Development and Application of High-Content Biological Screening for Modulators of NET Production.

Neutrophil extracellular traps (NETs) are DNA-based antimicrobial web-like structures whose release is predominantly mediated by reactive oxygen species (ROS); their purpose is to combat infections. However, unbalanced NET production and clearance is involved in tissue injury, circulation of auto-antibodies and development of several chronic diseases. Currently, there is lack of agreement regarding the high-throughput methods available for NET investigation. This study, therefore, aimed to develop and optimize a high-content analysis (HCA) approach, which can be applied for the assay of NET production and for the screening of compounds involved in the modulation of NET release. A suitable paraformaldehyde fixation protocol was established to enable HCA of neutrophils and NETs. Bespoke and in-built bioinformatics algorithms were validated by comparison with standard low-throughput approaches for application in HCA of NETs. Subsequently, the optimized protocol was applied to high-content screening (HCS) of a pharmaceutically derived compound library to identify modulators of NETosis. Of 56 compounds assessed, 8 were identified from HCS for further characterization of their effects on NET formation as being either inducers, inhibitors or biphasic modulators. The effects of these compounds on naïve neutrophils were evaluated by using specific assays for the induction of ROS and NET production, while their modulatory activity was validated in phorbol 12-myristate 13-acetate-stimulated neutrophils. Results indicated the involvement of glutathione reductase, Src family kinases, molecular-target-of-Rapamycin, and mitogen-activated-protein-kinase pathways in NET release. The compounds and pathways identified may provide targets for novel therapeutic approaches for treating NET-associated pathologies.

High content and high throughput screening to assess the angiogenic and neurogenic actions of mesenchymal stem cells in vitro.

Mesenchymal stem cells are universally regarded across many fields of medicine, as one of the most promising cell types for use in cell-based therapies. Although not yet fully understood, the therapeutic effects of these cells are largely attributed to the trophic actions of growth factors and cytokines present in the cell secretome. Specifically, the angiogenic and neurogenic properties of these cells make them attractive for the repair of vascularised and innervated tissues. In this study, we investigate the effect of mesenchymal stem cell conditioned media on in vitro assays of angiogenesis and nerve growth. We describe the use of two state of the art high content and high throughput cell analysis systems and compare them against manual analysis techniques. Mesenchymal stem cell secretomes stimulated angiogenesis and nerve growth in vitro in a donor dependant manner. Levels of neuroregulin, platelet-derived growth factor-AA and glial-derived neurotrophic factor, positively correlated with the observed angiogenic effects of these cells. High content and high throughput cell analysis systems such as the ones used in this study, may provide rapid screening tools to assist not only with patient selection but the identification of predictive therapeutic markers to support clinical outcome monitoring for patients treated with stem cell therapies.

Application of high-content analysis to the study of post-translational modifications of the cytoskeleton.

Cytokeratins 8 and 18 have recently been identified as acetylated. The acetylation of other cytoskeletal proteins has been reported as linked to stabilility. As colorectal cells exist bathed in pharmacologically active levels of the HDACi butyrate, we sought to apply state-of-the-art High Content Analytical approaches to identify the effect of butyrate upon the cytoskeleton of colorectal cells. We observed butyrate caused an increase in acetylation at three distinct residues of cytokeratin 8 (K10, K471, and K482) and that the kinetics of altered acetylation were distinct, implying either separate HDACs, or a heirachy of acetylation. This change in acetylation was associated with a breakdown in the cytokeratin cytoskeleton, implying a functional role for cytokeratin acetylation.

Religious perspectives on withdrawal of treatment from patients with multiple organ failure.

Religious or spiritual values often influence health care decision-making by patients and their families, particularly in times of crisis. Though religious values might seem to be irrelevant where continuing treatment is judged to be "futile", such clinical assessments should instead serve to open a dialogue about values and beliefs. The six major religious traditions in Australia have some similar values and principles about death and provision of care for the dying, but differ in their processes of ethical reasoning, cosmologies, and key moral concepts. Engaging with religious traditions on the common ground of basic values (such as human dignity, care, the sacredness of human life, non-violence, compassion, and selflessness) promotes negotiation of the manner in which care is provided, even where conflicts exist.