DNA microarray analysis of hypothermia-exposed murine lungs for identification of forensic biomarkers.

We used DNA microarray technology to analyze the pulmonary transcriptome of mice killed by hypothermia. This analysis identified significant differential regulation of 4094 genes; specifically, 1699 genes were upregulated, and 2395 were downregulated in response to hypothermia. The gene encoding cathelicidin antimicrobial peptide was the most upregulated gene, and that encoding BAI1-associated protein 2-like 1 was the most downregulated. Gene-set analysis identified significant hypothermia-induced variations in 101 pathways, and we discovered that pathways related to immunity are involved in the pulmonary pathogenesis of hypothermia. The present findings demonstrate some of the acute pulmonary responses to hypothermia and indicate several pulmonary genes as candidate forensic biomarkers of hypothermia. Furthermore, the present findings suggest that host defense is induced in hypothermic lungs. The present microarray data may facilitate the development of protein analyses for human forensics by immunohistochemistry, western blotting and enzyme-linked immunosorbent assay and may be beneficial in clinical research of hypothermia.

Time course analysis of large-scale gene expression in incised muscle using correspondence analysis.

Studying the time course of gene expression in injured skeletal muscle would help to estimate the timing of injuries. In this study, we investigated large-scale gene expression in incision-injured mouse skeletal muscle by DNA microarray using correspondence analysis (CA). Biceps femoris muscle samples were collected 6, 12, and 24 hours after injury, and RNA was extracted and prepared for microarray analysis. On a 2-dimensional plot by CA, the genes (row score coordinate) located farther from each time series (column score coordinate) had more upregulation at particular times. Each gene was situated in 6 subdivided triangular areas according to the magnitude of the relationship of the fold change (FC) value at each time point compared to the control. In each area, genes for which the ratios of two particular FC values were close to 1 were distributed along the two border lines. There was a tendency for genes whose FC values were almost equal to be distributed near the intersection of these 6 areas. Therefore, the gene marker candidates for estimation of the timing of injuries were detectable according to the location on the CA plot. Moreover, gene sets created by a specific gene and its surrounding genes were composed of genes that showed similar or identical fluctuation patterns to the specific gene. In various analyses on these sets, significant gene ontology term and pathway activity may reflect changes in specific genes. In conclusion, analyses of gene sets based on CA plots is effective for investigation of the time-dependent fluctuation in gene expression after injury.

A quantitative morphological analysis of three-dimensional CT coxal bone images of contemporary Japanese using homologous models for sex and age estimation.

Sexual dimorphisms and age-dependent morphological features of the human coxal bone were quantitatively analyzed using homologous models created from three-dimensional (3D) computed tomography images of the pelvis (male: 514 samples, female: 388 samples, age 16-100). Bilateral average coxal images of each sex and age decade were generated separately through principle component analyses (PCA). By measuring average point-to-point distances of 8472 corresponding points (average corresponding point differences [ACPDs]) between each homologous coxal image and the average images, the sex of more than 93% of the samples was correctly assigned. Some principal components (PCs) detected in PCA of the homologous models of the samples correlated fairly well with age and are affected by features of the curvature of the iliac crest, the arcuate line and the greater sciatic notch. Moreover, separate PCA using the average images of each age decade successfully detected the first PCs, which were strongly correlated with age. However, neither multiple regression analysis using PCs related to age nor comparison of ACPDs with the average images of each age decade could produce accurate results for age decade assignment of unknown (blind) samples. Therefore, more detailed analysis of age-dependent morphological features would be necessary for actual age estimation. In addition, some laterality or left and right shape difference of the coxal bone images was also elucidated, and was more significant in females. Analysis of 3D structures using homologous models and PCA appears to be a potential technique to detect subsistent morphological changes of bones.

Time-Dependent Changes in Local and Serum Levels of Inflammatory Cytokines as Markers for Incised Wound Aging of Skeletal Muscles.

