Making the Digital Humanities More Open

BrailleSC will undertake its second stage of development by designing and deploying a WordPress-based accessibility tool that will create braille content for endusers who are blind or low vision. Specifically, we plan to extend the use of Anthologize--a free and open source plug-in for WordPress that currently translates any RSS text into PDF, ePub, HTML, or TEI--to include the conversion of text to braille. As a result, we will not only make it easy for content creators to convert a text into braille, thereby extending humanities content to hundreds of thousands of visually disabled readers, but we will also experiment with making braille available visually through the WordPress interface. In partnership with the Maryland Institute for Technology in the Humanities (MITH) at the University of Maryland, College Park, we will continue to model the ways in which digital humanities projects should be designed and implemented with the needs of disabled users in mind

The Cell-Surface Marker Sushi Containing Domain 2 Facilitates Establishment of Human Naive Pluripotent Stem Cells.

Recently naive human pluripotent stem cells (hPSCs) have been described that relate to an earlier stage of development than conventional hPSCs. Naive hPSCs remain challenging to generate and authenticate, however. Here we report that Sushi Containing Domain 2 (SUSD2) is a robust cell-surface marker of naive hPSCs in the embryo and in vitro. SUSD2 transcripts are enriched in the pre-implantation epiblast of human blastocysts and immunostaining shows localization of SUSD2 to KLF17-positive epiblast cells. SUSD2 mRNA is strongly expressed in naive hPSCs but is negligible in other hPSCs. SUSD2 immunostaining of live or fixed cells provides unambiguous discrimination of naive versus conventional hPSCs. SUSD2 staining or flow cytometry enable monitoring of naive hPSCs in maintenance culture, and their isolation and quantification during resetting of conventional hPSCs or somatic cell reprogramming. Thus SUSD2 is a powerful non-invasive tool for reliable identification and purification of the naive hPSC phenotype.This research was funded by the Medical Research Council of the United Kingdom (G1001028 and MR/P00072X/1) and European Commission Framework 7 (HEALTH-F4-2013-602423, PluriMes). The Cambridge Stem Cell Institute receives core support from the Wellcome Trust and the Medical Research Council. AS is a Medical Research Council Professor

Integrated analysis of single-cell embryo data yields a unified transcriptome signature for the human pre-implantation epiblast.

Single-cell profiling techniques create opportunities to delineate cell fate progression in mammalian development. Recent studies have provided transcriptome data from human pre-implantation embryos, in total comprising nearly 2000 individual cells. Interpretation of these data is confounded by biological factors, such as variable embryo staging and cell-type ambiguity, as well as technical challenges in the collective analysis of datasets produced with different sample preparation and sequencing protocols. Here, we address these issues to assemble a complete gene expression time course spanning human pre-implantation embryogenesis. We identify key transcriptional features over developmental time and elucidate lineage-specific regulatory networks. We resolve post-hoc cell-type assignment in the blastocyst, and define robust transcriptional prototypes that capture epiblast and primitive endoderm lineages. Examination of human pluripotent stem cell transcriptomes in this framework identifies culture conditions that sustain a naïve state pertaining to the inner cell mass. Our approach thus clarifies understanding both of lineage segregation in the early human embryo and of in vitro stem cell identity, and provides an analytical resource for comparative molecular embryology.This work was supported by UK Biotechnology and Biological Sciences Research Council (BBSRC) research grant RG53615, UK Medical Research Council (MRC) programme grant G1001028, and institutional funding from the MRC and Wellcome Trust. AS is an MRC Professor

Basolateral and central amygdala differentially recruit and maintain dorsolateral striatum-dependent cocaine-seeking habits.

