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Although dental radiography is a valuable tool for age estimation in forensic anthropology and odontology, very limited radiological data are available regarding tooth development in healthy newborn babies during the first month of life.This study aimed to describe the radiological findings of tooth development in babies aged 0 days to 1 month.We analyzed the postmortem findings of five newborn babies with no known natural cause of death who had undergone autopsy, computed tomography (CT), and dental radiography. We estimated the gestational age for the babies aged 0 days and analyzed the condition of mandibular symphysis, existence of tooth germs, and presence or absence of calcification of the first permanent molars of all the babies.The calcified form of 20 deciduous teeth, tooth germs of the permanent upper and lower first molars, and non-calcified mandibular symphysis were observed in each case. However, calcification of the first permanent molar was observed in only two 1-month-old babies.The dental radiographic findings and anthropometric measurements of non-skeletonized, non-mummified term babies confirmed calcification of all the deciduous teeth and the first permanent molar at the age of 0 days and 1 month, respectively.

A novel method coupling spin column extraction with high-performance liquid chromatography–mass spectrometry was developed for simultaneous extraction of β-blockers and calcium channel blockers fro...

We compared three-dimensional (3D) CT images of stabbing victims subjected to volume-rendering (VR) or global illumination-rendering (GIR), a new technique now available for the reconstruction of 3D CT images. It simulates the complete interactions of photons with the scanned object, thereby providing photorealistic images. The diagnostic value of the images was also compared with that of macroscopic photographs. We used postmortem 3D CT images of 14 stabbing victims who had undergone autopsy and CT studies. The 3D CT images were subjected to GIR or VR and the 3D effect and the smoothness of the skin surface were graded on a 5-point scale. We also compared the 3D CT images of 37 stab wounds with macroscopic photographs. The maximum diameter of the wounds was measured on VR and GIR images and compared with the diameter recorded at autopsy. The overall image-quality scores and the ability to assess the stab wounds were significantly better on GIR than VR images (median scores: VR = 3 vs GIR = 4, p

A simple and cost-effective method for analyzing valproic acid (VPA) in biological samples was developed. VPA was extracted in methyl tertiary-butyl ether (MTBE) and derivatized using trimethylsilyldiazomethane. The MTBE extract was analyzed by gas chromatography-mass spectrometry (GC-MS). The extraction recovery in human whole blood and urine was over 90 %, with good linearity in the range of 1.0 to 250 µg/mL of VPA. The RSD for 2.0, 20, and 200 μg/mL VPA in whole blood ranged from 0.9 to 4.7 % for intra-day and 1.5 to 5.9 % for inter-day. The RSD for 2.0, 20, and 200 μg/mL VPA in urine ranged from 1.9 to 2.6 % for intra-day and 1.2 to 2.9 % for inter-day. As a preliminary cross-validation study, a cross-check was conducted using blinded concentration samples. The results demonstrated that the assay data of the two laboratories were comparable.

BackgroundMesenchymal stem cells (MSCs) repair injured tissue in a paracrine manner. To enhance their therapeutic properties, preconditioning with various factors has been researched. We have previously showed that MSCs cultured in serum-free medium (SF-MSCs) promote their immunosuppressive ability, thereby enhancing their anti-fibrotic effect. Here, we examined whether serum-free medium and hypoxic preconditioning synergistically enhance the therapeutic effects of MSCs on renal fibrosis in rats with ischemia–reperfusion injury (IRI).MethodsSF-MSCs were incubated under 1% O2conditions (hypo-SF-MSCs) or 21% O2conditions (normo-SF-MSCs) for 24 h before collection. After IRI procedure, hypo-SF-MSCs or normo-SF-MSCs were injected through the abdominal aorta. At 7 or 21 days post-injection, the rats were killed and their kidneys were collected to evaluate inflammation and fibrosis. In in vitro experiments, we investigated whether hypo-SF-MSCs enhanced secretion of anti-fibrotic humoral factors using transforming growth factor (TGF)-β1-stimulated HK-2 cells incubated with conditioned medium from hypo-SF-MSCs or normo-SF-MSCs.ResultsNormo-SF-MSCs showed attenuation of senescence, which increased their proliferative capacity. Although no significant difference in cellular senescence was found between normo-SF-MSCs and hypo-SF-MSCs, hypo-SF-MSCs further increased their proliferative capacity compared with normo-SF-MSCs. Additionally, administration of hypo-SF-MSCs more strongly ameliorated renal fibrosis than that of normo-SF-MSCs. Moreover, although hypo-SF-MSCs strongly attenuated infiltration of inflammatory cells compared with the control rats, which were treated with PBS, this attenuation was almost equal between normo-SF-MSCs and hypo-SF-MSCs. In vitro experiments revealed that hypo-SF-MSCs more significantly inhibited transforming growth factor (TGF)-β/Smad signaling compared with normo-SF-MSCs. Moreover, hypoxic preconditioning increased hepatocyte growth factor (HGF) secretion even under serum-free conditions, whereas knockdown of HGF in hypo-SF-MSCs attenuated inhibition of TGF-β/Smad signaling.ConclusionsThese results indicate that administration of ex vivo-expanded, hypoxia-preconditioned SF-MSCs may be a useful cell therapy to prevent renal fibrosis.

