Moss scoring guide

This Microsoft Word document provides a guide and examples for non-Arabidopsis growth cover scoring which was used to estimate interspecific competition

Code

This file contains the R code used to visualize and analyze the data

Fecundity data

This text file contains both individual and cage level fitness data

Census data

This text file contains the raw juvenile, vegetative adults, and reproductive adults census counts for the entire experiment

Competition data

This text file contains interspecific competition scores for each growth cage

Mineral Density Volume Gradients in Normal and Diseased Human Tissues

<div><p>Clinical computed tomography provides a single mineral density (MD) value for heterogeneous calcified tissues containing early and late stage pathologic formations. The novel aspect of this study is that, it extends current quantitative methods of mapping mineral density gradients to three dimensions, discretizes early and late mineralized stages, identifies elemental distribution in discretized volumes, and correlates measured MD with respective calcium (Ca) to phosphorus (P) and Ca to zinc (Zn) elemental ratios. To accomplish this, MD variations identified using polychromatic radiation from a high resolution micro-computed tomography (micro-CT) benchtop unit were correlated with elemental mapping obtained from a microprobe X-ray fluorescence (XRF) using synchrotron monochromatic radiation. Digital segmentation of tomograms from normal and diseased tissues (N=5 per group; 40-60 year old males) contained significant mineral density variations (enamel: 2820-3095mg/cc, bone: 570-1415mg/cc, cementum: 1240-1340mg/cc, dentin: 1480-1590mg/cc, cementum affected by periodontitis: 1100-1220mg/cc, hypomineralized carious dentin: 345-1450mg/cc, hypermineralized carious dentin: 1815-2740mg/cc, and dental calculus: 1290-1770mg/cc). A plausible linear correlation between segmented MD volumes and elemental ratios within these volumes was established, and Ca/P ratios for dentin (1.49), hypomineralized dentin (0.32-0.46), cementum (1.51), and bone (1.68) were observed. Furthermore, varying Ca/Zn ratios were distinguished in adapted compared to normal tissues, such as in bone (855-2765) and in cementum (595-990), highlighting Zn as an influential element in prompting observed adaptive properties. Hence, results provide insights on mineral density gradients with elemental concentrations and elemental footprints that in turn could aid in elucidating mechanistic processes for pathologic formations.</p></div

Segmented regions of different mineral densities in dentin.

<p>A) TOP PANEL: Virtual section of healthy dentin illustrates a constant HU line profile. LOWER PANEL: Mapped on the micro-CT virtual slice of diseased dentin is the HU gradient along a distance of 1400 μm; B) Segmented volumes within diseased dentin are represented in 2D and 3D. The mineral density range within normal dentin is 1400–1520 mg/cc. Segmented mineral density volumes illustrated two distinct zones of lower mineral content dentin: 400–600 mg/cc and 600–1000 mg/cc; C) Ca and P XRF area maps show concentration gradients (mmol/cm³) within diseased dentin regions in a 5 μm thick specimen. Ca and P maps show the region of interest indicated by the black dotted box on the inset, which shows a Ca map of the entire specimen. The bicolor map of Ca and P shows Ca deficiency (purple or blue) in the hypomineralized region (top portion at lesion site) and Ca rich regions (pink) gradually away from the lesion site.</p

Additional XRF analyses of elemental gradients in cementum, dentin and alveolar bone.

