Exploring Morin as an anti-quorum sensing agent (anti-QSA) against resistant strains of Staphylococcus aureus.

Staphylococcus aureus an opportunistic pathogen, causes biofilm-associated infections like nosocomial and chronic disease, where quorum sensing plays vital role. Biofilm-associated infections are provocative due to antibiotic resistance and become major public health threat to human. Accordingly, there is an urgent need to identify novel compounds and groundbreaking methods to pervade and clear biofilm-associated infections. It has been suggested that naturally occurring small molecules especially flavonoids could be used as a new source of anti-biofilm drugs. Thus, an attempt to evaluate the effectiveness of Morin against Staphylococcus aureus resistant strains, through in-vitro (antibacterial, anti-biofilm, microscopic analysis, anti-motility, anti-spreading, cell viability and EPS production assay) and in-silico studies (Docking and Molecular dynamic simulation). Interestingly, in vitro and microscopic analysis revealed Morin to have very less or negligible antibiotic activity against methicillin resistant and vancomycin resistant Staphylococcus aureus, however, Morin exhibited significant biofilm inhibition, reduced motility and spreading, and EPS production of both the resistant strains. Further, studies on the molecular interaction of Morin with global regulatory protein (SarA) of Staphylococcus aureus, suggested the effective role of Morin as an anti-quorum sensing agent and interpolating the further studies to suit it as drug candidate against the pathogenic bacteria.

Live Cell Adhesion, Migration, and Invasion Assays.

Cell migration is a fundamental procedure involved in many physiological processes such as embryological development, tissue formation, immune defense or inflammation, and cancer progression. Here, we provide four in vitro assays that describe step-by-step cell adhesion, migration and invasion strategies, and their corresponding image data quantification. These methods include the following: two-dimensional wound healing assays, two-dimensional individual cell-tracking experiments by live cell imaging, and three-dimensional spreading and transwell assays. These optimized assays will facilitate physiological and cellular characterization of cell adhesion and motility, which may be used for fast screening of specific therapeutic drugs for adhesion function, novel strategies in pathophysiological diagnosis, and assaying new molecules involved in migration and invasion metastatic properties of cancer cells.

Analysis of Replication Dynamics Using the Single-Molecule DNA Fiber Spreading Assay.

DNA replication is a fundamental process of life. Any perturbation of this process by endogenous or exogenous factors impacts on genomic stability and thereby on carcinogenesis. More recently, the replication machinery has been discovered as an interesting target for cancer therapeutic strategies. Given its high biological and clinical relevance, technologies for the analysis of DNA replication have attracted major attention. The so-called DNA fiber spreading technique is a powerful tool to directly monitor various aspects of the replication process by sequential incorporation of halogenated nucleotide analogs which later can be fluorescently stained and analyzed. This chapter outlines the use of the DNA fiber spreading technique for the analysis of replication dynamics and replication structures.

In vitro Cell Migration, Invasion, and Adhesion Assays: From Cell Imaging to Data Analysis.

Cell migration is a key procedure involved in many biological processes including embryological development, tissue formation, immune defense or inflammation, and cancer progression. How physical, chemical, and molecular aspects can affect cell motility is a challenge to understand migratory cells behavior. In vitro assays are excellent approaches to extrapolate to in vivo situations and study live cells behavior. Here we present four in vitro protocols that describe step-by-step cell migration, invasion and adhesion strategies and their corresponding image data quantification. These current protocols are based on two-dimensional wound healing assays (comparing traditional pipette tip-scratch assay vs. culture insert assay), 2D individual cell-tracking experiments by live cell imaging and three-dimensional spreading and transwell assays. All together, they cover different phenotypes and hallmarks of cell motility and adhesion, providing orthogonal information that can be used either individually or collectively in many different experimental setups. These optimized protocols will facilitate physiological and cellular characterization of these processes, which may be used for fast screening of specific therapeutic cancer drugs for migratory function, novel strategies in cancer diagnosis, and for assaying new molecules involved in adhesion and invasion metastatic properties of cancer cells.