Although laryngohyoid fracture indicates the applied neck pressure and is an important finding in hanging individuals, the reported rate varies widely and its true incidence remains controversial. We used computed tomography (CT) studies to investigate the incidence of laryngohyoid fracture in hanging individuals and identify factors contributing to such fractures. Considered for inclusion in this study were 107 attempted or successful hanging individuals subjected to CT studies between 2005 and 2019. After excluding 19 whose images were inadequate for evaluation, 88 subjects were included. Body suspension was complete in 20, partial in 49, and unknown in 19; 54 (61.4%) individuals died. Two radiologists performed image analysis and recorded the presence and site of laryngohyoid fractures. Multiple logistic regression analysis was used for factor analysis of laryngohyoid fractures; it included the gender, the age (< or ≧ 40 years), the type of suspension (complete or incomplete), and the outcome (death or survival). Of the 88 subjects, 35 (39.8%) presented with laryngohyoid fractures on CT images; the superior horn of the thyroid cartilage was fractured in 32 (91.4%) of the 35. Age was the only factor significantly related to laryngohyoid fracture (odds ratio = 2.85, 95% confidence interval = 1.08–7.52). In hanging individuals, the incidence of laryngohyoid fracture on CT images was 39.8%. The superior horn of the thyroid cartilage was the most frequent fracture site. • The incidence of laryngohyoid fracture on CT images of hanging individuals was almost 40%; the superior horn of the thyroid cartilage was the most frequent fracture site. • In older hanging individuals, attention must be paid to laryngohyoid fractures on CT images.

Although organophosphorus agents are used worldwide as pesticides, there have been many reports of pesticide poisoning. Nerve agents are organophosphorus agents that interfere with neurotransmission and have been used as chemical weapons in wars. These agents mainly irreversibly inhibit the action of acetylcholinesterase, an enzyme that breaks down acetylcholine, a neurotransmitter, and are believed to cause acute symptoms of poisoning. However, in recent years, the presence of subacute, delayed toxicity independent of acetylcholinesterase inhibition has been reported for some organophosphorus agents. We analyzed the subacute and delayed toxicity of bis(isopropylmethyl)phosphonate (BIMP), which has the same phosphonate group as sarin. BIMP rounded out the morphology of the cells and reduced the proportion of cells in the G1 phase of the cell cycle over time. No DNA damage was observed, suggesting that BIMP may affect cell division.

Zinc homeostasis in cells depends on zinc transporters, which are divided into 2 families: ZnT (SLC30A) and ZIP (SLC39A). In this study, we examined the effect of 20 single nucleotide polymorphisms (SNPs) in 10 genes encoding zinc transporters on blood zinc concentration in Japanese subjects ( n = 102). Blood zinc levels were determined by microwave plasma-atomic emission spectrometry, and SNPs were analyzed by polymerase chain reaction followed by restriction fragment length polymorphism analysis. Among the 20 SNPs examined, 3 SNPs (SLC30A3 rs11126936, SLC39A8 rs233804, and SLC39A14 rs4872479) were significantly associated with blood zinc concentration. Individuals with genotype TT and TG in rs11126936 showed significantly higher blood zinc concentrations than those with GG. As for rs233804, individuals harboring the A allele had significantly higher blood zinc concentrations than those without this allele. Furthermore, the genotype TT and TG in rs4872479 had significantly higher blood zinc concentrations than those with GG. Among these three SNPs, combination of SLC30A3 rs11126936 and SLC39A8 rs233804 may strongly affect blood zinc levels. This study is the first comprehensive investigation of the effect of SNPs in genes encoding zinc transporters on blood zinc concentration. Adverse effects of zinc deficiency are reported and above 3 SNPs may be related to genetic susceptibility to zinc deficiency.

We have previously reported that ischemic animal models treated with a respiratory inhibitor, rotenon, show an increased voluntary alcohol intake. Although it is clear that ischemic brain, as a result of reduced-blood flow, shows pathological events and/or neuro-degenerations apparently, little is known of causal relationship between the mechanism of neural dysfunction and voluntary alcohol consumption. Authors have investigated effects of permanent two-vessel occlusion (p2VO) on rat voluntary alcohol drinking behavior. In first experiment the p2VO-treated rats showed an increase of voluntary alcohol drinking behavior, as compared with sham controls. Using brain microdialysis technique, increases of only nucleus accumbens (ACC) dopamine (DA) releases were suppressed in the p2VO-treated rats significantly, following the high K+ (40 mM) perfusion through the microdialysis probe membrane. Alcohol (200 mM) perfusion-induced DA and serotonin (5-HT) releases in the ACC of the p2VO-treated rats were suppressed significantly in the second experiment, as compared with the sham-treated rats. In third experiment p2VO-treated rats showed significant decreases of the contents of DA, not 5-HT, in the ACC, caudate-putamen (C/P), ventral tegmental area-substantia nigra (VT/SN) and lateral hypothalamus (LH). Dopaminergic neurons in the ACC showed more functional vulnerability against the p2VO treatments, as compared with the serotonergic neurons. An increase of alcohol intake in the p2VO-treated rats means the compensation for the neural degeneration of the dopaminergic system in the ACC consisted brain rewarding system. It was likely suggested that neural disturbance of higher functions involved with incomplete global brain ischemia leads the risk of an abnormal alcohol drinking in human.

