Structural complexity of glycans derived from the diversities in composition, linage, configuration, and branching considerably complicates structural analysis. Nanopore-based single-molecule sensing offers the potential to elucidate glycan structure and even sequence glycan. However, the small molecular size and low charge density of glycans have restricted direct nanopore detection of glycan. Here we show that glycan sensing can be achieved using a wild-type aerolysin nanopore by introducing a facile glycan derivatization strategy. The glycan molecule can induce impressive current blockages when moving through the nanopore after being connected with an aromatic group-containing tag (plus a carrier group for the neutral glycan). The obtained nanopore data permit the identification of glycan regio- and stereoisomers, glycans with variable monosaccharide numbers, and distinct branched glycans, either independently or with the use of machine learning methods. The presented nanopore sensing strategy for glycans paves the way towards nanopore glycan profiling and potentially sequencing.

The programmed cell death (PDCD) family plays a significant role in the regulation of cell survival and apoptotic cell death. However, the evolution, distribution and role of the PDCD family in lampreys have not been revealed. Thus, we identified the PDCD gene family in the lamprey genome and classified the genes into five subfamilies based on orthologs of the genes, conserved synteny, functional domains, phylogenetic tree, and conserved motifs. The distribution of the lamprey PDCD family and the immune response of the PDCD family in lampreys stimulated by different pathogens were also demonstrated. In addition, we investigated the molecular function of lamprey PDCD2, PDCD5, and PDCD10. Our studies showed that the recombinant lamprey PDCD5 protein and transfection of the L-PDCD5 gene induced cell apoptosis, upregulated the expression of the associated X protein (BAX) and TP53 and downregulated the expression of B cell lymphoma 2 (BCL-2) independent of Caspase 3. In contrast, lamprey PDCD10 suppressed apoptosis in response to cis-diaminedichloro-platinum (II) stimuli. Our phylogenetic and functional data not only provide a better understanding of the evolution of lamprey PDCD genes but also reveal the conservation of PDCD genes in apoptosis. Overall, our results provide a novel perspective on lamprey immune regulation mediated by the PDCD family.

In previous research, we found that cell secretion from the adult lamprey supraneural body tissues possesses cytocidal activity against tumor cells, but the protein with cytocidal activity was unidentified. A novel lamprey immune protein (LIP) as defense molecule was first purified and identified in jawless vertebrates (cyclostomes) using hydroxyapatite column and Q Sepharose Fast Flow column. After LIP stimulation, morphological changes of tumor cells were analysed and measured whether in vivo or in vitro. LIP induces remarkable morphological changes in tumor cells, including cell blebbing, cytoskeletal alterations, mitochondrial fragmentation and endoplasmic reticulum vacuolation, and most of the cytoplasmic and organelle proteins are released following treatment with LIP. LIP evokes an elevation of intracellular calcium and inflammatory molecule levels. Our analysis of the cytotoxic mechanism suggests that LIP can upregulate the expression of caspase 1, RIPK1, RIP3 to trigger pyroptosis and necroptosis. To examine the effect of LIP in vivo, tumor xenograft experiments were performed, and the results indicated that LIP inhibits tumor growth without damage to mice. In addition, the cytotoxic action of LIP depended on the phosphatidylserine (PS) content of the cell membrane. These observations suggest that LIP plays a crucial role in tumor cell survival and growth. The findings will also help to elucidate the mechanisms of host defense in lamprey.