Summary The known molecular heterogeneity among pluripotent embryonic stem cells (ESCs) in culture may be maladaptive or a beneficial determinant of normal biology. In other systems such as hair and intestine, normal and dynamic heterogeneity among stem cells is necessary for balancing stem cell division and quiescence in order to maintain the stem cell pool. In ESCs, Sox2, Oct4 and Nanog (the SON genes) critically regulate pluripotency but their role in stem cell heterogeneity or pool maintenance is not clear. We previously reported an additional level of gene expression, the unexpected differential expression of mRNA coding region (CDS) and cognate 3′untranslated region (3UTR) components, often revealing novel developmental patterns. Here we show dramatic, patterned, concentric, differential expression of pluripotent gene 3UTR and CDS mRNA components in both undifferentiated ESCs in culture and in developing human and mouse embryos. This differential expression reveals robust pluripotent ESC heterogeneity that provides the underpinnings of a previously unrecognized ESC cell niche structure. A hallmark of the niche is the presence of cells expressing high levels of Nanog 3UTR but only low or no Nanog CDS at structure perimeters, We used a novel bioinformatic approach combining the 3UTR to CDS ratio and gene expression, and identified unique ESC clusters. High Nanog 3UTR cells have lower cell cycle activity and higher quiescence than other ESCs and express a distinct complement of cellular histone marks. These quiescent perimeter cells exchange with interior domains, opening the possibility that they act as cell replenishment reservoirs in the niche or the embryo. Functionally, the Nanog 3UTR, independent of its CDS, is critical, as its selective deletion decreases ESC cell division while increasing trophectoderm markers. Lastly, self-organization of ESCs according to pluripotent gene differential 3UTR and CDS expression is modified by activin signaling, indicating extrinsic modulation of the niche structure. Pluripotent embryonic stem cells, actively display regulated, dynamic differential expression of SON gene mRNA components, allowing the formation of a heterogenous, ordered, stem cell niche bordered by quiescent hub stem cells, thus illuminating the role of SON genes in pluripotent embryonic stem cell pool maintenance. More generally this suggests that differential mRNA component expression may widely define cell types that exist in non-permanent, different states in either development or disease.

Mature mRNA molecules are expected to be comprised of a 5'UTR, a 3'UTR and a coding region (CDS). Unexpectedly, however, there have been multiple recent reports of widespread differential expression of mRNA 3'UTRs and their cognate coding regions (CDS), reflecting the expression of isolated 3'UTRs (i3'UTRs); these i3'UTRs can be highly expressed, often in reciprocal patterns to their cognate CDS. As with other long non-coding (lncRNAs), isolated 3'UTRs are likely to play an important role in gene regulation, but little is known about the contexts in which they are deployed. To illuminate the functions of i3'UTRs, here we carry out in vitro, in vivo and in silico analyses of differential 3'UTR/CDS mRNA ratio usage across tissues, development and cell state changes both for a select list of developmentally important genes as well as by unbiased transcriptome-wide analyses. Across two developmental paradigms we find a distinct switch from high i3'UTR expression for stem cell related genes in proliferating cells to high CDS for these genes in newly differentiated cells. Unbiased transcriptome analysis across multiple gene sets shows that regardless of tissue, genes with high 3'UTR to CDS ratios belong predominantly to gene ontology categories related to cell-type specific functions. In contrast, the gene ontology categories of genes with low 3'UTR to CDS ratios are similar across tissues and relate to common cellular functions. We further show that, at least for some genes, traditional transcriptional start site genomic elements correspond to identified RNAseq 3'UTR peak regions, suggesting that some i3'UTRs may be generated by de novo transcription. Our results provide critical information from which detailed hypotheses for individual i3'UTRs can be tested, with a common theme that i3'UTRs appear poised to regulate cell-specific gene expression and state.