Wound age estimation is an important research field in forensic pathology. The expression levels of cytokines in the incised skeletal muscle were analyzed using a mouse model to explore the applicability for wound aging. A 5-mm long incisional wound was made at the biceps femoris muscle, and the muscle and serum were sampled at 6, 12, 24 and 48 hours after injury. Using a multiplex bead-based immunoassay, we measured the tissue levels of nine cytokines (IL-1ß, IL-6, IL-7, CCL2, CCL3, CCL4, CXCL1, CXCL2, and CXCL10), which are all involved in the pathways of inflammatory response and tissue injury. Immunoassay of post-injury muscle samples revealed significant increases in the levels of six cytokines, except for CCL3, CCL4 and IL-7, at 6 hours after injury. The elevated tissue levels of these six cytokines were maintained during 48 hours after injury, although the levels of IL-6 and CXCL1 were significantly decreased at 12 hours. In case of CCL3, its tissue levels were increased only at 12 hours. By contrast, CCL4 and IL-7 levels were increased only at 48 hours. Moreover, serum levels of most cytokines, except for CXCL1, remained unchanged during 24 hours after injury, followed by significant increases at 48 hours. Serum CXCL1 levels were increased at 6 hours and then decreased to the basal levels. Thus, the significant increase in the muscle levels of CXCL1 and IL-7 was observed at 6 and 48 hours after injury, respectively. Measuring muscle CXCL1 and IL-7 levels is helpful for estimating incised wound aging.

An Autopsy Case of Sudden Death in Neurofibromatosis Type 1 With Pheochromocytoma and Myocarditis.

An autopsy case of sudden death in a 33-year-old man with neurofibromatosis type 1 (von Recklinghausen disease), pheochromocytoma, and myocarditis is reported. The decedent was found in his bedroom in cardiopulmonary arrest. Polypoid, elastic dermal papules on the neck, chest, abdomen, and back, and flat dark-brown macules on the chest and abdomen were observed. Flat, ovoid, dark-brown freckles were present in both axillae. Examination of the right adrenal gland revealed a tumor measuring 5 cm × 5 cm × 3 cm. Microscopic examination of the myocardium revealed moderate neutrophilic and lymphocytic infiltrates. In the right adrenal gland, tumor cells contained abundant granular eosinophilic cytoplasm and exhibited cell-nesting with an alveolar pattern (Zellballen). Polygonal cells were seen together with rich vascular networks. Immunohistochemical analyses showed cells diffusely positive for chromogranin A and dopamine ß-hydroxylase. Furthermore, blood from the right heart and the right common iliac vein contained high concentrations of serum epinephrine, norepinephrine, and dopamine. Death was attributed to adrenal crisis: circulatory failure caused by excessive catecholamines produced by the pheochromocytoma. In addition, myocarditis, which had been induced chronically by catecholamines, would have also contributed adversely to the clinical course. Pheochromocytoma and myocarditis should be considered when sudden death occurs in the setting of neurofibromatosis type 1.

Pediatric autopsy case of asphyxia due to salmon egg (ikura) aspiration.

Here we report an autopsy case of asphyxia due to aspiration of a salmon egg (ikura) into the airway. The patient was a 19-month-old girl. During breakfast, she put salmon eggs into her mouth, and began to walk. She slipped, fell down, and collapsed. She was pronounced dead following 2 h of resuscitation. The body was autopsied 28 h after death. The gastric contents consisted of rice, orange sections, and white salmon eggs. The lungs were deeply congested and over-inflated. In the right lung, areas of atelectasis in the upper and middle lobes were seen. A yellow salmon egg (8 mm in diameter) was found in the trachea. Although fish eggs are consumed throughout the world, reports of this sort are limited. The aspiration of fish eggs is under-acknowledged and underreported. The importance of preventive measures needs to be emphasized to parents and caregivers.

Simultaneous time course analysis of multiple markers based on DNA microarray in incised wound in skeletal muscle for wound aging.

