Quantitative analysis of global 5-methyl- and 5-hydroxymethylcytosine in TET1 expressed HEK293T cells.

DNA methylation at the 5-position of cytosine bases (5-methylcytosine, 5mC) in genomic DNA is representative epigenetic modification and is involved in many cellular processes, including gene expression and embryonic development. The hydroxylation of 5mC provide 5-hydroxymethylcytosine (5hmC), the so-called sixth base rediscovered recently in mammalian cells, is also considered to act as an epigenetic regulator. We report herein the immunochemical assessment of 5hmC achieved by an enzyme-linked immunosorbent assay (ELISA) using our linker technology. The keys to this assay are 1) the immobilization of genomic DNA with the bifunctional linker molecule, and 2) quantitative analysis by using guaranteed standard samples containing defined amounts of 5hmC. We succeeded in the sensitive and quantitative detection of 5hmC as well as 5mC in HEK293T cells transfected with TET1, and also monitored the effect of ascorbate on the TET1 catalyzed conversion of 5mC to 5hmC. Our linker technology enables the rapid and stable immobilization of genomic samples and thus contributes to the realization of a reproducible 5hmC evaluation method.

Sleep Disturbance of Evacuees in Minamisanriku Town after Great East Japan Earthquake: Risk Factors and Treatment.

In 2011, Minamisanriku Town lost all of its medical facilities during the Great East Japan Earthquake. Using 10,459 anonymized disaster medical records of affected people in Minamisanriku Town, we assessed the prevalence and risk factors of sleep disturbance, which is known to exacerbate non-communicable diseases (NCDs) and anxiety disorder. Because sleep disturbance is a part of mental health issues, we divided the patients into two groups: patients (n = 492) with mental health issues other than sleep disturbance and the remaining (n = 9,967) with other comorbidities. Out of 492 patients with mental health issues, 295 patients (60.0%, 114 male, 158 female and 23 unknown) had sleep disturbance who might have required specific treatments. Out of the remaining 9,967 patients, 1,203 patients (12.1%, 361 male and 769 female and 73 unknown) had sleep disturbance. Univariate and multivariate analyses of the 9,967 patients revealed that the odds ratio (OR) of sleep disturbance was higher for female (OR 1.95), elderly persons over 60 (OR 16.15) and residing in evacuation centers (OR 1.36). Patients with two or more NCD had higher risk (OR 1.42). Importantly, sleep disturbance affects younger patients without NCD residing in evacuation center. Emergency medical teams most frequently prescribed benzodiazepines both for sleep induction and anxiolysis. In addition to high risk groups (female, older, with other mental health issues, residing in evacuation center), it is important to survey sleep disturbance in younger and healthier populations especially in evacuation centers and to provide psychosocial and medical support for them.

Medical Needs in Minamisanriku Town after the Great East Japan Earthquake.

The medical records of service in disaster provided at a place other than a medical facility are defined as disaster medical records (DMRs). In this epidemiological study, to clarify medical need characteristics and trends after disaster, we analyzed the all anonymized DMRs of Minamisanriku Town that lost medical facilities in 2011 Great East Japan Earthquake and its consequent tsunami. After screening of duplicated or irrelevant documents, there were 10,464 DMRs with 18,532 diagnoses from March 11 through May 13. From 34 diagnostic groups according to International Classification of Diseases (ICD)-10, we integrated diagnostic groups into five modules that might require treatment concepts of different types: non-communicable disease (NCD), infectious disease, mental health issue, trauma, and maternal and child health (MCH). Age and sex distributions of the patients were similar to those of population before the disaster. The largest diagnostic module was NCD (68%), followed by infectious disease (21%), mental health issues (6%), trauma (4%), and MCH (0.2%). The age-specific rate of NCD exhibited a similar or suppressed level from that of nationwide survey, with higher rate of pollinosis among young population. Infectious disease increased in most age groups but there was no apparent outbreak because of early interventions. Sleep deprivation was twice as frequent in middle-aged women, compared with men. Trauma and MCH were less frequent, but each exhibited a unique time trend. Trauma onset was continuously recorded, while MCH visits were concentrated on a specific day. The medical need after disaster dynamically changes, and appropriate anticipatory countermeasures are necessary.

