A method for determining the origin of crude drugs derived from animals using MinION, a compact next-generation sequencer.

We evaluated whether MinION, an inexpensive portable sequencer, can be used to identify the origin of crude drugs derived from animals. Standard and nonstandard crude drugs with different species of origin were examined. In addition, standards mixed with nonstandard samples were used. As a target gene, cytochrome c oxidase I was amplified and sequenced. The fast mode results had a slightly lower match ratio than high-accuracy mode, but the animals of origin were correctly determined by BLAST for all samples. For antler velvet derived from Rangifer tarandus, even when the sequences were aligned based on Cervus elaphus, the animal of origin was determined correctly. Minor contents could be detected from mixtures of two animals, if the mixtures contained at least 19:1 mtDNA when the coverage allele-fraction threshold was 0.05. By contrast, in fast mode, two sequences could not be separated due to the low accuracy of the base-calling for each read. For fieldwork, the species of origin of crude drugs could be identified with only simple DNA extraction and library preparation. Therefore, MinION appears to be a convenient tool for identifying the origins of crude drugs derived from animals.

Development of an Okinawa panel for biogeographic inference of Okinawans.

BACKGROUND: The Precision ID Ancestry Panel with 165 SNP markers was unable to differentiate between mainland Japanese and Okinawa Japanese or to distinguish either of them from other East Asian populations. AIM: An Okinawa panel was developed with the aim of further separating Okinawa Japanese individuals from mainland Japanese and other Asian groups. Seventy-five SNPs were selected using the most informative markers from the literature. Further, 22 SNPs were selected to separate Okinawa Japanese at minimum SNPs. SUBJECTS AND METHODS: Samples were collected from 48 unrelated individuals from mainland Japan and 46 unrelated residents of the Okinawa prefecture. Data were evaluated by STRUCTURE, principal component, and GenoGeographer analyses. RESULTS: The 22 SNP set had similar levels of differentiation in STRUCTURE and PCA analyses as the 75 SNP set. GenoGeographer analysis showed that, out of the 46 Okinawa Japanese individuals, the 75 SNP and 22 SNP sets correctly assigned the Okinawan population as the most likely population of origin for 32 and 31 individuals, respectively. CONCLUSION: Neither SNP set could completely differentiate between Okinawa Japanese and other Asian groups, however, these sets should be useful for crime investigation, when the sample, cost and time are limited.

Hepatic invasion of a gastric ulcer confirmed by endoscopy and surgery.

A 66-year-old woman who had been suffering from chronic anorexia for two years was transported to the hospital after being unable to consume food for three days. She had no hematemesis or abdominal pain and had no history of taking nonsteroidal anti-inflammatory drugs. Blood tests showed marked anemia with hemoglobin of 3.3 g/dL, and esophagogastroduodenoscopy revealed a large ulcer lesion in the lesser curvature of the gastric body and a liver-like mass protruding from the ulcer base. Biopsy of the mass showed proliferation of cells showing irregular cord-like structures, suggestive of normal liver tissue or hepatocellular carcinoma. Computed tomography scan showed no obvious free air in the abdomen. Despite conservative treatment, the patient developed hematemesis and progressive anemia, and surgery was performed (total gastrectomy with partial hepatectomy). Surgical specimen showed an ulcer lesion with fibrosis and loss of wall structure in all layers of the stomach, and liver adhesion with fibrosis deep in the ulcer, but no malignant findings. With the advent of powerful gastric acid secretion inhibitors, gastric ulcer invasion into the liver is now very rare, and this case is thus a valuable example showing very clear images.

Estimating individual mtDNA haplotypes in mixed DNA samples by combining MinION and MiSeq.

