The insides of cells can be viewed as a treasure trove of targets for therapeutic intervention of diseases or as deposits for contrasting agents. Increasingly the molecules that need to be delivered to the inside of cells for these purposes are macromolecular and membrane impermeable. Cell penetrating peptides (CPPs) have proven abilities to deliver a range of macromolecular cargo into cells thus raising their profile as potential delivery vectors for wide-ranging applications. There is evidence to suggest that CPPs first enter cells through endocytosis and that cytosolic delivery is mediated across endolysosomal membranes. Their capacity to do this, over direct plasma membrane translocation, is likely to depend on the nature and size of the cargo. Cells use a range of endocytic routes to facilitate entry from well characterised pathways regulated by clathrin to more recently discovered and less characterised pathways regulated by clathrin independent mechanisms. These are likely to determine the intracellular fate of cell delivery vectors including those based on cell penetrating peptides. Thus gaining accurate knowledge of their endocytic uptake and traffic is an important characterisation criteria for progress in this field. This review describes the different endocytic pathways that have been identified in mammalian cells and specific reports that have studied the uptake mechanisms and endocytic traffic of cell penetrating peptides and their associated cargo. These cargoes range from short peptides to an increasing library of nanoparticles such as quantum dots, liposomes and polymeric dendrimers. The studies highlight the effectiveness of cell penetrating peptides for delivering these entities into a diverse array of cell types using different endocytic pathways. This is shown using microscopy based colocalisation analysis with the few specific endocytic probes available, and chemical inhibitors of endocytosis that suffer from lack of specificity. Overall, more specific probes, inhibitors and novel technologies are required for accurate characterisation of cellular dynamics of cell penetrating peptide conjugates thus allowing them to reach their full potential as vectors for therapeutics and other payloads.

Telomere erosion, dysfunction, and fusion can lead to a state of cellular crisis characterized by large-scale genome instability. We investigated the impact of a telomere-driven crisis on the structural integrity of the genome by undertaking whole-genome sequence analyses of clonal populations of cells that had escaped crisis. Quantification of large-scale structural variants revealed patterns of rearrangement consistent with chromothripsis but formed in the absence of functional nonhomologous end-joining pathways. Rearrangements frequently consisted of short fragments with complex mutational patterns, with a repair topology that deviated from randomness showing preferential repair to local regions or exchange between specific loci. We find evidence of telomere involvement with an enrichment of fold-back inversions demarcating clusters of rearrangements. Our data suggest that chromothriptic rearrangements caused by a telomere crisis arise via a replicative repair process involving template switching.

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Telomeres are progressively eroded during repeated rounds of cell division due to the end replication problem but also undergo additional more substantial stochastic shortening events. In most cases, shortened telomeres induce a cell-cycle arrest or trigger apoptosis, although for those cells that bypass such signals during tumour progression, a critical length threshold is reached at which telomere dysfunction may ensue. Dysfunction of the telomere nucleoprotein complex can expose free chromosome ends to the DNA double-strand break (DSB) repair machinery, leading to telomere fusion with both telomeric and non-telomeric loci. The consequences of telomere fusions in promoting genome instability have long been appreciated through the breakage–fusion–bridge (BFB) cycle mechanism, although recent studies using high-throughput sequencing technologies have uncovered evidence of involvement in a wider spectrum of genomic rearrangements including chromothripsis. A critical step in cancer progression is the transition of a clone to immortality, through the stabilisation of the telomere repeat array. This can be achieved via the reactivation of telomerase, or the induction of the alternative lengthening of telomeres (ALT) pathway. Whilst telomere dysfunction may promote genome instability and tumour progression, by limiting the replicative potential of a cell and enforcing senescence, telomere shortening can act as a tumour suppressor mechanism. However, the burden of senescent cells has also been implicated as a driver of ageing and age-related pathology, and in the promotion of cancer through inflammatory signalling. Considering the critical role of telomere length in governing cancer biology, we review questions related to the prognostic value of studying the dynamics of telomere shortening and fusion, and discuss mechanisms and consequences of telomere-induced genome rearrangements.

Structural variation (SV) plays a fundamental role in genome evolution and can underlie inherited or acquired diseases such as cancer. Long-read sequencing technologies have led to improvements in the characterization of structural variants (SVs), although paired-end sequencing offers better scalability. Here, we present dysgu, which calls SVs or indels using paired-end or long reads. Dysgu detects signals from alignment gaps, discordant and supplementary mappings, and generates consensus contigs, before classifying events using machine learning. Additional SVs are identified by remapping of anomalous sequences. Dysgu outperforms existing state-of-the-art tools using paired-end or long-reads, offering high sensitivity and precision whilst being among the fastest tools to run. We find that combining low coverage paired-end and long-reads is competitive in terms of performance with long-reads at higher coverage values.