A number of N-alkyl indole or indazole-3-carbonyl analogs, with modified chemical structures, are distributed throughout the world as synthetic cannabinoids. Like synthetic cannabinoids, cathinone analogs are also abused and cause serious problems worldwide. Acute deaths caused by overdoses of these drugs have been reported. Various analytical methods that can cope with the rapid changes in chemical structures are required for routine analysis and screening of these drugs in seized and biological materials for forensic and clinical purposes. Although many chromatographic methods to analyze each drug have been published, there are only a few articles summarizing these analytical methods. This review presents the various colorimetric detections, immunochemical assays, gas chromatographic–mass spectrometric methods, and liquid chromatographic–mass spectrometric methods proposed for the analysis of synthetic cannabinoids and cathinones.

Upon acquirement of pulmonary circulation, the ancestral heart may have been remodelled coincidently with, or accompanied by, the production and rearrangement of progenitor cells. However, the progenitor populations that give rise to the left ventricle (LV) and sinus venosus (SV) are still ambiguous. Here we show that the expression of Secreted frizzled-related protein Sfrp5 in the mouse identifies common progenitors for the outflow tract (OFT), LV, atrium and SV but not the right ventricle (RV). Sfrp5 expression begins at the lateral sides of the cardiac crescent, excluding early differentiating regions, and continues in the venous pole, which gives rise to the SV. Lineage-tracing analysis revealed that descendants of Sfrp5-expressing cells at E7.5 contribute not only to the SV but also to the LV, atria and OFT and are found also in the dorsal splanchnic mesoderm accompanied by the expression of the secondary heart field marker, Islet1. These findings provide insight into the arrangement of cardiac progenitors for systemic circulation., It is unclear which progenitors define different regions of the heart. Here, the authors find Secreted frizzled-related protein 5 is expressed in murine progenitor cells for the outflow tract, first heart field, and sinus venosus, but not the right ventricle, and Wnt inhibition prevents progenitor proliferation.

Monolithic silica in MonoSpin for solid-phase extraction of drugs from whole blood samples was developed to facilitate high-throughput analysis. Monolithic silica of various pore sizes and octadecyl contents were synthesized, and their effects on recovery rates were evaluated. The silica monolith M18-200 (20μm through-pore size, 10.4nm mesopore size, and 17.3% carbon content) achieved the best recovery of the target analytes in whole blood samples. The extraction proceeded with centrifugal force at 1000rpm for 2min, and the eluate was directly injected into the liquid chromatography-mass spectrometry system without any tedious steps such as evaporation of extraction solvents. Under the optimized condition, low detection limits of 0.5-2.0ngmL-1 and calibration ranges up to 1000ngmL-1 were obtained. The recoveries of the target drugs in the whole blood were 76-108% with relative standard deviation of less than 14.3%. These results indicate that the developed method based on monolithic silica is convenient, highly efficient, and applicable for detecting drugs in whole blood samples.

A simple and precise drug screening method was developed for the detection of abused drugs in whole blood by using the Oratect® III device that is usually employed for the detection of drugs in saliva. Whole blood was acidified with phosphoric acid, following which the hemolyzed solution was filtered through the ultrafiltration column Vivaspin 2 Hydrosart®. The filtrate was then tested for the presence of drugs using Oratect III. The detection limit of the device for methamphetamine, amphetamine, morphine, codeine, dihydrocodeine, diazepam, alprazolam, estazolam, and prazepam in whole blood was 125, 125, 50, 50, 50, 25, 60, 15, and 75ng/mL, respectively. The concentration range detected was between therapeutic and toxic drug levels; therefore, the proposed method can be applied for detecting the presence of abused drugs in blood. Our method is a novel, optimized technique for use in forensic laboratories to screen whole blood for drugs of abuse.

For detection of a history of drug abused, we developed a simple method for extracting pyrrolidinophenone-type designer drugs in human hair by using a MonoSpin(®) C18 column. Target drugs were extracted from a single alkaline-digested hair segment (length, 10mm; weight, ca 0.1mg). The analytes extracted were then analyzed by high-performance liquid chromatography-mass spectrometry without evaporation of the eluent after MonoSpin extraction. Linearity from 0.5 to 500ng/mg was observed for all the tested drugs using an internal standard method (correlation coefficients >0.998) and the limit of detection was 0.2ng/mg. The recoveries were between 0.7 and 11.1%. The coefficients for intraday and interday variations at 4, 40, 200, and 400ng/mg in hair were between 0.7 and 11.1%. This method was successfully applied to the identification of these designer drugs in segmented human hair from drug abusers and indicated their history of drug abuse. The results were consistent with the patients' statements, indicating that this rapid method can be used to detect a history of drug abuse.

The organophosphorus compound sarin irreversibly inhibits acetylcholinesterase. We examined the acute cardiovascular effects of a sarin-like organophosphorus agent, bis(isopropyl methyl)phosphonate (BIMP), in anaesthetized, artificially ventilated rats. Intravenous administration of BIMP (0.8mg/kg; the LD50 value) induced a long-lasting increase in blood pressure and tended to increase heart rate. In rats pretreated with the non-selective muscarinic-receptor antagonist atropine, BIMP significantly increased both heart rate and blood pressure. In atropine-treated rats, hexamethonium (antagonist of ganglionic nicotinic receptors) greatly attenuated the BIMP-induced increase in blood pressure without changing the BIMP-induced increase in heart rate. In rats treated with atropine plus hexamethonium, intravenous phentolamine (non-selective α-adrenergic receptor antagonist) plus propranolol (non-selective β-adrenergic receptor antagonist) completely blocked the BIMP-induced increases in blood pressure and heart rate. In atropine-treated rats, the reversible acetylcholinesterase inhibitor neostigmine (1mg/kg) induced a transient increase in blood pressure, but had no effect on heart rate. These results suggest that in anaesthetized rats, BIMP induces powerful stimulation of sympathetic as well as parasympathetic nerves and thereby modulates heart rate and blood pressure. They may also indicate that an action independent of acetylcholinesterase inhibition contributes to the acute cardiovascular responses induced by BIMP.

