Lysine to arginine mutagenesis of chlorotoxin enhances its cellular uptake

Chlorotoxin (CTX), a disulfide-rich peptide from the scorpion Leiurus qu'mquestriatus, has several promising biopharmaceutical properties, including preferential affinity for certain cancer cells, high serum stability, and cell penetration. These properties underpin its potential for use as a drug design scaffold, especially for the treatment of cancer; indeed, several analogs of CTX have reached clinical trials. Here, we focus on its ability to internalize into cells-a trait associated with a privileged subclass of peptides called cell-penetrating peptides-and whether it can be improved through conservative substitutions. Mutants of CTX were made using solid-phase peptide synthesis and internalization into human cervical carcinoma (HeLa) cells was monitored by fluorescence and confocal microscopy. CTX_M1 (ie, [K15R/K23R]CTX) and CTX_M2 (ie, [K15R/K23R/Y29W]CTX) mutants showed at least a twofold improvement in uptake compared to CTX. We further showed that these mutants internalize into HeLa cells largely via an energy-dependent mechanism. Importantly, the mutants have high stability, remaining intact in serum for over 24 h; thus, retaining the characteristic stability of their parent peptide. Overall, we have shown that simple conservative substitutions can enhance the cellular uptake of CTX, suggesting that such type of mutations might be useful for improving uptake of other peptide toxins

Agentic learning: the pedagogical implications of young trans people’s online learning strategies

This paper proposes anew conceptualisation of learning in the age of the internet, increasing systemic rigidity of formal education and intensified media manipulation and partiality. Using empirical data and drawing on Social Activity Method it elaborates the different strategies young trans people recruit in their self-learning and contends that these constitute a type of learning where the control of pedagogy, the learning environment and the subject matter lies to a significant extent, with the learner, taking place in spaces free from the influence of hegemonic transphobia. This type of learning appears to constitute an effective but complex one. As, in this instance, the learning is taking place in a wider cultural environment where the subject matter is often suppressed and subject to ideological misrepresentation by hegemonic control of the public sphere, this study suggests that learning by providing learners with greater control over pedagogy and learning environment is effective

Pre-formulation and systematic evaluation of amino acid assisted permeability of insulin across in vitro buccal cell layers

The aim of this work was to investigate alternative safe and effective permeation enhancers for buccal peptide delivery. Basic amino acids improved insulin solubility in water while 200 and 400 µg/mL lysine significantly increased insulin solubility in HBSS. Permeability data showed a significant improvement in insulin permeation especially for 10 µg/mL of lysine (p < 0.05) and 10 µg/mL histidine (p < 0.001), 100 µg/mL of glutamic acid (p < 0.05) and 200 µg/mL of glutamic acid and aspartic acid (p < 0.001) without affecting cell integrity; in contrast to sodium deoxycholate which enhanced insulin permeability but was toxic to the cells. It was hypothesized that both amino acids and insulin were ionised at buccal cavity pH and able to form stable ion pairs which penetrated the cells as one entity; while possibly triggering amino acid nutrient transporters on cell surfaces. Evidence of these transport mechanisms was seen with reduction of insulin transport at suboptimal temperatures as well as with basal-to-apical vectoral transport, and confocal imaging of transcellular insulin transport. These results obtained for insulin is the first indication of a possible amino acid mediated transport of insulin via formation of insulin-amino acid neutral complexes by the ion pairing mechanism

In Vivo Methods to Study Uptake of Nanoparticles into the Brain

Several in vivo techniques have been developed to study and measure the uptake of CNS compounds into the brain. With these techniques, various parameters can be determined after drug administration, including the blood-to-brain influx constant (Kin), the permeability-surface area (PS) product, and the brain uptake index (BUI). These techniques have been mostly used for drugs that are expected to enter the brain via transmembrane diffusion or by carrier-mediated transcytosis. Drugs that have limitations in entering the brain via such pathways have been encapsulated in nanoparticles (based on lipids or synthetic polymers) to enhance brain uptake. Nanoparticles are different from CNS compounds in size, composition and uptake mechanisms. This has led to different methods and approaches to study brain uptake in vivo. Here we discuss the techniques generally used to measure nanoparticle uptake in addition to the techniques used for CNS compounds. Techniques include visualization methods, behavioral tests, and quantitative methods

Molecular Characterization of Circulating Tumor Cells to Study Cancer Immunoevasion

Cancer cells leaving the primary tumor immunosuppressive microenvironment become vulnerable to active immune surveillance and require mechanisms of immunoevasion to survive in the circulation. Studies have identified several pathways by which circulating tumor cells (CTCs) might escape the immune system/immunotherapy attack. The PD-1/PD-L1 axis is an immune checkpoint regulator, playing a major role in maintaining self-tolerance. It is now well recognized that tumor cells co-opt the PD-1/PD-L1 axis of immune regulation to interfere with cytotoxic T lymphocyte function. Transcriptional changes in CTCs, leading to the upregulation of PD-L1, might enable them to survive in circulation. Very recent data revealed a previously unappreciated role of epithelial-mesenchymal transition (EMT) in reprogramming the immune response in the local tumor microenvironment and a mutual regulation between EMT and immunoevasion is becoming apparent. In this chapter, we will describe in detail both EpCAM-dependent and -independent approaches that allow the identification of PD-L1 expression and EMT-like features in circulating tumor cells

Trans inclusive Higher Education : strategies to support trans, non-binary and gender diverse students and staff

There are significant challenges for trans and gender diverse people in higher education, both as learners and as members of staff. Our TransEDU research found that 86% of research participants had encountered barriers to their learning or work which they directly attributed to their trans or gender diverse status (Lawrence & Mckendry, Supporting transgender and non-binary students and staff in further and higher education: Practical advice for colleges and universities. Jessica Kingsley, 2019; Mckendry and Lawrence, TransEDU Scotland: Researching the experience of trans and gender diverse applicants, students and staff in Scotland’s colleges and universities. Research report, 2017a). This chapter will therefore explore two strategies, as case studies. Both have the potential to subvert current patterns of isolation, raise awareness and empower students and staff to be allies: the creation of champion groups or networks to advance and implement inclusion work; and the designation of a trained and well-publicised named contact for trans people within institutions