11 research outputs found
Comparison of causal factors associated with fire and the post-fire environment accounting for fatality rates in prescribed fires and wildfires from 1963–2014.
<p>Comparison of causal factors associated with fire and the post-fire environment accounting for fatality rates in prescribed fires and wildfires from 1963–2014.</p
Relative risk of different management techniques used by private land managers, based on occupational fatality rates as proxies.
<p>The fatality rate represents the number of fatal occupational injuries per 100,000 full-time equivalent workers. Mean fatality rates and 95% confidence intervals for each occupational proxy for years 2006–2013.</p
Number of fatal injuries in wildland fire from 1922–2013.
<p>(A) Fatal injuries are separated into 4 categories: those directly resulting from fire, those resulting from the use of vehicles and transportation, medical related, and environmental related. Trend in number of fatal injuries over time for each category is represented by locally weighted scatterplot smoothing (LOWESS) of the number of fatal injuries in each category over time.</p
Occupational proxies used to establish the relative fatality risk associated with private citizen use of different management techniques, based on data compiled for different occupations from the Census Database.
<p>Occupational proxies used to establish the relative fatality risk associated with private citizen use of different management techniques, based on data compiled for different occupations from the Census Database.</p
Machine-related and non-mechanical causes of wildland fire-related fatalities based on our classification scheme used to summarize the Wildland Fire Accidents Database.
<p>Machine-related and non-mechanical causes of wildland fire-related fatalities based on our classification scheme used to summarize the Wildland Fire Accidents Database.</p
Data Collection and Refinement Statistics.
<p>Data for the highest-resolution shell are given in parentheses.</p><p><i>R</i><sub><i>merge</i></sub> = Σ<sub><i>h</i></sub>Σ<sub><i>i</i></sub>|<i>I</i><sub><i>h</i>,<i>i</i></sub> − ⟨<i>I</i><sub><i>h</i></sub>⟩|/Σ<sub><i>h</i></sub>Σ<sub><i>i</i></sub><i>I</i><sub><i>h</i>,<i>i</i></sub>, where the outer sum (h) is over the unique reflections and the inner sum (i) is over the set of independent observations of each unique reflection.</p><p><i>R</i><sub><i>work</i></sub> = ∑<sub><i>hkl</i></sub>||<i>F</i><sub><i>obs</i></sub>| − |<i>F</i><sub><i>calc</i></sub>||/∑<i>F</i><sub><i>obs</i></sub>, where F<sub>obs</sub> and F<sub>calc</sub> are the observed and calculated structure factors of the respective reflections hkl.</p><p>R<sub>free</sub> is equivalent to R<sub>work</sub> but is calculated on a random set of reflections corresponding to 5% of all reflections and that are excluded from refinement.</p><p>*As defined by MolProbity [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144673#pone.0144673.ref052" target="_blank">52</a>]</p><p>Data Collection and Refinement Statistics.</p
Kinetic characterization of Bub1 complexes.
<p>(<b>A</b>-<b>C</b>) Bub1<sup>kinase</sup> and Bub1:Bub3 complex exhibit similar catalytic activity toward H2A (<b>A</b>) and Bor:Sur substrates (<b>B</b>) and hydrolyze ATP at similar rates (<b>C</b>). The mutation D917N abrogates ATPase activity (<b>C</b>). The kinase activity was determined using the ADP-Glo<sup>TM</sup> Kinase Assay and is plotted as a function of substrate concentration to allow fitting according to the Michaelis-Menten equation with R<sup>2</sup> = 0.99 (Bub1<sup>kinase</sup> on Bor:Sur R<sup>2</sup> = 0.97). KD–kinase dead. Error bars represent SD of a mean of at least 2 independent experiments. (<b>D</b>) Kinetic parameters of the Michaelis-Menten fits as determined in (<b>A</b>-<b>C</b>). (<b>E</b>) H2A contained in H3- or CENP-A nucleosomes is efficiently phosphorylated by Bub1<sup>kinase</sup> and Bub1:Bub3. (<b>F</b>) The kinase activity, plotted as a function of substrate concentration, allows fitting with the Michaelis-Menten equation with R<sup>2</sup> = 0.95 (H3) and 0.99 (CENP-A). Error bars represent SD of a mean of at least two independent experiments. (<b>G-H</b>) EMSA assays showing DNA and nucleosome binding of Bub1<sup>kinase</sup> and H3- or CENP-A nucleosomes. MN–mononucleosomes.</p
Reconstitution of Bub1 kinase and BubR1 pseudo-kinase.
