Evaluation of 3D/2D Imaging and Image Processing Techniques for the Monitoring of Seed Imbibition

Seed imbibition is a very important process in plant biology by which, thanks to a simple water income, a dry seed may turn into a developing organism. In natural conditions, this process occurs in the soil, e.g., with difficult access for a direct observation. Monitoring the seed imbibition with non-invasive imaging techniques is therefore an important and possibly challenging task if one tries to perform it in natural conditions. In this report, we describe a set of four different imaging techniques that enable to addressing this task either in 3D or in 2D. For each technique, the following items are proposed. A detailed experimental protocol is provided to acquire images of the imbibition process. With the illustration of real data, the significance of the physical quantities measured in terms of their relation to the income of water in the seed is presented. Complete image analysis pipelines are then proposed to extract dynamic information on the imbibition process from such monitoring experiments. A final discussion compares the advantages and current limitations of each technique in addition to elements concerning the associated throughput and cost. These are criteria especially relevant in the field of plant phenotyping where large populations of plants are imaged to produce quantitatively significative traits after image processin

The G-Quadruplex Ligand Telomestatin Impairs Binding of Topoisomerase IIIα to G-Quadruplex-Forming Oligonucleotides and Uncaps Telomeres in ALT Cells

In Alternative Lengthening of Telomeres (ALT) cell lines, specific nuclear bodies called APBs (ALT-associated PML bodies) concentrate telomeric DNA, shelterin components and recombination factors associated with telomere recombination. Topoisomerase IIIα (Topo III) is an essential telomeric-associated factor in ALT cells. We show here that the binding of Topo III to telomeric G-overhang is modulated by G-quadruplex formation. Topo III binding to G-quadruplex-forming oligonucleotides was strongly inhibited by telomestatin, a potent and specific G-quadruplex ligand. In ALT cells, telomestatin treatment resulted in the depletion of the Topo III/BLM/TRF2 complex and the disruption of APBs and led to the segregation of PML, shelterin components and Topo III. Interestingly, a DNA damage response was observed at telomeres in telomestatin-treated cells. These data indicate the importance of G-quadruplex stabilization during telomere maintenance in ALT cells. The function of TRF2/Topo III/BLM in the resolution of replication intermediates at telomeres is discussed

Isolation and detection of <it>Mycobacterium avium </it>subsp. <it>paratuberculosis </it>(MAP) from cattle in Ireland using both traditional culture and molecular based methods

<p>Abstract</p> <p>Background</p> <p><it>Mycobacterium avium </it>subsp. <it>paratuberculosis </it>(MAP) causes a chronic gastroenteritis affecting many species. Johne's disease is one of the most widespread and economically important disease of ruminants. Since 1992 and the opening of the European market, the exposure and the transmission of MAP in cattle herds considerably increased. Improvements in diagnostic strategies for Ireland and elsewhere are urgently required. In total, 290 cattle from seven Irish herds with either a history or a strong likelihood of paratuberculosis infection were selected by a veterinary team over 2 years. Faecal samples (290) were collected and screened for MAP by a conventional culture method and two PCR assays. In order to further evaluate the usefulness of molecular testing, a nested PCR was also assessed.</p> <p>Results</p> <p><it>M. paratuberculosis </it>was isolated and cultured from 23 faecal samples (7.9%) on solid medium. From a molecular perspective, 105 faecal samples (36%) were PCR positive for MAP specific DNA. A complete correlation (100%) was observed between the results of both molecular targets (IS900 and ISMAP02). Sensitivity was increased by ~10% with the inclusion of a nested PCR for ISMAP02 (29 further samples were positive). When culturing and PCR were retrospectively compared, every culture positive faecal sample also yielded a PCR positive result for both targets. Alternatively, however not every PCR positive sample (n = 105, 36%) produced a corresponding culture isolate. Interestingly though when analysed collectively at the herd level, the correlation between culture and PCR results was 100% (ie every herd which recorded at least 1 early PCR +ve result later yielded culture positive samples within that herd).</p> <p>Conclusion</p> <p>PCR on bovine faecal samples is a fast reliable test and should be applied routinely when screening for MAP within herds suspected of paratuberculosis. Nested PCR increases the threshold limit of detection for MAP DNA by approximately 10% but proved to be problematic in this study. Although slow and impractical, culturing is still regarded as one of the most reliable methods for detecting MAP among infected cattle.</p

Mitochondrial topoisomerase I is critical for mitochondrial integrity and cellular energy metabolism.

Mitochondria contain their own DNA genome (mtDNA), as well as specific DNA replication and protein synthesis machineries. Relaxation of the circular, double-stranded mtDNA relies on the presence of topoisomerase activity. Three different topoisomerases have been identified in mitochondria: Top1mt, Top3α and a truncated form of Top2β.The present study shows the importance of Top1mt in mitochondrial homeostasis. Here we show that Top1mt-/- murine embryonic fibroblasts (MEF) exhibit dysfunctional mitochondrial respiration, which leads decreased ATP production and compensation by increased glycolysis and fatty acid oxidation. ROS production in Top1mt-/- MEF cells is involved in nuclear DNA damage and induction of autophagy. Lack of Top1mt also triggers oxidative stress and DNA damage associated with lipid peroxidation and mitophagy in Top1mt-/- mice.Together, our data implicate Top1mt for mitochondrial integrity and energy metabolism. The compensation mechanism described here contributes to the survival of Top1mt-/- cells and mice despite alterations of mitochondrial functions and metabolism. Therefore, this study supports a novel model for cellular adaptation to mitochondrial damage

Enhanced autophagy in Top1mt−/− cells and mice liver.