We tried to estimate individual mtDNA haplotypes in mixed DNA samples by combining MinION and MiSeq. The BAM files produced by MiSeq were viewed using Integrative Genomics Viewer (IGV) to verify mixed bases. By sorting the reads according to base type for each mixed base, partial haplotypes were determined. Then, the BAM files produced by MinKNOW were viewed using IGV. To determine haplotypes with IGV, only mixed bases determined by MiSeq were used as target bases. By sorting the reads according to base type for each target base, each contributor's haplotype was estimated. In mixed samples from two contributors, even a haplotype with a minor contribution of 5% could be distinguished from the haplotype of the major contributor. In mixed samples of three contributors (mixture ratios of 1:1:1 and 4:2:1), each haplotype could also be distinguished. Sequences of C-stretches were determined very inaccurately in the MinION analysis. Although the analysis method was simple, each haplotype was correctly detected in all mixed samples with two or three contributors in various mixture ratios by combining MinION and MiSeq. This should be useful for identifying contributors to mixed samples.

Increased plasma glutamate in non-smokers with vasospastic angina pectoris is associated with plasma cystine and antioxidant capacity.

Objectives. Endothelial dysfunction caused by oxidative stress plays an important role in the development of vasospastic angina pectoris (VSAP). Glutamate causes endothelial dysfunction by generating oxidative stress, and it inhibits cystine import into endothelial cells via the cystine/glutamate antiporter (XC-), which leads to depletion of antioxidant glutathione. However, whether glutamate and cystine are implicated in the pathogenesis of VSAP remains unclear. We investigated plasma glutamate and cystine levels, oxidative stress markers and antioxidant capacity in non-smoker patients with VSAP to determine whether glutamate and cystine are associated with the development of VSAP. We assessed 49 non-smokers assigned to groups with (n = 27) and without (n = 22) VSAP, and also measured plasma glutamate, cystine, nitrotyrosine, reactive oxygen metabolites and biological antioxidant potential. Results. Plasma glutamate and cystine values were significantly higher in the group with, than without VSAP (59.8 ± 25.7 vs. 43.5 ± 18.7 µmol/L, p = .016 and 35.3 ± 14.2 vs. 25.2 ± 9.1 µmol/L, p = .0056, respectively). Plasma glutamate and cystine values were significantly and positively associated (r = 0.32, p = .027). Levels of the oxidative stress markers nitrotyrosine and reactive oxygen metabolites, and biological antioxidant potential of as a measure of antioxidant capacity, did not significantly differ between the two groups. However, glutamate and biological antioxidant potential values were significantly and negatively associated (r = -0.3, p = .036). Conclusion. Plasma glutamate levels were increased in patients with VSAP who did not smoke, and they were positively associated with plasma cystine and negatively associated with the biological antioxidant potential levels.

Glyceraldehyde-derived advanced glycation end-products are associated with left ventricular ejection fraction and brain natriuretic peptide in patients with diabetic adverse cardiac remodeling.

Objectives: Glyceraldehyde-derived advanced glycation end-products (Glycer-AGEs) have a strong binding affinity for their cognate receptor and elicit oxidative stress and inflammation. However, it remains unknown whether the levels of Glycer-AGEs correlate with the severity of cardiac function and heart failure in patients with diabetic adverse cardiac remodeling (DbCR). Fourteen heart failure patients with type 2 diabetes mellitus (DM) without other cardiac disorders (DbCR group) were enrolled. Another 14 patients with idiopathic dilated cardiomyopathy (DCM) without DM were served as a control (DCM group). All patients were assessed for serum Glycer-AGEs, nitrotyrosine (NT), and tumor necrosis factor alpha (TNFα) and for plasma brain natriuretic peptide (BNP). The left ventricular ejection fraction (LVEF) was evaluated by echocardiography. Results: The mean serum levels of Glycer-AGEs, NT, and TNFα in the DbCR group were significantly higher than those in the DCM group (for Glycer-AGEs, p = .0073; for NT, p = .005; for TNFα, p < .0001, respectively). In the patients with DbCR, the levels of serum Glycer-AGEs and TNFα were closely associated with LVEF and BNP values. Conclusions: Both Glycer-AGEs and TNFα showed close associations with LVEF and the levels of BNP in patients with DbCR. Glycer-AGEs and TNFα may play a pathological role in the development of DbCR.

Dissecting lesions in a culprit artery of a hemorrhagic focus in the basal ganglia: Histopathological analysis by serial sectioning.

