especially when comparing mtDNA from hair samples, we recommend the analysis of as many samples as possible in order to minimize the possibility that the detection of a rare polymorphism in a single sample would be considered an exclusion when it is really a match. The observation that the transmission of a mtDNA heteroplasmy from one individual to her offspring is likely to differ among the first-generation offspring and between that generation and subsequent generations lends further credence to the bottleneck theory of inheritance of human mtDNA.

A procedure for the analysis of mitochondrial DNA (mtDNA) using capillary electrophoresis instead of former gel-based methods is described. The procedure requires less manual manipulation in terms of electrophoresis and, therefore, reduces the chance of either human- or gel-related failures. Thus, the method is suitable for performing mtDNA typing from limited amounts of forensic samples. We also performed mtDNA typing of hair samples using this method, and were successful in typing from both hair roots and hair shafts as well as saliva and nail samples. The amount of PCR product indicated that the amount of mtDNA in the tip side of the hair shaft was less than that in the root side. However, one hair sample showed equal amounts of PCR products in both the tip and the root side. For the analysis of a sample derived from an individual with heteroplasmic mtDNA, the proportions of heteroplasmy from the saliva and nail samples were different from those from hair samples. For analyses of hairs from the same individual, each region of the hair showed different proportions of heteroplasmy and the results indicated the possibility of the different sequences in the same hair sample. Therefore, mtDNA analysis of hair samples will require additional investigation of procedures for heteroplasmic mtDNA. These results strongly suggest that the application of the developed method for hair samples will require careful treatment of the samples and a rigorous analysis of the results.

A 23-year-old woman was rescued from an accidental fire in a state of cardiopulmonary arrest. Based on the diagnosis of carbon monoxide (CO) poisoning, she received hyperbaric oxygen therapy and survived in a vegetative state. After 1 and a half years, she died without recovering from the vegetative state. At autopsy, the brain was observed to be moderately softened with a severely atrophied appearance and ventricular enlargement. In addition, a characteristic damage of hypoxic-ischemic leukoencephalopathy was also observed clearly in both the bilateral globus pallidus and cerebral white matter, which are typical findings of past acute CO poisoning. A long-term vegetative state causes the brain to soften and liquefy because of reactive gliosis and autolytic change. The cause of death becomes difficult to diagnose only from the autopsy findings in general. This case is rare in that the past acute CO poisoning could be diagnosed from the remaining typical cerebral findings even after a long-term vegetative state.

We describe a new procedure for the multiplex PCR amplification of the three short tandem repeat (STR) loci; TH01, CSF1PO and TPOX with small amplicon lengths (the Small TCT Multiplex). The accuracy of the Small TCT Multiplex was verified by typing 100 Japanese samples that had been previously typed using an AmpFlSTR Green I PCR amplification kit (Green I Kit). The results using the Small TCT Multiplex were consistent with those obtained by the Green I Kit. STR typing using the Small TCT Multiplex was examined from 36 bloodstain samples that had been left for 1 to 25 years at room temperature and compared to that using the Green I Kit. The Small TCT Multiplex was superior to the Green I Kit for STR typing especially in the case of bloodstain samples that had aged for more than 8 years.

Six short tandem repeat loci (DYS19, DYS388, DYS390, DYS391, DS392 and DYS393) on the nonrecombining portion of the human Y chromosome were examined to investigate the usefulness of Y-chromosome haplotyping for human identification purposes in the Japanese and Taiwanese populations. In addition to STR loci, a Y-chromosome Alu insertion polymorphism (YAP) was tested in the Japanese samples.

Hemorrhagic shock has often been cited as a cause of death in forensic practical cases, but it is usually difficult to prove by autopsy findings alone. Our experimental studies demonstrated that hemorrhagic shock has promoted the endovascular recruitment of activated neutrophils and finally led to dysfunction of the shock organs. In this study, activated neutrophils were studied as a marker of death from hemorrhagic shock in the organs of autopsy cases in our department. Morphological changes in the heart, lung, liver, and kidney were examined in cases of death from hemorrhagic shock. The frequency of appearance of activated neutrophils was measured by immunohistochemical staining in the organs. The commonly observed morphological changes of hemorrhagic shock, which were not always expressed in any cases, could not account well for the cause of death. However, the expression of activated neutrophils in the organs elevated significantly at a middle and long antemortem interval. The appearance of activated neutrophils in the shock organs might be useful as a morphological marker of the cause of death from hemorrhagic shock in forensic practice cases.

