Slow-growth phenotype of the Cet1p-Ceg1p mislocalization strains.

<p>The yeast strain HC101, harboring <i>CEN HIS3 CET1-GFP</i> N-terminal deletion mutants (without <i>CEN TRP1 hCAP1</i>) were grown in YPD medium and put on agar plates. These plates were incubated at 25, 30, or 37°C for 2 days.</p

Importance of the Cet1p-Ceg1p interaction for nuclear localization of the Cet1p-Ceg1p complex.

<p>(A) GST pull-down assay. A GST pull-down assay was performed using GST (or GST-Ceg1p OB domain) and Cet1p (or Cet1(4A)p) translated in rabbit reticulocyte lysate with [³⁵S]-methionine. GST pull-downs were separated by SDS-PAGE and detected with an imaging plate (left) and CBB staining (right). (B) Triphosphatase activity of Cet1(4A)p. The reaction was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078000#s2" target="_blank">Materials and Methods</a> using [γ-³²P]-terminated poly(A), and either 2.5, 5, 10, 20, 40, 80 nM Cet1(4A)p or Cet1p. The reaction products were analyzed by PEI-cellulose TLC. The autoradiogram of the thin-layer plate is shown. (C) Localization of Ceg1p-GFP in cells expressing Cet1(4A)p. The yeast strain <i>cet1</i>Δ<i>ceg1</i>Δ was transformed with both <i>2 μ URA3 CET1</i> or <i>CET1(4A)</i> and <i>CEN HIS3 CEG1-GFP</i> plasmids. The cell nucleus was stained with DAPI. (D) Localization of Cet1(4A)p-GFP. The yeast strain <i>cet1</i>Δ<i>ceg1</i>Δ was transformed with both <i>CEN HIS3 CET1(4A)-GFP</i> and <i>2 μ URA3 CEG1</i> plasmids. The cell nucleus was stained with DAPI. (E) Lethal phenotype of the yeast strain expressing Cet1(4A)p and Ceg1p-GFP. The yeast strain used in (C) and control strains were streaked on agar plates with or without 0.075% 5-FAA. These plates were incubated at 30°C for 2 days. (F) Localization of Ceg1p-GFP in cells expressing Cet1p triphosphatase active site mutant. The yeast strain <i>cet1</i>Δ<i>ceg1</i>Δ was transformed with <i>CEN HIS3 CET1(E305,307A)-GFP</i> or both <i>2 μ URA3 CET1(E305,307A)</i> and <i>CEN HIS3 CEG1-GFP</i> plasmids. The cell nucleus was stained with DAPI.</p

Characterization of the amino acid sequence required for nuclear localization of the Cet1p-Ceg1p complex.

<p>(A) Cet1p N-terminal deletion mutants. Localization results shown in (B) are summarized. N: localized in the nucleus. N+C: localized in both the nucleus and the cytoplasm. (B) Localization of Cet1p-GFP mutants in the <i>cet1</i>Δ<i>ceg1</i>Δ strain. The yeast strain <i>cet1</i>Δ<i>ceg1</i>Δ was transformed with the <i>CEN HIS3 CET1-GFP</i> N-terminal deletion mutant plasmid indicated in (A). The cell nucleus was stained with DAPI. (C) Amino acid sequence between residues 217 and 253 of Cet1p and Cet1p N-terminal deletion mutants. The underline indicates the WAQKW motif for Ceg1p binding. (D) Localization of the Cet1p-GFP N-terminal truncated mutants. The yeast strain <i>cet1</i>Δ<i>ceg1</i>Δ was transformed with the <i>CEN HIS3 CET1-GFP</i> N-terminal deletion mutant plasmid indicated in (C). The cell nucleus was stained with DAPI. (E) Localization of Ceg1p-GFP in cells expressing Cet1p N-terminal deletion mutants. The yeast strain <i>cet1</i>Δ<i>ceg1</i>Δ was transformed with both a <i>2 μ URA3 CET1</i> N-terminal deletion mutant plasmid and the <i>CEN HIS3 CEG1-GFP</i> plasmid. The cell nucleus was stained with DAPI. (F) Interaction between His-Cet1(228–549)p and GST-Ceg1. GST pull-down assay was performed using GST or GST-Ceg1p OB domain and Cet1p, Cet1(4A)p, or Cet1(228–549)p translated in rabbit reticulocyte lysate with [³⁵S]-methionine. GST pull-downs were separated by SDS-PAGE and detected with an imaging plate (left) and CBB staining (right).</p