We report the time-course profile of urinary excretion of α-pyrrolidinovalerophenone (α-PVP) and α-pyrrolidinobutiophenone (α-PBP) after intravenous injection in a human. The subject was a man aged in his forties, who intravenously injected unknown amounts of so-called unregulated drugs with the intention to commit suicide. The urinary concentrations of the drugs were analyzed by MonoSpin® extraction and gas chromatography–mass spectrometry. α-PVP, α-PBP, and their 2′-oxo metabolites of these compounds were identified in the urine. The highest concentration of α-PVP and α-PBP in urine was 1.2 and 1.6 μg/ml, respectively. The excretion half-lives of α-PVP and α-PBP were calculated to be 22 and 11 h, respectively. To our knowledge, this is the first report on the urinary excretion profiles of α-PVP and α-PBP in a human.

We report a fatal case of 3,4-methylenedioxypyrovalerone (MDPV) poisoning. A 35-year-old woman was taken to a nearby hospital after she was found unconscious. MDPV (1,200 ng/ml) and α-pyrrolidinobutiophenone (α-PBP; 200 ng/ml) were detected in her cardiac blood. Hair segments from the victim were also analyzed to confirm chronic drug use. MDPV and α-pyrrolidinovalerophenone (α-PVP) were found in many of the segments tested with the highest MDPV concentration at 22 ng/10-mm hair. We concluded that the woman died of acute MDPV poisoning, and that she had abused MDPV and α-PVP for a long time. To our knowledge, this is the first report dealing with MDPV in human hair segments, and with α-PBP in human specimens.

Organophosphorus (OP) compounds such as sarin are toxic agents that irreversibly inhibit the enzyme acetylcholinesterase. A recent study showed that OP compounds also have multiple toxicity mechanisms, and another suggested that endoplasmic reticulum (ER) dysfunction contributes to OP toxicity. However, the signaling pathway and mechanisms involved are poorly understood. We examined whether the sarin-like OP agent bis(isopropyl methyl)phosphonate (BIMP), which exhibits toxicity similar to that of sarin, induced ER stress in human astrocytoma CCF-STTG1 cells. Our results demonstrate that BIMP exposure reduced cell viability. Moreover, it induced changes in mitochondrial membrane potential and increased cleavage of caspase 3. Treatment with BIMP increased the mRNA levels of the ER stress marker genes binding immunoglobulin protein (BiP) and the transcription factor C/EBP homologous protein (CHOP). Furthermore, BIMP increased the protein expressions and phosphorylation of BiP, CHOP, and protein kinase RNA-like ER kinase and the phosphorylation of eukaryotic translation initiation factor 2. Compared to BIMP treatment alone, pretreatment with the CHOP siRNA, siCHOP, decreased BIMP-dependent CHOP expression and improved CCF-STTG1 cell viability. Our findings suggest that BIMP induced mitochondrial dysfunction and apoptotic cell death event mediated by ER stress in CCF-STTG1 cells and that treatment targeted at managing ER stress has the potential to attenuate the toxicity of OP nerve agents.

Organophosphorus compounds, such as sarin, are highly toxic nerve agents that inhibit acetylcholinesterase (AChE), but not cholinesterase, via multiple mechanisms. Recent studies have shown that organophosphorus compounds increase cyclooxygenase-2 (COX-2) expression and induce neurotoxicity. In this study, we examined the toxicity of the sarin-like organophosphorus agent bis(isopropyl methyl)phosphonate (BIMP) and the effects of BIMP on COX-2 expression in SK-N-SH human neuroblastoma cells. Exposure to BIMP changed cell morphology and induced caspase-dependent apoptotic cell death accompanied by cleavage of caspase 3, caspase 9, and poly (ADP-ribose) polymerase (PARP). It also increased COX-2 expression, while pretreatment with a COX inhibitor, ibuprofen, decreased BIMP-dependent cell death and COX-2 expression in SK-N-SH cells. Thus, our findings suggest that BIMP induces apoptotic cell death and upregulates COX-2 expression.

