A Novel Method for Visualizing Melanosome and Melanin Distribution in Human Skin Tissues.

Melanin incorporated into keratinocytes plays an important role in photoprotection; however, abnormal melanin accumulation causes hyperpigmentary disorders. To understand the mechanism behind the accumulation of excess melanin in the skin, it is essential to clarify the spatial distribution of melanosomes or melanin in the epidermis. Although several markers have been used to detect melanosomes or melanin, no suitable markers to determine the precise localization of melanin in the epidermis have been reported. In this study, we showed that melanocore-interacting Kif1c-tail (M-INK), a recently developed fluorescent probe for visualizing mature melanosomes, binds to purified melanin in vitro, and applied it for detecting melanin in human skin tissues. Frozen skin sections from different phototypes were co-stained for the hemagglutinin (HA)-tagged M-INK probe and markers of melanocytes or keratinocytes, and a wide distribution of melanin was observed in the epidermis. Analysis of the different skin phototypes indicated that the fluorescent signals of HA-M-INK correlated well with skin color. The reconstruction of three-dimensional images of epidermal sheets enabled us to observe the spatial distribution of melanin in the epidermis. Thus, the HA-M-INK probe is an ideal tool to individually visualize melanin (or melanosome) distribution in melanocytes and in keratinocytes in skin tissues.

Individual identification using the RapidHIT™ ID system for forensic samples.

The RapidHIT™ ID system produces GlobalFiler™ analysis results after a short operating time. This device is effective because it automatically extracts DNA from oral mucosal cells or from blood stains and saliva collected at a crime scene, with subsequent polymerase chain reaction performed to produce a DNA profile. Two types of dedicated cartridges are available for RapidHIT™ ID: the RapidHIT™ ID ACE GlobalFiler Express sample cartridge for oral cells and other samples and the RapidINTEL™ sample cartridge for minute samples, such as blood stains. Previously validated specimens include oral mucosa cells and blood stains left at crime scenes. There have been no reports of blood and nail clipping samples collected from the postmortem bodies at the time of death. This report summarizes the results of using the RapidHIT™ ID system by collecting a variety of actual forensic samples from postmortem bodies at different stages of decomposition, which were subsequently analyzed using these cartridges.

Extensive gastric necrosis secondary to acute gastric dilatation: A case report.

We report a case of sudden death in a patient who developed extensive gastric necrosis secondary to acute gastric dilatation. A 36-year-old man with mental retardation (but without difficulties in activities of daily living), developed an illness after a meal out with friends, necessitating 3 hospital visits. He returned home after receiving drug therapy; however, his condition deteriorated, and he was transferred to our hospital via ambulance. Whole-body computed tomography performed upon admission revealed gastric dilatation. A stomach tube was inserted, and 2000 mL of gastric aspirate was obtained. The patient died approximately 5 h later despite receiving treatment. Autopsy revealed 1000 mL of gastric contents and extensive gastric necrosis. He was diagnosed with extensive gastric necrosis secondary to acute gastric dilatation.

Vulvar reconstruction should be performed using gluteal-fold perforator flap because of less morbidities and complications.

The authors present a case of bilateral vulvar defects after abrasion of malignant skin neoplasm, reconstructed with a gluteal-fold perforator flap, resulting in a successful outcome.

Cutaneous malignant rhabdoid tumor in the palm of an adult.

Malignant rhabdoid tumor is a rare tumor occurring mostly in the neonatal kidneys and central nervous system. Cutaneous malignant rhabdoid tumors are extremely rare in adults. The aim of the study was to report on the clinical, histologic, and immunophenotypic characteristics of this cutaneous malignant rhabdoid tumor which developed in an adult. A 27-year-old male complained of a right palm neoplasm that had been present for 6 months, which was initially diagnosed as an epithelioid sarcoma by biopsy. However, detailed investigation with immunohistochemistry enabled us to make a diagnosis of a rhabdoid tumor. The patient underwent radical abrasion, chemotherapy, and irradiation, and has survived for 1 year without relapse. Only 20 adult cases have been reported thus far in the English literature. We are reporting the 21(st) case, who remains disease-free at 12 months. Complete resection and local irradiation may increase survival, because there is no standard and reliable curative chemotherapeutic regimen.

Vascularized bone graft is a better option for the reconstruction of maxillary defects.