Preliminary results investigating the microstructure, bonding and effect of beryllium oxide formation on retention in the JET ITER-like wall beryllium tiles, are presented. The tiles have been investigated by several techniques: Scanning Electron Microscopy (SEM) equipped with Energy Dispersive X-ray (EDX), Transmission Electron microscopy (TEM) equipped with EDX and Electron Energy Loss Spectroscopy (EELS), Raman Spectroscopy and Thermal Desorption Spectroscopy (TDS). This paper focuses on results from melted materials of the dump plate tiles in JET. From our results and the literature, it is concluded, beryllium can form micron deep oxide islands contrary to the nanometric oxides predicted under vacuum conditions. The deepest oxides analyzed were up to 2-micron thicknesses. The beryllium Deuteroxide (BeOxDy) bond was found with Raman Spectroscopy. Application of EELS confirmed the oxide presence and stoichiometry. Literature suggests these oxides form at temperatures greater than 700 °C where self-diffusion of beryllium ions through the surface oxide layer can occur. Further oxidation is made possible between oxygen plasma impurities and the beryllium ions now present at the wall surface. Under Ultra High Vacuum (UHV) nanometric Beryllium oxide layers are formed and passivate at room temperature. After continual cyclic heating (to the point of melt formation) in the presence of oxygen impurities from the plasma, oxide growth to the levels seen experimentally (approximately two microns) is proposed. This retention mechanism is not considered to contribute dramatically to overall retention in JET, due to low levels of melt formation. However, this mechanism, thought the result of operation environment and melt formation, could be of wider concern to ITER, dependent on wall temperatures.

Beryllium oxide (BeO) and deuteroxide (BeOxDy) have been found on the melted zone of a beryllium tile extracted from the upper dump plate of JET-ILW (2011–2012 campaign). Results have been obtained using Raman microscopy, which is sensitive to both the chemical bond and crystal structure, with a micrometric lateral resolution. BeO is found with a wurtzite crystal structure. BeOxDy is found as three different types which are not the β-phase but behaves as molecular species like Be(OD)2, O(Be-D)2 and DBeOD. The presence of a small amount of trapped D2O is also suspected. Our results therefore strongly suggest that D trapping occurs after melting through the formation of deuteroxides. The temperature increase favors the formation of crystal BeO which favors deuterium trapping through OD bonding.

The ITER Remote Experimentation Centre (REC) project (one of the three sub-projects of the International Fusion Energy Research Centre (IFERC)) is progressing under the agreement between the Government of Japan and the European Atomic Energy Community for the joint implementation of the Broader Approach (BA) activities in the field of fusion energy research. The objectives of the REC activity are to identify the functions and solve the technical issues for the construction of the REC for ITER at Rokkasho, and to develop the remote experiment system and verify the functions required for remote experimentation by using the Satellite Tokamak (JT-60SA) facilities to facilitate the future exploitation of ITER and JT-60SA. The functions of REC will be tested, and the total system will be demonstrated using JT-60SA and existing facilities in the EU, such as JET and WEST. The hardware of the REC has been prepared in Rokkasho Japan, which has the remote experiment room with a large video wall to show the plasma and operation status, IT equipment and a storage system by the reuse of the Helios supercomputer tape library. A broadband network infrastructure of 10Gbps has been installed connected to SINETS. Using this network system, fast data transfer from ITER to REC was examined in 2016, and the transfer of the data volumes expected for the initial ITER experiments has been demonstrated. A secure remote experimentation system has been developed, using JT-60SA, that has functions for preparing and setting of shot parameters, viewing the status of control data, streaming of the plasma status, data-exchange function of shot events, and monitoring of the facility operation. Remote data analysis techniques, data visualisation software, a documentation management and experiment planning system and numerical simulation codes for the preparation and performance estimation of discharges have also been developed.

This paper presents the first application to real JET data of the new equilibrium code NICE which enables the consistent resolution of the inverse equilibrium reconstruction problem in the framework of non-linear free-boundary equilibrium coupled to the Stokes model equation for polarimetry. The conducted numerical experiments enable first of all to validate NICE by comparing it to the well-established EFIT code on 4 selected high performance shots. Secondly the results indicate that the fit to polarimetry measurements clearly benefits from the use of Stokes vector measurements compared to the classical case of Faraday measurements, and that the reconstructed p' and ff' profiles are better constrained with smaller error bars and are closer to the profiles reconstructed by EFTM, the EFIT JET code using internal MSE constraints.

