Mitochondrial membrane potential (ΔΨm) is critical for maintaining the physiological function of the respiratory chain to generate ATP. A significant loss of ΔΨm renders cells depleted of energy with subsequent death. Reactive oxygen species (ROS) are important signaling molecules, but their accumulation in pathological conditions leads to oxidative stress. The two major sources of ROS in cells are environmental toxins and the process of oxidative phosphorylation. Mitochondrial dysfunction and oxidative stress have been implicated in the pathophysiology of many diseases; therefore, the ability to determine ΔΨm and ROS can provide important clues about the physiological status of the cell and the function of the mitochondria.Several fluorescent probes (Rhodamine 123, TMRM, TMRE, JC-1) can be used to determine Δψm in a variety of cell types, and many fluorescence indicators (Dihydroethidium, Dihydrorhodamine 123, H 2 DCF-DA) can be used to determine ROS. Nearly all of the available fluorescence probes used to assess ΔΨm or ROS are single-wavelength indicators, which increase or decrease their fluorescence intensity proportional to a stimulus that increases or decreases the levels of ΔΨm or ROS. Thus, it is imperative to measure the fluorescence intensity of these probes at the baseline level and after the application of a specific stimulus. This allows one to determine the percentage of change in fluorescence intensity between the baseline level and a stimulus. This change in fluorescence intensity reflects the change in relative levels of ΔΨm or ROS. In this video, we demonstrate how to apply the fluorescence indicator, TMRM, in rat cortical neurons to determine the percentage change in TMRM fluorescence intensity between the baseline level and after applying FCCP, a mitochondrial uncoupler. The lower levels of TMRM fluorescence resulting from FCCP treatment reflect the depolarization of mitochondrial membrane potential. We also show how to apply the fluorescence probe H 2 DCF-DA to assess the level of ROS in cortical neurons, first at baseline and then after application of H 2 O 2 . This protocol (with minor modifications) can be also used to determine changes in ∆Ψm and ROS in different cell types and in neurons isolated from other brain regions. Video LinkThe video component of this article can be found at https://www.jove.com/video/2704/ Protocol 1. Cell culture 1. Cortical neurons are isolated and grown using previously described techniques and plated on culture dishes with a glass bottom (MatTek Corporation, Ashland, MA ) coated with poly-D-lysine and laminin 1 . Preparing the stock solutions for the fluorescent probes TMRM and H 2 DCF-DA1. Prepare a 10-mM stock solution of TMRM by dissolving 5.0 mg of TMRM in 1 ml of anhydrous dimethylsulfoxide. Vortex it for 1 min. Then, make aliquots and store them at -20°C, protect from light, and use within one month. 2. Next, prepare a 10-mM stock solution of H 2 DCF-DA by dissolving 4.87 mg of H 2 DCF-DA in 1 ml of anhydrous DMSO. Similarly, vortex it for 1 m...
Epithelial-to-mesenchymal transition (EMT) plays an important role in prostate cancer (PCa) metastasis. The transcription/translation regulatory Y-box binding protein-1 (YB-1) is known to be associated with cancer metastasis. We observed that YB-1 expression increased with tumor grade and showed an inverse relationship with E-cadherin in a human PCa tissue array. Forced YB-1 expression induced a mesenchymal morphology that was associated with down regulation of epithelial markers. Silencing of YB-1 reversed mesenchymal features and decreased cell proliferation, migration and invasion in PCa cells. YB-1 is activated directly via Akt mediated phosphorylation at Ser102 within the cold shock domain (CSD). We next identified fisetin as an inhibitor of YB-1 activation. Computational docking and molecular dynamics suggested that fisetin binds on the residues from β1 - β4 strands of CSD, hindering Akt's interaction with YB-1. Calculated free binding energy ranged from −11.9845 to −9.6273 kcal/mol. Plasmon Surface Resonance studies showed that fisetin binds to YB-1 with an affinity of approximately 35 μM, with both slow association and dissociation. Fisetin also inhibited EGF induced YB-1 phosphorylation and markers of EMT both in vitro and in vivo. Collectively our data suggest that YB-1 induces EMT in PCa and identify fisetin as an inhibitor of its activation.
Grain amaranth is an underutilized crop with high nutritional quality from the Americas. Emerging genomic and biotechnological tools are becoming available that allow the integration of novel breeding techniques for rapid improvement of amaranth and other underutilized crops. Out of thousands of edible plants, only three cereals-maize, wheat and rice-are the major food sources for a majority of people worldwide. While these crops provide high amounts of calories, they are low in protein and other essential nutrients. The dependence on only few crops, with often narrow genetic basis, leads to a high vulnerability of modern cropping systems to the predicted climate change and accompanying weather extremes. Broadening our food sources through the integration of so-called orphan crops can help to mitigate the effects of environmental change and improve qualitative food security. Thousands of traditional crops are known, but have received little attention in the last century and breeding efforts were limited. Amaranth is such an underutilized pseudocereal that is of particular interest because of its balanced amino acid and micronutrient profiles. Additionally, the C photosynthetic pathway and ability to withstand environmental stress make the crop a suitable choice for future agricultural systems. Despite the potential of amaranth, efforts of genetic improvement lag considerably behind those of major crops. The progress in novel breeding methods and molecular techniques developed in model plants and major crops allow a rapid improvement of underutilized crops. Here, we review the history of amaranth and recent advances in genomic tools and give a concrete perspective how novel breeding techniques can be implemented into breeding programs. Our perspectives are transferable to many underutilized crops. The implementation of these could improve the nutritional quality and climate resilience of future cropping systems.
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