The immortal strand theory postulates stem cells protect themselves from DNA replication-associated mutations and subsequent cancer risk through selective segregation of template DNA strands. Stem cells self-renew by asymmetric cellular division. During asymmetric division, stem cells maintain their template DNA strands, while the newly synthesized DNA strands segregate to newly formed daughter cells. Previous studies have demonstrated that self-renewing mammary stem cells originate in the expanding mammary ducts during puberty-associated allometric growth. In this study, we labeled newly forming mammary stem cells with the thymidine analog 5-ethynl-2'-deoxyuridine for 2 weeks during allometric ductal expansion. Cells that incorporate and retain the nuclear label following extended chase periods are termed label-retaining cells (LRCs). A second nuclear label, 5-bromodeoxyuridine, was administered before euthanasia to identify cells traversing the cell cycle. Mammary cells collected following euthanasia were sorted based on nuclear label retention. Members of the Notch and Wnt signaling pathways were found differentially expressed by mammary LRCs. These pathways are involved in the regulation of stem cells in the mouse mammary gland. Upon further analysis, we found that in contrast to non-LRCs, Notch1 and Notch2 are expressed and localized in the nuclei of the LRCs. Expression of Notch-inducible genes, Hes1 and Hey2, was elevated in LRCs. Inhibition of Notch1 by shRNA reduced colony forming potential and label retention by mammary epithelial cells in vitro. These results indicate that genes are differentially regulated in the LRC population of mammary glands and Notch1 mediates asymmetric cell division of mammary progenitor cells.

Overexpression of the oncoprotein erbB2/HER2 is present in 20-30% of breast cancer patients and inversely correlates with patient survival. Reports have demonstrated the deterministic power of the mammary microenvironment where the normal mammary microenvironment redirects cells of non-mammary origin or tumor-derived cells to adopt a mammary phenotype in an in vivo model. This phenomenon is termed tumor cell redirection. Tumor-derived cells that overexpress the erbB2 oncoprotein lose their tumor-forming capacity in this model. In this model, phosphorylation of erbB2 is attenuated thus reducing the tumor cell's tumor-forming potential. In this report, we describe our results using an in vitro model based on the in vivo model mentioned previously. Tumor-derived cells are mixed in predetermined ratios with normal mammary epithelial cells prior to seeding in vitro. In this in vitro model, the tumor-derived cells are redirected as determined by attenuated phosphorylation of the receptor and reduced sphere and colony formation. These results match those observed in the in vivo model. This in vitro model will allow expanded experimental options in the future to determine additional aspects of tumor cell redirection that can be translated to other types of cancer.

e Plasmodium falciparum, the deadliest species of malaria parasites, is dependent on glycolysis for the generation of ATP during the pathogenic red blood cell stage. Hexokinase (HK) catalyzes the first step in glycolysis, transferring the ␥-phosphoryl group of ATP to glucose to yield glucose-6-phosphate. Here, we describe the validation of a high-throughput assay for screening smallmolecule collections to identify inhibitors of the P. falciparum HK (PfHK). The assay, which employed an ADP-Glo reporter system in a 1,536-well-plate format, was robust with a signal-to-background ratio of 3.4 ؎ 1.2, a coefficient of variation of 6.8% ؎ 2.9%, and a Z=-factor of 0.75 ؎ 0.08. Using this assay, we screened 57,654 molecules from multiple small-molecule collections. Confirmed hits were resolved into four clusters on the basis of structural relatedness. Multiple singleton hits were also identified. The most potent inhibitors had 50% inhibitory concentrations as low as ϳ1 M, and several were found to have lowmicromolar 50% effective concentrations against asexual intraerythrocytic-stage P. falciparum parasites. These molecules additionally demonstrated limited toxicity against a panel of mammalian cells. The identification of PfHK inhibitors with antiparasitic activity using this validated screening assay is encouraging, as it justifies additional HTS campaigns with more structurally amenable libraries for the identification of potential leads for future therapeutic development.

Background Reduced miR‐133a was previously found to be associated with thoracic aortic ( TA ) dilation, as seen in aneurysm disease. Because wall tension increases with vessel diameter (Law of Laplace), this study tested the hypothesis that elevated tension led to the reduction of miR‐133a in the TA . Methods and Results Elevated tension (1.5 g; 150 mm Hg) applied to murine TA ex vivo reduced miR‐133a tissue abundance compared with TA held at normotension (0.7 g; 70 mm Hg). Cellular miR‐133a levels were reduced with biaxial stretch of isolated murine TA fibroblasts, whereas smooth muscle cells were not affected. Mechanisms contributing to the loss of miR‐133a abundance were further investigated in TA fibroblasts. Biaxial stretch did not reduce primary miR‐133a transcription and had no effect on the expression/abundance of 3 micro RNA ‐specific exoribonucleases. Remarkably, biaxial stretch increased exosome secretion, and exosomes isolated from TA fibroblasts contained more miR‐133a. Inhibition of exosome secretion prevented the biaxial stretch‐induced reduction of miR‐133a. Subsequently, 2 in vivo models of hypertension were used to determine the effect of elevated wall tension on miR‐133a abundance in the TA : wild‐type mice with osmotic pump–mediated angiotensin II infusion and angiotensin II –independent spontaneously hypertensive mice. Interestingly, the abundance of miR‐133a was decreased in TA tissue and increased in the plasma in both models of hypertension compared with a normotensive control group. Furthermore, miR‐133a was elevated in the plasma of hypertensive human subjects, compared with normotensive patients. Conclusions Taken together, these results identified exosome secretion as a tension‐sensitive mechanism by which miR‐133a abundance was reduced in TA fibroblasts.

Based on our results we conclude that AREG is involved in regulating the proliferation and migration of erbB2/HER2-positive breast cancer cells.