In a hypertensive hemorrhagic focus of the basal ganglia, the culprit arteries have been reported to be associated with dissecting lesions, whose topographical relationship to the rupture sites remains to be clarified. Herein we describe multiple dissecting lesions in the culprit artery of hypertensive hemorrhage of the basal ganglia. A 1.0 × 0.8 × 0.8 cm-sized bleeding globe was confirmed at a left lenticulostriate artery and histologically analyzed by serial sectioning. Three independent dissecting lesions were identified in the culprit artery. They were situated near the bifurcations, ranging from 240 to 3200 µm in length. The dissections mainly occurred between the intima and media with disruption of the internal elastic lamina (IEL), forming a fresh thrombus within the false lumen. Two rupture sites causing the cerebral hematoma were confirmed away from the dissecting lesions. One was situated close but not adjacent to the longest dissecting lesion; the other, measuring approximately 150 µm in diameter, was adjacent to the bifurcation of an artery. The histopathological findings suggest that the dissecting lesion resulted from medial detachment following IEL disruption in the process of arterial rupture of the culprit artery. We conclude that this was a secondary manifestation during the rupture rather than a cause of the arterial rupture.

Recurrent Cerebral Venous Thrombosis Treated with Direct Oral Anticoagulants in a Japanese Man with Hereditary Protein C Deficiency.

We herein report a case involving a 32-year-old Japanese man with recurrent cerebral venous thrombosis due to hereditary protein C deficiency. He was admitted to our hospital with impaired consciousness. Brain magnetic resonance imaging demonstrated high intensities diffusely along the bilateral sulci and magnetic resonance venography revealed left transverse sinus and superior sagittal sinus stenoses. His father had a history of cerebral infarction and venous thrombosis. The protein C activity level examined by chromogenic synthetic substrate assay was markedly reduced. He was diagnosed with protein C deficiency, and a genetic analysis revealed a heterozygous mutation at exon 3 c.199G>A,p.Glu67Lys on the protein C gene. Four months later, at his second admission, he had transient aphasia, and his protein C activity was under 10%. We switched warfarin to the direct oral anticoagulants edoxaban. He remains fully recovered with no adverse events after the administration of edoxaban for a year. Direct oral anticoagulants may be a new tool for treating cerebral venous thrombosis due to hereditary protein C deficiency.

Estimation of the number of contributors to mixed samples of DNA by mitochondrial DNA analyses using massively parallel sequencing.

We evaluated whether the number of contributors to mixed DNA samples can be estimated by analyzing the D-loop of mitochondrial DNA using massively parallel sequencing. The A- (positions 16,209-16,400) and B- (positions 30-284) amplicons in hypervariable regions 1 and 2, respectively, were sequenced using MiSeq with 2 × 251 cycles. Sequence extraction and trimming were performed using CLC Genomics Workbench 11 and the number of observed haplotypes was counted for each amplicon type using Microsoft Excel. The haplotype ratios were calculated by dividing the number of counted reads of the corresponding haplotype by the total number of sequence reads. Haplotypes that were over the threshold (5%) were defined as positive haplotypes. The number of larger positive haplotypes in either of the two amplicon types was defined as the number of contributors. Samples were collected from seven individuals. Seventeen mixed samples were prepared by mixing DNA from two to five contributors at various ratios. The number of contributors was correctly estimated from almost all of the mixed samples containing equal amounts of DNA from two to five people. In mixed samples of two or three people, the minor components were detected down to a ratio of 20:1 or 8:2:1. However, heteroplasmy, base deletions, and sharing of the same haplotypes caused incorrect estimations of the number of contributors. Although this method still has room for improvement, it may be useful for estimating the number of contributors in a mixed sample, as it does not rely on forensic mathematics.

Long-term outcomes and safety of radical transmediastinal esophagectomy with preoperative docetaxel, cisplatin, and 5-fluorouracil combination chemotherapy for locally advanced squamous cell carcinoma of the thoracic esophagus.