In the development of addiction, drug seeking becomes habitual and controlled by drug-associated cues, and the neural locus of control over behaviour shifts from the ventral to the dorsolateral striatum. The neural mechanisms underlying this functional transition from recreational drug use to drug-seeking habits are unknown. Here we combined functional disconnections and electrophysiological recordings of the amygdalo-striatal networks in rats trained to seek cocaine to demonstrate that functional shifts within the striatum are driven by transitions from the basolateral (BLA) to the central (CeN) amygdala. Thus, while the recruitment of dorsolateral striatum dopamine-dependent control over cocaine seeking is triggered by the BLA, its long-term maintenance depends instead on the CeN. These data demonstrate that limbic cortical areas both tune the function of cognitive territories of the striatum and thereby underpin maladaptive cocaine-seeking habits.This work was supported by the Fondation pour la Recherche Médicale (FRM), the United Kingdom Medical Research Council (MRC) Grant 9536855 to BJE, the AXA research fund to ABR, an INSERM Avenir and an Agence Nationale de la Recherche (ANR) grant ANR12 SAMA00201 to DB. Research was conducted within both the MRC/Wellcome Trust Behavioral and Clinical Neuroscience Institute of Cambridge and the Inserm team “Psychobiology of Compulsive Disorders”, University of Poitiers.This is the final version of the article. It was first available from NPG via http://dx.doi.org/10.1038/ncomms1008

Capture of Mouse and Human Stem Cells with Features of Formative Pluripotency.

Pluripotent cells emerge as a naive founder population in the blastocyst, acquire capacity for germline and soma formation, and then undergo lineage priming. Mouse embryonic stem cells (ESCs) and epiblast-derived stem cells (EpiSCs) represent the initial naive and final primed phases of pluripotency, respectively. Here, we investigate the intermediate formative stage. Using minimal exposure to specification cues, we derive stem cells from formative mouse epiblast. Unlike ESCs or EpiSCs, formative stem (FS) cells respond directly to germ cell induction. They colonize somatic tissues and germline in chimeras. Whole-transcriptome analyses show similarity to pre-gastrulation formative epiblast. Signal responsiveness and chromatin accessibility features reflect lineage capacitation. Furthermore, FS cells show distinct transcription factor dependencies, relying critically on Otx2. Finally, FS cell culture conditions applied to human naive cells or embryos support expansion of similar stem cells, consistent with a conserved staging post on the trajectory of mammalian pluripotency.The Cambridge Stem Cell Institute receives core funding from Wellcome and the Medical Research Council. This research was funded by the Biotechnology and Biological Sciences Research Council and the Medical Research Council of the United Kingdom. AS is a Medical Research Council Professor

Gorab is a Golgi protein required for structure and duplication of Drosophila centrioles

We demonstrate that a Drosophila Golgi protein, Gorab, is present not only in the trans-Golgi but also in the centriole cartwheel where, complexed to Sas6, it is required for centriole duplication. In addition to centriole defects, flies lacking Gorab are uncoordinated due to defects in sensory cilia, which lose their nine-fold symmetry. We demonstrate the separation of centriole and Golgi functions of Drosophila Gorab in two ways: first, we have created Gorab variants that are unable to localize to trans-Golgi but can still rescue the centriole and cilia defects of gorab null flies; second, we show that expression of C-terminally tagged Gorab disrupts Golgi functions in cytokinesis of male meiosis, a dominant phenotype overcome by mutations preventing Golgi targeting. Our findings suggest that during animal evolution, a Golgi protein has arisen with a second, apparently independent, role in centriole duplication

OCT4 induces embryonic pluripotency via STAT3 signaling and metabolic mechanisms.

OCT4 is a fundamental component of the molecular circuitry governing pluripotency in vivo and in vitro. To determine how OCT4 establishes and protects the pluripotent lineage in the embryo, we used comparative single-cell transcriptomics and quantitative immunofluorescence on control and OCT4 null blastocyst inner cell masses at two developmental stages. Surprisingly, activation of most pluripotency-associated transcription factors in the early mouse embryo occurs independently of OCT4, with the exception of the JAK/STAT signaling machinery. Concurrently, OCT4 null inner cell masses ectopically activate a subset of trophectoderm-associated genes. Inspection of metabolic pathways implicates the regulation of rate-limiting glycolytic enzymes by OCT4, consistent with a role in sustaining glycolysis. Furthermore, up-regulation of the lysosomal pathway was specifically detected in OCT4 null embryos. This finding implicates a requirement for OCT4 in the production of normal trophectoderm. Collectively, our findings uncover regulation of cellular metabolism and biophysical properties as mechanisms by which OCT4 instructs pluripotency.This work was supported by the University of Cambridge, BBSRC project grant RG74277, BB/R018588/1 and MR/R017735/1 to HS and LB respectively, MRC PhD studentship for AK and a core support grant from the Wellcome Trust and MRC to the Wellcome Trust – Medical Research Council Cambridge Stem Cell Institute