A 21-year-old man, in whom abnormal nodules had been detected in bilateral lung fields with no clinical symptoms for two years, was admitted to the hospital with sudden cardiopulmonary arrest. Acute pulmonary embolism with a large embolus was diagnosed, but the patient died soon after admission. When the examination was compared with X-ray taken 4 days earlier by chance, a large tumor was now detected in the right heart and was suspected to be the cause of this complication after his death. Necropsy found a 3.8 x 3.5 x 1.0 cm myxoid tumor arising in the right atrium and a large fragment of this type of tumor was at the pulmonary trunk. Many old myxoma fragments were noted in the bilateral peripheral branch of the pulmonary artery. It was concluded that the abnormal nodules were old pulmonary fragments and the cause of death was pulmonary embolism of a large fragment originated from the atrial myxoma. An asymptomatic right atrial myxoma is extremely rare but nevertheless possible to unexpected death like this case.

Three Kinds of DNA analyses; AmpliType® PM typing, TH01 typing and mitochondrial DNA (mtDNA) analysis were evaluated to establish the corroborative method for forensic hair comparison. Five scalp telogen hairs were collected from 12 Japanese males ranging in age from 26 to 33 years. DNA was extracted from five hair shafts of 5cm in length and from five hair roots of 3mm in length taken from each subject. The PM typing was performed using the AmpliType® PM PCR Amplification and Typing Kit. The TH01 typing was carried out using Quick-TypeTM HUMTH01 and was detected by silver staining. For the mtDNA analysis, the sequencings of the two hypervariable regions (HV1 and HV2) of control region were performed by using the ABI PRISMTM Dye Terminator Cycle Sequencing Ready Reaction Kit with AmpliTaq® DNA polymerase FS and ABI PRISMTM model 377 DNA sequencer. In the telogen hair, the hair root was a suitable sample for both PM and TH01 typing compared to the hair shaft samples (about 50% of the subjects could be detected from the hair root samples). The PM typing and TH01 typing showed almost equal detectability. In the mtDNA analysis, the PCR amplifications of HV1 and HV2 were successfully performed in all twelve subjects by employing new primer, and the sequences of the PCR products from two subjects were determined. This result suggests that the mtDNA analysis can be applied for hair comparison in cases where the genomic DNA typing is not available. However, further study for the reliability and reproducibility of the mtDNA analysis should be performed.

Nucleotide sequences of 2 hypervariable regions (HV1, HV2) within the control region of human mitochondrial DNA (mtDNA) were analyzed from 55 unrelated Japanese. About 700 nucleotides were sequenced by using the nested PCR and the solid-phase direct sequencing methods. Comparison of these sequences with Anderson's reference sequence revealed 97 mutation types within 93 positions, and 11 positions of them were novel. Fifty five samples analyzed were classified into 52 different sequences, while 3 pairs have shown the same sequences. Comparison of the Japanese sequences to those reported from other populations indicated many differences in such a point that the substitutions at 16,223 and 73 in Japanese were more frequent than those in Caucasian, while the substitutions at 16,126 and 16,311 in Japanese were less frequent than those in Caucasian. Twenty one of 55 samples analyzed showed a T-to-C transition at the position 16,189 of the C-stretch region in the HV1 region. This replacement caused the blurred bands on the sequence image, which resulted in the ambiguity of exact number of cytosine in the C-stretch region of HV1. For this ambiguity, the number of cytosine in the C-stretch region should not be currently taken into account in forensic practices of individualization of evidence samples. Regardless of such problem, the polymorphisms of HV1 and HV2 regions are highly useful for individual identification.

