Stable gene replacement in barley by targeted double-strand break induction

Gene targeting is becoming an important tool for precision genome engineering in plants. During gene replacement, a variant of gene targeting, transformed DNA integrates into the genome by homologous recombination (HR) to replace resident sequences. We have analysed gene targeting in barley (Hordeum vulgare) using a model system based on double-strand break (DSB) induction by the meganuclease I-SceI and a transgenic, artificial target locus. In the plants we obtained, the donor construct was inserted at the target locus by homology-directed DNA integration in at least two transformants obtained in a single experiment and was stably inherited as a single Mendelian trait. Both events were produced by one-sided integration. Our data suggest that gene replacement can be achieved in barley with a frequency suitable for routine application. The use of a codon-optimized nuclease and co-transfer of the nuclease gene together with the donor construct are probably the components important for efficient gene targeting. Such an approach, employing the recently developed synthetic nucleases/nickases that allow DSB induction at almost any sequence of a genome of interest, sets the stage for precision genome engineering as a routine tool even for important crops such as barley

Temporal lobe white matter asymmetry and language laterality in epilepsy patients.

Recent studies using diffusion tensor imaging (DTI) have advanced our knowledge of the organization of white matter subserving language function. It remains unclear, however, how DTI may be used to predict accurately a key feature of language organization: its asymmetric representation in one cerebral hemisphere. In this study of epilepsy patients with unambiguous lateralization on Wada testing (19 left and 4 right lateralized subjects; no bilateral subjects), the predictive value of DTI for classifying the dominant hemisphere for language was assessed relative to the existing standard-the intra-carotid Amytal (Wada) procedure. Our specific hypothesis is that language laterality in both unilateral left- and right-hemisphere language dominant subjects may be predicted by hemispheric asymmetry in the relative density of three white matter pathways terminating in the temporal lobe implicated in different aspects of language function: the arcuate (AF), uncinate (UF), and inferior longitudinal fasciculi (ILF). Laterality indices computed from asymmetry of high anisotropy AF pathways, but not the other pathways, classified the majority (19 of 23) of patients using the Wada results as the standard. A logistic regression model incorporating information from DTI of the AF, fMRI activity in Broca\u27s area, and handedness was able to classify 22 of 23 (95.6%) patients correctly according to their Wada score. We conclude that evaluation of highly anisotropic components of the AF alone has significant predictive power for determining language laterality, and that this markedly asymmetric distribution in the dominant hemisphere may reflect enhanced connectivity between frontal and temporal sites to support fluent language processes. Given the small sample reported in this preliminary study, future research should assess this method on a larger group of patients, including subjects with bi-hemispheric dominance

The Large Enriched Germanium Experiment for Neutrinoless Double Beta Decay (LEGEND)

The observation of neutrinoless double-beta decay (0${\nu}{\beta}{\beta}$) would show that lepton number is violated, reveal that neutrinos are Majorana particles, and provide information on neutrino mass. A discovery-capable experiment covering the inverted ordering region, with effective Majorana neutrino masses of 15 - 50 meV, will require a tonne-scale experiment with excellent energy resolution and extremely low backgrounds, at the level of $\sim$0.1 count /(FWHM$\cdot$t$\cdot$yr) in the region of the signal. The current generation $^{76}$Ge experiments GERDA and the MAJORANA DEMONSTRATOR utilizing high purity Germanium detectors with an intrinsic energy resolution of 0.12%, have achieved the lowest backgrounds by over an order of magnitude in the 0${\nu}{\beta}{\beta}$ signal region of all 0${\nu}{\beta}{\beta}$ experiments. Building on this success, the LEGEND collaboration has been formed to pursue a tonne-scale $^{76}$Ge experiment. The collaboration aims to develop a phased 0${\nu}{\beta}{\beta}$ experimental program with discovery potential at a half-life approaching or at $10^{28}$ years, using existing resources as appropriate to expedite physics results.Comment: Proceedings of the MEDEX'17 meeting (Prague, May 29 - June 2, 2017

