Child injury in an urban Australian indigenous community: the safe koori kids intervention

Objective To design and evaluate an intervention targeting urban indigenous Australian children in order to increase their self-effi cacy, knowledge and attitudes towards safety. Methods The Safe Koori Kids intervention was developed and delivered to 790 children primary school aged children (13% indigenous) in 24 middle and upper primary classes across fi ve schools in Sydney, Australia. The intervention, consisting of fi ve safety modules, was evaluated using a mixed-methods approach. A pre-test post-test research design was applied to evaluate changes in key outcomes namely child self-effi cacy, knowledge and attitudes towards safety. Qualitative and quantitative data were collected from teachers. Findings There was a signifi cant increase (p\u3c0.05) in self-effi cacy among children from pre- to post-intervention for both Indigenous (6%) and non-Indigenous children (2%). Safety knowledge among Indigenous children increased from pre- to post intervention by 17% (p\u3c0.01) and non-Indigenous children by 15%, (p\u3c0.01). However, there were no signifi cant improvements in attitudes towards safety (indigenous children 2%, p=0.288, non-Indigenous children 1%, p=0.0721). Overall, Indigenous children scored lower than non-Indigenous children post intervention on self-effi cacy (75%:77%), knowledge (56%:63%) and attitudes towards safety (79%:84%). Teacher focus groups provided further evidence of the programs impact on children\u27s safety knowledge and attitudes. Conclusions The study contributes to our limited knowledge about effective child injury prevention for disadvantaged Indigenous minorities in high income countries. This is the fi rst intervention of its type in an urban indigenous setting in Australia which has positively contributed to the resilience of indigenous children and families with respect to safety and their environment

Characterization of ring-like F-actin structure as a mechanical partner for spindle positioning in mitosis.

Proper spindle positioning and orientation are essential for accurate mitosis which requires dynamic interactions between microtubule and actin filament (F-actin). Although mounting evidence demonstrates the role of F-actin in cortical cytoskeleton dynamics, it remains elusive as to the structure and function of F-actin-based networks in spindle geometry. Here we showed a ring-like F-actin structure surrounding the mitotic spindle which forms since metaphase and maintains in MG132-arrested metaphase HeLa cells. This cytoplasmic F-actin structure is relatively isotropic and less dynamic. Our computational modeling of spindle position process suggests a possible mechanism by which the ring-like F-actin structure can regulate astral microtubule dynamics and thus mitotic spindle orientation. We further demonstrated that inhibiting Plk1, Mps1 or Myosin, and disruption of microtubules or F-actin polymerization perturbs the formation of the ring-like F-actin structure and alters spindle position and symmetric division. These findings reveal a previously unrecognized but important link between mitotic spindle and ring-like F-actin network in accurate mitosis and enables the development of a method to theoretically illustrate the relationship between mitotic spindle and cytoplasmic F-actin

Autophosphorylation negatively regulates Mps1 kinetochore localization.

<p>(A) and (E) Representative immunofluorescence images of prometaphase cells expressing different LAP-tagged Mps1 constructs. At 36 hours after co-transfection with the Mps1 shRNA and indicated plasmids, cells were fixed and co-stained for ACA (red) and DNA (blue). Scale bar represents 10 µm. (B) and (F) Bar graph showing quantification of the ratios of kinetochore signal to cytoplasmic signal of different Mps1 constructs as indicated. Bars indicate mean ±SE from 3 independent experiments. In each experiment, 5 cells were measured (>60 kinetochores per cell). Statistics significance was determined by an unpaired Student's t test. (C) and (G) Western blot showing the comparable expression of different Mps1 constructs as indicated. 24 hours after transfection into 293T cells, cell lysates were prepared. After separation by SDS-PAGE, samples were probed with the indicated antibodies. (D) Schematic showing Mps1 autophosphorylation sites. 8 autophosphorylation sites outside of kinase domain are shown in red. 2 autophosphorylation sites within activation loop are shown in black.</p

Autophosphorylation of Thr12 and Ser15 is dispensable for Mps1 kinetochore localization and SAC function.

