[Identification of Descarbonsildenafil in an Adulterated Dietary Supplement and Evaluation of Its Inhibitory Activity for Phosphodiesterase Type 5 (PDE5)].

Some illegal dietary supplements contain phosphodiesterase type 5 (PDE5) inhibitors, such as sildenafil, for exerting "therapeutic" effects in erectile dysfunction. This is apparently dangerous, and thus, should be appropriately regulated. Identification of descarbonsildenafil was first reported in Singapore in a coffee sample labeled to exert male sexual performance enhancement effects. However, it is unclear whether the compound possesses PDE5 inhibitory activity. We encountered during our survey of dietary supplements, a sexual enhancement product commercially available in Tokyo, in which a peak presumed to be of descarbonsildenafil was detected by LC-UV and electrospray ionization-tandem MS (ESI-MS/MS). The compound was isolated and identified as descarbonsildenafil with liquid chromatography-quadrupole time-of-flight-mass spectrometry (LC-QTOF-MS), NMR, and X-ray crystal structural analysis. In addition, descarbonsildenafil showed PDE5 inhibitory activity in PDE5 inhibition assay, and its IC50 value for PDE5A1 was found to be 30 nmol/L. The results of INADEQUATE NMR and X-ray crystal structural analysis in this study provide information for the identification of descarbonsildenafil. Since this study indicates that this compound is a PDE5 inhibitor having adequate activity, it is regulated as a drug component in Japan.

Severe Cardiac Rupture by Only One Blow to the Chest in a Young Boy: An Autopsy Case.

Cardiac rupture by blunt chest trauma is commonly seen after motor vehicle accidents and falls; however, it is rarely caused by a blow to the chest. We herein report an autopsy case of a high school boy who sustained severe right ventricular rupture by only one knee kick to the chest during a quarrel. He was hospitalized and developed cardiopulmonary arrest. Emergency surgery was performed, but the patient died. The autopsy revealed no external severe trauma or deformation, but the side wall of the right ventricle contained a large V-shaped laceration. The other thoracic organs had no injuries. This case illustrates that severe cardiac rupture can occur by only one blow to the chest. Blunt cardiac injuries can occur even if no severe injuries are present on the body surface. We should consider the possibility of severe cardiac injuries regardless of the presence of external injuries.

Lipid-Lowering Therapy With Ezetimibe Decreases Spontaneous Atherothrombotic Occlusions in a Rabbit Model of Plaque Erosion: A Role of Serum Oxysterols.

OBJECTIVE: Plaque erosion is increasing its importance as one of the mechanisms of acute coronary syndromes in this statin era. However, the clinical efficacy of currently used lipid-lowering agents in the prevention of thrombotic complications associated with plaque erosion has not been clarified. Therefore, we examined the therapeutic effects of ezetimibe or rosuvastatin monotherapy on spontaneous atherothrombotic occlusion. APPROACH AND RESULTS: Femoral arteries of Japanese white rabbits, fed a high-cholesterol diet, were injured by balloon catheter, and then angiotensin II was continuously administrated. In 94% of these arteries, spontaneous thrombotic occlusions were observed after 5 weeks (median) of balloon injury. Histochemical analyses indicated that the injured arteries had similar pathological features to human plaque erosions; (1) spontaneous thrombotic occlusion, (2) lack of endothelial cells, and (3) tissue factor expression in vascular smooth muscle cells. Ezetimibe (1.0 mg/kg per day), but not rosuvastatin (0.6 mg/kg per day), significantly decreased thrombotic occlusion of arteries accompanied with accelerated re-endothelialization and the decreases of serum oxysterols despite the comparable on-treatment serum cholesterol levels. The 7-ketocholesterol inhibited the migration of human umbilical vein endothelial cells. Both 7-ketocholesterol and 27-hydroxycholesterol increased tissue factor expression in cultured rat vascular smooth muscle cells. Tissue factor expression was also induced by serum from vehicle- or rosuvastatin-treated rabbits, but the induction was attenuated with serum from ezetimibe-treated rabbits. CONCLUSIONS: We have established a novel rabbit model of spontaneous atherothromobotic occlusion without plaque rupture that is feasible to test the therapeutic effects of various pharmacotherapies. Ezetimibe may decrease atherothrombotic complications after superficial plaque erosion by reducing serum oxysterols.

