Data_Sheet_1_Viral Communities of Shark Bay Modern Stromatolites.xlsx

<p>Single stranded DNA viruses have been previously shown to populate the oceans on a global scale, and are endemic in microbialites of both marine and freshwater systems. We undertook for the first time direct viral metagenomic shotgun sequencing to explore the diversity of viruses in the modern stromatolites of Shark Bay Australia. The data indicate that Shark Bay marine stromatolites have similar diversity of ssDNA viruses to that of Highbourne Cay, Bahamas. ssDNA viruses in cluster uniquely in Shark Bay and Highbourne Cay, potentially due to enrichment by phi29-mediated amplification bias. Further, pyrosequencing data was assembled from the Shark Bay systems into two putative viral genomes that are related to Genomoviridae family of ssDNA viruses. In addition, the cellular fraction was shown to be enriched for antiviral defense genes including CRISPR-Cas, BREX (bacteriophage exclusion), and DISARM (defense island system associated with restriction-modification), a potentially novel finding for these systems. This is the first evidence for viruses in the Shark Bay stromatolites, and these viruses may play key roles in modulating microbial diversity as well as potentially impacting ecosystem function through infection and the recycling of key nutrients.</p

Morphometric Parameters Investigated in µCT Analysis (n = 7).

*<p>Significantly less than ground control, p<0.05. Means are reported ± standard deviation.</p

mRNA Gene Expression Levels Altered Following Spaceflight.

<p>mRNA Gene Expression Levels Altered Following Spaceflight.</p

Spaceflight Causes Overexpression of the Cell Cycle Arrest Molecule, p21, Independently of p53 Activation.

<p>RT-PCR analysis revealed significant alterations in many cell cycle molecules including a 3.31 fold up-regulation of p21 and down-regulation of p53 (<b>G</b>). Immunohistochemical analysis localized this overexpression of p21 to osteoblasts along the periosteal surface of the proximal femur (<b>A</b>, ground control, <b>B</b>, flight). Interestingly, we also observed p21-positive nuclei in cross-sections and longitudinal sections of muscle fibers adjacent to the femur (<b>C–D</b>, ground control, <b>E–F</b>, flight). *indicates p<0.05, # indicates p<0.01.</p

Spaceflight Alters mRNA Expression of Genes Associated with Osteogenic Growth and Mitogenic Signal Transduction Pathways.

<p>RT-PCR analysis of revealed altered expression levels of key genes involved in osteogenic growth and proliferation including growth factors, Bmp4 and Tgfβ2, and transcription factors Vdr and Sox9 (A). Analysis of key mitogenic signal transduction pathways revealed alterations in gene expression of the MAPK pathway, whilst Pi3K and Akt signaling molecules were not changed statistically. We also observed significant up-regulation of the NFκB inhibitor, NFκBIa/IκBα (B).</p

Spaceflight Causes Up-Regulation of Matrix Degradation Molecules.

<p>RT-PCR analysis of ilium revealed significant up-regulation of matrix degradation molecules MMP1a, MMP3, and MMP10 as well as small changes in a number of extracellular matrix molecules in flight samples compared to ground controls (<b>A</b>). Immunohistochemical analysis localized over-expression of MMP10 to osteocytes in the shaft of the proximal femur in flight samples (<b>C</b>) but not in ground controls (<b>B</b>), indicating a role for osteocytes in lacunae degradation. * indicates p<0.05, # indicates p<0.01.</p

Micro-Computed Tomography (μCT) Analysis of Spaceflown Ischium.

<p>The ischium of the pelvis, shown in orange (<b>A</b>), was analyzed using μCT (720 slices = 4.89 mm). The anatomical markers used for μCT analysis were (1) caudal apex of obturator foramen, (2) dorsal-most point of the ventral ramus of ischium, and (3) the ischial tuberosity (<b>A</b>) (For full details on anatomical markers see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061372#pone.0061372-Schutz1" target="_blank">[74]</a>). The ischial cross-sectional geometry (<b>B</b>) was analyzed by length (a), the width at the midpoint (b) and at 1/3 distance from the obturator foramen (c); and the bend angle (d). Flight samples (<b>D</b>) exhibited a more open cross-sectional geometry compared to the ground control (<b>C</b>), indicating a possible reduction in the pull force applied to the bone. Ground control samples (<b>F</b>) also exhibited greater thickness (orange/red) then the flight samples (<b>G</b>), indicating a reduction in overall bone thickness in spaceflight samples.</p

Nano-Computed Tomography Analysis of Lacunar Enlargement Following Spaceflight.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061372#pone-0061372-g002" target="_blank">Figures 2<b>A </b>and<b> 2B</b></a> show X-ray phase contrast images of osteocytes viewed laterally and from the top, respectively and illustrate the flattened shape of osteocytic lacunae. For quantification of lacunar size we used X-Ray absorption images of lateral views of lacunae in ischial cortical bone from ground control (<b>C</b>) and spaceflight (<b>D</b>) animals (n = 7). We observed a 17% increase in lacunae area and a 14% increase in lacunae perimeter of flight animals compared to ground controls (<b>E</b>). We also found a 13% increase in lacunae canalicular diameter and a 9% decrease in lacunae circularity of flight animals compared to ground controls (<b>F</b>). However, bulk density analysis showed no statistical difference between flight and ground control animals that is in agreement with µCT analysis (<b>G</b>). *indicates p<0.05, #indicates p<0.01.</p

Measurements of the Ischial Cross-Section Cut at the Widest Point of the Descending Ramus (n = 7). Refer to Fig. 1.

*<p>Significantly less than ground control, p<0.05,^#Significantly less than ground control, p<0.01. Means are reported ± standard deviation.</p