Gene editing towards hypoimmunogenic gluten proteins in wheat

One to two per cent of the population has Coeliac Disease (CD), an immune reaction to gluten. Wheat grains contain gluten, a mixture of glutenin and gliadin proteins, which build a network that gives wheat bread its unique properties and quality. Most gliadins and part of the glutenins contain immunogenic epitopes, which are the actual trigger of the immune reaction. A gluten-free diet, excluding wheat, barley and rye, is currently the only remedy for coeliac patients. This diet is not easy to adhere to, partly because wheat gluten is added to many processed food products for their viscoelastic properties. In addition, gluten-free products typically require the inclusion of numerous additives to adjust their texture and taste, resulting in products that are often less healthy than gluten-based equivalents, and more expensive. Thus there is a need to develop healthier food products for coeliac patients. One can now use CRISPR/Cas to remove all gluten genes, which would produce a gluten-free wheat which is interesting for many people who want to eat gluten-free, but it would have an inferior baking quality. In this PhD thesis, Aurélie Jouanin describes an alternative use of gene editing with CRISPR/Cas9 to precisely modify gliadin genes and strip them of immunogenic epitopes, to develop wheat with safe gluten. As a proof of principle she generated wheat plants in which some gliadin genes were modified or removed. These edited wheat plants are not yet safe for CD patients, as there is a large number of gluten genes present in wheat and not all gluten genes have been targeted. She has therefore also developed high-throughput methods to determine which genes have been modified and which remain to be edited in future steps towards a safe wheat variety. The regulation of gene editing as Genetic Modification (GM) in Europe is currently a hot topic. She discusses the inconsistency of the European regulation of gene editing in plants by displaying the similarities of mutations in gliadin genes that are obtained using random γ-irradiation mutagenesis and those obtained by targeted mutagenesis using gene editing. The former is being exempted from GM regulation while the latter is being subjected to GM regulation, following the ruling of the European Court of Justice in July 2018. She advises the European Commission to review its position on the matter and to regulate gene editing based on scientific evidence regarding the generated products, and on the innovation principle as part of responsible research innovation initiatives. Finally, she discusses some recently developed CRISPR approaches that may result in faster development of wheat with gluten that do not cause an immune reaction. The benefits and potential risks related to gene-edited wheat with gluten that do not cause an immune reaction are discussed. The requirement for producing and processing these varieties are touched upon. New test methods for food products need to be developed, since the current gluten-free tests will not be able to distinguish gluten stripped of immunogenic epitopes from regular gluten.</p

Optimización de la regeneración in vitro del olmo utilizando explantes de hoja y evaluación del proceso en experimentos de transformación

We set up a regeneration process for the elm hybrid ‘Sapporo’ resistant to Dutch Elm Disease, using leaf discs from in vitro grown plants and tested it in transformation experiments. Two steps were needed : bud induction (3 weeks) followed by bud elongation (4 weeks). Bud meristems initiated near the cut ribs, from several tissues (phloem, procambium, rib upper parenchyma) except epidermis. Regeneration depended on the use of agarose (6 g/l LSM) and diluted MS medium (1/2). The best results (11-14 buds/explant) were obtained in presence of 0.1 μM TDZ and 0.06 μM IAA and when maltose (55-110 mM) or sorbitol (110 mM) were used as carbohydrate sources. During shoot elongation TDZ should be decreased to 0.01 μM or replaced by 1-2 μM BAP and 1.4 μM of GA3 added. At this stage, a mixture of 55 mM sorbitol and 27.5 mM maltose was required. Up to 7 shoots / explant developed and were easily rooted (96%) and acclimatized when sorbitol (27.5 mM) and active charcoal (2 g/l) were added to the rooting medium. Transformation experiments were performed with Agrobacterium tumefaciens disarmed strain GV3101-pMP90 (pKyGUSintron). Regeneration zones exhibited stable GUS expression. However, all shoots grown in vitro died on a selective rooting medium (50 mg/l kanamycin). When attempts for in vitro selection were made (12.5 mg/l kanamycin), some buds were initiated but elongation was prevented. Susceptibility to neomycin seemed very high, therefore, selection markers and selection steps must be revised carefully.Se ha desarrollado un procedimiento para la regeneraci&oacute;n del olmo h&iacute;brido &laquo;Sapporo&raquo; resistente a la grafiosis mediante el uso de discos foliares procedentes de plantas cultivadas in vitro y examinadas en ensayos de transformaci&oacute;n. Para ello se requirieron dos pasos: inducci&oacute;n de las yemas (3 semanas), seguido de la elongaci&oacute;n de las yemas (4 semanas). Los meristemas que dieron origen a las yemas se formaron en la cercan&iacute;a de las costillas de corte a partir de diversos tejidos (floema, proc&aacute;mbium, par&eacute;nquima) excepto la epidermis. La regeneraci&oacute;n dependi&oacute; del uso de agarosa (6 g/l LSM) y de medio MS diluido (1/2). Los mejores resultados (11-14 yemas por explante) se obtuvieron en presencia de 0,1 &mu;M de TDZ y 0,06 &mu;M de IAA y cuando la maltosa (55-110 mM) o el sorbitol (110 mM) se usaron como fuente de carbohidratos. Durante la elongaci&oacute;n del tallo, el TDZ hubo de ser disminuido a 0,01 &mu;M o sustituido por 1-2 &mu;M de BAP y 1,4 &mu;M de GA3. En esta fase fue necesaria una mezcla de 55 mM de sorbitol y 27,5 mM de maltosa. Se desarrollaron al menos 7 tallos por explante que f&aacute;cilmente enraizaron (96%) y se aclimataron cuando se a&ntilde;adi&oacute; sorbitol (27,5 mM) y carb&oacute;n activo (2 g/l) al medio de enraizamiento. Los ensayos de transformaci&oacute;n se desarrollaron con las cepas GV3101-pMP90 (pKyGUSintron) de Agrobacterium tumefaciens. Las zonas de regeneraci&oacute;n presentaron una expresi&oacute;n GUS estable. No obstante, todas las yemas cultivadas in vitro murieron en un medio selectivo de enraizamiento (50 mg/l de kanamicina). Cuando se intent&oacute; la selecci&oacute;n in vitro (12,5 mg/l de kanamicina), algunas yemas se iniciaron pero la elongaci&oacute;n fue impedida. La susceptibilidad a la neomicina parece ser muy alta, por lo que los marcadores y los pasos de selecci&oacute;n deben ser cuidadosamente revisados

