MiR203 mediates subversion of stem cell properties during mammary epithelial differentiation via repression of ΔNP63α and promotes mesenchymal-to-epithelial transition.

During reproductive life, the mammary epithelium undergoes consecutive cycles of proliferation, differentiation and apoptosis. Doing so relies on the retained proliferative capacity, prolonged lifespan and developmental potency of mammary stem cells (MaSCs). ΔNp63α, the predominant TP63 isoform in mammary epithelia, is robustly expressed in MaSCs and is required for preservation of self-renewing capacity in diverse epithelial structures. However, the mechanism(s) underlying subversion of this activity during forfeiture of self-renewing capacity are poorly understood. MicroRNAs (miRNAs) govern critical cellular functions including stem cell maintenance, development, cell cycle regulation and differentiation by disrupting translation of target mRNAs. Data presented here indicate that expression of miR203, a miRNA that targets ΔNp63α and ΔNp63ß is activated during luminal epithelial differentiation and that this pattern is observed in the murine mammary hierarchy. In addition, we present evidence that the transcription factor Zeb1 represses miR203 expression, thus enhancing ΔNp63α protein levels. Furthermore, ectopic miR203 suppresses ΔNp63α expression, proliferation and colony formation. The anti-clonogenic effects mediated by miR203 require suppression of ΔNp63α. In addition, ectopic miR203 promotes mesenchymal-to-epithelial transition and disrupts activities associated with epithelial stem cells. These studies support a model in which induction of miR203 mediates forfeiture of self-renewing capacity via suppression of ΔNp63α and may also have anti-tumorigenic activity through its reduction of EMT and cancer stem cell populations.

Parallel and orthogonal E-field alignment of single-walled carbon nanotubes by ac dielectrophoresis.

We designed planar electrodes, for dielectrophoretic manipulation of single-walled carbon nanotubes (SWNTs), built as metal-oxide-semiconductor nanogap capacitors with common substrate and oxide thicknesses of 17 and 150 nm. Such design generates high electric fields (10(9) V m(-1)) and also the fringing field is curved due to the conducting substrate, unlike fields generated by conventionally used planar electrodes. Scanning electron microscopy images showed SWNTs aligned parallel and perpendicular to the electrodes. Raman spectroscopic mapping was used to produce separate images of the metallic (m-SWNT) and semiconducting (s-SWNT) nanotube density distributions. As expected, parallel alignment of the m-SWNTs with the E-field was found; however, also a perpendicular alignment of s-SWNTs was observed. Such orthogonal alignment of s-SWNTs is a rare observation and has not been experimentally reported before in detail with Raman images. Due to the unique electrode design, we were able to obtain substantial separation of m-SWNTs and s-SWNTs. Numerical modeling of the electric field factor of the dielectrophoresis force was done, and it matched perfectly with the experimental results. The orthogonal alignment of s-SWNTs results from comparable values of parallel and perpendicular polarizability to the nanotube axis.

Early remodeling in an inducible animal model of retinal degeneration.

Photoreceptor degeneration is followed by significant morphological changes in the second-order retinal neurons in humans and in several genetic animal models. However, it is not clear whether similar changes occur when photoreceptor degeneration is induced nongenetically, raising the question whether these changes are a general effect of deafferentation independent of the cause of degeneration. We addressed this by inducing selective photoreceptor degeneration with N-methyl-N-nitrosourea (MNU) and studying its effects on inner retinal neurons in a mouse for up to 3 months, using immunocytochemistry and iontophoretic labeling. To develop objective measures of photoreceptor degeneration and of retinal remodeling, we measured several retinal proteins using immunoblot analysis, and quantified gross visual ability of the animal in a visual cliff test. The MNU-induced progressive degeneration of rods and cones was associated with declining levels of postsynaptic density 95 protein in the retina, and with deteriorating visual performance of the animal. Müller glial cells showed enhanced reactivity for glial fibrillary acidic protein as demonstrated by immunocytochemistry, which also reflected in increased levels of the protein as demonstrated by immunoblotting. Horizontal cells and rod bipolar cells progressively lost their dendritic processes, which correlated with a slight decline in the levels of calbindin and protein kinase C alpha respectively. Horizontal cell axons, immunoreactive for nonphosphorylated neurofilaments, showed sprouting into the inner nuclear layer. Ganglion cells and their synaptic inputs, probed by immunolocalizing beta-III-tubulin, neurofilaments, bassoon and synaptophysin, appeared to be unaffected. These results demonstrate that MNU-induced photoreceptor degeneration leads to retinal remodeling similar to that observed in genetic models, suggesting that the remodeling does not depend on the etiopathology that underlies photoreceptor degeneration.