Semiquantitative screening of trace combustion-derived volatile substances in the blood of fire victims using NeedlEx® headspace gas chromatography/mass spectrometry.

BACKGROUND: Various autopsy findings are used for the diagnosis of burning death, including inhalation injury, soot in the respiratory tract, vital reaction, blood carboxyhemoglobin (COHb), and blood cyanide. However, not all findings are necessarily complete, and autopsy diagnosis can be difficult in cases where there are few findings. In this study, we analyzed combustion-derived volatile substances (CVSs) in post-mortem blood in fire-related cases, focusing on those that occur when buildings and furniture thermally decompose during a fire, and evaluated these as potential new autopsy findings. METHODS: The blood CVSs of 61 fire-related cases from 2013 to 2015 were analyzed by a rapid and operationally simple method that employed a gas chromatograph-mass spectrometer coupled to a NeedlEx®, an extraction needle packed with a medium that selectively adsorbs volatile organic compounds. RESULTS: A total of 39 types of aliphatic and aromatic hydrocarbons were detected in the blood of fire victims. They could be detected even in post-mortem blood with COHb <10%, cyanide concentration <0.25µg/ml, and no soot in the respiratory tract. Styrene was detected in all cases except for one case of burning suicide with kerosene. The blood CVSs could be readily differentiated between the victims of building fires, self-immolation with kerosene, and self-immolation with gasoline: the presence of alpha-methylstyrene with a large amount styrene indicated a building fire; the detection of C9-C12 aliphatic hydrocarbons and C3 alkylbenzenes suggested a kerosene-related fire; and the detection of large amount of toluene and C3 alkylbenzenes indicated a gasoline-related fire. The number of blood CVSs detected for the building fire victims showed correlations with COHb level, cyanide concentration, and the amount of soot. With self-immolation, the number of blood CVSs detected showed weak correlation with COHb level, cyanide concentration, or amount of soot. CONCLUSION: Analysis of blood CVSs enabled the identification of the type of fire-related death, which is impossible by conventional autopsy findings.

A Translational Study of a New Therapeutic Approach for Acute Myocardial Infarction: Nanoparticle-Mediated Delivery of Pitavastatin into Reperfused Myocardium Reduces Ischemia-Reperfusion Injury in a Preclinical Porcine Model.

BACKGROUND: There is an unmet need to develop an innovative cardioprotective modality for acute myocardial infarction, for which interventional reperfusion therapy is hampered by ischemia-reperfusion (IR) injury. We recently reported that bioabsorbable poly(lactic acid/glycolic acid) (PLGA) nanoparticle-mediated treatment with pitavastatin (pitavastatin-NP) exerts a cardioprotective effect in a rat IR injury model by activating the PI3K-Akt pathway and inhibiting inflammation. To obtain preclinical proof-of-concept evidence, in this study, we examined the effect of pitavastatin-NP on myocardial IR injury in conscious and anesthetized pig models. METHODS AND RESULTS: Eighty-four Bama mini-pigs were surgically implanted with a pneumatic cuff occluder at the left circumflex coronary artery (LCx) and telemetry transmitters to continuously monitor electrocardiogram as well as to monitor arterial blood pressure and heart rate. The LCx was occluded for 60 minutes, followed by 24 hours of reperfusion under conscious conditions. Intravenous administration of pitavastatin-NP containing ≥ 8 mg/body of pitavastatin 5 minutes before reperfusion significantly reduced infarct size; by contrast, pitavastatin alone (8 mg/body) showed no therapeutic effects. Pitavastatin-NP produced anti-apoptotic effects on cultured cardiomyocytes in vitro. Cardiac magnetic resonance imaging performed 4 weeks after IR injury revealed that pitavastatin-NP reduced the extent of left ventricle remodeling. Importantly, pitavastatin-NP exerted no significant effects on blood pressure, heart rate, or serum biochemistry. Exploratory examinations in anesthetized pigs showed pharmacokinetic analysis and the effects of pitavastatin-NP on no-reflow phenomenon. CONCLUSIONS: NP-mediated delivery of pitavastatin to IR-injured myocardium exerts cardioprotective effects on IR injury without apparent adverse side effects in a preclinical conscious pig model. Thus, pitavastatin-NP represents a novel therapeutic modality for IR injury in acute myocardial infarction.

