Mammalian lectin arrays for screening host-microbe interactions.

Many members of the C-type lectin family of glycan-binding receptors have been ascribed roles in the recognition of microorganisms and serve as key receptors in the innate immune response to pathogens. Other mammalian receptors have become targets through which pathogens enter target cells. These receptor roles have often been documented with binding studies involving individual pairs of receptors and microorganisms. To provide a systematic overview of interactions between microbes and the large complement of C-type lectins, here we developed a lectin array and suitable protocols for labeling of microbes that could be used to probe this array. The array contains C-type lectins from cow, chosen as a model organism of agricultural interest for which the relevant pathogen-receptor interactions have not been previously investigated in detail. Screening with yeast cells and various strains of both Gram-positive and -negative bacteria revealed distinct binding patterns, which in some cases could be explained by binding to lipopolysaccharides or capsular polysaccharides, but in other cases they suggested the presence of novel glycan targets on many of the microorganisms. These results are consistent with interactions previously ascribed to the receptors, but they also highlight binding to additional sugar targets that have not previously been recognized. Our findings indicate that mammalian lectin arrays represent unique discovery tools for identifying both novel ligands and new receptor functions.

Fast single-cell biochemistry: theory, open source microscopy and applications.

Fluorescence lifetime sensing enables researchers to probe the physicochemical environment of a fluorophore providing a window through which we can observe the complex molecular make-up of the cell. Fluorescence lifetime imaging microscopy (FLIM) quantifies and maps cell biochemistry, a complex ensemble of dynamic processes. Unfortunately, typical high-resolution FLIM systems exhibit rather limited acquisition speeds, often insufficient to capture the time evolution of biochemical processes in living cells. Here, we describe the theoretical background that justifies the developments of high-speed single photon counting systems. We show that systems with low dead-times not only result in faster acquisition throughputs but also improved dynamic range and spatial resolution. We also share the implementation of hardware and software as an open platform, show applications of fast FLIM biochemical imaging on living cells and discuss strategies to balance precision and accuracy in FLIM. The recent innovations and commercialisation of fast time-domain FLIM systems are likely to popularise FLIM within the biomedical community, to impact biomedical research positively and to foster the adoption of other FLIM techniques as well. While supporting and indeed pursuing these developments, with this work we also aim to warn the community about the possible shortcomings of fast single photon counting techniques and to highlight strategies to acquire data of high quality.

Exploiting Synthetic Lethality and Network Biology to Overcome EGFR Inhibitor Resistance in Lung Cancer.

Despite the recent approval of third-generation therapies, overcoming resistance to epidermal growth factor receptor (EGFR) inhibitors remains a major challenge in non-small cell lung cancer. Conceptually, synthetic lethality holds the promise of identifying non-intuitive targets for tackling both acquired and intrinsic resistance in this setting. However, translating these laboratory findings into effective clinical strategies continues to be elusive. Here, we provide an overview of the synthetic lethal approaches that have been employed to study EGFR inhibitor resistance and review the oncogene and non-oncogene signalling mechanisms that have thus far been unveiled by synthetic lethality screens. We highlight the potential challenges associated with progressing these discoveries into the clinic including context dependency, signalling plasticity, and tumour heterogeneity, and we offer a perspective on emerging network biology and computational solutions to exploit these phenomena for cancer therapy and biomarker discovery. We conclude by presenting a number of tangible steps to bolster our understanding of fundamental synthetic lethality mechanisms and advance these findings beyond the confines of the laboratory.