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  • br Conclusions In Summary a multilayer screening


    Conclusions In Summary, a multilayer screening of xanthones of natural origin (Xanthone-NPs) was undertaken against Pf-DHFR. The Re-rank score of screened compounds was size normalized and interaction profile was generated using experimentally known inhibitors. Three compounds X5, X113A and X164B have contact footprints similar to known inhibitors included in the study. Based on common features of three compounds, good docking score and interaction profile to experimentally known inhibitors, 11 purchasable compounds were screened from ZINC database. We hope that these findings would be helpful in providing deeper insight to DHFR binding and in designing new and more potent inhibitors against malaria.
    Conflict of interest
    Introduction For a better understanding the functions and regulations of various DNA sequences, and for gene therapy, introduction of DNA into mammalian CHIR-090 become a powerful tool. Until now, many biological and nonbiological techniques have been developed to introduce DNA into cells for optimal expression of exogenous genes in targeted mammalian cells. Viral vectors have been used most extensively as a biological techniques for gene introduction. The use of nonbiological methods over biological methods is currently increasing due to their much lower or lack of pathogenic risk [1], [2], [3]. The nonbiological techniques can be divided into chemical and physical methods. When compared with biological and chemical methods, the major advantages of physical methods are their high transfection efficiency for multiple cell types, easy to standardize the process, less limit on gene size, and useful for ex vivo application [4]. There are many physical methods to use today, for example, microinjection, conventional needle injection, high-pressure needle injection (hydrodynamics), particle-bombardment (“gene gun”), electroporation, ultrasound and encapsulated microspheres [4]. Among these physical methods, electroporation is the most versatile method of DNA transfection because it has been shown to work for a wide variety of both animal and plant cells [5], [6]. Electroporation exploits the fact that high-voltage electrical fields can cause transient pore formation in the cell membrane [7], [8], [9], [10]. However, efficiency of electroporation was affected by a lot of factors, including the concentration of DNA, DNA/cell ratio, media for electroporation and recovery, voltage and capacitance, and salt concentration [10], [11], [12]. Moreover, electroporation conditions are host cell line dependent [13] and therefore optimal electroporation conditions must be established for each host cell line. DHFR-deficient Chinese hamster ovary (CHO DHFR−) cells are one of the most popular mammalian expression system for inducible amplification of transgene. Target gene is generally subcloned into vectors containing the dihydrofolate reductase (DHFR) gene which serves as the selective marker gene in CHO DHFR− cells, and amplification of stably integrated transgene is induced by stepwise treatment with methotrexate [14], [15]. There are a lot of different protocols for transferring exogenous genes into CHO DHFR− cells by electroporation, but systematical comparison of their transfection efficiency is lacking. In order to obtain larger number of stably transfected cell clones, we systematically evaluated different electroporation conditions to get higher transfection efficiency using plasmid DNA pSV-β-Gal containing the β-galactosidase gene as a reporter gene.
    Materials and methods
    Results Electroporation, the formation of holes or pores in the cell membrane by high-voltage electric shock, has been reported to be more efficient than the conventional calcium phosphate, DEAE-dextran methods when exogenous DNA was introduced into cells [6], [11]. In order to optimize the process of electroporation for introducing exogenous DNA into CHO DHFR− cells, the following parameters were examined: voltage, capacitance and pulse duration, number of pulse, and different types of electroporation buffers.