The phenomena of retention and de-trapping of deuterium (D) and tritium (T) in plasma facing components (PFC) and supporting structures must be understood in order to limit or control total T inventory in larger future fusion devices such as ITER, DEMO and commercial machines. The goal of this paper is to present details of the thermal desorption spectrometry (TDS) system applied in total fuel retention assessment of PFC at the Joint European Torus (JET). Examples of TDS results from beryllium (Be) wall tile samples exposed to JET plasma in PFC configuration mirroring the planned ITER PFC is shown for the first time. The method for quantifying D by comparison of results from a sample of known D content was confirmed acceptable. The D inventory calculations obtained from Ion Beam Analysis (IBA) and TDS agree well within an error associated with the extrapolation from very few data points to a large surface area.

Progress in understanding lower hybrid current drive (LHCD) at high density has been made through experiments and modeling, which is encouraging given the need for an efficient off-axis current profile control technique in burning plasma. By reducing the wall recycling of neutrals, the edge temperature is increased and the effect of parametric instability (PI) and collisional absorption (CA) is reduced, which is beneficial for increasing the current drive efficiency. Strong single pass absorption is preferred to prevent CA and high LH operating frequency is essential for wave propagation to the core region at high density, presumably to mitigate the effect of PI. The dimensionless parameter that characterizes LH wave accessibility and wave refraction for the experiments in this joint study is shown to bracket the region in parameter space where ITER LHCD experiments will operate in the steady state scenario phase. Further joint experiments and cross modeling are necessary to understand the LHCD physics in weak damping regimes which would increase confidence in predictions for ITER where the absorption is expected to be strong.

In order to extend the investigation of the plasma cleaning of ITER first mirrors, a set of molybdenum mirrors was coated in a laboratory with ITER-relevant contaminants, namely beryllium and tungsten. Different coating techniques as well as several contaminant compositions were used to ensure a large variety of films to clean, completing a previous study conducted on mirrors exposed in the JET ITER-like wall (tokamak deposits) []. Due to the toxicity of beryllium, the samples were treated in a vacuum chamber specially built for this purpose. The cleaning was performed using capacitively coupled RF plasma and evaluated by performing reflectivity measurements, scanning electron microscopy, x-ray photoelectron spectroscopy and ion beam analysis. The removal of all types of contaminants was achieved by using different plasma compositions (argon, helium and mixtures of the two) with various ion energies (from 200–600 eV) and in some cases the mirror’s reflectivity was restored towards initial values. Pure helium discharges were capable of removing mixed beryllium/tungsten layers and oxidized molybdenum. In addition, no significant increase in the diffuse reflectivity of the mirrors was observed for the helium cleaning, though this was the case for some samples cleaned with argon. Helium is therefore appropriate for cleaning all mirrors in ITER leading to a possible cleaning regime where the entire vessel is filled with He and all mirrors are cleaned simultaneously without damaging their surfaces.

Mature messenger RNAs (mRNAs) consist of coding sequence (CDS) and 5' and 3' UTRs, typically expected to show similar abundance within a given neuron. Examining mRNA from defined neurons, we unexpectedly show extremely common unbalanced expression of cognate 3' UTR and CDS sequences; many genes show high 3' UTR relative to CDS, others show high CDS to 3' UTR. In situ hybridization (19 of 19 genes) shows a broad range of 3' UTR-to-CDS expression ratios across neurons and tissues. Ratios may be spatially graded or change with developmental age but are consistent across animals. Further, for two genes examined, a 3' UTR-to-CDS ratio above a particular threshold in any given neuron correlated with reduced or undetectable protein expression. Our findings raise questions about the role of isolated 3' UTR sequences in regulation of protein expression and highlight the importance of separately examining 3' UTR and CDS sequences in gene expression analyses.