Analysis for short tandem repeat (STR) loci is widely performed in forensic laboratories for human identification that utilizes commercially available kits that employ fluorescently labeled primers and capillary electrophoresis. With only a few exceptions, the sequences of the primers included in a kit are not disclosed by the kit manufacturers. Therefore, we developed a simple method to determine the 5′ end of the binding site of the primers included in commercial kits. Our method requires only custom primers and human genome sequence data and routinely used equipment and consumables. One or two custom primers are added to the PCR reaction mixture containing kit primers and input human DNA prior to amplification, and PCR products are separated by capillary electrophoresis after amplification. With this method we can determine which primer of the pair is fluorescently labeled and the 5′ end of the binding site of primers based on the changes in an electropherogram that are caused by the addition of the custom primer(s), and the human genome sequence data. This method is also useful for the determination of the shortest possible lengths of labeled kit primers.

Inductively coupled plasma/mass spectrometry (ICP/MS) was used and evaluated for the practical analysis of the arsenic content of body fluid samples without ashing. The calibration curve for arsenic in blood solution was shown to have good sensitivity, linearity and reproducibility. Blood obtained from normal volunteers was determined by ICP/MS, and compared with that determined by atomic absorption spectrometry (AAS). The arsenic contents determined by ICP/MS showed a linear correlation with that determined by AAS. Furthermore, arsenic was determined in the blood and supernatant of stomach contents obtained from a suicide autopsy case resulting from ingestion of an arsenic compound. It was therefore concluded that ICP/MS could be used rapidly and conveniently in the field of forensic toxicology in acute metal poisoning cases.

Alcohol intoxication can exacerbate hemodynamic instability following hemorrhagic shock. Impairment of hormonal, neurohumoral, and immune responses can contribute to such instability; however, the relationship between blood alcohol levels and the progression of hemorrhagic shock accompanied with these responses has not been clearly demonstrated. Herein, we examined this relationship in rats treated with various dose of alcohol. After oral administration of alcohol and then hemorrhage, the recovery of mean blood pressure (MBP); increase in plasma level of norepinephrine, epinephrine, and vasopressin; and survival interval decreased in a dose-dependent manner as the blood alcohol level increased. There were no significant differences in the production of proinflammatory cytokines such as tumor necrosis factor (TNF)-α and interleukin (IL)-1β among the groups. The present results demonstrated alcohol aggravates hemorrhagic shock in a dose-dependent manner not by alerting the immune response, but by suppressing hormonal and neurohumoral responses, thereby inhibiting hemodynamic autoregulation and shortening the survival interval.

Performing short tandem repeat (STR) analysis from degraded DNA is a challenge for forensic biologists. For assessing the quality and quantity of DNA, we developed quantitative PCR assays to determine the extent of DNA degradation. Quantitative PCR assays using primers that generate two sizes of amplicons from the same region of genomic DNA were used to determine the extent of DNA degradation. These quantitative PCR assays were used with artificially degraded DNA and degraded DNA extracted from aged bloodstains. Increased DNA degradation correlated with a decrease in the number of detectable loci in STR analysis. The extent of DNA degradation and the number of loci detected by STR analysis varied depending on the method of degradation. The extent of degradation of DNA extracted from aged bloodstains correlated well with that of DNA artificially degraded by DNase I in the presence of Mn(2+). Thus, determination of the extent of DNA degradation was helpful for estimating the number of detectable loci. Furthermore, this estimation method is expected to save time and labor, and is particularly suitable when only a limited amount of DNA can be extracted from casework samples.

A 22-year-old man was found dead after he had continued to sit on a reclining chair for 2 years. He had consumed an unbalanced diet, kept wearing the same pair of socks and never washed himself for the term. His skin of bilateral crura developed into elephantiasis with severely festered ulcers on its surface. At autopsy, subcutaneous edema was significant in his lower limbs, and chronic circulatory disturbance of lymphoducts and veins was observed histologically. There were no crucial findings to account for chronic edema in the lower limbs. It has been reported that maintaining a seated posture obstructs both lymphoducts and veins because of bending the groin, decreases their return flow by inducing muscular atrophy, and causes subcutaneous edema in the lower limbs. Oligotrophia and dirt on his limbs might have exacerbated the chronic edema in elephantiasis. We concluded that a long-term abnormal life-style had caused fatal elephantiasis.