BACKGROUND: It is unknown whether transmediastinal esophagectomy (TME) is an acceptable surgical procedure for locally advanced esophageal squamous cell carcinoma (ESCC). Therefore, we investigated the feasibility of long-term survival after TME with neoadjuvant docetaxel, cisplatin, and 5-fluorouracil combination chemotherapy (DCF therapy). METHODS: This retrospective, observational study included locally advanced resectable ESCC. All patients received two cycles of preoperative DCF therapy (60 mg/m2 of docetaxel and cisplatin on day 1 and 700 mg/m2/day of 5-FU on days 1-5 in each cycle) followed by radical TME. The main outcomes were survival and the rate of adverse events of chemotherapy and surgery. RESULTS: Sixteen patients were included in this study. All patients received two cycles of DCF therapy, followed by surgery. The median follow-up duration of the 16 patients was 35.4 months. The 2-year overall survival (OS) was 93.3% (95% confidence interval [CI], 61.3-99.0), and the 3-year OS was 78.8% (95% CI, 47.3-92.7). The 2-year and 3-year relapse-free survivals were both 73.3% (95% CI, 43.6-89.1). Leukopenia and neutropenia occurred in most patients; however, they were controllable. Fifteen patients completed TME, and one was converted to open transthoracic esophagectomy because of tracheal injury. Three-field dissection was performed for 12 of 16 patients (75%), and R0 resection was achieved in 15 of 16 patients (93.8%). Three cases of grade IIIb chylothorax were observed. There was no mortality in this study. CONCLUSION: Combined neoadjuvant DCF and TME for locally advanced ESCC was safe and less invasive than traditional therapies and had a satisfactory long-term prognosis.

Allele frequencies and forensic genetic parameters of 10 supplementary and two CODIS loci in a Japanese population genotyped using an Investigator HDplex Kit.

A total of 344 unrelated Japanese adults were genotyped to determine allele frequencies and evaluate forensic parameters for 10 autosomal supplementary non-CODIS loci and 2 autosomal CODIS loci using an Investigator® HDplex Kit for complex relationship testing.

Laparoscopic Transhiatal Thoracic Duct Ligation for Chylothorax after Esophagectomy.

In esophagectomy for thoracic esophageal cancer, chylothorax may develop at a certain frequency. For chylothorax, conservative treatment is selected first, but if it is not improved, thoracic duct (TD) ligation is considered. In general, transthoracic approach is chosen to reach the TD. However, it is sometimes difficult to identify the TD due to adhesion in the thoracic cavity. Hence, we selected a laparoscopic transhiatal approach to the TD. We introduce the procedure of our laparoscopic transhiatal TD ligation technique.

The origin of p40-negative and CDX2-positive primary squamous cell carcinoma of the stomach: case report.

BACKGROUND: Primary gastric squamous cell carcinoma (SCC) is a very rare disease. The origin of this tumor remains unclear, although there are some hypotheses. A 60-year-old man consulted a previous physician complaining of upper abdominal pain. Esophagogastroduodenoscopy revealed type 2 gastric cancer, and the patient was referred to our hospital. After close examination, the patient was diagnosed as cStage IIA gastric adenocarcinoma, and distal gastrectomy was performed. Histochemical studies showed typical findings of SCC, and the tumor was surrounded by intestinal metaplasia. Immunohistochemical examination was positive for cytokeratin (CK) 5/6 and caudal-type homeobox protein 2 (CDX2) and negative for p63/p40. CONCLUSION: The results of immunostaining for CK5/6 supported that this tumor was SCC, but the question why p63/p40 were negative and CDX2 was positive still remained. Concerning about the origin of p63/p40 and CDX2, it was suggested that the tumor cells were not derived from ectopic squamous epithelium but from intestinal metaplasia. And tumor cells looked like homogeneous and squamous metaplasia was not observed. These findings supported the idea that these tumor cells arose from stem cells in the intestinal metaplasia of the stomach.

Sudden unexpected death due to coronary thrombosis associated with isolated necrotizing vasculitis in the coronary arteries of a young adult.

