Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and organs 1,2,6,7 . During development, toxic chemicals or genetic alterations can cause an increase in PCD or change PCD patterns resulting in developmental abnormalities and birth defects [3][4][5] . To understand the etiology of these defects, the study of embryos can be complemented with in vitro assays that use differentiating embryonic stem (ES) cells.Apoptosis is a well-studied form of PCD that involves both intrinsic and extrinsic signaling to activate the caspase enzyme cascade. Characteristic cell changes include membrane blebbing, nuclear shrinking, and DNA fragmentation. Other forms of PCD do not involve caspase activation and may be the end-result of prolonged autophagy. Regardless of the PCD pathway, dying cells need to be removed. In adults, the immune cells perform this function, while in embryos, where the immune system has not yet developed, removal occurs by an alternative mechanism. This mechanism involves neighboring cells (called "non-professional phagocytes") taking on a phagocytic role-they recognize the 'eat me' signal on the surface of the dying cell and engulf it [8][9][10] . After engulfment, the debris is brought to the lysosome for degradation. Thus regardless of PCD mechanism, an increase in lysosomal activity can be correlated with increased cell death.To study PCD, a simple assay to visualize lysosomes in thick tissues and multilayer differentiating cultures can be useful. LysoTracker dye is a highly soluble small molecule that is retained in acidic subcellular compartments such as the lysosome [11][12][13] . The dye is taken up by diffusion and through the circulation. Since penetration is not a hindrance, visualization of PCD in thick tissues and multi-layer cultures is possible 12,13 . In contrast, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) analysis 14 , is limited to small samples, histological sections, and monolayer cultures because the procedure requires the entry/permeability of a terminal transferase.In contrast to Aniline blue, which diffuses and is dissolved by solvents, LysoTracker Red DND-99 is fixable, bright, and stable. Staining can be visualized with standard fluorescent or confocal microscopy in whole-mount or section using aqueous or solvent-based mounting media 12,13 . Here we describe protocols using this dye to look at PCD in normal and sonic hedgehog null mouse embryos. In addition, we demonstrate analysis of PCD in differentiating ES cell cultures and present a simple quantification method. In summary, LysoTracker staining can be a great complement to other methods of detecting PCD. Video LinkThe video component of this article can be found at

Rationale: Brain arteriovenous malformations (AVMs) are abnormal tangles of vessels where arteries and veins directly connect without intervening capillary nets, increasing the risk of intracerebral hemorrhage and stroke. Current treatments are highly invasive and often not feasible. Thus, effective non-invasive treatments are needed. We previously showed that AVM brain endothelial cells (AVM-BEC) secreted higher vascular endothelial growth factor (VEGF) and lower thrombospondin-1 (TSP-1) levels than control BEC; and that miR-18a normalized AVM-BEC function and phenotype, although its mechanism remained unclear. Objective: To elucidate the mechanism of action and potential clinical application of miR-18a as an effective non-invasive treatment to selectively restore the phenotype and functionality of AVM vasculature. Methods and Results: The molecular pathways affected by miR-18a in patient-derived BECs and AVM-BECs were determined by western-blot, RT-qPCR, ELISA, co-IP, immunostaining, knockdown and overexpression studies, flow cytometry, and luciferase reporter assays. MiR-18a was shown to increase TSP-1 and decrease VEGF by reducing plasminogen activator inhibitor (PAI-1/SERPINE1) levels. Furthermore, miR-18a decreased the expression of bone morphogenetic protein 4 (BMP4) and hypoxia inducible factor 1α (HIF-1α), blocking the BMP4/activin-like kinase 2 (ALK2)/ALK1/ALK5 and Notch signaling pathways. As determined by Boyden chamber assays, miR-18a also reduced the abnormal AVM-BEC invasiveness, which correlated with a decrease in MMP2, MMP9 and ADAM10 levels. In vivo pharmacokinetic studies showed that miR-18a reaches the brain following intravenous (IV) and intranasal (IN) administration. IN co-delivery of miR-18a and NEO100, a good manufacturing practices (GMP)-quality form of perillyl alcohol (POH), improved the pharmacokinetic profile of miR-18a in the brain without affecting its pharmacologic properties. Ultra-high-resolution computed tomography angiography and immunostaining studies in an Mgp-/- AVM mouse model showed that miR-18a decreased abnormal cerebral vasculature, and restored the functionality of the bone marrow, lungs, spleen and liver. Conclusions: MiR-18a may have significant clinical value in preventing, reducing and potentially reversing AVM.

Glioblastoma multiforme is a malignant brain tumor noted for its extensive vascularity, aggressiveness, and highly invasive nature, suggesting that cell migration plays an important role in tumor progression. The poor prognosis in GBM is associated with a high rate of tumor recurrence, and resistance to the standard of care chemotherapy, temozolomide (TMZ). The novel compound NEO212, a conjugate of TMZ and perillyl alcohol (POH), has proven to be 10-fold more cytotoxic to glioma stem cells (GSC) than TMZ, and is active against TMZ-resistant tumor cells. In this study, we show that NEO212 decreases migration and invasion of primary cultures of patient-derived GSCs, in both mesenchymal USC02 and proneural USC04 populations. The mechanism by which NEO212 reduces migration and invasion appears to be independent of its DNA alkylating effects, which cause cytotoxicity during the first hours of treatment, and is associated with a decrease in the FAK/Src signaling pathway, an effect not exhibited by TMZ. NEO212 also decreases the production of matrix metalloproteinases MMP2 and MMP9, crucial for GSC invasion. Gene expression analysis of epithelial and mesenchymal markers suggests that NEO212 increases the expression of epithelial-like characteristics, suggesting a reversion of the epithelial-to-mesenchymal transition process. Furthermore, in an orthotopic glioma model, NEO212 decreases tumor progression by reducing invasion of GSCs, thereby increasing survival time of mice. These studies indicate that NEO212, in addition to cytotoxicity, can effectively reduce migration and invasion in GSCs, thus exhibiting significant clinical value in the reduction of invasion and malignant glioma progression..

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