Development of prevention strategies against bath-related deaths based on epidemiological surveys of inquest records in Kagoshima Prefecture.

Sudden death in the bathroom (bath-related death) occurs more frequently in Japan than in other countries. To clarify the epidemiological characteristics of bath-related deaths, we reviewed inquest records of deaths in Kagoshima Prefecture from 2006 to 2019. We identified 2689 cases of bath-related death. Of these cases, 90% were among people aged ≥ 65 years. The majority occurred in a home bathtub between 16:00 and 20:00. Most deaths (52.0%) occurred in winter (December-February), and there were extremely strong negative correlations with the environmental temperatures (maximum, minimum, and mean) on the day of death. We identified the environmental temperature during cold winter months that bath-related deaths are likely to occur in Kagoshima, although further investigation concerning the effects of other confounding factors is required. Forensic autopsies have only been performed in 29 cases and the cause of death was not diagnosed correctly in the majority of cases. Although autopsies are essential to elucidate the pathogenesis of the deaths, it is difficult to increase the rate of autopsies under the current Japanese death investigation system. Therefore, we suggest that the best way to prevent bath-related death is establishing an "Alert system" based on our results, and to have people refrain from bathing on dangerous days.

Human protein factory for converting the transcriptome into an in vitro-expressed proteome,.

Appropriate resources and expression technology necessary for human proteomics on a whole-proteome scale are being developed. We prepared a foundation for simple and efficient production of human proteins using the versatile Gateway vector system. We generated 33,275 human Gateway entry clones for protein synthesis, developed mRNA expression protocols for them and improved the wheat germ cell-free protein synthesis system. We applied this protein expression system to the in vitro expression of 13,364 human proteins and assessed their biological activity in two functional categories. Of the 75 tested phosphatases, 58 (77%) showed biological activity. Several cytokines containing disulfide bonds were produced in an active form in a nonreducing wheat germ cell-free expression system. We also manufactured protein microarrays by direct printing of unpurified in vitro-synthesized proteins and demonstrated their utility. Our 'human protein factory' infrastructure includes the resources and expression technology for in vitro proteome research.

The trend of resistance to antibiotics for ocular infection of Staphylococcus aureus, coagulase-negative staphylococci, and Corynebacterium compared with 10-years previous: A retrospective observational study.

OBJECTIVE: To retrospectively identify epidemiological trends of infection on the ocular surface and investigate trends of resistance to bacterial antibiotics compared with 10-years previous for Staphylococcus aureus, coagulase-negative staphylococci (CNS), and Corynebacterium in Japan. MATERIALS AND METHODS: Bacterial isolate samples were collected from the conjunctival sacs of eyes afflicted with conjunctivitis, keratitis, dacryocystitis, and hordeolum from September 2004 through November 2005 (n = 145 isolates) and September 2014 through November 2015 (n = 195 isolates) at the Baptist Eye Institute, Kyoto, Japan. The prevalence of methicillin-resistant S. aureus (MRSA), methicillin-resistant CNS (MR-CNS), and fluoroquinolone-resistant Corynebacterium were examined, and susceptibility of isolated bacteria to levofloxacin (LVFX), cefmenoxime (CMX), chloramphenicol (CP), erythromycin (EM), vancomycin (VCM), and arbekacin (ABK) were compared between both time periods using the disc susceptibility method. RESULTS: Over the 10-year period from initial to final examination, the prevalence of MRSA and MR-CNS significantly decreased from 52% to 22% (P < 0.05) and from 47% to 25% (P < 0.05), respectively, yet there was no change in the prevalence of fluoroquinolone-resistant Corynebacterium (60% and 54%; P = 0.38). Antibiotic-resistance trend analysis revealed that susceptibility to antibiotics in 2014-2015 was similar to that in 2004-2005. MRSA and MR-CNS were susceptible to CP (88%), VCM (100%), and ABK (100%), while fluoroquinolone-resistant Corynebacterium was susceptible to CMX (100%), VCM (100%), and ABK (96%). CONCLUSION: The prevalence of MRSA and MR-CNS significantly decreased between the two time periods, yet more than 50% of the Corynebacterium isolates were still resistant to LVFX. Although no increase in bacterial resistance to antibiotics was found, a cautionary use of fluoroquinolone eye drops should be considered.

Fail-Safe Therapy by Gamma-Ray Irradiation Against Tumor Formation by Human-Induced Pluripotent Stem Cell-Derived Neural Progenitors.