<p>(<b>A</b>) Expression of LC3-A/B (microtubule-associated protein1 light chain 3) by Western blotting in WT and Top1mt−/− cells. (<b>B</b>) LC3-A/B and DAPI staining by immunofluorescence in WT and Top1mt−/− cells (<b>C</b>) Transmission electronic microscopy showing autophagic vesicules in WT and Top1mt−/− cells. (<b>D</b>) Expression of LC3-A/B by Western blotting in WT and Top1mt−/− cells in presence or absence of 10 mM NAC antioxidant. (<b>E</b>) LC3-A/B and DAPI staining by immunofluorescence in Top1mt−/− mice liver and relative quantification. (<b>F</b>) Transmission electronic microscopy showing mitophagy in control and Top1mt−/− mouse liver. Histogram corresponding to the proportion of mitochondria totally surrounded by cytoplasmic membrane (autophagosome) related to the total number of mitochondria.</p

ATM-dependent DNA damage response pathway activation in Top1mt−/− cells.

<p>(<b>A</b>) γH2AX, pS1981-ATM and actin protein expression detected by Western blotting in WT ant Top1mt−/− cells. (<b>B</b>) γH2AX visualization by immunofluorescence and relative quantification in WT and Top1mt−/− cells. (<b>C</b>) Cell cycle analysis of WT and Top1mt−/− cells. (<b>D</b>) Detection of γH2AX by Western blotting after ROS inhibition with 10 mM of NAC for 3 h in WT and Top1mt−/− cells. (<b>F</b>) Immunostaining of intestine sections with γH2AX antibody in control and Top1mt−/− mice livers.</p

Mitochondrial dysfunction in Top1mt−/− cells.

<p>FACS analysis of ROS (<b>A</b>), mitochondrial membrane potential (ΔΨm) (<b>B</b>) and intracellular calcium content (<b>C</b>) in WT and Top1mt−/− cells. CM-H₂DCFDA, TMRM and Calcium Green dye fluorescence were plotted against cells numbers (count). Red line represents median of the histogram for WT samples. For each panel, quantification of mean fluorescence intensity is shown on the right referred as percentage of WT values. (<b>D</b>) Mitochondrial hyperfusion in Top1mt−/− cells visualized by mitotracker red staining. (<b>E</b>) SLP-2 (Stomatin like protein 2) expression by Western-blot (cropped figure) in WT and Top1mt−/− cells. Right plots represent means ± standard deviations of at least 3 experiments (quantification from 3 independent determinations).</p

Increase of fatty acid oxidation and lipogenesis in Top1mt-deficient cell line and Top1mt−/− mouse liver.

<p>(<b>A</b>) Oxygen consumption rate (OCR) in WT and Top1mt−/− cells (means ± standard deviations of 3 experiments). (<b>B</b>) Fatty acid oxidation dependent oxygen consumption rate in WT and Top1mt−/− cells after a 10 µg/ml C75 treatment for 3 h (means ± standard deviations of 3 experiments) (<b>C</b>) Representative experiment showing FASN (Fatty Acid Synthase) expression by Western blotting in WT and Top1mt−/− cells. (<b>D</b>) Glutathione concentration in WT and Top1mt−/− cells (means ± standard deviations of 3 experiments). (<b>E</b>) Malonaldehyde (MDA) measurement in WT and Top1mt −/− cells. (<b>F</b>) HNE (4-Hydroxynonenal) by immunofluorescence staining in control and Top1mt −/− mice liver. (<b>G</b>) Glutathione concentration in blood of control and Top1mt−/− mice (means ± standard deviations of 3 experiments). (<b>H</b>) Malonaldehyde concentration in blood of control and Top1mt−/− mice (means ± standard deviations of 3 experiments). (<b>I</b>) Representative Western blotting experiment showing FASN (Fatty Acid Synthase) expression in liver from control and Top1mt−/− mice.</p

Induction of the retrograde response in Top1mt−/− cells.

<p>(<b>A</b>) Gene expression of nuclear-encoded TFAM, PGC-1α, NRF-1 and POLG by RT-PCR in WT and Top1mt−/− cells (means ± standard deviations of 3 experiments). Gene expression was normalized to β2-microglobulin (β2M). Right panels show representative Western blots for Top1mt, PGC-1α, TFAM, POLG and actin protein levels in WT and Top1mt−/− cells. (<b>B</b>) Myc gene expression by RT-PCR normalized to β2M (mean ± standard deviation of 3 experiments) and c-myc protein level by Western blotting in WT and Top1mt−/− cells. (<b>C</b>) ND2, Cox1, Cox2; Cox3; ND4; ND5 and CytB expression by RT-PCR in WT and Top1mt−/− cells (means ± standard deviations of 3 experiments). (<b>D</b>) Determination of mitochondrial mass by Mitotracker Green staining. Representative histogram corresponding to Mitotracker Green fluorescence (x axis) plotted against cells numbers (count) and quantification of Mitotracker Green mean fluorescence intensity in cell population referred as percentage of WT values.</p