Toward male individualization with rapidly mutating y-chromosomal short tandem repeats.

Relevant for various areas of human genetics, Y-chromosomal short tandem repeats (Y-STRs) are commonly used for testing close paternal relationships among individuals and populations, and for male lineage identification. However, even the widely used 17-loci Yfiler set cannot resolve individuals and populations completely. Here, 52 centers generated quality-controlled data of 13 rapidly mutating (RM) Y-STRs in 14,644 related and unrelated males from 111 worldwide populations. Strikingly, >99% of the 12,272 unrelated males were completely individualized. Haplotype diversity was extremely high (global: 0.9999985, regional: 0.99836-0.9999988). Haplotype sharing between populations was almost absent except for six (0.05%) of the 12,156 haplotypes. Haplotype sharing within populations was generally rare (0.8% nonunique haplotypes), significantly lower in urban (0.9%) than rural (2.1%) and highest in endogamous groups (14.3%). Analysis of molecular variance revealed 99.98% of variation within populations, 0.018% among populations within groups, and 0.002% among groups. Of the 2,372 newly and 156 previously typed male relative pairs, 29% were differentiated including 27% of the 2,378 father-son pairs. Relative to Yfiler, haplotype diversity was increased in 86% of the populations tested and overall male relative differentiation was raised by 23.5%. Our study demonstrates the value of RM Y-STRs in identifying and separating unrelated and related males and provides a reference database.

DNA extraction for short tandem repeat typing from mixed samples using anti-human leukocyte CD45 and ABO blood group antibodies.

DNA testing from mixed cell samples can be difficult to use successfully in criminal investigations. Here, we present a method for the extraction of DNA from mixed bloodstains involving plural contributors, after antibody-microbead captured cell separation. This method, together with the multiplex short tandem repeat typing presented, has proven highly successful in the recovery of DNA profiles corresponding to the ABO blood type. Methodological steps include magnetic separation using leukocyte specific CD45 antibody-coated microbeads and centrifugal separation of leukocyte agglutination by ABO antibody. The detection results of variable mixed ratio showed that the target DNA was detected accurately as low as 1:512 mixed ratio, regardless of the large amount of the background DNA present. The method presented here is applicable to PCR-based identification for various kinds of mixed samples.

Concurrent urothelial carcinoma in the renal pelvis of an allograft kidney and native recipient bladder: evidence of donor origin.

A 44-year-old woman was admitted to the hospital for asymptomatic gross hematuria. At the age of 28, she underwent transplantation of a kidney from her father for end-stage renal disease secondary to rapidly progressive glomerulonephritis. She resumed peritoneal dialysis when the allograft kidney stopped functioning at the age of 42. Dialysis was continued for the next 2 years, when the hematuria occurred and she was readmitted. Radiologic evaluation and transurethral resection of the bladder tumor revealed a tumor of the renal pelvis of the allograft kidney (cT3N0M0) and multiple bladder tumors (cT1N0M0). Total cystectomy and allograft nephroureterectomy were performed. Histopathological examinations revealed high grade urothelial carcinoma in the renal pelvis of the allograft kidney (pT3) and native bladder (pT1). Fluorescence in situ hybridization of both specimens demonstrated that the renal pelvic tumors and bladder cancer possessed XY karyotypes. These results indicated that the urothelial carcinoma developed de novo in the renal pelvis of the allograft kidney and was implanted into the recipient's native bladder.