Central ghrelin is required for the rewarding properties of drug abuse. We investigated whether alcohol affects ghrelinergic, dopaminergic, and serotoninergic neurons and growth hormone secretagogue receptor 1A (GHS-R1A) levels in the reward system of the brain. Alcohol-naive C57BL/6J mice received 2g/kg ethanol (EtOH) intraperitoneally (i.p.). Plasma ghrelin levels decreased between 1 and 4h. We investigated the effects of EtOH administration on plasma ghrelin levels in two different animal models at 1, 3, and 10months of age. Plasma ghrelin levels decreased following the EtOH treatment in 1- and 3-month-old short-term (1-day) alcohol vapor-exposed (STA) mice. In contrast, EtOH administration increased plasma ghrelin levels in 1- and 3-month-old long-term (20-day) alcohol vapor-exposed (LTA) mice. In vivo ghrelin release in the lateral hypothalamus (LH) increased in STA and LTA mice after the i.p. administration of EtOH. EtOH increased in vivo dopamine (DA), but not serotonin (5-HT) release in the LH of STA mice, and increased in vivo DA and 5-HT release in the LH of LTA mice. GHS-R1A mRNA expression and GHS-R1A protein levels in the LH were increased in LTA mice. The number of GHS-R1A-immunoreactive cells was greater in the LH and amygdala of LTA mice. These results support the neurobiological correlation between the development of drinking behavior and activation of ghrelinergic and serotonergic neurons in the LH. The activation of ghrelinergic systems in the amygdala may also induce an increase in 5-HT release in the LH during long-term alcohol intake.

Abdominal aortic aneurysms (AAAs), which commonly occur among elderly individuals, are accompanied by a risk of rupture and subsequent high mortality. Establishment of medical therapies for the prevention of AAAs requires further understanding of the molecular pathogenesis of this condition. This report details the possible involvement of Osteoprotegerin (OPG) in the prevention of AAAs through inhibition of Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). In CaCl2-induced AAA models, both internal and external diameters were significantly increased with destruction of elastic fibers in the media in Opg knockout (KO) mice, as compared to wild-type mice. Moreover, up-regulation of TRAIL expression was observed in the media by immunohistochemical analyses. Using a culture system, both the TRAIL-induced expression of matrix metalloproteinase-9 in smooth muscle cells (SMCs) and the chemoattractive effect of TRAIL on SMCs were inhibited by OPG. These data suggest that Opg may play a preventive role in the development of AAA through its antagonistic effect on Trail.

A method coupling spin column extraction with gas chromatography-mass spectrometry was developed for the simultaneous extraction of acidic and basic drugs from urine. Benzodiazepines, local anaesthetics, antidepressants, and barbiturates were used as model drugs. Sample loading, washing, and elution of the target drugs were accomplished by centrifugation of the column. In this study, mixed-mode monolithic silica bonded with a C18 reversed-phase and a strong cation exchange phase was packed in a spin column. The pH of a urine sample (0.2 mL) was adjusted to 3 and the analytes adsorbed onto the column were eluted with 0.1 mL of MeOH containing 2% NH3; all the tested drugs were simultaneously extracted from urine. The recovery of the tested drugs was 65-123%. Up to a concentration of 2500 ng/mL of the target drugs in urine, a linear curve was observed (r(2)>0.996). The intra- and interday RSDs at three different concentrations in urine were 2.1-14.7%. For RSDs lower than 15%, the limits of detection were 1-25 ng/mL. The proposed method was successfully applied for clinical and forensic cases and the results thus obtained were in good agreement with those obtained by conventional methods.

A simple, sensitive, and specific method with gas chromatography-mass spectrometry was developed for simultaneous extraction and derivatization of amphetamines (APs) and 3,4-methylenedioxyamphetamines (MDAs) in human urine by using a monolithic silica spin column. All the procedures, such as sample loading, washing, and elution were performed by centrifugation. APs and MDAs in urine were adsorbed on the monolithic silica and derivatized with propyl chloroformate in the column. Methamphetamine-d(5) was used as an internal standard. The linear ranges were 0.01-5.0 microg mL(-1) for methamphetamine (MA) and 3,4-methylenedioxymethamphetamine (MDMA) and 0.02-5.0 microg mL(-1) for amphetamine (AP) and 3,4-methylenedioxyamphetamine (MDA) (coefficient of correlationor = 0.995). The recovery of APs and MDAs in urine was 84-94%, and the relative standard deviation of the intra- and interday reproducibility for urine samples containing 0.1, 1.0, and 4.0 microg mL(-1) of APs and MDAs ranged from 1.4% to 13.6%. The lowest detection limit (signal-to-noise ratioor = 3) in urine was 5 ng mL(-1) for MA and MDMA and 10 ng mL(-1) for AP and MDA. The proposed method can be used to perform simultaneous extraction and derivatization on spin columns that have been loaded with a small quantity of solvent by using centrifugation.

To overcome the limitations of solid-phase extraction, we developed a device comprising a spin column packed with octadecyl silane-bonded monolithic silica for extracting amphetamines and methylenedioxyamphetamines from urine. Urine (0.5mL), buffer (0.4mL), and methoxyphenamine (internal standard) were directly put into the preactivated column. The column was centrifuged (3000rpm, 5min) for sample loading and washed. The adsorbed analytes were eluted and analyzed by high-performance liquid chromatography, without evaporation. The results were as follows: linear curves (drug concentrations of 0.2-20microg/mL); correlation coefficients >0.99; detection limit, 0.1microg/mL. The proposed method is not only useful for drugs from biological materials but also highly reproducible for the analysis of these drugs in urine.

