ABSTRACT
The innovative design of a triarylborane (TB)-dye with one NMe2-alkylated (propargylated) group and one NMe2 group yielded a system that is both an NMe2 π-donor and an inductive NMe2-alkyl cationic acceptor. Consequently, the new TB-dye was highly sensitive to a "click" reaction with an azide-substituted lysine side chain (yielding TB-lysine), resulting in a bathochromic shift of emission of 100 nm. In addition, fluorene attached to the lysine C-terminus showed FRET with the TB-chromophore, also sensitive to interactions with targets. Both the TB-dye and TB-lysine showed high affinities towards both DNA and proteins, reporting binding by an opposite fluorimetric response for DNA/RNA (quenching) vs. BSA (increase). Thus, the novel TB-dye is an ideal fluorimetric probe for orthogonal incorporation into bio-targets by "click" reactions due to fluorescence reporting of the progress of the "click" reaction and further sensing of the binding site composition. The TB-dye is moderately toxic to human cell lines after 2-3 days of exposure, but efficiently enters cells in 90 min, being non-toxic at short exposure. The most important product of the "click" reaction, TB-lysine, was non-toxic to cells and showed equal distribution between mitochondria and lysosomes. Further studies would focus particularly on the very convenient monitoring of the progress of "click" conjugation of the TB-dye with biorelevant targets inside living cells by confocal microscopy.
ABSTRACT
The low seroprevalent human adenovirus type 26 (HAdV26)-based vaccine vector was the first adenovirus-based vector to receive marketing authorization from European Commission. HAdV26-based vaccine vectors induce durable humoral and cellular immune responses and, as such, represent a highly valuable tool for fighting infectious diseases. Despite well-described immunogenicity in vivo, the basic biology of HAdV26 still needs some refinement. The aim of this study was to determine the pro-inflammatory cytokine profile of epithelial cells infected with HAdV26 and then investigate the underlying molecular mechanism. The expression of studied genes and proteins was assessed by quantitative polymerase chain reaction, western blot, and enzyme-linked immunosorbent assay. Confocal microscopy was used to visualize HAdV26 cell uptake. We found that HAdV26 infection in human epithelial cells triggers the expression of pro-inflammatory cytokines and chemokines, namely IL-6, IL-8, IL-1ß, and TNF-α, with the most pronounced difference shown for IL-6. We investigated the underlying molecular mechanism and observed that HAdV26-induced IL-6 gene expression is αvß3 integrin dependent and NF-κB mediated. Our findings provide new data regarding pro-inflammatory cytokine and chemokine expression in HAdV26-infected epithelial cells, as well as details concerning HAdV26-induced host signaling pathways. Information obtained within this research increases our current knowledge of HAdV26 basic biology and, as such, can contribute to further development of HAdV26-based vaccine vectors.