Cell replacement therapy holds great promise for Parkinson's disease (PD), but residual undifferentiated cells and immature neural progenitors in the therapy may cause tumor formation. Although cell sorting could effectively exclude these proliferative cells, from the viewpoint of clinical application, there exists no adequate coping strategy in the case of their contamination. In this study, we analyzed a component of proliferative cells in the grafts of human-induced pluripotent stem cell-derived neural progenitors and investigated the effect of radiation therapy on tumor formation. In our differentiating protocol, analyses of neural progenitors (day 19) revealed that the proliferating cells expressed early neural markers (SOX1, PAX6) or a dopaminergic neuron progenitor marker (FOXA2). When grafted into the rat striatum, these immature neurons gradually became postmitotic in the brain, and the rosette structures disappeared at 14 weeks. However, at 4-8 weeks, the SOX1(+)PAX6(+) cells formed rosette structures in the grafts, suggesting their tumorigenic potential. Therefore, to develop a fail-safe therapy against tumor formation, we investigated the effect of radiation therapy. At 4 weeks posttransplantation, when KI67(+) cells comprised the highest ratio, radiation therapy with (137)Cs Gammacell Exactor for tumor-bearing immunodeficient rats showed a significant decrease in graft volume and percentage of SOX1(+)KI67(+) cells in the graft, thus demonstrating the preventive effect of gamma-ray irradiation against tumorigenicity. These results give us critical criteria for the safety of future cell replacement therapy for PD.

Integrin ß4 is a major target antigen in pure ocular mucous membrane pemphigoid.

Previous studies of ocular mucous membrane pemphigoid (OMMP) have identified several components of the basement membrane zone to be autoantigens, including integrin ß4. However, there are no extensive or definitive reported studies that address this, particularly in pure OMMP. To clarify the major autoantigens in pure OMMP. In this study, we examined sera from 43 pure OMMP patients for both IgG and IgA antibodies using newly developed immunoblotting analyses with a hemidesmosome-rich fraction and various recombinant proteins of integrin α6ß4, in addition to our routine immune-serological tests. Using a hemidesmosome-rich fraction, sera from patients with pure OMMP demonstrated reactivity of IgG and/or IgA antibodies to integrin ß4, BP180 and laminin-332. The reactivity of pure OMMP sera to integrin ß4 was further confirmed by immunoblotting using integrin ß4 recombinant proteins. Using concentrated supernatant of HaCaT cells, only one serum sample showed positive IgG and IgA reactivity to LAD-1, the ectodomain of BP180. None of the pure OMMP sera reacted with any autoantigens on immunoblotting using normal human epidermal or dermal extracts, or purified human laminin-332. Integrin ß4 was considered to be the major and specific autoantigen for pure OMMP. The new methods established in this study are useful for detection of various autoantigens, particularly integrin ß4.

A comparative study on the observation of spontaneous nystagmus with Frenzel glasses and an infrared CCD camera.

OBJECTIVES: To compare the usefulness of a CCD camera with infrared illumination (IR-CCD camera) over Frenzel glasses (F Glasses) for the observation of spontaneous nystagmus, the incidence and direction of nystagmus, and the frequency, amplitude and slow phase of spontaneous nystagmus. METHODS: One hundred vertiginous patients, fifty-three females and forty-seven males participated in this study. Before undergoing routine neurotological examination, their eye movements were recorded by electronystagmogram (ENG) in conjunction with observations of eye movements under F glasses and through an IR-CCD camera. The data was collected from patients who exhibited spontaneous nystagmus either under F glasses or the IR-CCD camera. RESULTS: Thirty-three patients showed spontaneous nystagmus under F glasses. On the other hand, under the IR-CCD camera, all patients examined exhibited spontaneous nystagmus. The frequency of nystagmus was not significantly different between these two systems. However, the amplitude and slow phase velocity exhibited significantly larger values under the IR-CCD camera in patients with spontaneous nystagmus both under the IR-CCD camera and F glasses. CONCLUSION: From these observations and evidence, the IR-CCD camera can be recommended as a more useful system and powerful tool for neurotological examination than F glasses.

Intracellular thiol redox status regulates lymphangiogenesis and dictates corneal limbal graft survival.

