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Proton beams are promising means for treating tumours. Such charged particles stop at a defined depth, where the ionization density is maximum. As the dose deposit beyond this distal edge is very low, proton therapy minimises the damage to normal tissue compared to photon therapy. Nevertheless, inherent range uncertainties cast doubts on the irradiation of tumours close to organs at risk and lead to the application of conservative safety margins. This constrains significantly the potential benefits of protons over photons. In this context, several research groups are developing experimental tools for range verification based on the detection of prompt gammas, a nuclear by-product of the proton irradiation. At OncoRay and Helmholtz-Zentrum Dresden-Rossendorf, detector components have been characterised in realistic radiation environments as a step towards a clinical Compton camera. Corresponding experimental methods and results obtained during the ENTERVISION training network are reviewed. On the other hand, a novel method based on timing spectroscopy has been proposed as an alternative to collimated imaging systems. The first tests of the timing method at a clinical proton accelerator are summarised, its applicability in a clinical environment for challenging the current safety margins is assessed, and the factors limiting its precision are discussed.

Meiosis is a form of specialized cell division that generates gametes, allowing recombination of alleles and halving the chromosome number. Arabidopsis and maize are the plant models that have been most extensively studied to determine the genes involved in meiosis. Here we present an RNA-seq study in which gene expression in male meiocytes isolated during prophase I was compared to that in somatic tissues of the sunflower HA89 line. We sampled more than 490 million gene tags from these libraries, assembled them de novo into a sunflower transcriptome. We obtained expression data for 36,304 sunflower genes, of which 19,574 (54%) were differentially expressed between meiocytes and somatic tissue. We also determined the functional categories and metabolic pathways that are differentially expressed in these libraries. As expected, we found large differences between the meiotic and somatic transcriptomes, which is in accordance with previous studies in Arabidopsis and maize. Furthermore, most of the previously implicated meiotic genes were abundantly and differentially expressed in meiocytes and a large repertoire of transcription factors and genes related to silencing are expressed in the sunflower meiocytes. We detected transcription factors which appear to be exclusively expressed in meiocytes. Our results allow for a better understanding of the conservation and differences in the meiotic transcriptome of plants.

The mechanisms of epithelial wound healing are not completely understood, especially in the context of proteases and their receptors. It was recently shown that activation of protease-activated receptor-2 (PAR2) on intestinal epithelial cells induced the expression of cyclooxygenase-2 (COX-2), which has protective functions in the gastrointestinal tract. It was hypothesized that PAR2-induced COX-2 could enhance wound healing in intestinal epithelial cells. Caco2 cells were used to model epithelial wound healing of circular wounds. Cellular proliferation was studied with a 5-ethynyl-2'-deoxyuridine assay, and migration was studied during wound healing in the absence of proliferation. Immunofluorescence was used to visualize E-cadherin and F-actin, and the cellular transcription profile during wound healing and PAR2 activation was explored with RNA sequencing. PAR2 activation inhibited Caco2 wound healing by reducing cell migration, independently of COX-2 activity. Interestingly, even though migration was reduced, proliferation was increased. When the actin dynamics and cell-cell junctions were investigated, PAR2 activation was found to induce actin cabling and prevent the internalization of E-cadherin. To further investigate the effect of PAR2 on transcriptionally dependent wound healing, RNA sequencing was performed. This analysis revealed that PAR2 activation, in the absence of wounding, induced a similar transcriptional profile compared with wounding alone. These findings represent a novel effect of PAR2 activation on the mechanisms of epithelial cell wound healing that could influence the resolution of intestinal inflammation., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

Cancer cell lines have been the mainstay of intestinal epithelial experimentation for decades, due primarily to their immortality and ease of culture. However, because of the inherent biological abnormalities of cancer cell lines, many cellular biologists are currently transitioning away from these models and toward more representative primary cells. This has been particularly challenging, but recent advances in the generation of intestinal organoids have brought the routine use of primary cells within reach of most epithelial biologists. Nevertheless, even with the proliferation of publications that use primary intestinal epithelial cells, there is still a considerable amount of trial and error required for laboratories to establish a consistent and reliable method to culture three-dimensional (3D) intestinal organoids and primary epithelial monolayers. We aim to minimize the time other laboratories spend troubleshooting the technique and present a standard method for culturing primary epithelial cells. Therefore, we have described our optimized, high-yield, cost-effective protocol to grow 3D murine colonoids for more than 20 passages and our detailed methods to culture these cells as confluent monolayers for at least 14 days, enabling a wide variety of potential future experiments. By supporting and expanding on the current literature of primary epithelial culture optimization and detailed use in experiments, we hope to help enable the widespread adoption of these innovative methods and allow consistency of results obtained across laboratories and institutions. NEW & NOTEWORTHY Primary intestinal epithelial monolayers are notoriously difficult to maintain culture, even with the recent advances in the field. We describe, in detail, the protocols required to maintain three-dimensional cultures of murine colonoids and passage these primary epithelial cells to confluent monolayers in a standardized, high-yield and cost-effective manner., (Copyright © 2017 the American Physiological Society.)