Coronary arteritis is an uncommon cause of sudden death in non-atherosclerotic coronary diseases, and is mostly associated with systemic vasculitis or systemic autoimmune diseases; therefore, sudden death due to isolated coronary arteritis rarely occurs. The case described in this report is that of a 34-year-old man with no significant personal medical history who died suddenly after presenting with nausea. Postmortem examination revealed a significant infiltration of lymphocytes predominantly on the adventitia and periadventitial tissues of the coronary arteries in the epicardium. The lymphocytic infiltrate partially extended to the thickened intima with fibrosis, destructing the media and internal elastic lamina, and the lumen was occluded by a thrombus in the left main stem and left anterior descending branch. The arterial walls exhibited focal fibrinoid necrosis with regression in the intima and fibrous scars with angiogenesis in the media and adventitia. Focal myocardial infarction was detected in the left ventricle as a fibrotic change of the myocardium. No findings associated with vasculitis were discerned in the aorta, other peripheral arteries, or major organs. Laboratory tests of postmortem blood samples returned negative results for antinuclear antibodies, cryoglobulin, immunoglobulin G4, and cytoplasmic anti-neutrophil cytoplasmic antibodies for myeloperoxidase and proteinase 3. These autopsy findings suggest that the sudden death was caused by isolated necrotizing vasculitis that is assumed to be polyarteritis nodosa localized at the coronary arteries. However, pathological characteristics may not be exactly the same between isolated necrotizing vasculitis in the coronary arteries and polyarteritis nodosa.

Deaths Associated With Brotizolam Poisoning From a Single Drug Overdose: Four Reported Cases.

Brotizolam is a short-acting hypnotic in the benzodiazepine family, and fatal poisonings by an overdose of brotizolam are rare. This report describes 4 cases of deaths associated with brotizolam poisoning from a single drug overdose. The ages ranged from 51 to 90 years, and the postmortem interval between death and tissue sampling was 1.5 to 2.5 days. These deaths were classified as 1 homicide and 3 suicides. The concentration of the brotizolam ranged from 0.05 to 0.21 mg/L in the blood samples. Ethanol, which could cause mild alcohol intoxication, was detected in the blood samples from 2 cases. Postmortem examinations did not find any significant pathologic conditions, except for a case of death by drowning in a bathtub due to brotizolam poisoning. These 4 cases suggest that a brotizolam overdose should not be underestimated in terms of its fatal effects, particularly when situations involve alcohol intoxication, injury subsequent to the poisoning, or underlying medical conditions including aging.

The Relationship Between Cephalogram Analysis and Oxygen Desaturation Index During Sleep in Patients Submitted for Mandibular Setback Surgery.

OBJECTIVES: The aim of this study was to examine the relationship between morphologic factors of mandibular protrusion patients and clinical indices of obstructive sleep apnea (OSA). METHODS: Fifty-two Japanese patients divided into 2 groups: 1 jaw surgery group (30 patients) and 2 jaw surgery group (22 patients). Morphologic changes were studied using cephalograms taken before surgery and 1 year after surgery. Functional changes studied using impulse oscillometry and pulse oximetry during sleep, both of which are clinically useful measures in assessing OSA, taken before surgery and 1 year after surgery. RESULT: Lower face cage area significantly decreased in 1 jaw group than in 2 jaw group patients. Positive significant correlation was found between changes in 3% oxygen desaturation index (ODI) and changes of tongue area and vertical position of the hyoid bone in 1 jaw surgery group. Multiple regression analysis indicates that tongue area and airway area were independently significant predictors of 3% ODI in 1 jaw group patients. CONCLUSION: In 2 jaw surgery, maxillary surgery compensated for the effect of mandibular setback surgery. Mandibular setback surgery to mandibular protrusion patients was performed within the range of adequate movement distance, but precautions for risk of postoperative obstructive sleep apnea syndrome should be considered.

Fatal Intracranial Aneurysms and Dissections Causing Subarachnoid Hemorrhage: An Epidemiological and Pathological Analysis of 607 Legal Autopsy Cases.

