Plastid genome evolution of the non-photosynthetic liverwort Aneura mirabilis (Malmb.) Wickett & Goffinet (Aneuraceae)

Aneura mirabilis is a parasitic liverwort in the Aneuraceae that obtains fixed atmospheric carbon from a host tree, using a fungal symbiont that is simultaneously ectomycorrhizal on the tree. Originally placed in its own genus, Cryptothallus, I determined in chapter one that the parasitic liverwort is nested within a Glade of morphologically indistinguishable photosynthetic liverworts and should be considered to be a distinct species of the genus Aneura. The annotated, complete plastid genome sequence of A. mirabilis, presented in chapter two, revealed the complete functional loss of the chlororespiration (ndh) genes, pseudogenes for major subunits of photosystem I, photosystem II, and the cytochrome b6f complex, and an inversion of psbE and petL. Aside from this inversion, the order of genes is congruent with the order of genes in Marchantia polymorpha, the only other liverwort plastid genome sequence available. The designation of pseudogenes was made using genomic comparisons with the phylogenetically distant liverwort M. polymorpha. In order to correlate functional gene losses with the evolution of a heterotrophic life history, we sampled several populations of A. mirabilis and its photosynthetic sister groups and sequenced plastid regions homologous with the losses detected in the plastid genome sequence. A functional gene loss or the psbE- petL inversion was never detected in a photosynthetic liverwort. All the gene losses and the psbE-petL inversion were detected in every population of the nonphotosynthetic liverwort sampled. The rates of synonymous and non-synonymous substitutions were estimated for eight pseudogenes and six genes to detect whether they are evolving under relaxed purifying selection. Here we present evidence that A. mirabilis has recently acquired an obligately heterotrophic life history, and that large deletions, and structural rearrangements may play an important role in the functional reduction of the plastid genome, rather than a relaxation of a synonymous substitution bias, early in the shift to parasitism.

Baseline characteristics of knowledge workers (KW) participation by study design for trials 1 and 2.

<p>(0.05c = study design 1—no previous experience; 0.05c_500_90% = study design 2—moderate experience)</p><p>Baseline characteristics of knowledge workers (KW) participation by study design for trials 1 and 2.</p

ROC curves for each study design in trials 1 and 2.

<p>ROC curves for each study design in trials 1 and 2.</p

Cardiovascular Risks Associated with Low Dose Ionizing Particle Radiation

<div><p>Previous epidemiologic data demonstrate that cardiovascular (CV) morbidity and mortality may occur decades after ionizing radiation exposure. With increased use of proton and carbon ion radiotherapy and concerns about space radiation exposures to astronauts on future long-duration exploration-type missions, the long-term effects and risks of low-dose charged particle irradiation on the CV system must be better appreciated. Here we report on the long-term effects of whole-body proton (¹H; 0.5 Gy, 1 GeV) and iron ion (⁵⁶Fe; 0.15 Gy, 1GeV/nucleon) irradiation with and without an acute myocardial ischemia (AMI) event in mice. We show that cardiac function of proton-irradiated mice initially improves at 1 month but declines by 10 months post-irradiation. In AMI-induced mice, prior proton irradiation improved cardiac function restoration and enhanced cardiac remodeling. This was associated with increased pro-survival gene expression in cardiac tissues. In contrast, cardiac function was significantly declined in ⁵⁶Fe ion-irradiated mice at 1 and 3 months but recovered at 10 months. In addition, ⁵⁶Fe ion-irradiation led to poorer cardiac function and more adverse remodeling in AMI-induced mice, and was associated with decreased angiogenesis and pro-survival factors in cardiac tissues at any time point examined up to 10 months. This is the first study reporting CV effects following low dose proton and iron ion irradiation during normal aging and post-AMI. Understanding the biological effects of charged particle radiation qualities on the CV system is necessary both for the mitigation of space exploration CV risks and for understanding of long-term CV effects following charged particle radiotherapy.</p></div

ECHO, HEMO Measurements of Cardiac Functions and Cardiac Remodeling in IR + Aging + AMI Model.