<p>(A) Representative immunofluorescence images of prometaphase cells expressing different LAP-tagged Mps1 constructs. At 36 hours after co-transfection with the Mps1 shRNA and indicated plasmids, cells were fixed and co-stained for ACA (red), DNA (blue) and Mad2 (shown as gray scale images). (B) Bar graph showing quantification of the relative Mad2 kinetochore signal intensity in LAP-Mps1^WT or Mps1^KD expressing cells. Bars indicate mean ±SE from 5 cells measured (at least 20 kinetochores per cell). (C) and (E) Representative immunofluorescence images of prometaphase cells expressing different LAP-tagged Mps1 constructs. At 36 hours after co-transfection with the Mps1 shRNA and indicated plasmids, cells were fixed and co-stained for ACA (red), DNA (blue) and Mad1 (C, shown as gray scale images) or Mad2 (E, showing as gray scale images). Scale bar represents 10 µm. (D) and (F) Bar graph showing quantification of the Mad1 (D) or Mad2 (F) kinetochore signal in cells treated as in (C) or (E). Bars indicate mean ±SE from 5 cells measured (at least 20 kinetochores per cell). A.U. means arbitrary unit. (G) Representative immunofluorescence images of prometaphase cells expressing different GFP-tagged Mps1 truncations. At 36 hours after co-transfection with the Mps1 shRNA and indicated plasmids, cells were fixed and co-stained for ACA (red) and DNA (blue). Scale bar represents 10 µm. (H) Bar graph showing quantification of the kinetochore signal of indicated Mps1 truncation protein as in (G). Bars indicate mean ±SE from 5 cells measured (at least 20 kinetochores per cell).</p

Phospho-mimetic Mps1^8D impairs the kinetochore recruitment of BubR1 and Mad2.

<p>(A) Representative immunofluorescence images of prometaphase cells co-transfected with Mps1 shRNA and GFP vector at a 3∶1 ratio. Cells were fixed and co-stained for BubR1 (red) and DNA (blue). Scale bar represents 10 µm. (B) and (C) Representative immunofluorescence images of prometaphase cells expressing different LAP-tagged Mps1 constructs. At 36 hours after co-transfection with the Mps1 shRNA and indicated plasmids, cells were fixed and co-stained for BubR1 (red) (B) or Mad2 (C), and DNA (blue). Scale bar represents 10 µm. (D) Bar graph showing quantification of kinetochore signal of BubR1 and Mad2 in cells expressing different Mps1 constructs as indicated. Bars indicate mean ±SE from 3 independent experiments. In each experiment, 5 cells were measured (>60 kinetochores per cell). * <i>P</i><0.001 versus shMock or Mps1^WT and Mps1^8A rescue groups.</p

Identification of a ring-like F-actin structure during mitosis.

<p>(A) The distribution of F-actin in different phases of mitosis in HeLa cells. Asynchronized cells were fixed and co-stained for F-actin (red), microtubule (green) and DNA (blue). A ring-like F-actin structure appears during metaphase and anaphase and was indicated by the arrows (<b><i>a, b, c</i></b>). Scale bar, 5 µm. (B) The representative immunofluorescence images of two cells with cytoplasmic F-actin. Cell A represents the cells with slightly rotated spindles, and Cell B represents the cells with distinctly rotated spindle. The boxed areas are shown magnified in the right panels. Scale bar, 5 µm. (C) Multiple-layer images of Cell A and Cell B. The plane passing through the midpoint of spindle poles and parellel to substrate is chosen as Z = 0, and the images in Fig. 1B is taken at the plane Z = 0. The distance between layer Z and layer Z+1 is 0.2 µm. Scale bar, 5 µm. (D) Astral microtubules and cytoplasmic F-actin structure cross and distribute differently in cytoplasm. The yellow arrows indicate the detailed distribution of astral microtubules. The cytoplasmic F-actin structure distributes around the spindle continuously. The boxed areas are shown magnified 16 times in the bottom panels. Scale bar, 5 µm.</p

Modeling of spindle positioning and hypothesis of the function of ring-like F-actin structure.