PURPOSE: Compounds regulating intracellular thiol redox status, such as N,N-diacetyl-L-cystine dimethylester (NM(2)), were shown to prolong corneal graft survival in a penetrating keratoplasty (PKP) model. However, the effect of NM(2) on hemangiogenesis and lymphangiogenesis has not been investigated. The effect of manipulating ambient thiol redox status on riskier (higher rejection rate) transplantation models, such as limbal graft survival and hemangiogenesis and lymphangiogenesis in a corneal suture model, were investigated. METHODS: C57BL/10 mice that received BALB/c corneas were treated by subconjunctival injection of NM(2), and limbal graft survival was assessed. Sutured C57BL/6 received daily intraperitoneal injections of NM(2), glutathione diethylester (GSH-OEt), or PBS. Lymphatic endothelial cell (LEC) and peritoneal mps were treated with NM(2) or GSHOEt, and then VEGFR3, neuropilin-2, podoplanin, and LYVE-1 expression were analyzed. Supernatants were collected for analysis of TNF-alpha and VEGF-A levels by ELISA. RESULTS: Significantly less cellular infiltration was detected in mice with corneal limbal transplant-treated NM(2)-treated mice. Hemangiogenesis and lymphangiogenesis were suppressed in the NM(2)-treated mice. NM(2) treatment of mps led to reduced levels of VEGFR3, neuropilin-2, podoplanin, and LYVE-1 expression compared with PBS- or GSHOEt- treated mps, lower levels of TNF-alpha, and increased secretion of VEGF. Moreover, NM(2)-treated LECs had reduced levels of LYVE-1 and Prox-1. CONCLUSIONS: Reduction of ambient redox status reduced inflammatory cell infiltrates. Consequently, reduced inflammatory response might have contributed to both the observed prolonged corneal limbal graft survival and the attenuated hemangiogenesis and lymphangiogenesis in cornea.

Lattice corneal dystrophy type IV (p.Leu527Arg) is caused by a founder mutation of the TGFBI gene in a single Japanese ancestor.

PURPOSE: Lattice corneal dystrophy (LCD) type IV (LCD4) is a late-onset corneal dystrophy with amyloid deposition at the deep stromal layer of cornea. As with other corneal dystrophies, this LCD subtype is also caused by a mutation (p. Leu527Arg) of the transforming growth factor, beta-induced (TGFBI) gene. Although LCD type I has been reported worldwide, LCD4 has been reported only in the Japanese population. In the present study, a haplotype analysis was performed to investigate whether this LCD subtype is caused by a founder mutation. METHODS: Genomic DNA samples were extracted from 13 unrelated patients with LCD4. As a control, genomic DNA samples from 96 normal volunteers were also analyzed. For the haplotype analysis, the samples were amplified by polymerase chain reaction (PCR), TA-cloned, isothermally amplified, and subjected to a 1-base primer extension assay against a mutation site (c.1580T>G) and six known single-nucleotide polymorphisms (SNPs; rs4669, rs2072239, rs7727725, rs17689879, rs6871571, and rs3792900), which are located adjacent to the mutation site. RESULTS: The haplotype analysis revealed that all the disease-carrying alleles from the 13 LCD4 patients shared an identical haplotype, whereas non-disease-carrying alleles from the normal volunteers and the LCD4 patients exhibited four haplotypes. There was a statistically significant difference in the haplotype distribution between the disease-carrying and the non-disease-carrying alleles. CONCLUSIONS: The findings of this study strongly indicate that LCD4 was caused by a founder mutation of the TGFBI gene that occurred in a single Japanese ancestor.

High-throughput kinase assay based on surface plasmon resonance suitable for native protein substrates.

We report a novel in vitro high-throughput (HTP) kinase assay using surface plasmon resonance (SPR). In vitro tyrosine phosphorylation was performed in a microtiter plate, after which the substrate was captured with an antibody on a sensor chip and phosphotyrosine (pTyr) was detected with an anti-pTyr antibody. The capture and pTyr detection steps were performed using a Biacore A100, which is a sensitive and high-performance flow-cell-based SPR biosensor. This system allowed multiple sample processing (1000 samples/day) and high-quality data sampling. We compared the abilities of the HTP-SPR method and a standard radioisotope assay by measuring the phosphorylation of several substrate proteins by the Fyn tyrosine kinase. Similar results were obtained with both methods, suggesting that the HTP-SPR method is reliable. Therefore, the HTP-SPR method described in this study can be a powerful tool for a variety of screening analyses, such as kinase activity screening, kinase substrate profiling, and kinase HTP screening of kinase inhibitors.