Metabolic Fate of [14C]Diuron and [14C]Linuron in Wheat (Triticum aestivum) and Radish (Raphanus sativus)

Metabolism of xenobiotics in plants usually occurs in three phases, phase I (primary metabolism), phase II (conjugation processes), and phase III (storage). The uptake and metabolism of [14C]diuron and [14C]linuron were investigated in wheat and radish. Seeds were sown in quartz sand and irrigated with a nutrient solution of either radioactive herbicide. Plants were harvested after two weeks, and metabolites were extracted and then analyzed by radio-reverse-high-performance liquid chromatography (HPLC). Uptake of the two molecules was higher in radish compared to wheat. Translocation of parent compounds and related metabolites from roots to aerial plant parts was important, especially for radish. A large proportion of extractable residues were found in radish whereas nonextractable residues amounted to 30% in wheat, mainly associated with roots. Chemical structure of metabolites was thereafter identified by acid, alkaline, and enzymatic hydrolyses followed by electrospray ionization mass spectrometry (ESI-MS) and proton nuclear magnetic resonance spectroscopy (1H NMR). This study highlighted the presence of diuron and linuron metabolites conjugated to sugars in addition to N-demethylation and N-demethoxylation products

Semiconductor charge qubit relaxation due to two-phonon processes

We theoretically study the relaxation of electron orbital states of a double quantum dot system due to two-phonon processes. In particular, we calculate how the relaxation rates depend on the separation distance between the quantum dots, the strength of quantum dot confinement, and the lattice temperature. Enhancement of the rates by specific inter-dot distances and lattice temperatures, and the relative strength of different scattering channels are discussed. Our results show that although at low temperatures ($T \sim 1$ K) two-phonon processes are almost four orders of magnitude weaker compared to one-phonon processes in relaxing electron orbital states, at room temperature they are as important as one-phonon processes.Comment: Submitted to PR

Démographie du Puffin cendré Calonectris diomedea de Selvagem Grande (30° 09' n, 15° 52' w)

The breeding numbers of the Cory ' s Shearwaters Calonectris diomedea of Selvagem Grande (30° 09' N, 15 ° 52' W) have shown important variations during the last three decades, decreasing from about 100,000 birds at the beginning of the seventies to 5,000 only in 1 977 following depredations, and increasing regularly since then to reach nowadays about 30,000. The mean age at first breeding is 8.9 years. Every year, 11.8 % of the breeders temporarily stop breeding, these sabbatical leaves lasting 1.7 years on average and 7 years at most. 56.6 % of the eggs laid produce fledglings, with significant variations between years. The mean annual survival rate of the adults is comparatively low, 92.7 %, higher for females (93.5 %) than for males (91.9 %) and varying significantly between years (min. : 84.1 %, max.: 98.0 % ). The survival rate of the immatures between fledging and first breeding is 32.8 % at !east. Selvagem Grande population, which numbered an important proportion of young inexperienced birds at the beginning of the eighties, following the depredations of the seventies, has gained age and experience during the eighties and the nineties. But the relatively low annual survival rate of the adults, which is probably linked to the development of new fishing methods, is an alarming sign for the future of the population of Cory' s Shearwaters, notwithstanding the conservation actions engaged in the breeding localitiesLes effectifs des Puffins cendrés Calonectris diomedea de Selvagem Grande (30° 09' N, 15° 52' W) ont montré au cours des trois dernières décennies des variations considérables, passant de 100 000 reproducteurs environ au début des années 70 à 5 000 seulement en 1977 à la suite de déprédations, et augmentant régulièrement depuis lors pour atteindre aujourd'hui environ 30 000. La reproduction s'établit en moyenne à 8,9 années. Chaque année, 11,8 % des reproducteurs ne nichent pas, les interruptions temporaires de la reproduction durant en moyenne 1,7 années et au maximum 7 ans. 56,6 % des oeufs pondus produisent des poussins en fin de croissance, des variations significatives existant d'une année à l'autre. Le taux de survie annuel des adultes est relativement faible, 92,7 %. Il est plus élevé chez les femelles (93,5 %) que chez les mâles (91,9 %) et il varie significativement d'une année à l'autre (min.: 84,1 %, max. : 98,0 %). Enfin, le taux de survie des immatures entre l'envol en fin de croissance et la première reproduction est au moins égal à 32,8 %. La population de Selvagem Grande, qui comptait une proportion importante de jeunes oiseaux inexpérimentés au début des années 80, à la suite des massacres de reproducteurs de la fin-des années 70, a acquis de l'âge et de l'expérience au cours des décennies 80 et 90. Mais la relative faiblesse du taux de survie annuel des adultes, qu'il faut sans doute mettre en relation avec le développement de nouvelles méthodes de pêche, est un signe inquiétant pour l'avenir des populations de Puffins cendrés, malgré les mesures de protection prises sur les lieux de reproduction