Assessment of incised wound age in skeletal muscles is important because fatal injuries are often complicated with muscle involvement. Transcriptome of injured skeletal muscle along with histopathological and immunohistochemistry staining, were analyzed to explore the biological effect of incised injuries using a mouse incised injury model. An incisional wound was made at the biceps femoris muscle of anesthetized mice, and the muscles were sampled at 6, 12, 24, 36 and 48h post-injury. DNA microarray analysis using RNA extracted from the muscle samples of 12h post-injury identified 3,655 upregulated and 3,583 downregulated genes. Referring to the results of the gene ontology and gene expression pathway analysis, time course expression of five cytokines, namely chemokine (C-C motif) ligand 4 (CCL4), chemokine (C-X-C motif) ligand 5 (CXCL5), interleukin-1 beta (IL-1ß), interleukin- 6 (IL-6) and interleukin-7 (IL-7), were analyzed by quantative reverse transcription PCR (qRT-PCR). CXCL5 was the most upregulated gene throughout the post-injury period with higher expression from 6 through 36h post injury. Upregulation of CCL4 and IL-1ß was also persisted until 36h post injury. IL-6 mRNA was highly and rapidly expressed at 6h post-injury followed by significant decrease at 12h. Unlike other four cytokines, IL-7 showed slow and steady increasing over time until 48h post-injury. Immunohistochemical staining of post-injury samples showed gradual mild increase of staining intensity proportional to increasing time points especially around the wound edges. The present study highlights the unique dynamics of each cytokine and reflects their roles in the process of muscle wound healing, and suggests the potential of them as a tool for forensic wound age estimation.

RNA Editing Modulates Human Hepatic Aryl Hydrocarbon Receptor Expression by Creating MicroRNA Recognition Sequence.

Adenosine to inosine (A-to-I) RNA editing is the most frequent type of post-transcriptional nucleotide conversion in humans, and it is catalyzed by adenosine deaminase acting on RNA (ADAR) enzymes. In this study we investigated the effect of RNA editing on human aryl hydrocarbon receptor (AhR) expression because the AhR transcript potentially forms double-stranded structures, which are targets of ADAR enzymes. In human hepatocellular carcinoma-derived Huh-7 cells, the ADAR1 knockdown reduced the RNA editing levels in the 3'-untranslated region (3'-UTR) of the AhR transcript and increased the AhR protein levels. The ADAR1 knockdown enhanced the ligand-mediated induction of CYP1A1, a gene downstream of AhR. We investigated the possibility that A-to-I RNA editing creates miRNA targeting sites in the AhR mRNA and found that the miR-378-dependent down-regulation of AhR was abolished by ADAR1 knockdown. These results indicated that the ADAR1-mediated down-regulation of AhR could be attributed to the creation of a miR-378 recognition site in the AhR 3'-UTR. The interindividual differences in the RNA editing levels within the AhR 3'-UTR in a panel of 32 human liver samples were relatively small, whereas the differences in ADAR1 expression were large (220-fold). In the human liver samples a significant inverse association was observed between the miR-378 and AhR protein levels, suggesting that the RNA-editing-dependent down-regulation of AhR by miR-378 contributes to the variability in the constitutive hepatic expression of AhR. In conclusion, this study uncovered for the first time that A-to-I RNA editing modulates the potency of xenobiotic metabolism in the human liver.

Single-Nucleotide Polymorphisms in Cytochrome P450 2E1 (CYP2E1) 3'-Untranslated Region Affect the Regulation of CYP2E1 by miR-570.

Human cytochrome P450 2E1 (CYP2E1) catalyzes the metabolism of numerous xenobiotics, including acetaminophen and ethanol. CYP2E1 expression is known to be extensively regulated by post-transcriptional and post-translational mechanisms. A previous study had reported that a single-nucleotide polymorphism (SNP) 1561A>G in the 3'-untranslated region (3'-UTR) of CYP2E1 leads to a decreased CYP2E1 mRNA level in human peripheral blood mononuclear cells. In this study, we examined the possibility that microRNA(s) (miR) may be involved in the SNP-mediated modulation of CYP2E1 expression. Genotyping and sequencing analyses revealed that another SNP, 1556T>A, in the 3'-UTR was in complete linkage disequilibrium with the SNP 1561A>G. We termed the alleles with 1556T and 1561A or 1556A and 1561G haplotype I or II, respectively. A luciferase assay revealed that miR-570 recognizes the CYP2E1 3'-UTR of haplotype I but not haplotype II. Human embryonic kidney 293 (HEK293) cell lines stably expressing human CYP2E1 that included the 3'-UTR of haplotype I or II (HEK/2E1(I) or HEK/2E1(II) cells, respectively) were established. Overexpression of miR-570 significantly decreased the CYP2E1 protein level in the HEK/2E1(I) cells but not in the HEK/2E1(II) cells. In seven human livers with diplotype I/I, the CYP2E1 protein levels were inversely correlated with the miR-570 levels, but no relationship was observed in 25 human livers with diplotypes I/II and II/II. Collectively, it was demonstrated that human CYP2E1 was regulated by miR-570 in a genotype-dependent manner. This report describes the first proof that SNP in 3'-UTR of human P450 affects binding of miRNA to modulate the expression in the liver.

