Supplemental_Material – Supplemental material for Lateral Gene Transfer Between Protozoa-Related Giant Viruses of Family Mimiviridae and Chlamydiae

<p>Supplemental material, Supplemental_Material for Lateral Gene Transfer Between Protozoa-Related Giant Viruses of Family Mimiviridae and Chlamydiae by Takanori Watanabe, Sumire Yamazaki, Chinatsu Maita, Mizue Matushita, Junji Matsuo, Torahiko Okubo and Hiroyuki Yamaguchi in Evolutionary Bioinformatics</p

In the United States, two-thirds of the nonelderly

(2003), the cost of EHI has increased by over 59 percent since 2000 with no accompanying increase in the scale or scope of benefits. These increases in health insurance premiums may have significant effects on labor markets, including changes in the number of jobs, hours worked per employee, wages, and compensation packages. Indeed, it is possible that a significant portion of the increase in the uninsured population may be a consequence of employers shedding this benefit as health insurance premiums rise. Understanding how labor-market characteristics affect adjustments to increased health insurance costs is of vital policy importance. Some proposals to cover the uninsured rely on “employer mandates ” requiring employers to cover eligible workers. Other proposals provide tax credits for the purchase of non-employer health insurance. The effects of these proposals on employment, wages, and health insurance coverage will be driven by the elasticities of labor supply and demand, institutional constraints on wages and compensation packages, and how much workers value the increase in health insurance costs. Since employers provide such coverage voluntarily, if workers fully value these benefits and are able to sort between firm

Sapporo underground pedestrian space and air sampling locations.

<p>(A) Representative features showing the pedestrian with walkers. (B) Map showing the pedestrian structure with the location of public spaces or entrances/exits. The photo and the map were obtained with permission from the following website (<a href="http://www.sapporo-chikamichi.jp/" target="_blank">http://www.sapporo-chikamichi.jp/</a>). (C) Image taken during air sampling. As mentioned in the text, the sampler was approximately 3 m from the wall. (D) Air sampler filter opening. The opening was adjusted to be 1.5 m from the floor to align with pedestrian head. The filter consisting of PTFE-binding glass fiber filter was pointed toward the center of the pedestrian passageway.</p

Changes in walker numbers and environmental factors (dust particle numbers) in the underground pedestrian space.

<p>(A) Changes in walker numbers. (B) Changes in particle numbers (particle size Δ0.5: 0.3–0.5μm). (C) Changes in numbers of particles (particle size Δ1.0: 0.5–1.0μm). (D) Changes in numbers of particles (particle size Δ5.0: 1.0–5.0μm). Left and right panels show daily and monthly changes, respectively. Asterisks indicate a significant difference (<i>p</i><0.05) between bars connected by lines.</p

Comparison of Pearson's correlation coefficient among factors and phylum types in the underground pedestrian space.

<p>(A) Matrix showing comparison of Pearson's correlation coefficient among factors. Values show the correlation coefficient. Bold values showing positive and negative correlation with colors indicate statistical significance (a correlation coefficient value of >0.5 or <−0.05 with a <i>p</i>-value < 0.05). (B) Clustering of the phylum type consisting of distinct factors. All values of the factors with distinct ranges were adjusted to an equivalent range from “0” to “1”. Factors include Δ0.5 (particle size: 0.3–0.5μm), Δ1.0 (particle size: 0.5–1.0μm), Δ5.0 (particle size: 1.0–5.0μm), Temp (temperature: °C), Humidity (%), AP (atmospheric pressures: hpa), CFU-SCD (CFU numbers per m³ estimated by SCD plate culture), CFU-R2A (CFU numbers per m³ estimated by R2A plate culture), CFU-Total (total CFU numbers per m³ estimated by SCD and R2A plate cultures), Walker (walker numbers per 10 min) and OTU (OTU numbers estimated by 16S rDNA sequence analysis and BLAST search). These data were similarly processed with a setting (>80% filtering and Spearman rank correlation). After replaced to the files with a ‘cdt’ extension in the software of Cluster 3.0 for visualizing on the TreeViewX.</p

Changes in environmental factors (temperature, humidity and atmospheric pressure) in the underground pedestrian space.

