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  1. The social amoeba Dictyostelium discoideum is a commonly used eukaryotic model organism for the study of cell division, chemotaxis, differentiation, phagocytosis, and other cellular processes. Electroporation is an effective and efficient method for delivering plasmid DNA into D. discoideum, an invaluable tool for studying intracellular processes. The technology is readily available but often prohibitively expensive. Although several custom-built electroporation devices have been developed, none deliver the specific 8.5kV/cm exponentially decaying waveform required for D. discoideum transformation. The present study examined whether a simple, inexpensive device can be built to produce this waveform through a simple resistor-capacitor (RC) circuit. A pulse generator RC circuit was built incorporating inexpensive electronic components and a 3D printed cuvette chamber. All four possible combinations of custom-built and commercial pulse generators and custom-built and commercial cuvette chambers were used to transform D. discoideum cells with a plasmid encoding green fluorescent protein (GFP). There were no significant differences in the number of surviving cells immediately following or 24 hours post-transformation between the systems. All combinations of custom-built and commercial systems achieved comparably high transformation efficiency shown by percent of cells expressing GFP six days after the transformation. Since the waveform-specific electroporation system we present here can be built by non-experts with easily obtainable materials and 3D printing, we envision this device to benefit investigators in areas with low research budgets and educators in multiple STEM fields. 
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  2. Abstract

    Understanding the molecular evolution of the SARS‐CoV‐2 virus as it continues to spread in communities around the globe is important for mitigation and future pandemic preparedness. Three‐dimensional structures of SARS‐CoV‐2 proteins and those of other coronavirusess archived in the Protein Data Bank were used to analyze viral proteome evolution during the first 6 months of the COVID‐19 pandemic. Analyses of spatial locations, chemical properties, and structural and energetic impacts of the observed amino acid changes in >48 000 viral isolates revealed how each one of 29 viral proteins have undergone amino acid changes. Catalytic residues in active sites and binding residues in protein–protein interfaces showed modest, but significant, numbers of substitutions, highlighting the mutational robustness of the viral proteome. Energetics calculations showed that the impact of substitutions on the thermodynamic stability of the proteome follows a universal bi‐Gaussian distribution. Detailed results are presented for potential drug discovery targets and the four structural proteins that comprise the virion, highlighting substitutions with the potential to impact protein structure, enzyme activity, and protein–protein and protein–nucleic acid interfaces. Characterizing the evolution of the virus in three dimensions provides testable insights into viral protein function and should aid in structure‐based drug discovery efforts as well as the prospective identification of amino acid substitutions with potential for drug resistance.

     
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