Immobilization of DNA with nitrogen mustard-biotin conjugate for global epigenetic analysis.

We report the quantitative analysis of 5-methylcytosine, a representative epigenetic modification in genomic DNA, with an enzyme-linked immunosorbent assay (ELISA). We synthesized a novel hetero-bifunctional linker molecule consisting of nitrogen mustard and biotin to capture DNA on the surface of biosensing devices. The molecule can successfully immobilize genomic DNA on a streptavidin coated 96-well microplate, which was then employed for immunochemical epigenetic assessment. We achieved the sensitive and quantitative detection of 5-mC in genomic DNA samples. The CpG methylation ratios obtained from our system for mouse brain and mouse small intestine genomes were 79% and 82%, respectively. These numbers are in good agreement with the previously reported methylation ratio of 75-85%, which was identified by whole genome bisulfite sequencing. Accordingly, the present technology using our novel bifunctional linker molecule provides a fast, easy, and inexpensive method for epigenetic assessment, without the need for any conventional bisulfite treatment, polymerase chain reaction (PCR), or sequencing.

Agent-Based Modeling and Simulation of Nosocomial Infection among Healthcare Workers during Ebola Virus Disease Outbreak in Sierra Leone.

Healthcare workers (HCWs) are often exposed to nosocomial infection when caring for patients with Ebola Virus Disease (EVD). During the 2014-2016 EVD outbreak in West Africa, more than 200 HCWs died of EVD in Sierra Leone. To determine the factors that are important for preventing infection among HCWs during EVD outbreak, we used agent-based modeling and simulation (ABMS) by focusing on education, training and performance of HCWs. Here, we assumed 1, 000 HCWs as "agents" to analyze their behavior within a given condition and selected four parameters (P1-P4) that are important in the prevention of infection: "initially educated HCWs (P1)", "initially educated trained (P2)", "probability of seeking training (P3)" and "probability of appropriate care procedure (P4)." After varying each parameter from 0% to 100%, P3 and P4 showed a greater effect on reducing the number of HCWs infected during EVD outbreak, compared with the other two parameters. The numbers of infected HCWs were decreased from 897 to 26 and from 1,000 to 59, respectively, when P3 or P4 was increased from 0% to 100%. When P2 was increased from 0% to 100%, the number of HCWs infected was decreased from 166 to 44. Paradoxically, the number of HCWs infected was increased from 56 to 109, when P1 was increased, indicating that initial education alone cannot prevent nosocomial infection. Our results indicate that effective training and appropriate care procedure play an important role in preventing infection. The present model is useful to manage nosocomial infection among HCWs during EVD outbreak.

Nation-Wide Implementation of Disaster Medical Coordinators in Japan.

In the 2011 Great East Japan Earthquake (GEJE), successful medical and public health coordination by pre-assigned disaster medical coordinators saved many affected people, though the coordination itself had difficulties. This study aims to clarify the implementation and the challenges of disaster medical coordinators in Japan. We performed questionnaire surveillance in 2012 and 2014 to all prefectural government on assignment of disaster medical coordinators, their expected roles and supporting system. Out of all 47 prefectures, assignment or planning of disaster medical coordinators jumped up from four (8.5%) to 43 (91.5%) by the end of 2015. The most expected role is the coordination with Japan Disaster Medical Assistant Team (DMAT) and with other early responders. The evacuation center management, public health coordination and preparedness before disaster are less frequently expected. The supporting materials, human resource, and tools for communication vary according to the prefecture. Successful implementation requires the effort of health and governmental stakeholders. The coordination between prefectural and local coordinators and the coordination between medical and public health authorities still need to be improved. The roles of disaster medical coordinators depend on the local context and types of hazards. Education and training to build fundamental capacity is necessary. In conclusion, Japanese disaster medical system rapidly implemented disaster medical coordinator after GEJE. Their roles and standardization are challenging, but education, training and systematic support by the local government will enhance the effective preparedness and response of the health sector in disasters.

Phosphoproteomic analysis of kinase-deficient mice reveals multiple TAK1 targets in osteoclast differentiation.