BACKGROUND: There are no detailed reports, in terms of epidemiology and pathology, on intracranial aneurysms and on dissections that were found in unexpected fatal subarachnoid hemorrhage (SAH) cases. In this report we analyzed, based on large-sized medicolegal autopsy cases, the detailed epidemiology and pathological aspects of both lesions. METHODS: We analyzed 607 autopsy cases of unexpected fatal SAHs including 496 aneurysms and 111 dissections. RESULTS: The following results were obtained: (1) Patients who died of dissections were younger than those who died of aneurysms; (2) symptom prevalence rates of aneurysms were 31.9%, appearing to be lower than those in previous studies; (3) a significantly higher prevalence of clinical symptoms was found in patients with dissections (60.5%) than patients with aneurysms; (4) hypertensive cardiomegaly was indicated in more than 80%, while no obvious difference in incidence in hypertensive cardiomegaly was noted between aneurysms and dissections; (5) aneurysms were found to occur much more frequently in the anterior communicating artery (31.9%) and vertebral arteries (7.5%), while dissections were found much more commonly in vertebral arteries (93.7%); and (6) the size of aneurysms was much smaller in general than that previously regarded as a risk factor of rupturing. CONCLUSIONS: These data might help in the prompt intervention in SAH and also in the prevention of lethal SAH in clinical settings.

Regioselective Rearrangement of 4,4-Disubstituted 2-Hydroxycyclohexa-2,5-Dienones under Deoxyfluorination Conditions.

The dienone-phenol rearrangement is a useful tool for the synthesis of highly substituted phenols. In our previous study of the rearrangement of 4,4-disubstituted 2-hydroxycyclohexa-2,5-dienone under deoxyfluorination conditions, bond migration proceeded with very poor regioselectivity. In this paper, an acid-mediated rearrangement of O-perfluoroalkylsulfonyl difluorides with regioselective migration toward the ß'-carbon is reported. This method allowed the synthesis of a fluorinated analog of allocolchicinoids with improved total yield. Successful application to other substrates was also demonstrated.

Identification of a common single nucleotide polymorphism at the primer binding site of D2S1360 that causes heterozygote peak imbalance when using the Investigator HDplex Kit.

Phenomena known as null alleles and peak imbalance can occur because of mutations in the primer binding sites used for DNA typing. In these cases, an accurate statistical evaluation of DNA typing is difficult. The estimated likelihood ratio is incorrectly calculated because of the null allele and allele dropout caused by mutation-induced peak imbalance. Although a number of studies have attempted to uncover examples of these phenomena, few reports are available on the human identification kit manufactured by Qiagen. In this study, 196 Japanese individuals who were heterozygous at D2S1360 were genotyped using an Investigator HDplex Kit with optimal amounts of DNA. A peak imbalance was frequently observed at the D2S1360 locus. We performed a sequencing analysis of the area surrounding the D2S1360 repeat motif to identify the cause for peak imbalance. A point mutation (G>A transition) 136 nucleotides upstream from the D2S1360 repeat motif was discovered in a number of samples. The allele frequency of the mutation was 0.0566 in the Japanese population. Therefore, human identification or kinship testing using the Investigator HDplex Kit requires caution because of the higher frequency of single nucleotide polymorphisms at the primer binding site of D2S1360 locus in the Japanese population.

Identification of canine saliva using mRNA-based assay.

A dog saliva analysis in addition to a bite-mark analysis may be important for evidence when a crime involves a dog bite. In this study, the utility of detecting canine saliva-specific mRNAs to identify canine saliva was evaluated. Canine saliva swabs (n = 20), urine swabs (n = 20), body surface swabs (n = 20), whole blood samples (n = 10), human saliva (n = 20), human skin surface swabs (n = 20), and human whole blood (n = 20) were tested. The saliva-specific genes encoding statherin (STATH), carbonic anhydrase VI (CA-VI), and dog allergens (Canf1 and Canf2) were analyzed as candidate genes. Moreover, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as confirmation of canine mRNA extraction. STATH, CA-VI, Canf1, Canf2, and GAPDH mRNAs were detected in 19/20, 1/20, 11/20, 4/20, and 20/20 saliva samples, respectively. The STATH, CA-VI, Canf1, Canf2, and GAPDH mRNAs did not exhibit cross-reactivity with samples of human origin. This mRNA-based assay was also able to detect canine saliva in mock forensic samples. The results of this study indicated that the detection of STATH mRNA is useful for the identification of canine saliva, and GAPDH is a suitable marker for canine mRNA extraction.