CYP2A7 pseudogene transcript affects CYP2A6 expression in human liver by acting as a decoy for miR-126.

Human cytochrome P450 (CYP)2A6 is responsible for the metabolic activation of tobacco-related nitrosamines, as well as the metabolism of nicotine and some pharmaceutical drugs. There are large interindividual differences in CYP2A6 activity and expression, largely attributed to genetic polymorphisms. However, the variability was observed within homozygotes of the wild-type CYP2A6 gene. In this study, we investigated the possibility that CYP2A6 might be regulated by microRNA. A luciferase assay revealed that a microRNA recognition element (MRE) of miR-126* found in the 3'-untranslated region (UTR) of CYP2A6 mRNA is functional. We established two HEK293 cell lines stably expressing CYP2A6, with one including and the other excluding the full-length 3'-UTR (HEK/2A6+UTR and HEK/2A6 cells, respectively). Overexpression of miR-126* markedly decreased CYP2A6 protein levels, enzyme activity, and mRNA level in HEK/2A6+UTR cells, whereas it marginally decreased those in HEK/2A6 cells, indicating that the 3'-UTR including the MRE is functional for the downregulation of CYP2A6 by miR-126*. The inhibition of miR-126* increased CYP2A6 protein levels in primary human hepatocytes, suggesting that miR-126* downregulates endogenous CYP2A6 expression. In 20 human liver samples, the expression ratios of CYP2A6 and a pseudogene transcript CYP2A7 mRNA were highly variable (CYP2A7/CYP2A6: 0.1 to 12). Interestingly, we found that CYP2A7 was another target of miR-126* and restored the miR-126*-dependent downregulation of CYP2A6 by acting as a decoy for miR-126*. In conclusion, this study demonstrates that human CYP2A6 is post-transcriptionally regulated by miR-126* and that CYP2A7 affects CYP2A6 expression by competing for miR-126* binding.

Retinoid X receptor α in human liver is regulated by miR-34a.

Retinoid X receptor α (RXRα) forms a heterodimer with numerous nuclear receptors to regulate drug- or lipid-metabolizing enzymes. In this study, we investigated whether human RXRα is regulated by microRNAs. Two potential recognition elements of miR-34a were identified in the RXRα mRNA: one in the coding region and the other in the 3'-untranslated region (3'-UTR). Luciferase assays revealed that miR-34a recognizes the element in the coding region. The overexpression of miR-34a in HepG2 cells significantly decreased the endogenous RXRα protein and mRNA levels. The stability of RXRα mRNA was decreased by the overexpression of miR-34a, indicating that miR-34a negatively regulates RXRα expression by facilitating mRNA degradation. We found that the miR-34a-dependent down-regulation of RXRα decreases the induction of CYP26 and the transactivity of CYP3A4. miR-34a has been reported to be up-regulated by p53, which has an ability to promote liver fibrosis. The p53 activation resulted in an increase of the miR-34a level and a decrease of the RXRα protein level. In addition, the miR-34a levels in eight fibrotic livers were higher than those in six normal livers, and the reverse trend was found for the RXRα protein levels. An inverse correlation was observed between the miR-34a and the RXRα protein levels in the 14 samples. Taken together, the data show that miR-34a negatively regulates RXRα expression in human liver, and affects the expression of its downstream genes. This miR-34a-dependent regulation might be the underlying mechanism responsible for the decreased expression of the RXRα protein in fibrotic livers.

DNA microarray analysis of the mouse adrenal gland for the detection of hypothermia biomarkers: potential usefulness for forensic investigation.