TAK1 (encoded by Map3k7) is a mitogen-activated protein kinase kinase kinase (MAP3K), which activates the transcription factors AP-1 and NF-κB in response to receptor activator of NF-κB ligand (RANKL) stimulation, thus constituting a key regulator of osteoclast differentiation. Here we report the functional relevance of the kinase activity of TAK1 in the late stage of osteoclast differentiation in vivo using Ctsk-Cre mice and TAK1 mutant mice in which the TAK1 kinase domain was flanked by loxP. The Map3k7(flox/kd)Ctsk(Cre/+) mice displayed a severe osteopetrotic phenotype due to a marked decrease in osteoclast number. RANKL-induced activation of MAPK and NF-κB was impaired in the late stage of osteoclast differentiation. The absence of suppressive effect of an administered NF-κB inhibitor on the late stage of osteoclastogenesis led us to investigate unknown TAK1 targets in osteoclast differentiation. We performed a phosphoproteomic analysis of RANKL-stimulated osteoclast precursor cells from Map3k7(flox/kd)Ctsk(Cre/+) mice, revealing multiple targets regulated by TAK1 during osteoclastogenesis. Thus, TAK1 functions as a critical regulator of the phosophorylation status of various cellular proteins that govern osteoclastogenesis.

Immune complexes regulate bone metabolism through FcRγ signalling.

Autoantibody production and immune complex (IC) formation are frequently observed in autoimmune diseases associated with bone loss. However, it has been poorly understood whether ICs regulate bone metabolism directly. Here we show that the level of osteoclastogenesis is determined by the strength of FcRγ signalling, which is dependent on the relative expression of positive and negative FcγRs (FcγRI/III/IV and IIB, respectively) as well as the availability of their ligands, ICs. Under physiological conditions, unexpectedly, FcγRIII inhibits osteoclastogenesis by depriving other osteoclastogenic Ig-like receptors of FcRγ. Fcgr2b(-/-) mice lose bone upon the onset of a hypergammaglobulinemia or the administration of IgG1 ICs, which act mainly through FcγRIII. The IgG2 IC activates osteoclastogenesis by binding to FcγRI and FcγRIV, which is induced under inflammatory conditions. These results demonstrate a link between the adaptive immunity and bone, suggesting a regulatory role for ICs in bone resorption in general, and not only in inflammatory diseases.

Inhibitor of NF-kappa B kinases alpha and beta are both essential for high mobility group box 1-mediated chemotaxis [corrected].

Inhibitor of NF-kappaB kinases beta (IKKbeta) and alpha (IKKalpha) activate distinct NF-kappaB signaling modules. The IKKbeta/canonical NF-kappaB pathway rapidly responds to stress-like conditions, whereas the IKKalpha/noncanonical pathway controls adaptive immunity. Moreover, IKKalpha can attenuate IKKbeta-initiated inflammatory responses. High mobility group box 1 (HMGB1), a chromatin protein, is an extracellular signal of tissue damage-attracting cells in inflammation, tissue regeneration, and scar formation. We show that IKKalpha and IKKbeta are each critically important for HMGB1-elicited chemotaxis of fibroblasts, macrophages, and neutrophils in vitro and neutrophils in vivo. By time-lapse microscopy we dissected different parameters of the HMGB1 migration response and found that IKKalpha and IKKbeta are each essential to polarize cells toward HMGB1 and that each kinase also differentially affects cellular velocity in a time-dependent manner. In addition, HMGB1 modestly induces noncanonical IKKalpha-dependent p52 nuclear translocation and p52/RelB target gene expression. Akin to IKKalpha and IKKbeta, p52 and RelB are also required for HMGB1 chemotaxis, and p52 is essential for cellular orientation toward an HMGB1 gradient. RAGE, a ubiquitously expressed HMGB1 receptor, is required for HMGB1 chemotaxis. Moreover, IKKbeta, but not IKKalpha, is required for HMGB1 to induce RAGE mRNA, suggesting that RAGE is at least one IKKbeta target involved in HMGB1 migration responses, and in accord with these results enforced RAGE expression rescues the HMGB1 migration defect of IKKbeta, but not IKKalpha, null cells. Thus, proinflammatory HMGB1 chemotactic responses mechanistically require the differential collaboration of both IKK-dependent NF-kappaB signaling pathways.