RESUMO
A live cell is a complex, yet extremely important container. Understanding the dynamics in a selected intracellular component is a challenging task. We have recently made significant progress in this direction using a confocal microscope as a tool. The smallest size of the focused spot in a confocal microscope is â¼0.2 µm (200 nm). This is nearly one hundred times smaller than the size of a live cell. Thus, one can selectively study different intracellular components/organelles in a live cell. In this paper, we discuss how one can image different intracellular components/organelles, record fluorescence spectra and decay at different locations, ascertain local polarity and viscosity, and monitor the dynamics of solvation, proton transfer, red-ox and other phenomena at specified locations/organelles inside a cell. We will highlight how this knowledge enriched us in differentiating between cancer and non-cancer cells, 3D tumor spheroids and towards drug delivery.
Assuntos
Microscopia Confocal , Organelas/química , Peptídeos beta-Amiloides/química , Linhagem Celular , Cumarínicos/química , Corantes Fluorescentes/química , Humanos , Organelas/patologia , Prótons , Esferoides Celulares/química , Esferoides Celulares/patologia , ViscosidadeRESUMO
The transdifferentiation of human mesenchymal stem cells (hMSC) to functional neurons is crucial for the development of future neuro-regenerative therapeutics. Currently, transdifferentiation of hMSCs to neurons requires a "chemical cocktail" along with neural growth factors. The role of the individual molecules present in a "chemical cocktail" is poorly understood and may cause unwanted toxicity or adverse effects. Toward, this goal, we have showcased the discovery of an imidazole-based "single-molecule" transdifferentiation initiator SG-145C. This discovery was achieved via screening of a small molecule library through extensive in silico studies to shortlist the best-fitting molecules. This discovery evolved through a careful selection to target Glycogen synthase kinase-3ß (GSK-3ß), which is one of the important proteins responsible for neurogenesis. Rigorous computational experiments, as well as extensive biological assays, confirmed that SG-145C has significant potential to transdifferentiate hMSCs to neurons. Interestingly, our results suggest that SG-145C can inhibit the proteasomal degradation of phosphorylated ß-catenin, in turn promoting transdifferentiation of hMSCs into neurons via the Wnt pathway.
RESUMO
Alzheimer's disease (AD) exhibits a multitude of syndromes which add up to its complex nature. In AD, amyloid plaques are deposited along with abnormal accumulation of transition-metal ions. These transition-metal ions are redox-active and help to induce the formation of various polymorphic forms of amyloid-ß. Amyloid oligomeric and fibrillar aggregates are the main cause for neuronal toxicity. Another reason for neuronal toxicity arises from generation of reactive oxygen species (ROS) catalyzed by redox-active metal ions through Fenton's reaction. In this direction, an Aß inhibitor possessing the metal chelation property will be the most promising approach against multifaceted AD. Herein, a rhodamine-B-based compound (Rh-BT) has been designed and synthesized. Rhodamine was attached with benzothiazole as a recognition unit for amyloid-ß aggregates. The molecule can effectively capture redox metal ions from the Aß-Cu2+ complex as well as inhibit Aß self-assembly such as toxic oligomeric and fibrillar aggregates. Various biophysical assays show that Rh-BT interacts with the Aß peptide, is capable of decreasing metal-induced ROS generation, and inhibits Aß-Cu2+-induced cytotoxicity. All these results support the multifunctional nature of Rh-BT, which has an Aß-specific recognition unit. In addition to the above properties, Rh-BT also exhibits good serum stability in vivo and blood-brain barrier permeability. Therefore, Rh-BT can be considered as a potent multifunctional therapeutic for the treatment of AD.