Assessment at the boundaries: service learning as case study

This is the accepted version of the following article: Shay, S. 2008. Assessment at the boundaries: service learning as case study. British Educational Research Journal. 34(4): 525-540. DOI: 10.1080/01411920701609406., which has been published in final form at http://dx.doi.org/10.1080/01411920701609406.This article explores the value systems which inform assessment practices in higher education, specifically how particular forms of knowledge valued in the curriculum shape and constrain assessment practices. The data for this article is drawn from two courses which participated in a service learning research and development project at the University of Cape Town. Drawing on Pierre Bourdieu and Basil Bernstein, the article argues that the location of these courses—within the field of higher education and a particular kind of institution, faculty and department—shapes their assessment systems, practices and outcomes in certain ways. What is valued in this field (Bourdieu) is a form of knowledge production which requires students 'to step out of the particularities'. This form of knowledge operates as a regulative discourse, constituting what counts as legitimate. Using the assessment system as a 'window', this article explores how these service learning courses constitute and are constituted by the regulative discourse of the field. While the constraints of the field are powerful, this project offers some hopeful signs of forms of curriculum, pedagogy and assessment that, at the very least, name and challenge these underlying value systems

Development of methods for the preparation of radiopure ⁸²Se sources for the SuperNEMO neutrinoless double-beta decay experiment

A radiochemical method for producing 82Se sources with an ultra-low level of contamination of natural radionuclides (40K, decay products of 232Th and 238U) has been developed based on cation-exchange chromatographic purification with reverse removal of impurities. It includes chromatographic separation (purification), reduction, conditioning (which includes decantation, centrifugation, washing, grinding, and drying), and 82Se foil production. The conditioning stage, during which highly dispersed elemental selenium is obtained by the reduction of purified selenious acid (H2SeO3) with sulfur dioxide (SO2) represents the crucial step in the preparation of radiopure 82Se samples. The natural selenium (600 g) was first produced in this procedure in order to refine the method. The technique developed was then used to produce 2.5 kg of radiopure enriched selenium (82Se). The produced 82Se samples were wrapped in polyethylene (12 μm thick) and radionuclides present in the sample were analyzed with the BiPo-3 detector. The radiopurity of the plastic materials (chromatographic column material and polypropylene chemical vessels), which were used at all stages, was determined by instrumental neutron activation analysis. The radiopurity of the 82Se foils was checked by measurements with the BiPo-3 spectrometer, which confirmed the high purity of the final product. The measured contamination level for 208Tl was 8-54 μBq/kg, and for 214Bi the detection limit of 600 μBq/kg has been reached.</p

Effect of Mouth Rinsing and Ingestion of Carbohydrate Solutions on Mood and Perceptual Responses During Exercise

Background: The aim of this study was to investigate whether mouth rinsing or ingesting carbohydrate (CHO) solutions impact on perceptual responses during exercise. Methods: Nine moderately trained male cyclists underwent a 90-min glycogen-reducing exercise, and consumed a low CHO meal, prior to completing an overnight fast. A 1-h cycle time trial was performed the following morning. Four trials, each separated by 7days, were conducted in a randomized, counterbalanced study design: 15% CHO mouth rinse (CHOR), 7.5% CHO ingestion (CHOI), placebo mouth rinse (PLAR) and placebo ingestion (PLAI). Solution volumes (1.5ml·g-1 ingestion trials and 0.33ml·kg-1 rinsing trials) were provided after every 12.5% of completed exercise. Perceptual scales were used to assess affective valence (feeling scale, FS), arousal (felt arousal scale, FAS), exertion (ratings of perceived exertion, RPE) and mood (profile of mood states, POMS) before, during and immediately after exercise. Results: There was no difference in RPE (CHOI, 14.0±9; CHOR, 14.2±.7; PLAI, 14.6±1.8; PLAR, 14.6±2.0; P=0.35), FS (CHOI, 0.0±1.7; CHOR, -0.2±1.5; PLAI, -0.8±1.4; PLAR, -0.8±1.6; P0.15), or FAS (CHOI, 3.6±1.1; CHOR, 3.5±1.0; PLAI, 3.4±1.4; PLAR, 3.3±1.3; P=725) scores between trials. While overall POMS score did not appear to differ between trials, the 'vigour' subscale indicated that CHOI may facilitate the maintenance of 'vigour' scores over time, in comparison to the steady decline witnessed in other trials (P=0.04). There was no difference in time trial performance between trials (CHOI, 65.3±4.8min; CHOR, 68.4±3.9min; PLAI, 68.7±5.3min; PLAR, 68.3±5.2min; P=0.21) but power output was higher in CHOI (231.0±33.2 W) relative to other trials (221-223.6 W; Plt0.01). Conclusions: In a CHO-reduced state, mouth rinsing with a CHO solution did not impact on perceptual responses during high-intensity exercise in trained cyclists and triathletes. On the other hand CHO ingestion improved perceived ratings of vigour and increased power output during exercise