There are two main subgroups of midbrain dopaminergic (DA) neurons: the more medially located ventral tegmental area (VTA) DA neurons, which have axons that innervate the ventral-lateral (VL) striatum, and the more laterally located substantia nigra (SN) DA neurons, which preferentially degenerate in Parkinson's disease (PD) and have axons that project to the dorsal-medial (DM) striatum. DA axonal projections in the striatum are not discretely localized and they arborize widely, however they do not stray from one zone to the other so that VTA axons remain in the VL zone and SN axons in the DM zone. Here we provide evidence that Netrin-1 acts in a novel fashion to topographically pattern midbrain DA axons into these two striatal zones by means of a gradient of Netrin-1 in the striatum and by differential attraction of the axons to Netrin-1. Midbrain DA neurons are attracted to the striatum in culture and this attraction is blocked by an anti-DCC (Netrin receptor) antibody. Mechanistically, outgrowth of both VTA and SN DA axons is stimulated by Netrin-1, but the two populations of DA axons respond optimally to overlapping but distinct concentrations of Netrin-1, with SN axons preferring lower concentrations and VTA axons preferring higher concentrations. In vivo this differential preference is closely mirrored by differences in Netrin-1 expression in their respective striatal target fields. In vivo in mice lacking Netrin-1, DA axons that reach the striatum fail to segregate into two terminal zones and to fully innervate the striatum. Our results reveal novel actions for Netrin-1 and provide evidence for a mechanism through which DA axons can selectively innervate one of two terminal zones in the striatum but have free reign to arborize widely within a terminal zone.

IntroductionThe genetics underlying the idiopath-ic hypercalciuria leading to calcium-containing renal stones remains elus-ive. The discovery of rare monogenic tubulopathies, often leading to hype-rcalciuria, has increased our underst-anding of tubular physiology and pat-ho-physiology. However, insights int-o idiopathic calcium stone formation have not been gained from these dis-orders. The aim of this study is to ex-amine CYP24A1 mutations in cohorts of patients with calcium nephrolithi-asis.Materials and MethodsWe examined two cohorts of stone-forming patients for mutations in CYP24A1, which encodes the vitamin D24-hydroxylase enzyme. The first cohort had a biochemical phenotype of suppressed parathyroid hormone and high normal serum calcium, whilst the second cohort had a hypercalciuria phenotype. We did not identify bi-allelic sequence variants in CYP24A1 in our cohorts.ResultsIn cohort 1, we identified 9 known s-equence variants. In cohort 2 we ide-ntified 7 known sequence variants.ConclusionCYP24A1 mutations remain a rare cause of calcium nephrolithiasis and hypercalciuria.

Joubert syndrome (JBTS) is an inherited ciliopathy leading to a cerebellum-retinal-renal syndrome.Recent genetic advances have allowed positional cloning and identification of numerous JBTS genes.CEP290, one of the JBTS genes identified, (alias NPHP6) encodes a centrosomal protein and accounts for 7% of patients with Joubert syndrome.We have identified a murine Embryonic Stem (ES) cell line containing a Cep290 "gene trap" using data base searches.ES cells were cultured before injecting into murine blastocysts to create chimaeric mice.Chimeras were bred to produce viable, healthy heterozygous mutant mice.Heterozygous mutant mice have been intercrossed to produce mice homozygous for the Cep290 truncating mutation.Cep290-/animals (homozygous for the gene trap Cep290) exhibit a cortico-medullary cystic kidney disease commencing from birth.Histological examination reveals that these cysts are collecting duct in origin, staining positively for aquaporin-2 and -3.In this study the cilia were investigated in cystic Cep290-/animals using Electron Microscopy (EM) analysis.Scanning electron microscopy (SEM) identified that cilia were evident within renal tubules in Cep290-/animals.Once cilia were identified in Cep290-/animals Transmission Electron Microscopy (TEM) was carried out to investigate cross sections of the collecting duct cilium in cystic and non-cystic kidneys.TEM analysis identified tubular basement membrane disruptions in Cep290-/animals.The Cep290-/mouse described provides an excellent model to investigate the mechanisms involved in cyst formation and to test novel therapeutic agents.

Dopaminergic neurons derived from human embryonic stem cells will be useful in future transplantation studies of Parkinson's disease patients. As newly generated neurons must integrate and reconnect with host cells, the ability of hESC-derived neurons to respond to axon guidance cues will be critical. Both Netrin-1 and Slit-2 guide rodent embryonic dopaminergic (DA) neurons in vitro and in vivo, but very little is known about the response of hESC-derived DA neurons to any axonal guidance cues. Here we examined the ability of Netrin-1 and Slit-2 to affect human ESC DA axons in vitro. hESC DA neurons mature over time in culture with the developmental profile of DA neurons in vivo, including expression of the DA neuron markers FoxA2, En-1 and Nurr-1, and receptors for both Netrin and Slit. hESC DA neurons respond to exogenous Netrin-1 and Slit-2, showing an increased responsiveness to Netrin-1 as the neurons mature in culture. These responses were maintained in the presence of pro-inflammatory cytokines that might be encountered in the diseased brain. These studies are the first to evaluate and confirm that suitably matured human ES-derived DA neurons can respond appropriately to axon guidance cues.