<p>(<b>A</b>) Diagrammatic representation of the experimental design to evaluate the effect of acute, low-dose, whole body 50 cGy 1 GeV ¹H and 15 cGy 1 GeV/n ⁵⁶Fe IR in the hearts of 8–10 months old C57BL/6NT over 10 months in <b>Radiation + Aging + AMI</b>. IR-induced alterations in cardiac function were assessed by echocardiography (ECHO), hemodynamic (HEMO) and morphometric/histologic measurements and activation of signaling pathways by protein analyses. Acute myocardial infarct (AMI) was induced by ligation of the left anterior descending (LAD) coronary artery 1, 3 and 10 months post-IR, and mice were monitored over 28 days post-AMI. <i>ECHO analysis of cardiac function in the hearts of full-body ¹H-IR, ⁵⁶Fe-IR and non-IR control mice 1, 3 and 10 months post-IR in IR+Aging+AMI model for:</i> EF% 1 month (<b>B</b>), 3 months (<b>D</b>), 10 months (<b>F</b>), PWth (mm) 1 month (<b>C</b>), 3 months (<b>E</b>) and 10 months (<b>G</b>). Results in all graphs (<b>B–G</b>) are presented as mean ± SEM; <i>n</i> = 6–8 animals per time point/group. Non-IR control – solid green line, ¹H-IR - dashed blue line and ⁵⁶Fe-IR - dotted red line. <i>HEMO measurements and analysis of cardiac function in the hearts of full-body ¹H-IR, ⁵⁶Fe-IR and non-IR control mice 1, 3 and 10 months post-IR for:</i> (<b>H</b>) LV ESP (mmHg), (<b>I</b>) LV EDP (mmHg), (<b>J</b>) LV dP/dt_Max and dP/dt_Min (mmHg/sec). Results in all graphs (<b>H–J</b>) are presented as mean ± SEM; <i>n</i> = 6–8 animals per time point/group for non-IR control - solid black bars, ¹H-IR - solid grey bars and ⁵⁶Fe-IR - solid white bars. Statistical significance was assigned when <i>P</i><0.05. <i>Cardiac Remodeling 1, 3 and 10 months post-IR and 28 days after AMI:</i> Cardiac fibrosis was measured in the heart tissue post-AMI using Masson's Trichrome staining - blue is fibrosis and dotted line indicates the infarct scar size. Measurements represent midline length of the infarct when >50% of the LV was involved (mm) and every 3^rd section of the adjacent 8 µm size section were measured and infarct size was reconstructed as described before. Insets are representative images of ¹H-IR, ⁵⁶Fe-IR and non-IR control mice 1 month (<b>K</b>), 3 months (<b>L</b>) and 10 months (<b>M</b>) post-IR and 28 days after AMI. Graphic representation of the infarct size/scar (mm²) 1 month (<b>K</b>), 3 months (<b>L</b>) and 10 months (<b>M</b>) post-IR and 28 days after AMI. Results in all graphs are presented as mean ± SEM; <i>N</i> = 6–8 animals per time point/group for non-IR control - solid black bars, ¹H-IR - solid grey bars and ⁵⁶Fe-IR - solid white bars. Statistical significance was assigned when <i>P</i><0.05.</p

IR-induced Changes in Signaling Pathways 1, 3 and 10 months post-IR.

<p>Representative western blot scans of heart tissue homogenates from sham controls, ¹H- and ⁵⁶Fe-IR mice at 1, 3 and 10 months post-IR in <b>IR+Aging model</b>. Bands represent phosphorylated (p), total (T) and loading control for the following proteins - NCX and GAPDH (<b>A, I, Q</b>), SERCA2a and GAPDH (<b>C, K, S</b>), p-NFATc4, T-NFATc4 and β-actinin (<b>E, M, U</b>), p-p38, T-p38 and GAPDH (<b>G, O, W</b>). Quantification and graphic representation of total protein levels and phosphorylation using densitometric analysis of phospho-band intensities after adjusting for corresponding GAPDH/β-actinin and total band intensities of heart tissue homogenates at 1, 3 and 10 months post-IR for the following proteins - NCX (<b>B, J, R</b>), SERCA2a (<b>D, L, T</b>), p-NFATc4 (<b>F, N, V</b>), p-p38 (<b>H and P</b>). Results in all graphs represent mean ± SEM of the pooled data from <i>n</i> = 6–8 animals per time point/group for non-IR control (solid black bars), ¹H-IR (solid grey bars) and ⁵⁶Fe-IR (solid white bars) groups. Statistical significance was assigned when <i>P</i><0.05.</p

Formation and the Decay of <i>p</i>-H2AX Foci, Inflammatory and Oxidative DNA damage Responses.