We read the article of Kinnunen et al., which evaluated the result of maxillary defects, and feel some objections. We present our considerations of their operative indication and thoughts based on our surgical experiences. Defects after palatectomy, which have left no dentition for the retention of an obturator, require vascularized bone-containing free flaps. Local flaps are available in only small defects of Class 1 and 2a. Most palatomaxillary defects following malignant tumor abrasion are classified as 2b, 2c, 3, or 4, which require microsurgical free flap transfer combined with bony reconstruction. Regarding bony reconstruction, non-vascularized bone grafts tend to be absorbed. Thus, we believe that bony reconstruction should be performed with vascularized bone. We agree with the authors' comment that PTMF may be useful in repairing defects due to complications in microvascular procedures in the palatal area. However, even when bone segment is required for salvage surgery, using a vascularized bone flap is more preferable. A parietal bone-fascial-periosteal flap based on the superficial temporal vessels is a suitable and reliable bone flap for the reconstruction of a maxillary defect following free skin flap transfer to the palate.

Simultaneous determination for oxicam non-steroidal anti-inflammatory drugs in human serum by liquid chromatography-tandem mass spectrometry.

A high-performance liquid chromatography-tandem mass spectrometry (LC/MS/MS) technique was developed for the simultaneous determination of five non-steroidal anti-inflammatory oxicam drugs (ampiroxicam, tenoxicam, piroxicam, meloxicam and lornoxicam) in human plasma. These five oxicam drugs and isoxicam (internal standard) were extracted from human plasma with an Oasis(®) MAX cartridge column and analysed on a Unison UK-C18 column (2.0 mm × 100 mm, 3 µm) with an acetonitrile:10mM formic ammonium buffer (pH 3.0) (50:50) mobile phase at 0.20 ml/min at 37°C. The analytes were detected using a tandem mass spectrometer, equipped with an electrospray ion source (ESI). The instrument was used in multiple-reaction-monitoring (MRM) mode. The extraction yields from a 200 µl human plasma sample (containing 10 ng of each drugs) with the Oasis(®) MAX cartridge column were 93.3-102.5%. The detection limits were 0.01-6.5 ng/ml (S/N=3). Our developed method is very useful for the simultaneous determination of five oxicam (non-steroidal anti-inflammatory) drugs in human plasma by LC/MS/MS.

ABO genotyping by TaqMan assay and allele frequencies in a Japanese population.

ABO genotyping have become common tools for forensic casework. We developed a new rapid ABO genotyping method using a fast real-time PCR system with the TaqMan® Sample-to-SNP™ Kit. Eight single nucleotide polymorphism (SNP) sites in the ABO gene (nt 261, 297, 467, 657, 703, 829, 930 and 1061) were selected to determine the ABO genotypes. ABO genotypes were easily determined by examining allelic discrimination patterns. This method enabled analyses to be completed in about 1h per plate with no postmortem change influences. The detection limit in each SNP site was examined as 100pg per reaction. ABO genotyping from 1000 Japanese individuals was also examined to determine the distribution of ABO genotypes and allele frequencies. Thus, 31 genotypes were clearly identified, and these were controlled by four common and seven rare alleles. The power of discrimination, heterozygosity and polymorphism information contents were 0.913, 0.775 and 0.812, respectively. Therefore, selecting these eight SNP sites could be useful for high specific ABO genotyping. This rapid, sensitive and accurate genotyping method is useful for forensic casework.

Simultaneous determination of 5 psychotropic drugs of various types in an autopsy case of acute multiple drug poisoning.

We attempted the simultaneous determination of 5 drugs, mirtazapine, sertraline, chlorpromazine, amoxapine and zolpidem, detected in a gas chromatography-mass spectrometry screening test in an autopsy case. The solid-phase extraction of the analytes from biological samples was achieved using Oasis(®)HLB cartridges (Waters, Milford, MA, USA). Gas chromatography was performed on a HP-5MS fused silica capillary column (30 m × 0.25 mm i.d., 0.25 µm film thickness, Agilent Technologies). The mass spectrometer was operated with an electron energy of 70 eV in electron impact mode. The qualitative and quantitative analyses were performed in full-scan mode and the selected ion monitoring mode, respectively. The total ion chromatogram showed good separation of these drugs. Linear graphs were obtained with good correlation coefficients for these drugs from 0.001 to 2.0 µg/mL (r(2)=0.9909-0.9986) using imipramine-d6 as an internal standard. The recoveries of these drugs were found to be 62.8-88.0% in spiked whole blood. Mirtazapine, sertraline, chlorpromazine, amoxapine and zolpidem were found in post-mortem samples of the deceased at concentrations of 2.67, 0.07, 0.25, 0.32 and 0.68 µg/mL, respectively. The concentration of mirtazapine was within the lethal level and those of amoxapine and zolpidem were within the toxic level. We diagnosed that the cause of death was acute multiple drug poisoning. The simple and practical procedure used in this study is useful for the simultaneous determination of psychotropic drugs of various types in post-mortem biological samples.