alpha-Synuclein (alpha-Syn) is a 14 kDa protein of unknown function that has been implicated in the pathophysiology of Parkinson's disease (PD). Here, we show that alpha-Syn-/- mice are viable and fertile, exhibit intact brain architecture, and possess a normal complement of dopaminergic cell bodies, fibers, and synapses. Nigrostriatal terminals of alpha-Syn-/- mice display a standard pattern of dopamine (DA) discharge and reuptake in response to simple electrical stimulation. However, they exhibit an increased release with paired stimuli that can be mimicked by elevated Ca2+. Concurrent with the altered DA release, alpha-Syn-/- mice display a reduction in striatal DA and an attenuation of DA-dependent locomotor response to amphetamine. These findings support the hypothesis that alpha-Syn is an essential presynaptic, activity-dependent negative regulator of DA neurotransmission.

Sonic Hedgehog (Shh) is a secreted protein that controls cell fate and mitogenesis in the developing nervous system. Here we show that a constitutively active form of Smoothened (Smo-M2) mimics concentration-dependent actions of Shh in the developing neural tube, including activation of ventral marker genes ( HNF3β , patched , Nkx2.2 , netrin-1 ), suppression of dorsal markers ( Pax-3 , Gli-3 , Ephrin A5 ) and induction of ventral neurons (dopaminergic, serotonergic) and ventrolateral motor neurons (Islet-1+, Islet-2+, HB9+) and interneurons (Engrailed-1+, CHX10+). Furthermore, Smo-M2's patterning activities were cell autonomous, occurring exclusively in cells expressing Smo-M2 . These findings suggest that Smo is a key signaling component in the Hh receptor and that Shh patterns the vertebrate nervous system as a morphogen, rather than through secondary relay signals.

The glial cell line-derived neurotrophic factor (GDNF) family of ligands binds to lipid anchored proteins termed GDNF family receptor (GFR)alphas, and then activates the RET receptor tyrosine kinase, by ligand GFRalpha. The binding of soluble GFRalphas to transfected cells suggested that different GFRalphas were dedicated to particular ligands, with GDNF acting primarily or entirely through GFRalpha1, and neurturin (NTN), through GFRalpha2. More recent evidence has suggested the possibility of cross-talk between these ligands and the two receptors. We examined here whether crosstalk between the GDNF ligands and the GFRalphas is biologically relevant, using midbrain dopaminergic, and parasympathetic, submandibular gland neurons. By biochemical and genetic addition and/or deletion of GFRalpha1 and 2, we show that in both neuronal cell types, robust biological activities of GDNF or NTN can be mediated by either GFRalpha1 or GFRalpha2, although GDNF is slightly more potent in dopaminergic (DA) neurons which normally express GFRalpha1, and NTN in submandibular neurons which normally express GFRalpha2. Throughout the body, GDNF and NTN are likely to have important biological actions on both GFRalpha1- and GFRalpha2-expressing cells.

Our limited life expectancy, and inevitable death, is brought about by the ongoing deterioration of the body's essential cell types, tissues, and organs. Thus, either a reliable source of replacement organs or cells or a method to continually induce the repair of endogenous organs would profoundly affect the current statistics of aging-related disease and death. The prospect of greater longevity, associated with an increased expectancy of good health, underlies the intense excitement surrounding work on embryonic stem cells. Stem cells are unique in that they have the capacity for unlimited self-renewal along with the ability to produce multiple different types of terminally differentiated descendants. Thus, they might serve as a multipotential source of replacement body parts in cardiovascular, autoimmune, Alzheimer's, or Parkinson's disease or for diabetes, osteoporosis, cancer, spinal cord injury, or birth defects. Identifying the extrinsic signals that control lineage specific differentiation of stem cells to allow the generation of specific tissues in vitro and, eventually in vivo, is a critical issue in stem cell research.