<p>(<b>A</b>) Diagrammatic representation of the experimental design to evaluate the effect of a single low-dose full body exposure to 50 cGy, 1 GeV ¹H and 15 cGy, 1 GeV/n ⁵⁶Fe ions on the formation and disappearance of <i>p</i>-H2AX foci, inflammation (CD68 staining), and oxidative DNA damage (8-oxo-deoxy-Guanosine ELISA) in the hearts of 8–10 months old (at the time of initial IR) C57BL/6NT mice over 28 days post-IR. CD68 staining and 8-oxo-dG ELISA were also performed at 2 and 3 months; (<b>B</b>) Graphic representation of mean <i>p</i>-H2AX foci/cell (cells with >1 foci) in non-EC (<i>p</i>-H2AX plus/ISB4 minus) cells and ECs (<i>p</i>-H2AX plus/ISB4 plus) in the heart tissue isolated from mice of sham-irradiated, ¹H-IR and ⁵⁶Fe-IR up to 28 days. In figures <b>B</b> and C the time points on the X-axis are represented in Log² scale. Dotted line represents the maximum <i>p</i>-H2AX foci/cell for non-ECs and dashed line represents maximum <i>p</i>-H2AX for ECs. *<i>P</i><0.05 for ¹H vs. ⁵⁶Fe. (<b>C</b>) Graphic representation of % change in CD68 positive (+) cells in the heart tissue of non-IR, ¹H-IR and ⁵⁶Fe-IR mice within hours and up to 12 weeks post-IR. CD68 (+) cells in control hearts was set at 100% (dash-dotted line). *<i>P</i><0.01 and **<i>P</i><0.001 for ¹H vs. ⁵⁶Fe. (<b>D</b>) Graphic representation of percent change in IR-induced 8-oxo-dG levels, quantified by ELISA in heart tissue isolated from sham-irradiated, ¹H-IR and ⁵⁶Fe-IR mice up to 12 weeks. The level of 8-oxo-dG in control hearts was set at 100% (dash-dotted line). *<i>P</i><0.005 for ¹H vs. ⁵⁶Fe All results are presented as mean ± SEM; <i>n</i> = 6–8 animals per time point/group for sham controls, ¹H-IR (solid blue line) and ⁵⁶Fe-IR (solid red line) groups. Statistical significance was assigned when <i>P</i><0.05.</p

ECHO and HEMO Measurements of Cardiac Functions and Evaluation of Cardiac Fibrosis in IR + Aging Model.

<p>(<b>A</b>) Diagrammatic representation of the experimental design to evaluate the effect of acute low-dose, whole body 50 cGy, 1 GeV ¹H and 15 cGy, 1 GeV/n ⁵⁶Fe IR in the hearts of 8–10 months old C57BL/6NT over 10 months post-IR. IR-induced alterations in <b>Radiation+Aging</b> model in cardiac function were assessed by echocardiography (ECHO), hemodynamic (HEMO) and morphometric/histologic measurements and activation of signaling pathways by protein analyses. <i>ECHO analysis of cardiac function in the hearts of full-body ¹H-IR, ⁵⁶Fe-IR and non-IR control mice 1, 3 and 10 months post-IR for:</i> (<b>B</b>) Ejection Fraction - EF%, (<b>C</b>) Posterior wall thickness - PWth (mm). <i>HEMO measurements and analysis of cardiac function in the hearts of full-body ¹H-IR, ⁵⁶Fe-IR and non-IR control mice 1, 3 and 10 months post-IR for:</i> (<b>D</b>) LV ESP (mmHg), (<b>E</b>) LV EDP (mmHg), (<b>F</b>) LV dP/dt_Max and dP/dt_Min (mmHg/sec). (<b>G</b>) Graphic representation of % fibrosis in the hearts of full-body ¹H-IR, ⁵⁶Fe-IR and non-IR control mice 1, 3 and 10 months post-IR evaluated with Masson's trichrome staining. Results in all graphs (<b>B–G</b>) are presented as mean ± SEM; <i>n</i> = 6–8 animals per time point/group for non-IR control (solid black bars), ¹H-IR (solid grey bars) and ⁵⁶Fe-IR (solid white bars). Statistical significance was assigned when <i>P</i><0.05.</p

Molecular Pathways 3 days post-AMI in ¹H-IR, ⁵⁶Fe-IR and non-IR control hearts at 10 months post-IR.

<p>Representative Western blot images of heart tissue homogenates from sham control, ¹H- and ⁵⁶Fe-IR mice at 10 months post-IR in <b>IR + Aging + AMI model</b>. Images represent phosphorylated (p), total (T) and loading control for the following proteins (<b>A</b>) VEGF-A and GAPDH, (<b>C</b>) p-Erk1/2, T-Erk1/2 and GAPDH, (<b>E</b>) p-Akt (T308), T-Akt and GAPDH, (<b>G</b>) p-Akt (S473), T-Akt and GAPDH, (<b>I</b>) p-p38, T-p38 and GAPDH. Quantification and graphic representation of total protein levels and phosphorylation using densitometric analysis of phospho-band intensities after adjusting for corresponding GAPDH/β-actinin and total band intensities of heart tissue homogenates at 10 months post-IR and 3 days after AMI for the following proteins - (<b>B</b>) VEGF-A, (<b>D</b>) p-Erk1/2, (<b>F</b>) p-Akt (T308), (<b>H</b>) p-Akt (S473), (<b>J</b>) <b>p</b>-p38. Results in all graphs are depicted as mean ± SEM values and represent a pooled data from <i>n</i> = 6–8 animals per time point/group for non-IR control (solid black bars), ¹H-IR (solid grey bars) and ⁵⁶Fe-IR (solid white bars) groups. Statistical significance was assigned when <i>P</i><0.05.</p