Tetanus following replantation of an amputated finger: a case report.

INTRODUCTION: Tetanus is an infectious disease caused by tetanus toxin produced by Clostridium tetani and induces severe neurological manifestations. We treated a patient who developed tetanus during hospitalization for replantation of an amputated finger. To the best of our knowledge, this is the first published case report of such an entity. CASE PRESENTATION: A 49-year-old Japanese man had an amputation of his right middle finger at the distal interphalangeal joint region in an accident at work. His middle finger was successfully replanted, but his fingertip was partially necrotized because of crushing and so additional reconstruction with a reverse digital arterial flap was performed 15 days after the injury. Tetanus developed 21 days after replantation of the middle finger, but symptoms remitted via rapid diagnosis and treatment. CONCLUSIONS: In replantation after finger trauma with exposure of nerve and blood vessel bundles, concern over injuring nerves and blood vessels may prevent irrigation and debridement from being performed sufficiently; these treatments may have been insufficiently performed in this patient. It is likely that the replanted middle finger partially adhered, and Clostridium tetani colonized the partially necrotized region. Even when there is only limited soil contamination, administration of tetanus toxoid and anti-tetanus immunoglobulin is necessary when the fingers are injured outdoors and the finger nerves and blood vessels are exposed. The drugs should be administered just after replantation if the finger has been amputated. However, if clinicians pay attention to the possibility of tetanus development, treatment can be rapidly initiated.

Evaluation of superficial and deep self-inflicted wrist and forearm lacerations.

PURPOSE: Self-inflicted wrist or forearm laceration is a specific type of injury presenting to emergency departments. Many investigators have described wrist-cutting from a psychiatric viewpoint. We hypothesized that the character of patients with deep wounds is different from those with superficial wounds. We investigated patients who cut their wrist or forearms as an act of self-mutilation from the viewpoint of wound severity. METHODS: We reviewed 31 patients with self-inflected wrist injuries who were treated in our medical center from 2004 through 2009. We divided them into 2 groups: deep (15 patients) and superficial (16 patients). We investigated differences in age and gender, sites of self-cutting, frequency of self-injury attempts, object used for wrist cutting, group psychiatric parameters, required wound treatments, and psychiatric history and follow-up. RESULTS: Younger patients were more likely to have injured themselves severely compared with older patients. Differences in clinical findings between deep and superficial injury groups included the following: (1) all male patients had deep injuries; (2) patients with superficial wounds were more likely to have cut themselves previously; (3) patients in the deep injury group tended to injure themselves at multiple sites; (4) patients in the deep injury group tended to perform self-cutting with any sharp-edged object at hand; (5) 50% of our patients had received no psychiatric care before being seen by us for their injury; and (6) one-third discontinued the psychiatric treatment prematurely. CONCLUSIONS: There are differences between patients who perform self-inflicted deep versus superficial wrist cutting. We also found that the ages and psychiatric diagnoses of our patients differed from previous reports. This is likely because the available literature includes only patients who received psychiatric care. We found that 50% of our patients had received no psychiatric care, which highlights the importance of hand surgeons treating these patients to initiate psychiatric consultation.

Analysis of the sarcomere protein gene mutation on cardiomyopathy - Mutations in the cardiac troponin I gene.

Developments in the molecular genetic studies of cardiomyopathy (CM) have led to discovery of a large number of mutations in the genes encoding the sarcomeric proteins. In this study, comprehensive screening of TNNI3 was performed in 36 consented autopsy cases diagnosed as CM, in order to evaluate the prevalence of gene mutations in sudden death caused by CM. In DCM cases, a new missense mutation Pro16Thr was detected. A single nucleotide polymorphism at -8 position of intron 3 (IVS 3 -8 T>A) was identified, which had a significant difference in allele frequency between DCM and control cases. From these results, it was indicated that this study contribute to genetic based diagnosis, risk stratification and prevention of sudden death caused by CM.