Nuclear accumulation of Cet1p-GFP in the <i>cet1</i>

<p>Δ<b><i>ceg1</i></b>Δ <b>strain.</b> (A) Complementation of <i>cet1</i>Δ and <i>ceg1</i>Δ by Cet1p-GFP and Ceg1p-GFP, respectively. Yeast strains HC101 and HC201 were transformed with a <i>CEN HIS3 CET1-GFP</i> and <i>CEN HIS3 CEG1-GFP</i> plasmid, respectively. His⁺ isolates and control strains were streaked on agar plates with or without 0.075% 5-FAA. These plates were incubated at 30°C for 2 days. (B) Localization of Cet1p-GFP and Ceg1p-GFP in the <i>cet1</i>Δ<i>ceg1</i>Δ strain. Yeast strain <i>cet1</i>Δ<i>ceg1</i>Δ was transformed with <i>CEN HIS3 CET1-GFP</i>, <i>CEN HIS3 CEG1-GFP</i>, or both <i>2 μ URA3 CET1</i> and <i>CEN HIS3 CEG1-GFP</i> plasmids. The yeast strains were grown in YPD medium and fixed by ethanol. The cell nucleus was stained with DAPI after fixation.</p

The influence of the Great East Japan earthquake on microscopic polyangiitis: A retrospective observational study

<div><p>Background</p><p>Antineutrophil cytoplasmic antibody-associated vasculitis is triggered by environmental factors, including silica dust exposure. Repeated tsunami waves brought a large volume of silica-containing sludge inland after the Great East Japan earthquake in 2011. We aimed to determine if the serious disaster influenced the clinical features of the microscopic polyangiitis.</p><p>Methods</p><p>This is an observational retrospective study conducted in a single institute. A total of 43 patients were included based on the CHCC2012 criteria for microscopic polyangiitis from 2007 to 2015. We used the Poisson regression model to determine the incidence of microscopic polyangiitis within the annual population of the medical district. The participants were selected during a 3-year period from before (N = 13) to after the disaster (N = 20). The differences of parameters and the overall survival between the groups were analyzed.</p><p>Results</p><p>The incidence of microscopic polyangiitis increased after the disaster (λ = 17.4/million/year [95%CI: 7.66–39.6] before the disaster and λ = 33.1/million/year [17.7–61.7] after the disaster, <i>P</i> = 0.044). A high Birmingham Activity Score was associated with a high incidence of microscopic polyangiitis after the disaster. The overall survival of the patients with microscopic polyangiitis declined significantly after the disaster.</p><p>Conclusions</p><p>The Great East Japan earthquake influenced the development of the microscopic polyangiitis in our restricted area. The patients who developed after the disaster had severe symptoms and a high mortality rate.</p></div

Comparison of overall survival between before and after the disaster.

<p>Survival curves were plotted to compare the mortality of each group using the Kaplan-Meier method. The dotted and solid lines indicate the time-to-events of participants before and after the disaster, respectively. The notches within the line represent the censors. The numbers at risk are illustrated below the chart. Overall survival was significantly worse in the group after the disaster (<i>P</i> = 0.029 by log rank test).</p

Change in the annual incidence of microscopic polyangiitis (MPA) in the Ishinomaki medical area.

<p>The incident cases were divided by the population at risk in the restricted medical area and plotted according to annual intervals. The white and black bars represent the incidence per 1 million people before and after the disaster, respectively. The mean incidence of MPA was 17.4 /million/year [95% Confidence Interval (CI): 7.66 to 39.61] before the disaster and 33.1 /million/year [95% CI: 17.73 to 61.74] after the disaster. The dashed line indicates the mean annual incidence of MPA in Japan. The incidence ratio and <i>P</i>-value were obtained using Poisson regression analysis (<i>P</i> = 0.044).</p

Baseline characteristics of 43 MPA patients in this study.

<p>Baseline characteristics of 43 MPA patients in this study.</p

Comparison of factors before and after the GEJE.

<p>Comparison of factors before and after the GEJE.</p

Flow diagram of screening for participants in this study.

<p>The crude number of patients with microscopic polyangiitis was counted on the basis of ICD-10 codes from medical records in our hospital. Each of the patients was diagnosed with MPA based on the CHCC2012 criteria. Drug-induced and recurrent cases were excluded. Abbreviations: ICD, international classification of diseases; RPGN, rapidly progressive glomerulonephritis; GPA, granulomatosis with polyangiitis; EGPA, eosinophilic granulomatosis with polyangiitis, CHCC; Chapel Hill Consensus Conference, MPA; microscopic polyangiitis.</p