Preferential Localization of Human Origins of DNA Replication at the 5′-Ends of Expressed Genes and at Evolutionarily Conserved DNA Sequences

Replication of mammalian genomes requires the activation of thousands of origins which are both spatially and temporally regulated by as yet unknown mechanisms. At the most fundamental level, our knowledge about the distribution pattern of origins in each of the chromosomes, among different cell types, and whether the physiological state of the cells alters this distribution is at present very limited.We have used standard λ-exonuclease resistant nascent DNA preparations in the size range of 0.7–1.5 kb obtained from the breast cancer cell line MCF–7 hybridized to a custom tiling array containing 50–60 nt probes evenly distributed among genic and non-genic regions covering about 1% of the human genome. A similar DNA preparation was used for high-throughput DNA sequencing. Array experiments were also performed with DNA obtained from BT-474 and H520 cell lines. By determining the sites showing nascent DNA enrichment, we have localized several thousand origins of DNA replication. Our major findings are: (a) both array and DNA sequencing assay methods produced essentially the same origin distribution profile; (b) origin distribution is largely conserved (>70%) in all cell lines tested; (c) origins are enriched at the 5′ends of expressed genes and at evolutionarily conserved intergenic sequences; and (d) ChIP on chip experiments in MCF-7 showed an enrichment of H3K4Me3 and RNA Polymerase II chromatin binding sites at origins of DNA replication.Our results suggest that the program for origin activation is largely conserved among different cell types. Also, our work supports recent studies connecting transcription initiation with replication, and in addition suggests that evolutionarily conserved intergenic sequences have the potential to participate in origin selection. Overall, our observations suggest that replication origin selection is a stochastic process significantly dependent upon local accessibility to replication factors

Cryogenic characterization of a LiAlO 2 crystal and new results on spin-dependent dark matter interactions with ordinary matter: CRESST Collaboration

In this work, a first cryogenic characterization of a scintillating LiAlO 2 single crystal is presented. The results achieved show that this material holds great potential as a target for direct dark matter search experiments. Three different detector modules obtained from one crystal grown at the Leibniz-Institut für Kristallzüchtung (IKZ) have been tested to study different properties at cryogenic temperatures. Firstly, two 2.8 g twin crystals were used to build different detector modules which were operated in an above-ground laboratory at the Max Planck Institute for Physics (MPP) in Munich, Germany. The first detector module was used to study the scintillation properties of LiAlO 2 at cryogenic temperatures. The second achieved an energy threshold of (213.02 ± 1.48) eV which allows setting a competitive limit on the spin-dependent dark matter particle-proton scattering cross section for dark matter particle masses between 350MeV/c2 and 1.50GeV/c2. Secondly, a detector module with a 373 g LiAlO 2 crystal as the main absorber was tested in an underground facility at the Laboratori Nazionali del Gran Sasso (LNGS): from this measurement it was possible to determine the radiopurity of the crystal and study the feasibility of using this material as a neutron flux monitor for low-background experiments. © 2020, The Author(s)

Lithium-Containing Crystals for Light Dark Matter Search Experiments

In the current direct dark matter search landscape, the leading experiments in the sub-GeV mass region mostly rely on cryogenic techniques which employ crystalline targets. One attractive type of crystals for these experiments is those containing lithium, due to the fact that 7Li is an ideal candidate to study spin-dependent dark matter interactions in the low mass region. Furthermore, 6Li can absorb neutrons, a challenging background for dark matter experiments, through a distinctive signature which allows the monitoring of the neutron flux directly on site. In this work, we show the results obtained with three different detectors based on LiAlO2, a target crystal never used before in cryogenic experiments