<p>(A) The overview of a mitotic cell from three dimensions. X-axis links two spindle poles when the spindle is in its equilibrium position, and Z-axis is vertical to the substrate and passes through the midpoint of spindle poles, and Y-axis is simultaneously vertical to X-axis and Z-axis. Different regions are colored for F-actin (red), microtubule (green), chromosomes (blue), cortex and medium (black). (B) The geometrical hypothesis of cell. A mitotic HeLa cell is supposed to be an ellipsoid with its height H and radius R. Then, the geometry can be described with a formula, and the point on the surface can be positioned with an alterable vector <b><i>D</i></b> and two parameters θ, φ. (C) The schematic diagram of spindle rotation in two sections, X-Y and Z-X. The geometrical center of spindle is supposed to be translated from the geometrical center of cell, and the coordinate of two spindle poles can be expressed with <b>A</b>, the distance from spindle pole to equate plate. The rotation in X-Y and Z-X sections can be described with the parameters, <b>Θ, Φ</b>. (D) The hypothesis of oscillation. The microtubule-motor complex is simplified as an oscillator, and the oscillation follows Hooke's Law of Elasticity and is influenced by the obstruction from cytoplasm. Here, <b><i>D</i></b>_-c represents the vector from spindle pole to cell cortex, which can be decomposed into a regular vector <b><i>D</i></b> and the position of one spindle pole. (E) The dynamic transition between effective and ineffective connections or binding state. and were supposed to be two rate constant for this transition. We also supposed that only the oscillators with its |<b><i>D</i></b>| in the interval [D₁, D₂] and a positive momentum make effective connections. Then the number of effective or ineffective connections can be expressed as displayed in the figure.</p

The perturbation of ring-like F-actin structure formation by chemical inhibitors and real-time imaging analyses.

<p>(A) The method to measure the apparent intensity of ring-like F-actin structure. A cell is classified into four regions named circle 1, 2, 3 and 4. Circle 1 includes the region of spindle-chromosome complex; circle 2 includes the region of ring-like F-actin structure just around the spindle, excluding the region inside circle 1; circle 3 includes the region of all cytoplasmic F-actin just inside the cortex, excluding the region inside circle 2; circle 4 includes the region of the whole cell body, excluding the region inside circle 3. The formulae to calculate apparent intensity are displayed here. (B) Representative images of a HeLa cell treated with DMSO and MG132. Cells were double blocked and released. MG132 was added at time point -5′, and DMSO was added at 0′. We acquired each image every 1 minute since -10', and the total time was 1 hour. Cells were transfected with GFP-tubulin (green) and mCherry-UtroCH (red) to label microtubules and F-actin, respectively. Scale bar, 5 µm. (C) Representative images of HeLa cells treated with DMSO, BI2536, Blebbistatin, Reversine and Lat B. We used the same method as (B). The influence of chemical treatment on the ring-like structure and spindle position can be observed and measured from the supplemental movies. Scale bar, 5 µm. (D) The apparent intensity of the ring-like structure in each cell measured from the living cell imaging. The curve of circle 3 represents the photo-bleaching along the time. We chose the apparent intensity of circle 3 as the normalization factor. We also chose the apparent intensity of circle 2 as the apprent intensity of ring-like F-actin structure.</p

Treatment of chemical compounds alters spindle position and symmetric division.

<p>(A) Measurements for the relative translation of the spindle from the cell centroid (D/R). We draw two circles to determine the centroids of spindle and cell on the Z = 0 plane. The first circle highlights the spindle while the other one outlines the cell. Coordinates of the centroids of the circles were used to calculate the offset distance between them. Dividing the distance by the radius of the cell gives the relative distance which is expressed as D/R ratio. (B)Measurements for the area ratio of the daughter cells. The two polygons go around each daughter cell. Then the area ration equals the larger area divided by the smaller area. (C) The D/R changes along time with the treatment of drugs. The method is mentioned in materials and methods. The spindle moved away from the cell centroid in BI2536, Reversine and Lat B groups. However, the shift was finally recovered in Blebbistatin group. The significances displayed here demonstrate the differences between subsequent time points and T = 0' by t-test. Red curves represent the mean value of D/R, and small red bars represent the ±3xSD for a 95% confidence interval. Small black points represent the values of D/R of each cell. (D) The curves presenting D/R along time by living cell imaging. The variation of D/R is enhanced in experimental groups comparing to the DMSO group. (E) The area ratio of daughter cells. BI2536 (p<0.001), Reversine (p<0.01) and Latruculin B (p<0.001) groups are significantly different from DMSO group, which means that the daughter cells of these groups intend to divide asymmetrically, but the Blebbistatin group is not (t-test p = 0.58 and F-test p = 0.94). Large red bars represent the mean values, and short red bars represent ±3xSD for a 95% confidence interval. Small black points represent the values of area ratio of each cell. (F)The rotating angle of spindles in cells treated with drugs. Reversine, BI2536 and Nocodazole groups are significantly different from DMSO group by t-test, which suggests that Mps1 and Plk1 play a role in spindle orientation. The cells were treated with MG132 for 2 hours and then the drugs for 1 hour, respectively.</p