We analyzed the adrenal gland transcriptome of mice killed by hypothermia using DNA microarray technology. A total of 4051 significantly expressed genes were identified; 2015 genes were upregulated and 2036 were downregulated.The FBJ osteosarcoma oncogene was the most upregulated,whereas stearoyl coenzyme A desaturase 3 was the most downregulated. Validation by quantitative polymerase chain reaction revealed that results obtained by both methods were consistent. In the gene set analysis, significant variations were found in nine pathways, and we suggest that transforming growth factor ß and tumor necrosis factor α would be involved in the pathogenesis of hypothermia. Gene functional category analysis demonstrated the most overexpressed categories in upregulated and downregulated genes were cellular process in biological process, binding in molecular function, and cell and cell part in cellular component. The present study demonstrated acute adrenal responses in hypothermia, and we suggest that understanding adrenal mRNA expression would be useful for hypothermia diagnosis. Furthermore, the present microarray data may also facilitate development of immunohistochemical analysis of human cases. In forensic practice, the combination of macroscopic and microscopic observations with molecular biological analyses would be conducive to more accurate diagnosis of hypothermia. Although this study is aimed at forensic practice, the present data regarding more than 20,000 genes of the adrenal gland would be beneficial to inform future clinical hypothermia research. From the viewpoint of adrenal gene activity, they could contribute to elucidating the pathophysiology of hypothermia.

Distribution of Aconitum alkaloids in autopsy cases of aconite poisoning.

Aconite is a well-known toxic-plant containing Aconitum alkaloids such as aconitines, benzoylaconines, and aconins. We describe here the distribution of Aconitum alkaloids detected by liquid chromatography-tandem mass spectrometry (LC/MS/MS) in three autopsy cases of suicide by aconite poisoning. Case 1: a male in his fifties had eaten aconite leaves. The concentrations of jesaconitine in cardiac blood, urine, and kidney were 12.1 ng/ml, 993.0 ng/ml, and 114.2 ng/g, respectively. Case 2: a female in her fifties had eaten aconite root. The aconite root in the stomach included a high level of mesaconitine. The concentrations of mesaconitine in cardiac blood, liver, and kidney were 69.1 ng/ml, 960.9 ng/g, and 776.9 ng/g, respectively. Case 3: a male in his sixties had drunk liquor in which aconite root had been soaked. The concentrations of mesaconitine and aconitine in cardiac blood were 259.5 and 228.5 ng/ml, respectively. The Aconitum alkaloid levels were very high in the liver. The absorption of ethanol and Aconitum alkaloids might have been increased because of his having undergone total gastrectomy. In all three cases, the Aconitum alkaloid levels were high in the liver and kidney and low in the heart and cerebrum. The level in the cerebrum was lower than that in blood. Data on the distribution of the Aconitum alkaloids in the body in cases of aconite poisoning is useful to elucidate various actions of aconite alkaloids.

Hypothalamic transcript profiling in hypothermia using SuperSAGE.

Understanding of molecular mechanisms underlying hypothermia is of primary importance in devising strategies to diagnose hypothermia. We investigated the hypothalamic transriptome in hypothermia. For transcriptomic analyses, SuperSAGE, an improved method of serial analysis of gene expression, was used. Totally, 62,208 and 54,084 tags were collected from the hypothalami of normal and hypothermia, respectively. And 367 transcripts were differentially expressed at a statistically significant level. That is, 157 and 210 transcripts among them were expressed at a higher level in normal and hypothermic hypothalami. Results obtained by SuperSAGE and quantitative PCR were consistent in 6 selected genes, although levels of differences detected by the 2 methods were not exactly the same. mRNA expressions in the hypothalamus were considered to be useful for hypothermic diagnosis. Various methods have been applied for gene expression analyses and biomarker detections. However in forensic pathology, SuperSAGE would be a promising method, especially in gene discoveries and transcriptomic analyses.

A novel polymorphic allele of human arylacetamide deacetylase leads to decreased enzyme activity.