<p>(<b>A</b>) Bub1 and BubR1 share a similar domain organization. Schematic description of the domains and constructs used in this manuscript: TPR–tetratricopeptide repeat, BD–binding domain, WD40 –an approximately 40-residue sequence repeat often terminating with a tryptophan (W) and aspartate (D) dipeptide. (<b>B</b>) Purified Bub1<sup>kinase</sup>, Bub1:Bub3, BubR1<sup>kinase</sup>, and BubR1:Bub3 were separated by SDS-PAGE. Their respective expected molecular mass is indicated. (<b>C</b>) Both Bub1<sup>kinase</sup> and BubR1<sup>kinase</sup> bind to mant-ATP. The change in fluorescence emission at 450 nm is plotted as a function of total mant-ATP concentration. The data were fitted with a one site binding equation using Origin 9.0, with R<sup>2</sup> = 0.99 for both curves. Error bars represent SD of a mean of at least 2 independent experiments. a.u.–arbitrary units. (<b>D</b>-<b>E</b>) ESI-MS spectra of purified Bub1<sup>kinase</sup> (<b>D</b>) or BubR1<sup>kinase</sup> (<b>E</b>) before and after incubation with ATP for auto-phosphorylation. Theoretical calculated masses are given in brackets under the measured masses. (<b>F</b>) BubR1 is not an active kinase. Maltose binding protein (MBP), H1, and the Borealin:Survivin complex (Bor:Sur) were incubated with 50 nM BubR1 constructs and ATP and analyzed on SDS PAGE visualizing phosphates using the Pro-Q® Diamond Phosphoprotein Gel Stain. 10 nM Bub1:Bub3 was used as a positive control. BR1<sup>kinase</sup>–BubR1<sup>kinase</sup>, BR1B3 –BubR1:Bub3.</p
Bub1<sup>kinase</sup> possesses conserved residues that recognize a substrate consensus sequence.
<p>(<b>A</b>) Alignment of phosphorylation sites found in Bub1-dependent phosphorylation reactions using the ClustalX algorithm in Jalview [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144673#pone.0144673.ref054" target="_blank">54</a>]. Numbers indicate boundaries of the protein segments shown; phosphorylated residues are denoted with an asterisk, the conservation of residues is highlighted using the ClustalX coloring scheme. (<b>B</b>) The structure of Bub1<sup>kinase</sup> (PDB ID 4QPM) was superimposed onto the structure of PKA bound to a pseudo-substrate inhibitor [PDB ID 1APM, reference [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144673#pone.0144673.ref055" target="_blank">55</a>]]. The image shows only Bub1 and the position of the pseudo-substrate peptide (yellow) after alignment of PKA to Bub1. Bub1 residues putatively involved in substrate recognition are depicted as sticks. (<b>C</b>) Bub1<sup>kinase</sup> sequence and structure conservation. The same residues as in (<b>B</b>) are shown in sticks. Conservation was determined by aligning Bub1<sup>kinase</sup> from 14 organisms with ConSurf [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144673#pone.0144673.ref056" target="_blank">56</a>], the scoring legend is depicted on the upper left. Conservation scores obtained for positions in the alignment that had less than 6 un-gapped amino acids were considered to be unreliable and colored light yellow in the graphic visualization output. Images were created with CCP4MG [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144673#pone.0144673.ref053" target="_blank">53</a>] and Pymol (Schrödinger LLC, Portland, OR). (<b>D</b>) Bub1<sup>kinase</sup>-dependent H2A phosphorylation can be reduced by mutating V115D, L116N, L117N on H2A. GST-H2A constructs were incubated with Bub1<sup>kinase</sup> and ATP, then analyzed by SDS PAGE, and phosphorylated proteins were specifically stained using Pro-Q® Diamond Phosphoprotein Gel Stain.</p
Biochemical characterization of Bub1 complexes.
<p>(<b>A</b>) Bub1:Bub3 (green) and BubR1:Bub3 (blue) co-elute in a stable complex from a size exclusion chromatography column (black) and can be assembled as a complex onto phosphorylated MBP-Knl1<sup>138-225</sup> (purple). A bar of the respective color indicates fractions analyzed on SDS PAGE (below). (<b>B</b>) Analytical ultracentrifugation. Normalized c(s) distribution curves for Bub1:Bub3 (green) and BubR1:Bub3 (blue). A predominant peak at 4.5 S (s<sub>20,w</sub> = 4.7) is apparent for Bub1:Bub3 and at 4.3 S (s<sub>20,w</sub> = 4.5) for BubR1:Bub3 indicating a single dominant sedimentation species that corresponds to a theoretical molecular weight of 150 kDa and 144 kDa, respectively. The theoretical mass of a 1:1 complex is indicated for both in brackets. Frictional ratios were determined as 2.16 for Bub1:Bub3 and 2.21 for BubR1:Bub3. (<b>C</b>) Complexes of Bub1 exhibit similar kinase activity toward a GST-H2A substrate. The kinase activity is plotted as a function of substrate concentration to allow fitting according to the Michaelis-Menten equation with R<sup>2</sup> = 0.99. Error bars represent SD of a mean of at least two independent experiments. (<b>D</b>) Kinetic parameters of the Michaelis-Menten fits as determined in (<b>C</b>). a.u.-arbitrary units.</p