Excessive autolytic inflammation accompanied by dysfunction of "shock organs" is recognized as arising from hemorrhagic shock due to the promotion of endovascular recruitment of neutrophils. Here, activated neutrophils in the organs of autopsy cases were evaluated as a marker of death from hemorrhagic shock. Morphologically-determined injury to the heart, lung, liver, and kidney was investigated in death from five major causes: hemorrhagic shock, head injury, exsanguination, asphyxia, and drowning. The frequency of activated neutrophils was assessed by immunohistochemical staining. When the antemortem interval was less than 2h, it was found that neither morphological damage nor neutrophil frequencies were significantly different after death due to any of these 5 causes. In contrast, at longer antemortem intervals up to 8h, the frequency of neutrophils in hemorrhagic shock was significantly greater than in head injury, whereas the degree of morphological damage was no different. Thus, the appearance of activated neutrophils in the primary organs could be useful to identify death caused by hemorrhagic shock after longer antemortem intervals.

We have developed a multiplex, single nucleotide polymorphism (SNP) typing system for forensic identification, based on the Invader assay. Using this system, fluorescence data for 21 SNP loci were collected and analyzed. We used endpoint genotyping, commonly used with the Invader assay, and also developed more reliable typing criteria because endpoint typing was occasionally unable to clearly identify the genotype of some loci. Analysis of the fluorescence data identified criteria that had high reproducibility for each genotype. One such criterion is the climbing angle of the curve resulting from two-dimensional plots of the two kinds of fluorescence used for the Invader assay. The climbing angle was observed at the time during the reaction when either or both of the fluorescence intensities increased most significantly. The angles were remarkably associated with homozygous genotypes. On the other hand, all heterozygote endpoint fluorescence ratios were highly reproducible and had very little aberration. These values enabled SNP typing to be more clearly defined compared with typing using only the endpoint fluorescence ratio, which is commonly used with the Invader assay. The values suggested in this study are easily calculated from raw fluorescence data and will be useful for multiplex SNP typing based on the Invader assay.

Hemorrhagic shock occasionally causes a fatal outcome following an outbreak of lung dysfunction, but the precise mechanism has not been clearly elucidated. Several studies have indicated that hemorrhagic shock causes a delayed vascular inflammatory decompensation and leads to inflammation-related organ dysfunction. Tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta are known as major proinflammatory cytokines that play an important role in excessive autolytic inflammation, finally inducing organ dysfunctions. In this study, the role of TNF-alpha and IL-1beta on lung dysfunction following hemorrhagic shock was examined by using FR167653, a potent inhibitor of TNF-alpha and IL-1beta production that acts by suppressing p38 mitogen-activated protein kinase (MAPK).Hemorrhagic shock was induced in anesthetized male rats by bleeding via a common carotid catheter for 20 minutes to 25% of total body blood volume without fluid resuscitation. Mean blood pressure, heart rate and arterial blood gas components were recorded up to 5 hours after the bleeding. The levels of TNF-alpha, IL-1beta and lactic dehydrogenase (LDH)-3 isozyme were measured in the serum of pulmonary venous blood. The lung tissue was excised for the assay of mRNA and for histopathological study.The expressions of mRNA for TNF-alpha and IL-1beta in the lung tissue and the concentrations of both cytokines in pulmonary serum increased after a hemorrhage. Inflammation-related injuries and function deterioration were observed in the lung following hemorrhagic shock. These hemorrhagic changes were inhibited by pretreatment with FR167653.TNF-alpha and IL-1beta play a key role in the development of inflammation-related lung dysfunction following hemorrhagic shock. Our model should be useful to explain the pathogenesis of lung dysfunction following hemorrhagic shock.

When mitochondrial DNA (mtDNA) heteroplasmies are detected, they often confound forensic identification, especially if they are the result of poor biological sampling. In this study, we determined the ratio of heteroplasmy in samples that were amplified from a very small amount of template mtDNA or a few cells using a highly sensitive nested polymerase chain reaction (PCR) procedure and a direct sequencing analysis. As a result, more than half of the detected sequences (i.e., 17/20, 15/20, and 14/20) showed homoplasmy derived from a variation in the heteroplasmy proportion when only 10 copies of template mtDNA samples were amplified and analyzed. Additionally, with products amplified from one or several white blood cells (WBCs), several previously undetected heteroplasmies were detected. These results indicate the risks associated with using highly sensitive mtDNA techniques in forensic investigations because of the variable proportions of heteroplasmy or nucleotide substitutions that can possibly be detected from a very small biological sample.