Single-cell analysis tools for drug discovery and development

The genetic, functional or compositional heterogeneity of healthy and diseased tissues presents major challenges in drug discovery and development. Such heterogeneity hinders the design of accurate disease models and can confound the interpretation of biomarker levels and of patient responses to specific therapies. The complex nature of virtually all tissues has motivated the development of tools for single-cell genomic, transcriptomic and multiplex proteomic analyses. Here, we review these tools and assess their advantages and limitations. Emerging applications of single cell analysis tools in drug discovery and development, particularly in the field of oncology, are discussed

Decreased thickness and integrity of the macular elastic layer of Bruch's membrane correspond to the distribution of lesions associated with age-related macular degeneration.

Age-related macular degeneration (AMD) is a leading cause of blindness in the elderly. In its severest form, choroidal neovessels breach the macular Bruch's membrane, an extracellular matrix compartment comprised of elastin and collagen laminae, and grow into the retina. We sought to determine whether structural properties of the elastic lamina (EL) correspond to the region of the macula that is predilected toward degeneration in AMD. Morphometric assessment of the macular and extramacular regions of 121 human donor eyes, with and without AMD, revealed a statistically significant difference in both the integrity (P < 0.0001) and thickness (P < 0.0001) of the EL between the macular and extramacular regions in donors of all ages. The EL was three to six times thinner and two to five times less abundant in the macula than in the periphery. The integrity of the macular EL was significantly lower in donors with early-stage AMD (P = 0.028), active choroidal neovascularization (P = 0.020), and disciform scars (P = 0.003), as compared to unaffected, age-matched controls. EL thickness was significantly lower only in individuals with disciform scars (P = 0.008). The largest gaps in macular EL integrity were significantly larger in all categories of AMD (each P < 0.0001), as compared to controls. EL integrity, thickness, and gap length in donors with geographic atrophy did not differ from those of controls. These structural properties of the macular EL correspond spatially to the distribution of macular lesions associated with AMD and may help to explain why the macula is more susceptible to degenerative events that occur in this disease

Influenza infection induces host DNA damage and dynamic DNA damage responses during tissue regeneration

Influenza viruses account for significant morbidity worldwide. Inflammatory responses, including excessive generation of reactive oxygen and nitrogen species (RONS), mediate lung injury in severe influenza infections. However, the molecular basis of inflammation-induced lung damage is not fully understood. Here, we studied influenza H1N1 infected cells in vitro, as well as H1N1 infected mice, and we monitored molecular and cellular responses over the course of 2 weeks in vivo. We show that influenza induces DNA damage to both, when cells are directly exposed to virus in vitro (measured using the comet assay) and also when cells are exposed to virus in vivo (estimated via γH2AX foci). We show that DNA damage, as well as responses to DNA damage persist in vivo until long after virus has been cleared, at times when there are inflammation associated RONS (measured by xanthine oxidase activity and oxidative products). The frequency of lung epithelial and immune cells with increased γH2AX foci is elevated in vivo, especially for dividing cells (Ki-67-positive) exposed to oxidative stress during tissue regeneration. Additionally, we observed a significant increase in apoptotic cells as well as increased levels of DNA double strand break (DSB) repair proteins Ku70, Ku86 and Rad51 during the regenerative phase. In conclusion, results show that influenza induces DNA damage both in vitro and in vivo, and that DNA damage responses are activated, raising the possibility that DNA repair capacity may be a determining factor for tissue recovery and disease outcome.Singapore National Research Foundatio