TA-p63-gamma regulates expression of DeltaN-p63 in a manner that is sensitive to p53.

Genetic analysis indicates that TP63 is required for establishment and preservation of self-renewing progenitors within the basal layer of several epithelial structures, however, the specific contributions of transactivating (TA-p63) and dominant-negative (DeltaN-p63) isoforms remain largely undefined. Recent studies have suggested a model in which TA-p63 plays an important role in the establishment of progenitor populations in which expression of DeltaN-p63 contributes to the preservation of self-renewing capacity. Our previous studies indicate that DeltaN-p63 is a transcriptional target of p53, however, the absence of overt epithelial deficiencies in p53-/- mice and reports of increased expression of DeltaN-p63 in p53-/- mice suggest p53-independent mechanisms also contribute to expression of DeltaN-p63. Here, we present data indicating that, prolonged loss of p53 leads to the activation of a p53-independent mechanism for transcriptional regulation of DeltaN-p63. This p53-independent mechanism is sensitive to ectopic p53 but not to a p53 mutant that lacks the transactivation domain. We further show that in cells in which p53 is expressed TA-p63-gamma protein is destabilized in a manner that is p53 dependent and sensitive to pharmacologic inhibition of the 26S proteosome. Consistent with this observation, we demonstrate that loss of p53 leads to the stabilization of TA-p63-gamma that is reversible by ectopic p53. Finally, we present evidence that disruption of TA-p63-gamma expression leads to decreased expression of DeltaN-p63 and that overexpression of TA-p63-gamma was sufficient to enhance the activity of the DeltaN-p63 promoter. Taken together, our studies indicate that TA-p63-gamma is capable of activating expression of DeltaN-p63 and that this mechanism may account for p53-independent expression of DeltaN-p63.

Changes in magnetocardiogram patterns of infarcted-reperfused myocardium after injection of superparamagnetic contrast media.

The changes in spatio-temporal patterns of magnetocardiograms were investigated following injection of superparamagnetic iron-oxide (SPIO) nanoparticles using a dog model of ischemia/reperfusion. Acute myocardial infarction was induced by ligation of the left anterior descending coronary in two anesthetized open-chest dogs. Following 60 min coronary occlusion and 30 min reperfusion, dogs were subjected to injections of SPIO at a dose of 0.56 mg Fe/kg using a catheter inserted into the left atrium. Magnetocardiograms were measured before coronary occlusion, 15 min after reperfusion and immediately after administration of the SPIO. Magnetic field maps of early reperfused myocardium showed spatio-temporal field distributions consistent with anterior myocardial infarction. Magnetic field distribution measured after injection of SPIO revealed additional spatio-temporal features most prominent during ventricular repolarization due to augmentation/fragmentation of the ST-segment detected at several sensor locations. No significant differences in MCG patterns were noted following contrast agent injections in a dog without coronary occlusion. In conclusion, preliminary experimental evidence appears to support the notion that superparamagnetic contrast agents increase the sensitivity of standard MCG and may have an important implication for magnetocardiography in the assessment of regional myocardial ischemia, infarction and perfusion.