<p>A) Correlation (density) plots for cementum, dentin, and alveolar bone show the degree of equivalency between Ca to P and Ca to Zn patterns. Ca/P line plots (insets on the correlation plots) reveal Ca/P ratio range within each tissue. Correlation plots indicate a spread of Ca and P x-ray fluorescence signals in cementum and bone, while distinct zones in hypomineralized dentin (1* and 2*) were found. The reverse was identified for Ca to Zn correlation plots, which show distinct zones in cementum (area maps indicating AC and C) and alveolar bone (area maps indicating AB and NB) due to a range in Zn concentration for a narrow variation in Ca. For Ca/Zn plots, insets indicate summated regions that contributed to the overall spread in Zn vs. Ca signals. XRF bicolor maps (Ca and Zn for cementum and bone, Ca and P for dentin; bottom panel in A) indicate segmented zones from which the correlation plots are derived (C = cellular, AC = acellular, HD = healthy dentin, AB = alveolar bone, NB = new bone). Regions 1* and 2* are both hypomineralized zones, 1* being at the lesion site with severe demineralization (Ca/P = 0.32 ± 0.05) and 2* being directly adjacent to the lesion site with slightly higher mineralization (Ca/P = 0.46 ± 0.04); B) Correlation between micro-CT (N = 5) and XRF data (N = 1) for bone, dentin, and cementum with a corresponding line plot of mean MD vs. Ca/P ratio are shown. No correlation was found between mineral density and the Ca/Zn ratio. Zn dominant regions identified using XRF were not identified using micro-CT For example, an adapted or new bone (NB), Zn rich (AC) and Zn poor (C) layers within cementum were not discerned using micro-CT. The bottom right plot illustrates spread between mineral density and Ca/P ratio and an apparent linearity within diseased and healthy dentin.</p

Segmented regions of different mineral densities in cementum.

<p>A) X-ray virtual sections of cementum from a periodontically infected tooth illustrates affected cementum with dental calculus (white arrows). Following segmentation (right panel), a narrow mineral density range was identified; B) Mineral gradients in dental calculus with micro-CT 2D virtual sections and 3D volume rendering of supragingival dental calculus (top panel) and subgingival dental calculus (bottom panel). Note that supragingival is porous compared to consistent stratification observed in subgingival while both contain similar ranges of mineral density values (MD Range: 1290–1770 mg/cc); C) Light microscope image at 10X showing various healthy tissues i.e. bone, PDL, cementum, CDJ, and dentin. XRF area maps illustrate concentration gradients in Ca, Zn, and P in mmol/cm³. Bottom panel is a bicolor map of Ca and Zn, with blue regions of Ca dominance and red regions of Zn dominance. Note: Same landmarks are shown in all panels. A line profile (white dotted line) in the Zn image map shows Zn concentration (mmol/cm³) vs. distance (mm). Line was drawn from bone to dentin, perpendicular to the Zn bands in cementum.</p

Average HU and MD ranges from experiments and literature.

<p>A) UPPER PANEL: Mineral density and HU ranges of hard and soft tissues and regions of overlap amongst tissues are shown. LOWER PANEL: A schematic of segmentation procedure for diseased tissues, where the upper portion of the 3D volume (dark blue, *) represents lesion volume and the lower portion (medium to light blue, **) is a gradient between lesion shifting to a normal healthy tissue. Mineral gradients through the volume of material are identified by computing mineral density from each 2D virtual slice (e.g. 1, 2, and 3 following procedures highlighted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121611#pone.0121611.g001" target="_blank">Fig 1B</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121611#pone.0121611.g001" target="_blank">1C</a>). Statistically distinct (p<0.05) hypomineralized zones(*) are decoupled from the observed mineral gradient (upper line plot) for the entire diseased region using segmentation (following procedure highlighted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121611#pone.0121611.g001" target="_blank">Fig 1D</a>). The bar chart distinguishes each segmented hypomineralized zone against the healthy zone (**), the combined value (average of both healthy and hypomineralized zones) and healthy dentin; B) Summary of mineral density and HU ranges with average values across hard and soft tissues, including healthy and diseased conditions; C) LEFT PANEL: Mineral density of healthy alveolar bone and dentin calculated by gravimetric and micro-CT. RIGHT PANEL: Calculated mineral density of healthy and diseased hard tissues using a micro-computed tomography unit. To attain mineral density from the data, an ROI through samples was taken for healthy tissues (similar to figs B and C), segmentation was applied for diseased tissues (fig D). Segmented data was compared with ashed mineral density. A = ashed, H = Healthy, D = Diseased, (1) = Hypomineralized, (2) = Hypermineralized, * = A statistically insignificant result (p>0.05), while all other groups not indicated with an asterisk are statistically different.</p