Vanadium accelerates polymerase chain reaction and expands the applicability of forensic DNA testing.

Polymerase chain reaction (PCR) has been rapidly established as one of the most widely used techniques in molecular biology. Because most DNA analysis is PCR-based, the analysis of unamplifiable DNA of poor quality or low quantity is nearly impossible. However, we observed that if an appropriate concentration of vanadium chloride is added to the standard reaction mixture, the enzymatic amplification of DNA could be enhanced. Using multiplex PCR with the addition of vanadium, DNA typing was possible from even trace amounts of DNA that we were unable to amplify using normal reaction conditions. This method might be an effective tool for not only criminal investigations and ancient DNA analysis, but also for nearly all fields using DNA technology.

Application of a novel multiplex polymerase chain reaction system for 12 X-chromosomal short tandem repeats to a Japanese population study.

The analysis of X-chromosomal short tandem repeat (X-STR) polymorphisms has been the focus of attention in several researches, mainly due to its applicability in the investigation of complex kinship cases. A new 12 X-STR multiplex system (GATA172D05, DXS7423, DSX6809, DXS10134, DXS7132, DXS9902, DXS6789, DXS10074, DXS8378, DXS9898, DXS10147, and GATA31E08) was developed and applied to a Japanese population study. DNA samples from 290 males and 160 females were successfully analyzed using the 12 X-STR multiplex system. No mutation was detected in the kinship cases involving 34 family trios. The combined powers of discrimination of the 12 X-STR loci in males and females were 0.999997 and 0.9999999996, respectively. We conclude that the combined analysis of 12 X-STR loci using this single multiplex polymerase chain reaction system is a powerful tool in forensic DNA testing.

[The succession of the Hippocratic corpus in modern Greece].

This paper examines how the Hippocratic corpus was passed on during the Enlightenment of modern Greece, introducing part of the latest Greek research on the history of medicine. Although classical studies at large had stagnated at the time under the rule of the Ottoman Empire, with the movement toward independence in the second half of the 18th century the Greeks raised their consciousness of the fact that they were the successors to their ancestral great achievements. From that time classical studies, including the history of medicine, had been activated. From some medical dissertations and books written by Greek doctors or researchers of those days, we will recognize that they made efforts to deepen the substance of modern Greek medicine, seeking the principles of medical practice from the ancient heritage.

Cell death and cell proliferation in cartilage layers in human anterior cruciate ligament tibial insertions after rupture.

The purpose of this study is to investigate cellular responses and histological changes of cartilaginous layers in human anterior cruciate ligament (ACL) tibial insertions after rupture compared with those in normal insertions. Fully 16 tibial insertions of ruptured ACLs were obtained during primary ACL reconstructions. We also obtained 16 normal ACL tibial insertions from cadavers. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) to detect apoptosis, proliferating cell nuclear antigen (PCNA) staining, and histological examination were performed. The percentage of TUNEL-positive chondrocytes in ruptured ACL insertions (30.2 +/- 15.6%) was higher than that in normal insertions (9.6 +/- 5.8%). The percentage of PCNA-positive chondrocytes was significantly different between ruptured ACL insertions (19.9 +/- 15.0%) and normal insertions (12.3 +/- 7.3%). The average thickness of the cartilage layer, the glycosaminoglycan-stained area, and the number of chondrocytes per millimeter in ruptured ACL insertions was smaller than those in normal insertions. The decrease in the number of chondrocytes owing to an imbalance between cell death and cell proliferation in the ACL insertions after rupture, as compared with normal insertions, may lead to histological changes of the cartilage layer in the insertions. An in-depth understanding of injured ACL insertion may help elucidate the etiology of histological changes and the function and significance of the existence of the cartilage layer of insertion. This understanding may help in developing optimal treatment protocols for ACL injuries if apoptosis and cell proliferation are controlled.

[Suicide case of carbon dioxide poisoning using dry ice].

