EJP18 peptide derived from the juxtamembrane domain of epidermal growth factor receptor represents a novel membrane-active cell-penetrating peptide.

Membrane-active peptides have been extensively studied to probe protein-membrane interactions, to act as antimicrobial agents and cell-penetrating peptides (CPPs) for the delivery of therapeutic agents to cells. Hundreds of membrane-active sequences acting as CPPs have now been described including bioportides that serve as single entity modifiers of cell physiology at the intracellular level. Translation of promising CPPs in pre-clinical studies have, however, been disappointing as only few identified delivery systems have progressed to clinical trials. To search for novel membrane-active peptides a sequence from the EGFR juxtamembrane region was identified (named EJP18), synthesised, and examined in its L- and D-form for its ability to mediate the delivery of a small fluorophore and whole proteins to cancer cell lines. Initial studies identified the peptide as being highly membrane-active causing extensive and rapid plasma membrane reorganisation, blebbing, and toxicity. At lower, non-toxic concentrations the peptides outperformed the well-characterised CPP octaarginine in cellular delivery capacity for a fluorophore or proteins that were associated with the peptide covalently or via ionic interactions. EJP18 thus represents a novel membrane-active peptide that may be used as a naturally derived model for biophysical protein-membrane interactions or for delivery of cargo into cells for therapeutic or diagnostic applications.

Contrasting roles for actin in the cellular uptake of cell penetrating peptide conjugates.

The increased need for macromolecular therapeutics, such as peptides, proteins and nucleotides, to reach intracellular targets necessitates more effective delivery vectors and a higher level of understanding of their mechanism of action. Cell penetrating peptides (CPPs) can transport a range of macromolecules into cells, either through direct plasma membrane translocation or endocytosis. All known endocytic pathways involve cell-cortex remodelling, a process shown to be regulated by reorganisation of the actin cytoskeleton. Here using flow cytometry, confocal microscopy and a variety of actin inhibitors we identify how actin disorganisation in different cell types differentially influences the cellular entry of three probes: the CPP octaarginine - Alexa488 conjugate (R8-Alexa488), octaarginine conjugated Enhanced Green Fluorescent Protein (EGFP-R8), and the fluid phase probe dextran. Disrupting actin organisation in A431 skin epithelial cells dramatically increases the uptake of EGFP-R8 and dextran, and contrasts strongly to inhibitory effects observed with transferrin and R8 attached to the fluorophore Alexa488. This demonstrates that uptake of the same CPP can occur via different endocytic processes depending on the conjugated fluorescent entity. Overall this study highlights how cargo influences cell uptake of this peptide and that the actin cytoskeleton may act as a gateway or barrier to endocytosis of drug delivery vectors.

Fluorescence labelling of extracellular vesicles using a novel thiol-based strategy for quantitative analysis of cellular delivery and intracellular traffic.

Extracellular vesicles, including exosomes, are naturally derived nanovesicles generated in and released by numerous cell types. As extracellular entities they have the capacity to interact with neighbouring cells and distant tissues and affect physiological processes as well as being implicated in numerous diseases including tumorigenesis and neurodegeneration. They are also under intense investigation as delivery vectors for biotherapeutics. The ways in which EVs interact with recipient cells to influence cell physiology and deliver a macromolecular payload are at the early stages of exploration. A significant challenge within these studies is the ability to label EVs directly or indirectly with fluorescent probes to allow visualization without compromising functionality. Here, we present a thiol-based fluorescence labelling method allowing comprehensive analysis of the cellular uptake of prostate cancer derived EVs in live cells using confocal microscopy. Labelling of the EVs in this way did not influence their size and had no effect on their ability to induce differentiation of lung fibroblasts to myofibroblasts. For endocytosis analyses, depletion of key endocytic proteins and the use of chemical inhibitors (Dynasore, EIPA, Rottlerin and IPA-3) indicated that fluid-phase endocytosis and/or macropinocytosis was involved in EV internalisation. Over a period of six hours EVs were observed to increasingly co-localise with lysosomes, indicating a possible termination point following internalisation. Overall this method provides new opportunities for analysing the cellular dynamics of EVs as biological entities affecting cell and whole body physiology as well as investigating their potential as drug delivery vectors.

Life disruption and generic complexity: a social linguistic analysis of narratives of cancer illness.