A simple, sensitive, and specific liquid chromatography–mass spectrometry (LC–MS) method for simultaneous determination of dibucaine and naphazoline from serum was developed and validated. The extraction procedure was performed using a monolithic silica spin column. Chromatographic separation of dibucaine and naphazoline was achieved on a C 18 reverse phase column with a mobile phase gradient (mobile phase A: 10 mM ammonium formate and mobile phase B: acetonitrile) at a flow rate of 0.2 mL/min. LC–MS was operated under the selective ion monitoring mode using the electrospray ionization technique in the positive mode. The retention times for naphazoline, dibucaine, and the internal standard (IS) were 6.7, 7.8, and 8.0 min, respectively. A linear graph was obtained for dibucaine and naphazoline with correlation coefficients >0.998 for all analytes by this method. The limit of quantification of dibucaine and naphazoline was 10 and 25 ng/mL, respectively. The mean recoveries were greater than 70%. Both compounds were stable under conditions of short-term storage, long-term storage as well as after freeze–thaw cycles. Monolithic spin column extraction and LC–MS analysis enabled the separation of dibucaine and naphazoline within 20 min.

Liver dysfunction involving toluene intoxicity includes elevation of transaminase level and delayed complications of liver failure, but its effect on hepatitis B virus (HBV) infection is as yet unknown. Here, we report a case of fulminant hepatitis B developed in a toluene abuser. A 23-year-old female toluene abuser was admitted to a local clinic because of nausea, vomiting, and dizziness, and a mild elevation of serum transaminase level was identified. She was treated as an outpatient, but continued toluene inhalation during follow-up. Five days later, she was found in a drowsy state of consciousness and taken to the emergency unit of our institution. Laboratory findings showed an alanine aminotransferase level of 4,659 IU, a remarkably prolonged prothrombin time, and she was diagnosed with fulminant hepatitis B. Intensive care was carried out, but she died the next day. Molecular analysis revealed that the HBV isolate was classified as genotype C, and nucleotide positions that are prone to fulminant hepatitis were A at 1,762 and G at 1,764 in the core promoter region, and G at 1,896 in codon 28 in the precore region. The long-term toluene inhalation could have contributed to drastic clinical course of acute hepatitis B in this patient.

Of the 8 genotypes of HBV (genotypes A-H), genotype G is unique in that it has an insertion in the core gene and two stop codons in the precore region preventing the synthesis of hepatitis B e antigen. Most individuals with genotype G are coinfected with other genotypes, typically genotype A. Mice with severe combined immunodeficiency disease carrying human hepatocytes were infected with HBV particles propagated in Huh7 cells in culture. Mice monoinfected with genotype G did not raise detectable HBV DNA in serum, although products of the core gene emerged 4 to 8 weeks after inoculation. When they were superinfected with genotype A at week 10, however, HBV DNA of genotype A developed, which was replaced almost completely by that of genotype G within 10 weeks. Such a rapid takeover was also observed in mice initially infected with genotype A or C and superinfected with genotype G. Similar viral dynamics occurred in mice simultaneously coinfected with genotypes G and A. Takeover was markedly enhanced in mice inoculated with a serum passage containing genotype G with a trace of genotype A. Coinfection of mice with genotypes G and A induced abundant cellular steatosis along with increased fibrosis in the liver, which was not detected in mice monoinfected with genotype A or G. Conclusion: Genotype G can monoinfect chimeric mice at very low levels, and its replication increases maredly when coinfected with other genotypes. Coinfection with genotype G could enhance fibrosis under immunocompromised states. (HEPATOLOGY 2007;45:929–937.)

We report a fatal case of hypermagnesemia caused by oral ingestion of milk containing magnesium chloride as a folk remedy. The autopsy of the 75-year-old woman showed no injury or illness as the cause of death. Neither alcohol nor controlled drugs were found by the toxicological analysis. Her posthumous serum magnesium concentration was 10.2 mg/dL. The concentration of magnesium in serum was higher than that of the previous reports. Therefore, the cause of death was considered to be hypermagnesemia. Recently, the prescription drugs, over-the-counter drugs, and health foods containing magnesium are consumed as supplements in the world, and their potential toxicity should be recognized.

The amiloride-sensitive epithelial Na+ channel (ENaC) regulates Na+ homeostasis into cells and across epithelia. So far, four homologous subunits of mammalian ENaC have been isolated and are denoted as alpha, beta, gamma, and delta. The chemical agents acting on ENaC are, however, largely unknown, except for amiloride and benzamil as ENaC inhibitors. In particular, there are no agonists currently known that are selective for ENaCdelta, which is mainly expressed in the brain. Here we demonstrate that capsazepine, a competitive antagonist for transient receptor potential vanilloid subfamily 1, potentiates the activity of human ENaCdeltabetagamma (hENaCdeltabetagamma) heteromultimer expressed in Xenopus oocytes. The inward currents at a holding potential of -60 mV in hENaCdeltabetagamma-expressing oocytes were markedly enhanced by the application of capsazepine (or =1 microM), and the capsazepine-induced current was mostly abolished by the addition of 100 microM amiloride. The stimulatory effects of capsazepine on the inward current were concentration-dependent with an EC50 value of 8 microM. Neither the application of other vanilloid compounds (capsaicin, resiniferatoxin, and olvanil) nor a structurally related compound (dopamine) modulated the inward current. Although hENaCdelta homomer was also significantly activated by capsazepine, unexpectedly, capsazepine had no effect on hENaCalpha and caused a slight decrease on the hENaCalphabetagamma current. In conclusion, capsazepine acts on ENaCdelta and acts together with protons. Other vanilloids tested do not have any effect. These findings identify capsazepine as the first known chemical activator of ENaCdelta.