We had a suicide case suspected to result in death from carbon dioxide poisoning by dry ice in the car. A clay cooking stove with charcoal was in the car, but the charcoal had no burning sign. CO hemoglobin saturation degree of the suicide victim's blood was 0%. Moreover, there were signs that 50 kg of dry ice was brought in the car. To clarify the cause of death, reproducibility testing was carried out by using a car under the same conditions. CO2 concentration in it increased to 22% and O2 concentration decreased to 16% within 20 minutes. From these observations, his death was considered to be caused by hypoxia and CO2 narcosis. CO2 in the suicide victim's blood was higher than those in the blood of healthy persons, and the same range was visible in the blood of fire victims. These data might support above supposition concerned with the cause of death. Blood analysis will be helpful in clearing the cause of death by CO2 poisoning.

Time-course changes of hematology and clinical chemistry values in pregnant rats.

The aim of this study is to report how pregnancy alters hematology and clinical chemistry values in rats. Female and male Sprague-Dawley rats were mated; the day of copulation was designated as Day 0. Hematology and clinical chemistry measurements were conducted on Days 7, 14, 17 and 21 in pregnant rats. Measurements were also conducted in non-pregnant rats. Red blood cells (RBC), hemoglobin (Hb), hematocrit (Ht), total protein and albumin decreased on Days 7, 14, 17 and 21; sodium, chloride and glucose decreased on Days 14, 17 and 21; iron decreased on Days 17 and 21; hemoglobin content of reticulocytes (CHr), calcium, inorganic phosphorus and the albumin/globulin ratio decreased on Day 21; and total cholesterol, phospholipid and high-density lipoprotein cholesterol decreased on Day 14 in pregnant rats compared with non-pregnant rats. Reticulocyte increased on Days 7, 14 and 17; mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, neutrophil count and rate increased on Days 14, 17 and 21; platelets, fibrinogen, triglyceride and free fatty acid increased on Days 17 and 21; and activated partial thromboplastin time was prolonged on Days 17 and 21 in pregnant rats compared with non-pregnant rats. The decreased RBC, Hb, Ht, CHr and iron in pregnant rats indicated that they suffered from iron deficiency anemia. These data can be used as background information for effective evaluation in reproductive toxicology studies.

Examination of mRNA expression in rat hearts and lungs for analysis of effects of exposure to concentrated ambient particles on cardiovascular function.

Epidemiological studies have suggested that fine particulate matter (f-PM) is associated with adverse effects on cardiovascular health. However, these effects on the cardiovascular system have not yet been fully elucidated. Using mRNA expression and correlation analyses, we designed the present study to elucidate (1) translocation of chemicals in inhaled f-PM to the heart, (2) induction of oxidative stress, one of the causes of cardiovascular diseases (CVDs), (3) mRNA expression related to CVDs, and (4) correlations among mRNA expression of various molecules and cardiovascular function. Wistar Kyoto male rats were exposed to concentrated ambient particles (CAPs, 0.6-1.5mg/m3) in Yokohama for 4 days (4.5h/day) or to filtered air for 3 days and CAPs for 1 day or to filtered air for 4 days. Messenger RNA expression and cardiovascular function were measured after the 4-day exposure. In samples of heart tissue, the mRNAs of cytochrome P450 (CYP) 1B1, a biomarker of exposure to chemicals; heme oxygenase-1 (HO-1), a marker of oxidative stress; and endothelin A (ET A) receptor, a receptor of vasoconstrictors, were up-regulated by CAPs; their levels were significantly correlated with the cumulative weight of CAPs in the exposure chamber. The up-regulation of ET A receptor mRNA was significantly correlated with the increase in HO-1 mRNA and weakly with the increase in mean blood pressure (Delta MBP). These results suggest the possibility that chemicals in CAPs might be translocated to the heart, where they induce oxidative stress and activate endothelin signaling, resulting in an increase in the blood pressure. The exposure to f-PM might thus affect cardiovascular function through activation of endothelin signaling.