Vpr-induced DNA double-strand breaks: molecular mechanism and biological relevance.

We focus on the role of Vpr in inducing DNA double-strand breaks (DSBs) in the host cell. Based on the summarized findings of Vpr-induced DSBs and the finding of Vpr in the plasma of HIV-1-positive patients, we discuss the roles of Vpr in viral infection, especially viral infection of resting macrophages. We also describe the possible involvement of Vpr in non-AIDS-defining cancers, which represent an emerging crisis in HIV-1-positive patients.

A case of V-A shunt catheters migration into the pulmonary artery.

A man in his sixties, who developed CPA at home, was transferred to the emergency center. Since CT images revealed a tube-shaped foreign body in the pulmonary artery, pulmonary embolism was initially suspected; however, this did not lead to a definite diagnosis. Autopsy revealed that the foreign body in the cadaver was a fragment of a V-A shunt catheter implanted about 30 years previously for the treatment of hydrocephalus. Although fibrous adhesion of a part of the catheter to the pulmonary artery wall was seen, suggesting that a fracture of the catheter had occurred a long time before, it was not known when the fracture had occurred. Since no pulmonary arterial obstruction secondary to the catheter or new thrombi, which had been initially suspected, were observed, the cause of death was determined to be ischemic cardiac failure. A fracture of a shunt catheter may be typically associated with some clinical manifestations, which are often found and treated. In this case, however, no symptoms appeared and the fracture of the shunt catheter remained untreated for a long time. This case was therefore considered to be extremely rare, and is an example of how a serious iatrogenic disease could occur.

Modulation of TNF-alpha-converting enzyme by the spike protein of SARS-CoV and ACE2 induces TNF-alpha production and facilitates viral entry.

Severe acute respiratory syndrome coronavirus (SARS-CoV) is a high-risk infectious pathogen. In the proposed model of respiratory failure, SARS-CoV down-regulates its receptor, angiotensin-converting enzyme 2 (ACE2), but the mechanism involved is unknown. We found that the spike protein of SARS-CoV (SARS-S) induced TNF-alpha-converting enzyme (TACE)-dependent shedding of the ACE2 ectodomain. The modulation of TACE activity by SARS-S depended on the cytoplasmic domain of ACE2, because deletion mutants of ACE2 lacking the carboxyl-terminal region did not induce ACE2 shedding or TNF-alpha production. In contrast, the spike protein of HNL63-CoV (NL63-S), a CoV that uses ACE2 as a receptor and mainly induces the common cold, caused neither of these cellular responses. Intriguingly, viral infection, judged by real-time RT-PCR analysis of SARS-CoV mRNA expression, was significantly attenuated by deletion of the cytoplasmic tail of ACE2 or knock-down of TACE expression by siRNA. These data suggest that cellular signals triggered by the interaction of SARS-CoV with ACE2 are positively involved in viral entry but lead to tissue damage. These findings may lead to the development of anti-SARS-CoV agents.

HIV-1 Vpr induces DNA double-strand breaks.

Recent observations imply that HIV-1 infection induces chromosomal DNA damage responses. However, the precise molecular mechanism and biological relevance are not fully understood. Here, we report that HIV-1 infection causes double-strand breaks in chromosomal DNA. We further found that Vpr, an accessory gene product of HIV-1, is a major factor responsible for HIV-1-induced double-strand breaks. The purified Vpr protein promotes double-strand breaks when incubated with isolated nuclei, although it does not exhibit endonuclease activity in vitro. A carboxyl-terminally truncated Vpr mutant that is defective in DNA-binding activity is less capable of Vpr-dependent double-strand break formation in isolated nuclei. The data suggest that double-strand breaks induced by Vpr depend on its DNA-binding activity and that Vpr may recruit unknown nuclear factor(s) with positive endonuclease activity to chromosomal DNA. This is the first direct evidence that Vpr induces double-strand breaks in HIV-1-infected cells. We discuss the possible roles of Vpr-induced DNA damage in HIV-1 infection and the involvement of Vpr in further acquired immunodeficiency syndrome-related tumor development.