Marcin Serocki

The cellular adaptive response to hypoxia relies on the expression of hypoxia‐inducible factors (HIFs), HIF‐1 and HIF‐2. HIFs regulate global gene expression changes during hypoxia that are necessary for restoring oxygen homeostasis and promoting cell survival. In the early stages of hypoxia, HIF‐1 is elevated, whereas at the later stages, HIF‐2 becomes the predominant form. What governs the transition between the two HIFs (the HIF switch) and the role of miRNAs in this regulation are not completely clear. Genome‐wide expression studies on the miRNA content of RNA‐induced silencing complexes (RISC) in HUVECs exposed to hypoxia compared to the global miRNA‐Seq analysis revealed very specific differences between these two populations. We analyzed the miRNA and mRNA composition of RISC at 2 h (mainly HIF‐1 driven), 8 h (HIF‐1 and HIF‐2 elevated), and 16 h (mainly HIF‐2 driven) in a gene ontology context. This allowed for determining the direct impact of the miRNAs in modulating the cellular signaling pathways involved in the hypoxic adaptive response. Our results indicate that the miRNA‐mRNA RISC components control the adaptive responses, and this does not always rely on the miRNA transcriptional elevations during hypoxia. Furthermore, we demonstrate that the hypoxic levels of the vast majority of HIF‐1‐dependent miRNAs (including miR‐210‐3p) are also HIF‐2 dependent and that HIF‐2 governs the expression of 11 specific miRNAs. In summary, the switch from HIF‐1 to HIF‐2 during hypoxia provides an important level of miRNA‐driven control in the adaptive pathways in endothelial cells.

Seven conjugates composed of well-known fluoroquinolone antibacterial agents, ciprofloxacin (CIP) or levofloxacin (LVX), and a cell-penetrating peptide transportan 10 (TP10-NH₂) were synthesised. The drugs were covalently bound to the peptide via an amide bond, methylenecarbonyl moiety, or a disulfide bridge. Conjugation of fluoroquinolones to TP10-NH₂ resulted in congeners demonstrating antifungal in vitro activity against human pathogenic yeasts of the Candida genus (MICs in the 6.25–100 µM range), whereas the components were poorly active. The antibacterial in vitro activity of most of the conjugates was lower than the activity of CIP or LVX, but the antibacterial effect of CIP-S-S-TP10-NH₂ was similar to the mother fluoroquinolone. Additionally, for two representative CIP and LVX conjugates, a rapid bactericidal effect was shown. Compared to fluoroquinolones, TP10-NH₂ and the majority of its conjugates generated a relatively low level of reactive oxygen species (ROS) in human embryonic kidney cells (HEK293) and human myeloid leukemia cells (HL-60). The conjugates exhibited cytotoxicity against three cell lines, HEK293, HepG2 (human liver cancer cell line), and LLC-PK1 (old male pig kidney cells), with IC₅₀ values in the 10–100 µM range and hemolytic activity. The mammalian toxicity was due to the intrinsic cytoplasmic membrane disruption activity of TP10-NH₂ since fluoroquinolones themselves were not cytotoxic. Nevertheless, the selectivity index values of the conjugates, both for the bacteria and human pathogenic yeasts, remained favourable.