A Novel Task for the Investigation of Action Acquisition

We present a behavioural task designed for the investigation of how novel instrumental actions are discovered and learnt. The task consists of free movement with a manipulandum, during which the full range of possible movements can be explored by the participant and recorded. A subset of these movements, the ‘target’, is set to trigger a reinforcing signal. The task is to discover what movements of the manipulandum evoke the reinforcement signal. Targets can be defined in spatial, temporal, or kinematic terms, can be a combination of these aspects, or can represent the concatenation of actions into a larger gesture. The task allows the study of how the specific elements of behaviour which cause the reinforcing signal are identified, refined and stored by the participant. The task provides a paradigm where the exploratory motive drives learning and as such we view it as in the tradition of Thorndike [1]. Most importantly it allows for repeated measures, since when a novel action is acquired the criterion for triggering reinforcement can be changed requiring a new action to be discovered. Here, we present data using both humans and rats as subjects, showing that our task is easily scalable in difficulty, adaptable across species, and produces a rich set of behavioural measures offering new and valuable insight into the action learning process

Early Developmental and Evolutionary Origins of Gene Body DNA Methylation Patterns in Mammalian Placentas

Over the last 20-80 million years the mammalian placenta has taken on a variety of morphologies through both divergent and convergent evolution. Recently we have shown that the human placenta genome has a unique epigenetic pattern of large partially methylated domains (PMDs) and highly methylated domains (HMDs) with gene body DNA methylation positively correlating with level of gene expression. In order to determine the evolutionary conservation of DNA methylation patterns and transcriptional regulatory programs in the placenta, we performed a genome-wide methylome (MethylC-seq) analysis of human, rhesus macaque, squirrel monkey, mouse, dog, horse, and cow placentas as well as opossum extraembryonic membrane. We found that, similar to human placenta, mammalian placentas and opossum extraembryonic membrane have globally lower levels of methylation compared to somatic tissues. Higher relative gene body methylation was the conserved feature across all mammalian placentas, despite differences in PMD/HMDs and absolute methylation levels. Specifically, higher methylation over the bodies of genes involved in mitosis, vesicle-mediated transport, protein phosphorylation, and chromatin modification was observed compared with the rest of the genome. As in human placenta, higher methylation is associated with higher gene expression and is predictive of genic location across species. Analysis of DNA methylation in oocytes and preimplantation embryos shows a conserved pattern of gene body methylation similar to the placenta. Intriguingly, mouse and cow oocytes and mouse early embryos have PMD/HMDs but their placentas do not, suggesting that PMD/HMDs are a feature of early preimplantation methylation patterns that become lost during placental development in some species and following implantation of the embryo

Early Developmental and Evolutionary Origins of Gene Body DNA Methylation Patterns in Mammalian Placentas.

Over the last 20-80 million years the mammalian placenta has taken on a variety of morphologies through both divergent and convergent evolution. Recently we have shown that the human placenta genome has a unique epigenetic pattern of large partially methylated domains (PMDs) and highly methylated domains (HMDs) with gene body DNA methylation positively correlating with level of gene expression. In order to determine the evolutionary conservation of DNA methylation patterns and transcriptional regulatory programs in the placenta, we performed a genome-wide methylome (MethylC-seq) analysis of human, rhesus macaque, squirrel monkey, mouse, dog, horse, and cow placentas as well as opossum extraembryonic membrane. We found that, similar to human placenta, mammalian placentas and opossum extraembryonic membrane have globally lower levels of methylation compared to somatic tissues. Higher relative gene body methylation was the conserved feature across all mammalian placentas, despite differences in PMD/HMDs and absolute methylation levels. Specifically, higher methylation over the bodies of genes involved in mitosis, vesicle-mediated transport, protein phosphorylation, and chromatin modification was observed compared with the rest of the genome. As in human placenta, higher methylation is associated with higher gene expression and is predictive of genic location across species. Analysis of DNA methylation in oocytes and preimplantation embryos shows a conserved pattern of gene body methylation similar to the placenta. Intriguingly, mouse and cow oocytes and mouse early embryos have PMD/HMDs but their placentas do not, suggesting that PMD/HMDs are a feature of early preimplantation methylation patterns that become lost during placental development in some species and following implantation of the embryo

Regions of high methylation in placentas cover gene bodies.

<p>(A) Heatmap of average methylation in the gene bodies (introns and exons, excluding CpG islands and promoters) of orthologous genes. Only the top few GO biological processes with Benjamini p-values below 1.0E-3 are shown. For a complete list see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005442#pgen.1005442.s020" target="_blank">S3 Table</a>. Branches A and B were combined because they contain similar GO terms. (B) Comparison of percent methylation between human placenta (red curve) and rhesus placenta (purple curve). Rhesus methylation data was lifted over to the human genome. Vertical purple lines show large chromosomal breaks in synteny between the two species. The fourth ring in shows regions of higher (blue) and lower (red) methylation in human placenta compared to rhesus. The fourth circle in shows the locations of human genes in black. (C) Spinograms showing the probability that a 5 kb window is in a gene given that window's average percent methylation. Bars are color-coded by percent methylation and bar widths show the percentage of windows with that methylation level. Bars furthest from the 0.5 blue line marker show the most information about gene location.</p

Genome-wide methylation patterns in mammalian placentas show both large-scale divergence and gene-specific similarities.

