Environmental chlamydiae alter the growth speed and motility of host acanthamoebae.

Symbiosis between living beings is an important driver of evolutionary novelty and ecological diversity; however, understanding the mechanisms underlying obligate mutualism remains a significant challenge. Regarding this, we have previously isolated two different Acanthamoeba strains harboring endosymbiotic bacteria, Protochlamydia (R18 symbiotic amoebae: R18WT) or Neochlamydia (S13 symbiotic amoebae; S13WT). In this study, we treated the symbiotic amoebae R18WT and S13WT with doxycycline (DOX) and rifampicin (RFP), respectively, to establish the aposymbiotic amoebae R18DOX and S13RFP, respectively. Subsequently, we compared the growth speed, motility, phagocytosis, pinocytosis, and morphology of the symbiotic and aposymbiotic amoebae. The growth speed of R18DOX was decreased, although that of S13RFP was increased. A marked change in motility was observed only for R18DOX amoebae. There was no difference in phagocytic and pinocytic activities between the symbiotic and aposymbiotic amoebae. Meanwhile, we observed a significant change in the phalloidin staining pattern and morphological changes in R18DOX (but not S13RFP) aposymbiotic amoebae, indicating a change in actin accumulation upon removal of the Protochlamydia. Infection of C3 (a reference strain) or S13RFP amoebae with Protochlamydia had a harmful effect on the host amoebae, but R18DOX amoebae re-infected with Protochlamydia showed recovery in both growth speed and motility. Taken together, we conclude that endosymbiont environmental chlamydiae alter the growth speed and/or motility of their host Acanthamoeba, possibly implying an close mutual relationship between amoebae and environmental chlamydiae

Development of a mobile laboratory system in hydrogen fuel cell buses and evaluation of the performance for COVID-19 RT-PCR testing

Abstract We designed and developed two new types of hydrogen fuel cell (HFC) buses (motorcoach and minibus) with a mobile laboratory system. Feasibility studies have been performed for mobile laboratory testing, particularly for the laboratory performance of COVID-19 RT-PCR (PCR). We evaluated the driving range capability, PCR sample size capacity, turnaround time (TAT), and analytical performance for the detection of SARS-CoV-2. Saliva samples were used for the current study, and the analytical performance was compared with that of the reference PCR. The estimated driving range and sample size capacity of the HFC and HFC minibus were 432 km and 2847 samples, respectively, for the HFC motorcoach and 313 km and 1949 samples for the HFC minibus. For the TAT, the median time between sample submission and completion of PCR was 86 min for the motorcoach and 76 min for the minibus, and the median time between sample submission and electronic reporting of the result to each visitor was 182 min for the motorcoach and 194 min for the minibus. A secondary analysis of 1574 HFC mobile laboratory testing samples was conducted, and all negative samples were found to be negative by reference PCR. Furthermore, all samples were confirmed to be positive by reference PCR or other molecular examinations

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

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

Cell death did not occur in PBMCs regardless of activation with PMA and ionomycin.

<p><b>A</b>) Representative DAPI staining images showing that the bacteria induced cell death in immortal Jurkat cells, but not in primary PBMCs. Data was estimated at 24 h after incubation. Magnification, ×1,000. <b>B and C</b>) Time-course changes of the percentage of dead PBMCs with (C) or without (B) PMA and ionomycin. PBMC cell death prevalence was estimated using a trypan blue exclusion assay. The plot shown represents the mean value of the data from individual donors performed in duplicate. The averages in parentheses at each time point are compared. *, <i>p</i><0.05; significantly different from each average for the “without treatment group” at each of the time points after incubation.</p

Enriched EB, but not RB or amoebal components, induced apoptosis in HEp-2 cells.

<p><b>A</b>) Representative images showing that only EB (MOI 90), but not RB (equivalent MOI 90) or amoeba components, induced apoptosis at 24 h after incubation. Magnification, ×200. <b>B</b>) Time-course changes of the percentage 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