Human arylacetamide deacetylase (AADAC) is responsible for the hydrolysis of clinically used drugs such as flutamide, phenacetin, and rifamycins. Our recent studies suggested that human AADAC is a relevant enzyme pharmacologically and toxicologically. To date, the genetic polymorphisms that affect enzyme activity in AADAC have been unknown. In this study, we found single-nucleotide polymorphisms in the human AADAC gene in a liver sample that showed remarkably low flutamide hydrolase activity. Among them, g.13651G > A (V281I) and g.14008T > C (X400Q) were nonsynonymous. The latter would be predicted to cause a C-terminal one-amino acid (glutamine) extension. The AADAC*2 allele (g.13651G > A) was found in all populations investigated in this study (European American, African American, Korean, and Japanese), at allelic frequencies of 52.6 to 63.5%, whereas the AADAC*3 allele (g.13651G > A/g.14008T > C) was found in European American (1.3%) and African American (2.0%) samples. COS7 cells expressing AADAC.1 (wild-type) exhibited flutamide, phenacetin, and rifampicin hydrolase activities with intrinsic clearance (CLint) values of 1.31 ± 0.06, 1.00 ± 0.02, and 0.39 ± 0.02 µl x min(-1) x unit(-1), respectively. AADAC.2, which is a protein produced from the AADAC*2 allele, showed moderately lower or similar CLint values, compared with AADAC.1, but AADAC.3 showed substantially lower CLint values (flutamide hydrolase, 0.21 ± 0.02 µl x min(-1) x unit(-1); phenacetin hydrolase, 0.12 ± 0.00 µl x min(-1) x unit(-1); rifampicin hydrolase, 0.03 ± 0.01 µl x min(-1) x unit(-1), respectively). Microsomes from a liver sample genotyped as AADAC*3/AADAC*3 showed decreased enzyme activities, compared with those genotyped as AADAC*1/AADAC*1, AADAC*1/AADAC*2, and AADAC*2/AADAC*2. In conclusion, we found an AADAC allele that yielded decreased enzyme activity. This study should provide useful information on interindividual variations in AADAC enzyme activity.

Aryl hydrocarbon receptor nuclear translocator in human liver is regulated by miR-24.

Aryl hydrocarbon receptor nuclear translocator (ARNT) forms a heterodimer with aryl hydrocarbon receptor or hypoxia inducible factor 1α to mediate biological responses to xenobiotic exposure and hypoxia. Although the regulation mechanism of the ARNT expression is largely unknown, earlier studies reported that the human ARNT protein level was decreased by hydrogen peroxide or reactive oxygen species. These stimuli increase the miR-24 level in various human cell lines. In silico analysis predicts that some microRNAs including miR-16 and miR-23b may bind to ARNT mRNA. This background prompted us to investigate whether human ARNT is regulated by microRNAs. Overexpression of miR-24 into HuH-7 and HepG2 cells significantly decreased the ARNT protein level, but not the ARNT mRNA level, indicating translational repression. However, overexpression of miR-16 or miR-23b caused no change in the ARNT expression. The miR-24-dependent down-regulation of ARNT decreased the expression of its downstream genes such as CYP1A1 and carbonic anhydrase IX. Luciferase assay was performed to determine the element on the ARNT mRNA to which miR-24 binds. Finally, it was demonstrated that the miR-24 levels in a panel of 26 human livers were inversely correlated with the protein levels or the translational efficiency of ARNT. Taken together, we found that miR-24 negatively regulates ARNT expression in human liver, affecting the expression of its downstream genes. miR-24 would be one of the factors underlying the mechanisms by which ARNT protein is decreased by reactive oxygen species.

A fatal case of hemoperitoneum after ultrasound-guided liver biopsy in a patient with intravascular large B-cell lymphoma.

An autopsy case of fatal complication of percutaneous liver biopsy targeting a rare type of large B-cell lymphoma is presented. A 79 year-old man was referred to the hematology unit of a hospital because of anemia with thrombocytopenia and hepatosplenomegaly. Results of inguinal lymph node biopsy were inconclusive. To investigate a mass lesion in the liver visualized by ultrasonography, image-guided liver biopsy was performed following platelet infusion. The patient became unresponsive 6h post procedure because of hypotension due to intraperitoneal hemorrhage of undetermined origin. Autopsy revealed hemoperitoneum but failed to identify any macroscopic intra- or extrahepatic vascular injuries. Histopathological investigation disclosed infiltration of atypical lymphocytes into the systemic organs including the lymph nodes, spleen, liver, and lungs. Prominent hemophagocytosis was also noted. The lymphoma cells were exclusively accumulated within vascular and sinusoidal structures, and diagnosed with immunohistochemistry as Asian variant of intravascular large B-cell lymphoma. Massive blood extravasation was presumed to originate directly from the markedly dilated liver sinusoids filled with erythrocytes, macrophages and tumor cells, under the condition of impaired hemostasis. Although the biopsy was thought to have been correctly performed, this case would be instructive for evaluation of the indications and risks associated with liver biopsy.