Retinal oxygenation and oxygen metabolism in Abyssinian cats with a hereditary retinal degeneration

PURPOSE. To investigate the effects of a hereditary retinal degeneration on retinal oxygenation and determine whether it is responsible for the severe attenuation of retinal circulation in hereditary photoreceptor degenerations. METHODS. Seven adult Abyssinian cats affected by hereditary retinal degeneration were studied. Oxygen microelectrodes were used to collect spatial profiles of retinal oxygenation in anesthetized animals. A one-dimensional model of oxygen diffusion was fitted to the data to quantify photoreceptor oxygen utilization (QO 2 ). RESULTS. Photoreceptor QO 2 progressively decreased until it reached zero in the end stage of the disease. Average inner retinal oxygen tension remained within normal limits at all disease stages, despite the observed progressive retinal vessel attenuation. Light affected photoreceptors normally, decreasing QO 2 by ϳ50% at all stages of the disease. CONCLUSIONS. Loss of photoreceptor metabolism allows choroidal oxygen to reach the inner retina, attenuating the retinal circulation in this animal model of retinitis pigmentosa (RP) and probably also in human RP. As the degeneration progresses, there is a strong relationship between changes in the a-wave of the ERG and changes in rod oxidative metabolism, indicating that these two functional measures change together. 2-6 We hypothesized that the pronounced vasoconstriction results from decreased oxygen utilization by the outer retina after photoreceptor loss. Because less choroidal oxygen is used by the photoreceptors, it should reach the inner retina, where oxygen is known to cause constriction of retinal vessels. 10 Vessels do not simply constrict. Eventually they are lost in animals with photoreceptor degenerations. 6 These studies suggest not only a physiological role of oxygen, but a trophic (or antitrophic) one as well. In the present study, we further examined the hypothesis that oxygen from the choroid is responsible for attenuation of the retinal circulation, by characterizing changes in retinal oxygenation and oxidative metabolism during the progression of a feline hereditary retinal degeneration in a colony of Abyssinian cats. 3 The autosomal recessive genetic defect in this animal is not yet known, but it exhibits a slow rod-cone degeneration similar to human RP. Using measurements of intraretinal oxygen tension (PO 2 ) in these animals and our mathematical model of oxygen diffusion and utilization, 13 A preliminary report of these results has been presented (Linsenmeier RA, et al. IOVS 2000;41:ARVO Abstract 4721). METHODS Animals Eight Abyssinian cats affected by a hereditary retinal degeneration were studied. These are the same cats in which an ERG study, reported in this issue, was also performed. 13 3,13 The hematocrit from one stage 3 animal was low because of surgical complications and continued to decrease during the experiment. In this animal, the earlier choroidal PO 2 and the inner retinal PO 2 , which were in the normal range, are included in the reported data, but oxygen consumption data and later choroidal PO 2 data from this animal were not used. Electrode problems prevented data collection in one stage 2 animal. Data from animals with retinal degeneration were compared to similar data obtained from normal cats

Metabolic control of DNA methylation in naive pluripotent cells.

Naive epiblast and embryonic stem cells (ESCs) give rise to all cells of adults. Such developmental plasticity is associated with genome hypomethylation. Here, we show that LIF-Stat3 signaling induces genomic hypomethylation via metabolic reconfiguration. Stat3-/- ESCs show decreased α-ketoglutarate production from glutamine, leading to increased Dnmt3a and Dnmt3b expression and DNA methylation. Notably, genome methylation is dynamically controlled through modulation of α-ketoglutarate availability or Stat3 activation in mitochondria. Alpha-ketoglutarate links metabolism to the epigenome by reducing the expression of Otx2 and its targets Dnmt3a and Dnmt3b. Genetic inactivation of Otx2 or Dnmt3a and Dnmt3b results in genomic hypomethylation even in the absence of active LIF-Stat3. Stat3-/- ESCs show increased methylation at imprinting control regions and altered expression of cognate transcripts. Single-cell analyses of Stat3-/- embryos confirmed the dysregulated expression of Otx2, Dnmt3a and Dnmt3b as well as imprinted genes. Several cancers display Stat3 overactivation and abnormal DNA methylation; therefore, the molecular module that we describe might be exploited under pathological conditions