Summary Transmitochondrial cybrids and multiple OMICs approaches were used to understand mitochondrial reprogramming and mitochondria-regulated cancer pathways in triple negative breast cancer (TNBC). Analysis of cybrids and established breast cancer (BC) cell lines showed that metastatic TNBC maintains high levels of ATP through fatty acid β-oxidation (FAO) and activates Src oncoprotein through autophosphorylation at Y419. Manipulation of FAO including the knocking down of carnitine palmitoyltransferase-1 (CPT1) and 2 (CPT2), the rate-limiting proteins of FAO, and analysis of patient-derived xenograft models, confirmed the role of mitochondrial FAO in Src activation and metastasis. Analysis of TCGA and other independent BC clinical data further reaffirmed the role of mitochondrial FAO and CPT genes in Src regulation and their significance in BC metastasis.

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells. This decrease of oxygen tension can be due to a reduced supply in oxygen (causes include insufficient blood vessel network, defective blood vessel, and anemia) or to an increased consumption of oxygen relative to the supply (caused by a sudden higher cell proliferation rate). Hypoxia can be physiologic or pathologic such as in solid cancers [1][2][3] , rheumatoid arthritis, atherosclerosis etc… Each tissues and cells have a different ability to adapt to this new condition. During hypoxia, hypoxia inducible factor alpha (HIF) is stabilized and regulates various genes such as those involved in angiogenesis or transport of oxygen 4 . The stabilization of this protein is a hallmark of hypoxia, therefore detecting HIF is routinely used to screen for hypoxia [5][6][7] .In this article, we propose two simple methods to induce hypoxia in mammalian cell cultures and simple tests to evaluate the hypoxic status of these cells. Video LinkThe video . This product is soluble in water (100 mg/ml), yielding a clear, red solution.1. Prepare a 25mM stock solution in sterile dd water, (prepare immediately before use) 2. Use CoCl 2 at the final concentration of 100μM in your regular cell culture media to induce hypoxia. 3. Add the CoCl 2 containing media to your cells and incubate the cultures for 24hours in a conventional incubator (37°C; 5% C0 2 ).The above concentration works for the cell lines we have tested but each cell line should be tested at various concentrations (to establish a dose-dependent curve) as well as at various incubation times in order to limit drug related toxicity and optimize the assay. 3. Place the cell culture in the hypoxic chamber. Also place a Petri dish containing sterile water in the chamber to provide adequate humidification of the cultures. 4. Place the "twin" cell culture in normoxia as control. 5. Make sure your trays are secured and not moving, and then close the chamber with its lid. Correctly position the steel ring clamp to ensure the hermetical closure of the chamber and close it. 6. To create hypoxia, attach the tubing to a "hypoxia tank" containing a 1% O 2 gas mixture. If you have a flow meter connected to your tank your chamber will be directly connected to it (gas tank-flow meter -chamber). We use a flow meter incorporated in our regulator. 7. It is important to remove most if not all oxygen present in the chamber and in your media, to do so flush the chamber by opening the gas tank at a flow rate of 20 liters per minutes for 7-10 minutes; then quickly turn off the gas flow and completely close the chamber by closing both white clamps. 8. Return the chamber to a conventional incubator for the desired period of time. If you use large cultures, allow media in cultures to de-gas for 1-2 hours and then repeat flush. Hypoxia induced in Modular Incubator Chamber

Thoracic aortic dissection (TAD) is a highly lethal vascular disease. In many patients with TAD, the aorta progressively dilates and ultimately ruptures. Dissection formation, progression, and rupture cannot be reliably prevented pharmacologically because the molecular mechanisms of aortic wall degeneration are poorly understood. The key histopathologic feature of TAD is medial degeneration, a process characterized by smooth muscle cell depletion and extracellular matrix degradation. These structural changes have a profound impact on the functional properties of the aortic wall and can result from excessive protease-mediated destruction of the extracellular matrix, altered signaling pathways, and altered gene expression. Review of the literature reveals differences in the processes that lead to ascending versus descending and sporadic versus hereditary TAD. These differences add to the complexity of this disease. Although tremendous progress has been made in diagnosing and treating TAD, a better understanding of the molecular, cellular, and genetic mechanisms that cause this disease is necessary to developing more effective preventative and therapeutic treatment strategies.

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive molecules contain an oxygen and include H2O2 (hydrogen peroxide), NO (nitric oxide), O2 -(oxide anion), peroxynitrite (ONOO -), hydrochlorous acid (HOCl), and hydroxyl radical (OH -).Oxidative species are produced not only under pathological situations (cancers, ischemic/reperfusion, neurologic and cardiovascular pathologies, infectious diseases, inflammatory diseases 1 , autoimmune diseases 2 , etc…) but also during physiological (non-pathological) situations such as cellular metabolism 3, 4 . Indeed, ROS play important roles in many cellular signaling pathways (proliferation, cell activation 5, 6 , migration 7 etc..). ROS can be detrimental (it is then referred to as "oxidative and nitrosative stress") when produced in high amounts in the intracellular compartments and cells generally respond to ROS by upregulating antioxidants such as superoxide dismutase (SOD) and catalase (CAT), glutathione peroxidase (GPx) and glutathione (GSH) that protects them by converting dangerous free radicals to harmless molecules (i.e. water). Vitamins C and E have also been described as ROS scavengers (antioxidants).Free radicals are beneficial in low amounts 3 . Macrophage and neutrophils-mediated immune responses involve the production and release of NO, which inhibits viruses, pathogens and tumor proliferation 8 . NO also reacts with other ROS and thus, also has a role as a detoxifier (ROS scavenger). Finally NO acts on vessels to regulate blood flow which is important for the adaptation of muscle to prolonged exercise 9,10 . Several publications have also demonstrated that ROS are involved in insulin sensitivity 11,12 .Numerous methods to evaluate ROS production are available. In this article we propose several simple, fast, and affordable assays; these assays have been validated by many publications and are routinely used to detect ROS or its effects in mammalian cells. While some of these assays detect multiple ROS, others detect only a single ROS. Video LinkThe video component of this article can be found at

Rationale Aortic aneurysm and dissection (AAD) are major diseases of the adult aorta caused by progressive medial degeneration of the aortic wall. Although the overproduction of destructive factors promotes tissue damage and disease progression, the role of protective pathways is unknown. Objective In this study, we examined the role of AKT2 in protecting the aorta from developing AAD. Methods and Results AKT2 and phospho-AKT levels were significantly downregulated in human thoracic AAD tissues, especially within the degenerative medial layer. Akt2-deficient mice showed abnormal elastic fibers and reduced medial thickness in the aortic wall. When challenged with angiotensin II (AngII), these mice developed aortic aneurysm, dissection, and rupture with features similar to those in humans, in both thoracic and abdominal segments. Aortas from Akt2-deficient mice displayed profound tissue destruction, apoptotic cell death, and inflammatory cell infiltration that were not observed in aortas from wild-type mice. Additionally, AngII-infused Akt2-deficient mice showed significantly elevated expression of matrix metalloproteinase (MMP)-9 and reduced expression of tissue inhibitor of metalloproteinase (TIMP)-1. In cultured human aortic vascular smooth muscle cells, AKT2 inhibited the expression of MMP-9 and stimulated the expression of TIMP-1 by preventing the binding of transcription factor forkhead box protein O1 (FOXO1) to the MMP-9 and TIMP-1 promoters. Conclusions Impaired AKT2 signaling may contribute to increased susceptibility to the development of AAD. Our findings provide evidence of a mechanism that underlies the protective effects of AKT2 on the aortic wall and that may serve as a therapeutic target in the prevention of AAD.