This paper draws on social linguistics to inquire into the meaning and function of complexity in illness narratives. According to social linguists, five different story-type genres occur in spoken English. These are illustrated and differentiated using examples drawn from 10 interviews with people who have undergone colectomy for colorectal cancer. In order to test a hypothesis that complexity in illness narratives is related to life disruption, the 10 accounts were ranked in terms of their generic complexity. Measures of life disruption were based on rankings furnished independently by two readers from different disciplines who were blind to the hypothesis being tested. These two rankings showed a high level of agreement (r(s) = 0.85, p<0.01). When the two life disruption rankings and the generic complexity ranking were compared, a high degree of concordance between the three rankings was observed (W = 0.91, p<0.01). No evidence was found of associations between generic complexity and gender, interviewer, surgical outcome in terms of stoma (p>0.05), age (p>0.7) nor time since diagnosis (p>0.1). We conclude that in this study, generic complexity was strongly and significantly related to life disruption. To explain the function of complexity in interaction, we characterise the illness narrative as a genre in its own right, and argue that illness narratives need to be considered both in terms of the work they do both on the listener and for the narrator. In the former case, complexity opens up a discursive space for the dynamic positioning of the interlocutor. In the latter case, we propose that complexity reflects the degree to which the process of re-ordering life by assigning meaning is occurring as the interaction unfolds. In both cases, complex narratives can thus be understood as "hard working" narratives.

Distal phenylalanine modification for enhancing cellular delivery of fluorophores, proteins and quantum dots by cell penetrating peptides.

For cell penetrating peptides (CPPs) to fulfil their promise as effective delivery vectors we need a better understanding of their mechanisms of cell binding and uptake. This is especially the case when they are linked to different types of cargo. Here we describe new studies based on our previous findings suggesting that, for peptide-CPP chimeras, distal hydrophobic residues upstream of the CPP sequence can have profound effects on the way they interact with cells. We studied peptides bearing an N-terminal Glycine or Phenylalanine linked via a neutral and flexible bridging group, SGSGSGSG, to three well-studied CPPs: octaarginine, penetratin and TP10. Using a combination of flow cytometry, live-cell imaging and image analysis we examined the effects of this single amino acid change on binding and uptake of Alexa488-fluorophore, bovine serum albumin and quantum dot cargoes. The influence of the glycine-phenylalanine switch for fluorophore delivery was most dramatic in TP10, increasing cellular uptake by 4.4 and 9.9 fold in non-adherent and adherent cells, respectively. Only penetratin showed effective uptake of bovine serum albumin with the phenylalanine variant showing an increase of 1.6 fold over the glycine variant. The uptake of quantum dots was most efficiently demonstrated by octaarginine, with the glycine variant increasing uptake 4.8 fold and the phenylalanine variant increasing uptake 9.5 fold over quantum dots alone. Overall the data demonstrate that hydrophobicity distal to the CPP could be utilised to enhance their capacity to bind to the cell membrane and deliver a range of macromolecules to the insides of cells.

Dosage effects of chromosomes of homoeologous groups 1 and 6 upon bread-making quality in hexaploid wheat.

The endosperm storage proteins, glutenin and gliadin, are major determinants of bread-making quality in hexaploid wheat. Genes encoding them are located on chromosomes of homoeologous groups 1 and 6. Aneuploid lines of these groups in spring wheat cultivar 'Chinese Spring' have been used to investigate the effect of varying the dosage of chromosomes and chromosome arms upon bread-making quality, where quality has been assessed using the SDS-sedimentation test. Differences between the group 1 chromosomes for quality were greater than those between the group 6 chromosomes. The chromosomes were ranked within homoeologous groups for their effect on quality as follows (>=better quality): 1D>1B>1A and 6A>6B=6D. The relationship of chromosome dosage with quality was principally linear for four of the chromosomes, but not for 6B and 6D. Increases in the dosage of 1B, 6A and, especially, 1D, were associated with significant improvements in quality, whereas increases in the dosage of 1A were associated with reductions in quality. The effects of 1A and 1D were such that the best genotype for quality was nullisomic 1A-tetrasomic 1D. For group 1, effects of the long arm appeared in general to be more important than effects of the short arm. For group 6, effects were found associated with the long arms as well as with the short arms, a surprising result in view of the absence of genes encoding storage proteins on the long arms. Significant interactions were found between chromosomes and genetic backgrounds, and between individual chromosomes. Analysis of trials grown over two years demonstrated that, although additive environmental differences over years and genotype x years interaction were present, they were relatively small in magnitude compared with purely genetic differences.