The amiloride-sensitive epithelial Na(+) channel (ENaC) controls Na(+) transport into cells and across epithelia. So far, four homologous subunits of mammalian ENaC have been isolated and are denoted as alpha, beta, gamma, and delta. ENaCdelta can associate with beta and gamma subunits and generate a constitutive current that is 2 orders of magnitude larger than that of homomeric ENaCdelta. However, the distribution pattern of ENaCdelta is not consistent with that of the beta and gamma subunits. ENaCdelta is expressed mainly in the brain in contrast to beta and gamma subunits, which are expressed in non-neuronal tissues. To explain this discrepancy, we searched for novel functional properties of homomeric ENaCdelta and investigated the detailed tissue distribution in humans. When human ENaCdelta was expressed in Xenopus oocytes and Chinese hamster ovary cells, a reduction of extracellular pH activated this channel (half-maximal pH for an activation of 5.0), and the acid-induced current was abolished by amiloride. The most striking finding was that the desensitization of the acid-evoked current was much slower (by approximately 10% 120 s later), dissociating from the kinetics of acid-sensing ion channels in the degenerin/epithelial Na(+) channel family, which were rapidly desensitized during acidification. RNA dot-blot analyses showed that ENaCdelta mRNA was widely distributed throughout the brain and was also expressed in the heart, kidney, and pancreas in humans. Northern blotting confirmed that ENaCdelta was expressed in the cerebellum and the hippocampus. In conclusion, human ENaCdelta activity is regulated by protons, indicating that it may contribute to the pH sensation and/or pH regulation in the human brain.

We report the case of a 3-year-20-day-old girl who died of starvation as a result of severe neglect. Her body weight had been 12 kg 70 days before her death, but was only 5 kg at the time of autopsy. From information supplied by her parents to police, we calculated her daily caloric intake and estimated the factors for physical activity. The daily recommended dietary allowance for the victim was calculated from 700 kcal/ day x the appropriate factor for physical activity. In the absence of enough food, body fat (7.2 kcal/g body fat) and protein (4 kcal/g protein) would have been used to compensate until death. The calculated body weight at the time of death was around 5 kg. The statements of the parents therefore appear to be true.

There are various toxic chemicals that cause cell death. However, in certain cases deleterious agents elicit various cellular responses prior to cell death. To determine the cellular mechanisms by which such cellular responses are induced is important, but sufficient attention has not been paid to this issue to date. In this study, we showed the characteristic effects of an organophosphorus (OP) agent, bis(pinacolyl methyl)phosphonate (BPMP), which we synthesized for the study of OP nerve agents, on cultured rat astrocytes. Morphologically, BPMP induced cytoplasmic vacuolation and stellation in the rat astrocytes. Cytoplasmic vacuolation is a cell pathological change observed, for example, in vacuolar degeneration, and stellation has been reported in astrocytic reactions against various stimuli. By pretreatment with cycloheximide, a protein synthesis inhibitor, stellation was inhibited, although vacuolation was not. Cell staining with a mitochondrion-selective dye indicated that the vacuolation probably occurs in the mitochondria that are swollen and vacuolatred in the center. Interestingly, the extracellular signal-regulated kinase (ERK) cascade inhibitor inhibited vacuolation and, to some extent, stellation. These results suggest that the ERK signaling cascade is important for the induction of mitochondrial vacuolation. We expect that a detailed study of these astrocytic reactions will provide us new perspectives regarding the variation and pathological significance of cell morphological changes, such as vacuolar degeneration, and also the mechanisms underlying various neurological disorders.

The authors report four cases in which severely damaged human remains were identified by mitochondrial DNA (mtDNA) sequencing. Degraded DNA was extracted from highly adipoceratous tissues using the phenol-chloroform method and polymerase chain reaction amplified for sequencing of two hypervariable regions, hypervariable region 1 and hypervariable region 2, of mitochondrial DNA. They also sequenced these regions of blood samples that were obtained from the presumptive mother or sister of the human remains. The sequencing results were compared with each other and with the Anderson's sequence. It was concluded from the sequence data that a lower part of a body in case 1 and some organs in case 2 were from the same woman, and a human head in case 3 and a female body in case 4 were from the relative of a presumptive mother and a sister, respectively.

A procedure utilizing Chelex 100, chelating resin, was adapted to extract DNA from dental pulp. The procedure was simple and rapid, involved no organic solvents, and did not require multiple tube transfers. The extraction of DNA from dental pulp using this method was as efficient, or more so, than using proteinase K and phenol-chloroform extraction. In this study, the Chelex method was used with amplification and typing at Y-chromosomal loci to determine the effects of temperature on the sex determination of the teeth. The extracted teeth were incinerated in a dental furnace for 2 minutes at 100 degrees C, 200 degrees C, 300 degrees C, 400 degrees C, and 500 degrees C. After the isolation of DNA from the dental pulp by the Chelex method, alphoid repeats, and short tandem repeats, the human Y chromosome (DYZ3), DYS19, SYS389, DYS390, and DYS393 could be amplified and typed in all samples incinerated at up to 300 degrees C for 2 minutes. The DYS389 locus in some samples could not be amplified at 300 degrees C for 2 minutes. An autopsy case is described in which genotypings of DYS19, DYS390, and DYS393 from dental pulp obtained from a burned body were needed. The data presented in this report suggest that Chelex 100-based DNA extraction, amplification, and typing are possible in burned teeth in forensic autopsy cases.