<p>(A) Phylogenetic tree of the species studied and the classification of their placenta types. Branch lengths are not to scale. (B) Density curves of average percent methylation in non-overlapping 20 kb windows in mammalian placentas. For comparison, the methylation distribution for brain tissue is shown in beige for some species and human cord blood is shown in aquamarine. The opossum brain sample was fetal whereas the other brain samples were postnatal. The interquartile range and medians are shown as black bars and white dots, respectively, for the placenta samples. Mouse brain data is from Hon et al. [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005442#pgen.1005442.ref019" target="_blank">19</a>] (GSE42836), human placenta data is from Schroeder et al [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005442#pgen.1005442.ref021" target="_blank">21</a>] (GSE25930). (C) Comparison of global methylation patterns in select species after liftOver to the human genome and smoothing (for full figure, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005442#pgen.1005442.s004" target="_blank">S4 Fig</a>). Human placenta PMDs, as determined by HMM, are shown in black bars at top. (D) Comparison of methylation patterns at the <i>CNTNAP2</i> locus in select species (for full figure, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005442#pgen.1005442.s005" target="_blank">S5 Fig</a>). Raw species CpG site methylation wig data were graphed on the UCSC Genome Browser without preprocessing. Black line represents 50% methylation. Genes of interest are orange. EEM = extra-embryonic membrane.</p

Gene methylation and expression in oocytes compared to placenta in mouse and cow.

<p>(A) Distribution of average methylation in 50 kb windows (first column) and gene bodies (second column) during early human, mouse, and cow development. The third column shows the relationship between average gene body methylation and gene expression in oocytes. Black lines show the marginal distribution of percent methylation in gene bodies. All protein-coding genes from each species were used. Human oocyte and sperm methylation data are from Okae et al. [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005442#pgen.1005442.ref027" target="_blank">27</a>] (JGAS00000000006), human oocyte expression data are from Reich et al. [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005442#pgen.1005442.ref034" target="_blank">34</a>] (GSE32689), mouse oocyte and sperm methylation and expression data are from Wang et al [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005442#pgen.1005442.ref024" target="_blank">24</a>] (GSE56697), and cow oocyte expression data are from Graf et al [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005442#pgen.1005442.ref033" target="_blank">33</a>] (GSE52415). (B) Heatmap of average gene body methylation in human, mouse, and cow oocytes. For a complete list of all GO and KEGG terms for each quadrant, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005442#pgen.1005442.s020" target="_blank">S3 Table</a>.</p

DataSheet_1_Fluorine labelling of therapeutic human tolerogenic dendritic cells for 19F-magnetic resonance imaging.pdf

Tolerogenic dendritic cell (tolDC) therapies aim to restore self-tolerance in patients suffering from autoimmune diseases. Phase 1 clinical trials with tolDC have shown the feasibility and safety of this approach, but have also highlighted a lack of understanding of their distribution in vivo. Fluorine-19 magnetic resonance imaging (19F-MRI) promises an attractive cell tracking method because it allows for detection of 19F-labelled cells in a non-invasive and longitudinal manner. Here, we tested the suitability of nanoparticles containing 19F (19F-NP) for labelling of therapeutic human tolDC for detection by 19F-MRI. We found that tolDC readily endocytosed 19F-NP with acceptable effects on cell viability and yield. The MRI signal-to-noise ratios obtained are more than sufficient for detection of the administered tolDC dose (10 million cells) at the injection site in vivo, depending on the tissue depth and the rate of cell dispersal. Importantly, 19F-NP labelling did not revert tolDC into immunogenic DC, as confirmed by their low expression of typical mature DC surface markers (CD83, CD86), low secretion of pro-inflammatory IL-12p70, and low capacity to induce IFN-γ in allogeneic CD4+ T cells. In addition, the capacity of tolDC to secrete anti-inflammatory IL-10 was not diminished by 19F-NP labelling. We conclude that 19F-NP is a suitable imaging agent for tolDC. With currently available technologies, this imaging approach does not yet approach the sensitivity required to detect small numbers of migrating cells, but could have important utility for determining the accuracy of injecting tolDC into the desired target tissue and their efflux rate.</p

DataSheet_2_Fluorine labelling of therapeutic human tolerogenic dendritic cells for 19F-magnetic resonance imaging.pdf

Tolerogenic dendritic cell (tolDC) therapies aim to restore self-tolerance in patients suffering from autoimmune diseases. Phase 1 clinical trials with tolDC have shown the feasibility and safety of this approach, but have also highlighted a lack of understanding of their distribution in vivo. Fluorine-19 magnetic resonance imaging (19F-MRI) promises an attractive cell tracking method because it allows for detection of 19F-labelled cells in a non-invasive and longitudinal manner. Here, we tested the suitability of nanoparticles containing 19F (19F-NP) for labelling of therapeutic human tolDC for detection by 19F-MRI. We found that tolDC readily endocytosed 19F-NP with acceptable effects on cell viability and yield. The MRI signal-to-noise ratios obtained are more than sufficient for detection of the administered tolDC dose (10 million cells) at the injection site in vivo, depending on the tissue depth and the rate of cell dispersal. Importantly, 19F-NP labelling did not revert tolDC into immunogenic DC, as confirmed by their low expression of typical mature DC surface markers (CD83, CD86), low secretion of pro-inflammatory IL-12p70, and low capacity to induce IFN-γ in allogeneic CD4+ T cells. In addition, the capacity of tolDC to secrete anti-inflammatory IL-10 was not diminished by 19F-NP labelling. We conclude that 19F-NP is a suitable imaging agent for tolDC. With currently available technologies, this imaging approach does not yet approach the sensitivity required to detect small numbers of migrating cells, but could have important utility for determining the accuracy of injecting tolDC into the desired target tissue and their efflux rate.</p