Application of computerised correction method for optical distortion of two-dimensional facial image in superimposition between three-dimensional and two-dimensional facial images.

The applicability of computerised correction of optical distortion to two-dimensional (2D)/three-dimensional (3D) facial image superimposition was investigated. Two-dimensional (2D) facial images of 10 male volunteers were taken with a commercially available closed circuit device (CCD) camera (reference camera) at four areas of the lens field: the centre, top, upper right and right. Correction was made by computer by calculating differences vis-à-vis the co-ordinates of dots on a test chart. Discrepancies in facial outlines between the 3D and 2D images decreased following correction in all lens fields and were below the threshold for true positive. The correction method was also tested using an actual surveillance camera and video recorder installed in a bank. The method was found to be effective for the correction of facial images, especially those taken in the top and right lens fields. Since the total error (observed error) remaining after correction appeared close to the random error (real error), systematic error was thought to be minimised by correction. Therefore, the present method was thought to display high fidelity, and could be useful for supplementary examination of conventional superimposition.

A case of Takayasu arteritis complicated by right atrium perforation and injuries of the right common iliac artery and vein caused by cannulation for percutaneous cardiopulmonary support.

We describe an autopsy case of Takayasu arteritis with right atrium perforation and injuries of the right common iliac artery and vein caused by cannulation for percutaneous cardiopulmonary support (PCPS). The decedent was an 8-year-old girl admitted for examination in respect to chest pain and syncope. During catheter angiography, she suddenly went into cardiac arrest. PCPS was attempted, whereupon bleeding into the abdominal cavity and an injury to the common iliac vein were observed. She was pronounced dead 78 hours after the initiation of PCPS. Autopsy revealed thickening of the aortic wall from the ascending aorta to the abdominal aorta, with narrowing of the proximal branches. Hemopericardium induced by right atrium perforation, and an injury of the right common iliac artery, were also found. Microscopic examinations of the aorta disclosed thickening of each layer of the vessel wall and inflammatory cell infiltration, mainly into the outer layer of the media. It was speculated that manipulation of the catheter and the underlying Takayasu arteritis caused cardiac arrest. It is also considered that hypovolemia was induced by the injuries of the right common iliac artery and vein caused during the insertion of the PCPS venous cannula. In addition, the right atrium was injured by the edge of the PCPS cannula, leading to hemopericardium. In pediatric cases involving PCPS, or in cases where cannulation is difficult, regular examination of the pericardial and abdominal cavities by echocardiography would provide useful information to prevent such accidents.

Human CYP2E1 is regulated by miR-378.

Human CYP2E1 is one of the pharmacologically and toxicologically important cytochrome P450 isoforms. Earlier studies have reported that the CYP2E1 expression is extensively regulated by post-transcriptional and post-translational mechanisms, but the molecular basis remains unclear. In the present study, we examined the possibility that microRNA may be involved in the post-transcriptional regulation of human CYP2E1. In silico analysis identified a potential recognition element of miR-378 (MRE378) in the 3'-untranslated region (UTR) of human CYP2E1 mRNA. Luciferase assays using HEK293 cells revealed that the reporter activity of the plasmid containing the MRE378 was decreased by co-transfection of precursor miR-378, indicating that miR-378 functionally recognized the MRE378. We established two HEK293 cell lines stably expressing human CYP2E1 including or excluding 3'-UTR. When the precursor miR-378 was transfected into the cells expressing human CYP2E1 including 3'-UTR, the CYP2E1 protein level and chlorzoxazone 6-hydroxylase activity were significantly decreased, but were not in the cells expressing CYP2E1 excluding 3'-UTR. In both cell lines, the CYP2E1 mRNA levels were decreased by overexpression of miR-378, but miR-378 did not affect the stability of CYP2E1 mRNA. In a panel of 25 human livers, no positive correlation was observed between the CYP2E1 protein and CYP2E1 mRNA levels, supporting the post-transcriptional regulation. Interestingly, the miR-378 levels were inversely correlated with the CYP2E1 protein levels and the translational efficiency of CYP2E1. In conclusion, we found that human CYP2E1 expression is regulated by miR-378, mainly via translational repression. This study could provide new insight into the unsolved mechanism of the post-transcriptional regulation of CYP2E1.