The distribution of allele frequency of X-chromosomal STR, DXS10011, from 99 unrelated Japanese, 72 male and 27 female, were determined by PCR amplification and PAGE. At the same time, haplotype frequencies of five Y-chromosomal STR loci, DYS19, DYS389I, DYS389II, DYS390 and DYS393 from male samples were determined.

Introduction The literature described that neural damage caused by ischemia definitely occurs in brain areas. However, few studies have shown real-time changes of extracellular monoamine levels at the time of transient ischemia. Methods We examined changes in the responses of dopamine (DA) and serotonin (5-HT) release in the nucleus accumbens (ACC) of rats treated with four-vessel occlusion (4VO) in experiment 1. In the second experiment, we investigated the selective neural vulnerabilities among the ACC, lateral hypothalamus (LH), and frontal cortex (FC) of rats treated with 4VO and four days of reperfusion. Results The extracellular levels of DA and 5-HT were remarkably increased 200- and 20-fold upon the 10-min clipping of both common carotid arteries in transient cerebral ischemia, respectively. Each increased monoamine release returned to the baseline levels immediately. The release of DA in the ACC and FC was significantly decreased in the rats treated with the coagulation of bilateral vertebral arteries (2VO), compared with that of sham-operated rats. K + -induced DA release in the ACC and FC of 4VO-treated rats was increased without alteration of DA content. Discussion Surviving dopaminergic neurons in the ACC and FC showed neural hyperfunction associated with the monoamine release, serotonergic neurons in particular these areas exhibiting functional resistance to the transient ischemic change. Conclusion It is suggested that the remarkable extracellular release of DA and 5-HT was not the cause of the ischemic delayed neural degeneration in each brain area, and that the functions of neurotransmitter release involved remarkable resistance to the transient ischemia.

Immunoassays are useful methods for the determination of regulated drugs in clinical and forensic laboratories. Although the Instant-View M-1 (IV M-1) immunoassay kit is frequently used to screen drugs in laboratories in Japan, basic information about the IV M-1 such as its specificity and reactivity is not available. In this study, we determined the specificity and cross-reactivity of IV M-1 for the detection of benzodiazepine-related drugs and their metabolites in urine. The IV M-1 could detect triazolobenzodiazepines such as triazolam in urine at concentrationsor = 300 ng/mL. However, thienodiazepines such as etizolam could not be detected because of lack of cross reactivity. A correlation was observed between the structure of the metabolites and the reactivity of the kit; 4-hydroxy metabolites of alprazolam and triazolam were detectable, whereas a-hydroxy metabolites were not. Furthermore, 7-amino metabolites such as nitrazepam could not be detected at any concentration, including high concentrations. The specificity and reactivity of various kits used for detection of drugs in urine are different. Therefore, it is necessary to consider the basic features of the kit used while assessing the results obtained.

Benzodiazepines are one of the most widely prescribed drugs for the treatment of a wide spectrum of clinical disorders. They are used as anticonvulsants, anxiolytics, hypnotics or muscle relaxants with different duration of action. In this paper, a simple and sensitive method for the determination of benzodiazepines in whole blood using solid-phase extraction and gas chromatography/mass spectrometry (GC/MS) is described. The drugs spiked in whole blood were extracted with an Oasis™ HLB solid-phase extraction cartridge (Waters), which contains a copolymer designed to have a hydrophilic–lipophilic balance. GC/MS analysis was performed using a Shimadzu QP-5000 equipped with a BPX5 capillary column (15 m×0.32 mm I.D., film thickness 0.25 μm, SGE). Nineteen benzodiazepines and two thienodiazepines were well separated from each other on their SIM chromatograms and also on the TIC with the exception of oxazolam to cloxazolam separation. The blank extract from whole blood gave no peaks that interfered with all benzodiazepines and thienodiazepines on the chromatogram. The calibration curves for selected benzodiazepines with fludiazepam as an internal standard showed excellent linearity over the concentration range 5–500 ng/ml blood with a correlation coefficients of >0.995. The detection limits ranged from 0.2 to 20 ng/ml blood. The method is simple and sensitive for the determination of benzodiazepines in whole blood and seems to be useful in the practice of forensic science.

Mitochondrial DNA (mtDNA) is a powerful tool for forensic casework when the resource is highly putrefied or very limited. It is important that mtDNA sequence databases continue to be generated and published, to extend mtDNA typing capability to additional populations and to increase the size of existing databases. The aim of this study was to analyze hypervariable region 1 (HV1) of mtDNA and establish a new database of mtDNA in Japanese population. Three hundred and ninety-four unrelated Japanese individuals were investigated. They were classified into 193 haplotypes based on HV1 sequence. Nucleotide substitutions were clustered in several positions and were 79% at 16,223, 50% at 16,362, and 29% at 16,189. Transition accounts for 81% of all the mutations and especially transition from cytosine to thymine (33%) was the most frequent substitution. 16,223–16,362 was observed in 34 individuals, following 16,129–16,223–16,362 in 29 individuals, and haplotype 16,223–16,278–16,362 in 14 individuals. Genetic diversity and random match probability in this population were estimated to 0.987% and 1.95%, respectively. In conclusion, HV1 of mitochondria sequence has a high discrimination power, but in addition to HV1, analysis of other region such as HV2 and HV3 are required in certain cases.