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  TOP SECRET PATRIOT

Project: NIMBUS ACUMEN SCIENCE TECHNOLOGIES SEPTEMBER 2562 – FEBRUARY 2562

Description: Enhancement of BRANDY reprogramming through protein transduction. The sequences that comprised the B3 cocktail – EDGE, MIRAGE, and PHANTOM, were extensively characterized and shown to be protein-encoding genes. The proteins were thoroughly investigated, and shown to have DNA-binding activity and a lack of activity or affinity for other cellular materials. The proteins were re-named EDGE, MIRAGE, and PHANTOM, whereas their encoding genes were re-named Edge, Mirage, and Phantom, in adherence with standard genetics protocols.

A human genome-wide screen was conducted to investigate binding sites for the B3 transcription factors, and NMR, X-ray crystallography, and amino acid sequencing integrated with computational analysis revealed extensively complex structures for the transcription factors, with numerous possible DNA-binding domains, and that the specificity of individual domain binding appeared to be regulated by the tertiary structure of the factors, where nearby domains all integrated together to effect the specific DNA-binding capabilities of each individual domain. This hierarchy of organization, previously unrecognized in any known species categorized, suggests that the EDGE, MIRAGE, and PHANTOM proteins are evolutionarily conserved and have an integral role in Flood physiology. Genome-wide screening of B3 DNA binding partners was inconclusive, although weak statistical correlation was observed with a number of known proto-oncogenes and pluripotency ESC-expressed genes, many DNA-binding domains on the B3 factors appeared inert. However, hybridization with purified protein with the LIBRARY revealed numerous binding partners, suggesting perhaps that the B3 factors serve as crucial regulators of Flood cellular processes, possibly including phenotype, self-renewal, differentiation, and de-differentiation.

Co-transduction of the BRANDY family lentiviral cocktail and the translated B3 factor products enhanced reprogramming efficiency >100,000-fold and further increased temporal kinetics to the minute-range. This suggests that the actual protein products of the B3 factors may be essential in upregulating infectious transmission and kinetics of infection during Flood parasitization.

In vitro translation of all BRANDY and SWIFT family sequences revealed that ~63% of BRANDY / SWIFT sequences are DNA-binding, perhaps indicative of transcription factor activity, while ~11% are RNA-binding, perhaps indicative of miRNA regulatory activity. Protein products EDGE and MIRAGE showed high affinity for all BRANDY sequences during LIBRARY hybridization while PHANTOM showed decreased DNA-binding affinity. This suggests that EDGE and MIRAGE are core proteins while PHANTOM is an accessory binding protein. Internal hybridization of B3 proteins revealed that EDGE, MIRAGE, and PHANTOM are all interactants with each other, and freeze-NMR revealed highly complex integral structures when purified B3 proteins are hybridized to LIBRARY, but not human, DNA. This suggests a complex synergistic capability of the three proteins that regulates their transcriptional regulatory function. A subset of sixteen (16) BRANDY sequences were shown to have especially high affinity for the highest-order EDGE/MIRAGE/PHANTOM transcriptional complex, and these sixteen sequences were code-named GAMBLE. Individual assays of the individual GAMBLE sequences revealed that they all encode respective protein products, most of which possess kinase, phosphatase, polymerase, or ionophore activities.

Co-transduction of EDGE, MIRAGE, and PHANTOM proteins along with the GAMBLE sequences resulted in a high infection efficiency amongst cultured cell lines (>93% transduction efficiency) and a novel plastic phenotype in the cultured cells, with varying stages of de-differentiation but not reorganization, similar to the early phases of BRANDY reprogramming. Sequential co-transduction of the B3 proteins with the GAMBLE sequence followed by lentiviral transduction of the remaining BRANDY library demonstrated even higher percentage of resultant phenotype than with B3 protein transduction alone, with efficiency increasing >10,000-fold compared to protein transduction alone, and also end-stage reprogramming phenotype for all cell lines, not such de-differentiation. The secondary lentiviral transduction including GAMBLE sequences did not improve reprogramming efficiency.