The level of human natural antibodies of immunoglobulin M isotype against Le^C in patients with breast cancer is lower than in healthy women. The epitope specificity of these antibodies has been characterized using a printed glycan array and enzyme-linked immunosorbent assay (ELISA), the antibodies being isolated from donors’ blood using Le^C-Sepharose (Le^C is Galβ1-3GlcNAcβ). The isolated antibodies recognize the disaccharide but do not bind to glycans terminated with Le^C, which implies the impossibility of binding to regular glycoproteins of non-malignant cells. The avidity (as dissociation constant value) of antibodies probed with a multivalent disaccharide is 10⁻⁹ M; the nanomolar level indicates that the concentration is sufficient for physiological binding to the cognate antigen. Testing of several breast cancer cell lines showed the strongest binding to ZR 75-1. Interestingly, only 7% of the cells were positive in a monolayer with a low density, increasing up to 96% at highest density. The enhanced interaction (instead of the expected inhibition) of antibodies with ZR 75-1 cells in the presence of Galβ1-3GlcNAcβ disaccharide, indicates that the target epitope of anti-Le^C antibodies is a molecular pattern with a carbohydrate constituent rather than a glycan.

Ribosome biogenesis is essential for the functioning of living cells. In higher eukaryotes, this multistep process is tightly controlled and involves a variety of specialized proteins and RNAs. This pool of so-called ribosome biogenesis factors includes diverse proteins with enzymatic and structural functions. Some of them have homologs in yeast S. cerevisiae, and their function can be inferred from the structural and biochemical data obtained for the yeast counterparts. The functions of human proteins RPF1 and ESF1 remain largely unclear, although RPF1 has been recently shown to participate in 60S biogenesis. Both proteins have drawn our attention since they contribute to the early stages of ribosome biogenesis, which are far less studied than the later stages. In this study, we employed the loss-of-function shRNA/siRNA-based approach to the human cell line HEK293 to determine the role of RPF1 and ESF1 in ribosome biogenesis. Downregulating RPF1 and ESF1 significantly changed the pattern of RNA products derived from 47S pre-rRNA. Our findings demonstrate that RPF1 and ESF1 are associated with different pre-ribosomal particles, pre-60S, and pre-40S particles, respectively. Our results allow for speculation about the particular steps of pre-rRNA processing, which highly rely on the RPF1 and ESF1 functions. We suggest that both factors are not directly involved in pre-rRNA cleavage but rather help pre-rRNA to acquire the conformation favoring its cleavage.

The rise in antimicrobial resistance represents a significant challenge to global health. The reason partially lies in an inappropriate use of conventional antibiotics and the subsequent rapid spread of multidrug-resistant pathogen strains. This emergency requires an urgent search for conceptually new antimicrobial agents. A viable alternative to conventional antibiotics is antimicrobial peptides (AMPs), which are ribosomally synthesized molecules with considerable potential as next-generation anti-infectious therapeutics. Previously, we have reported on the β-hairpin peptide Ap9, an analog of abarenicin from the marine polychaeta Abarenicola pacifica, with potent activity against key Gram-negative pathogens. Here, it is shown that Ap9 acts in a manner resembling polymyxin B, namely via interaction with lipopolysaccharide (LPS), and retains its activity against polymyxin-resistant isolates without observed cross-resistance, and causes insignificant damage in cytoplasmic membrane at bactericidal concentrations. NMR spectroscopy reveals that LPS binding induces a conformational rearrangement of Ap9, its dimer formation, and local structural remodeling of the peptide region (residues 8–12) into 3₁₀-helix. Bacterial resistance to Ap9 was found to be relatively low with a reduced susceptibility associated with infrequent genetic alterations, such as the mutation in lptD or the deletion in mlaA. Furthermore, Ap9 demonstrates a favorable tolerability, a wider therapeutic window than that of polymyxin B, and a sufficiently long half-life through the systemic use, as well as in vivo efficacy in murine models of Gram-negative infections, including sepsis caused by the mcr-1-harboring Escherichia coli strain. The obtained results point to Ap9 as a promising candidate for further preclinical studies aimed at development of an alternative to polymyxins.