<p>(A) Changes in temperature (°C). (B) Changes in humidity (%). (C) Changes in atmospheric pressure (hpa). Left and right panels show the daily and monthly changes, respectively. Asterisks indicate a significant difference (<i>p</i><0.05) between bars connected by lines.</p

A possible scenario showing bacterial features released from walkers and the built environment.

<p>Upper panel (small number of walkers) showing the number of bacteria released from individuals is minimal. Middle panel (moderate number of walkers) showing that although increasing walker occupancy facilitates the number of floating small dust particles with bacteria released from individuals, the number of bacteria is still minimal. Lower panel (high number of walkers) showing that increasing temperature and humidity in the presence of a high concentration of small dust particles causes the bacteria released from walkers to reach the maximum level.</p

Representative TEM images of HEp-2 cells treated with <i>Protochlamydia</i> or staurosporine.

<p>Cells were cultured with bacteria adjusted at MOI 90 or staurosporine for 6 h, and then fixed. <b>A</b>) Negative control cells without any treatment. <b>B</b>) Cells treated with staurosporine (10 µM). <b>C and D</b>) Representative images of HEp-2 cells treated with the bacteria.</p

Viable <i>Protochlamydia</i> induced apoptosis.

<p>Cells were cultured with or without the bacteria adjusted at MOI 90 [or either heat- or UV-killed bacteria (equivalent MOI 90)] or other chlamydiae for 24 h, and then cell morphological changes were also estimated by DAPI staining. The representative images were captured at 24 h after incubation. Magnification, ×200. <b>A</b>) Negative control cells without any treatment. <b>B</b>) Cells treated with viable bacteria. <b>C</b>) Cells treated with heat-killed bacteria. <b>D</b>) Cells treated with UV-killed bacteria. <b>E, F and G</b>) Cells treated with other viable chlamydiae adjusted at MOI 90 [<i>C. trachomatis</i> D (E), <i>C. trachomatis</i> L2 (F) and <i>P. acanthamoebae</i> (G)]. <b>H</b>) Time-course changes in the prevalence of apoptotic cells. The data shown represent the means + SD, obtained from at least three independent experiments performed in triplicate. *, <i>p</i><0.05; significantly different from each data immediately (0 h) after incubation.</p

Cell death in HEp-2 cells induced by the addition of <i>Protochlamydia</i> is apoptosis.

<p>Apoptotic cell death after the addition of <i>Protochlamydia</i> (MOI 90) was estimated using the TUNEL assay (A, B) and DAPI staining (experiments with caspase inhibitors) (C, D). Staurosporine (10 µM) was used as a positive control to induce apoptosis. <b>A</b>) Representative images of apoptotic cells with phase contrast images at 24 h after incubation. Green, apoptotic cells. Magnification, ×100. <b>B</b>) The prevalence of apoptotic cells estimated by TUNEL assay with time-course changes. The percentage of apoptotic cells was measured under a microscope by counting at least 200 cells in three random fields for each culture sample. The data shown represent the means + standard deviations (error bars) (SD), obtained from at least three independent experiments performed in triplicate. *, <i>p</i><0.05; significantly different from each data at immediately (0 h) after incubation. <b>C</b>) The prevalence of cells with condensed chromatin with time-cause changes in the presence or absence of the inhibitior, Z-VAD-FMK (general caspase inhibitor). The percentage of the cells was measured under a microscope by counting at least 200 cells in three random fields for each culture sample. The data shown represent the means + SD, obtained from at least three independent experiments performed in triplicate. *, <i>p</i><0.05; significantly different from each data at immediately (0 h) after incubation. <b>D</b>) The prevalence of cells with condensed chromatin at 24 h after incubation in the presence or absence of the inhibitor, Z-DEVE-FMK (specific caspase-3 inhibitor). The data shown represent the means + SD, obtained from at least three independent experiments performed in triplicate. *, <i>p</i><0.05; significantly different from each data without the treatment.</p