Uncovering the cytotoxic effects of air pollution with multi-modal imaging of in vitro respiratory models.

Annually, an estimated seven million deaths are linked to exposure to airborne pollutants. Despite extensive epidemiological evidence supporting clear associations between poor air quality and a range of short- and long-term health effects, there are considerable gaps in our understanding of the specific mechanisms by which pollutant exposure induces adverse biological responses at the cellular and tissue levels. The development of more complex, predictive, in vitro respiratory models, including two- and three-dimensional cell cultures, spheroids, organoids and tissue cultures, along with more realistic aerosol exposure systems, offers new opportunities to investigate the cytotoxic effects of airborne particulates under controlled laboratory conditions. Parallel advances in high-resolution microscopy have resulted in a range of in vitro imaging tools capable of visualizing and analysing biological systems across unprecedented scales of length, time and complexity. This article considers state-of-the-art in vitro respiratory models and aerosol exposure systems and how they can be interrogated using high-resolution microscopy techniques to investigate cell-pollutant interactions, from the uptake and trafficking of particles to structural and functional modification of subcellular organelles and cells. These data can provide a mechanistic basis from which to advance our understanding of the health effects of airborne particulate pollution and develop improved mitigation measures.

Folding-Mediated DNA Delivery by α-Helical Amphipathic Peptides.

The renaissance gene therapy experiences these days requires specialist biomaterials and a systemic understanding of major factors influencing their ability to deliver genetic material. Peptide transfection systems represent a major class of such biomaterials. Several peptidic reagents have been commercialized to date. However, a comparative assessment of peptide sequences alone without auxiliary support or excipients against a common determinant for their ability to complex and deliver DNA has been lacking. This study cross-compares commercial and experimental transfection reagents from the same family of helical amphiphiles. Factors defining the efficacy of DNA delivery including cell uptake and gene expression are assessed along with cytotoxicity and DNA complexation. The results show that despite differences in sequence composition, length, and origin, peptide reagents of the same structural family exhibit similar characteristics and limitations with common variability trends. The cross-comparison revealed that functional DNA delivery is independent of the peptide sequence used but is mediated by the ability of the reagents to co-fold with DNA. Peptide folding proved to be the common determinant for DNA complexation and delivery by peptidic transfection reagents.

Designer protein pseudo-capsids targeting intracellular bacteria.

The emergence of multidrug-resistant bacteria stimulates the search for antimicrobial materials capable of addressing challenges conventional antibiotics fail to address. The ability to target intracellular bacteria remains one of the most fundamental tasks for contemporary antimicrobial treatments. Here we report engineered protein pseudo-capsids targeting bacteria internalised in macrophages. Using a combination of live-cell imaging and single-cell electron microscopy analysis we show that these materials effectively disrupt the bacteria without affecting the host cells. The study offers a disruptive antimicrobial strategy demonstrating potential for developing principally more challenging mechanisms for bacteria to overcome.

An engineered, quantifiable in vitro model for analysing the effect of proteostasis-targeting drugs on tissue physical properties.

Cellular function depends on the maintenance of protein homeostasis (proteostasis) by regulated protein degradation. Chronic dysregulation of proteostasis is associated with neurodegenerative and age-related diseases, and drugs targeting components of the protein degradation apparatus are increasingly used in cancer therapies. However, as chronic imbalances rather than loss of function mediate their pathogenesis, research models that allow for the study of the complex effects of drugs on tissue properties in proteostasis-associated diseases are almost completely lacking. Here, to determine the functional effects of impaired proteostatic fine-tuning, we applied a combination of materials science characterisation techniques to a cell-derived, in vitro model of bone-like tissue formation in which we pharmacologically perturbed protein degradation. We show that low-level inhibition of VCP/p97 and the proteasome, two major components of the degradation machinery, have remarkably different effects on the bone-like material that human bone-marrow derived mesenchymal stromal cells (hMSC) form in vitro. Specifically, whilst proteasome inhibition mildly enhances tissue formation, Raman spectroscopic, atomic force microscopy-based indentation, and electron microscopy imaging reveal that VCP/p97 inhibition induces the formation of bone-like tissue that is softer, contains less protein, appears to have more crystalline mineral, and may involve aberrant micro- and ultra-structural tissue organisation. These observations contrast with findings from conventional osteogenic assays that failed to identify any effect on mineralisation. Taken together, these data suggest that mild proteostatic impairment in hMSC alters the bone-like material they form in ways that could explain some pathologies associated with VCP/p97-related diseases. They also demonstrate the utility of quantitative materials science approaches for tackling long-standing questions in biology and medicine, and could form the basis for preclinical drug testing platforms to develop therapies for diseases stemming from perturbed proteostasis or for cancer therapies targeting protein degradation. Our findings may also have important implications for the field of tissue engineering, as the manufacture of cell-derived biomaterial scaffolds may need to consider proteostasis to effectively replicate native tissues.