First Report of CRISPR/Cas9 Gene Editing in Castanea sativa Mill

CRISPR/Cas9 has emerged as the most important tool for genome engineering due to its simplicity, design flexibility, and high efficiency. This technology makes it possible to induce point mutations in one or some target sequences simultaneously, as well as to introduce new genetic variants by homology-directed recombination. However, this approach remains largely unexplored in forest species. In this study, we reported the first example of CRISPR/Cas9-mediated gene editing in Castanea genus. As a proof of concept, we targeted the gene encoding phytoene desaturase (pds), whose mutation disrupts chlorophyll biosynthesis allowing for the visual assessment of knockout efficiency. Globular and early torpedo-stage somatic embryos of Castanea sativa (European chestnut) were cocultured for 5 days with a CRISPR/Cas9 construct targeting two conserved gene regions of pds and subsequently cultured on a selection medium with kanamycin. After 8 weeks of subculture on selection medium, four kanamycin-resistant embryogenetic lines were isolated. Genotyping of these lines through target Sanger sequencing of amplicons revealed successful gene editing. Cotyledonary somatic embryos were maturated on maltose 3% and cold-stored at 4°C for 2 months. Subsequently, embryos were subjected to the germination process to produce albino plants. This study opens the way to the use of the CRISPR/Cas9 system in European chestnut for biotechnological application

Pain in amyotrophic lateral sclerosis: a population-based controlled study

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FUNCTIONAL CHARACTERIZATION OF A TOMATO GLUTHATHIONE S TRANSFERASE GENE AND ITS IMPLICATION IN THE PLANT RESPONSE TO ENVIRONMENTAL STRESSES

Mitigating the negative effects of abiotic stresses on crop productivity is pivotal in order to meet the global demand for food and other agricultural commodities. Abiotic stresses caused by deficiencies or excesses in environmental factors such as water, salt, light, and temperature can substantially reduce plant growth and productivity and even survival. Abiotic stresses were estimated to cause an overall yield loss of ~70% in key agricultural crops and global warming is even expected to further worsen food security. Abiotic stresses induce in plant cells increasing levels of reactive oxygen species (ROS) that are critical for stress signaling and mainly affect chloroplast protein synthesis and photosystem II repair. A plethora of antioxidants and antioxidant enzymes protect plant cells from oxidative stresses caused by an excess of ROS accumulation, thus engineering the cell redox cycle for enhancing the ROS-scavenging capacity might contribute to empower plant stress tolerance. Glutathione S-transferases (GSTs) is a multigene superfamily with diverse cellular mechanisms and metabolic functions and has been considered as one of the key members of plant stress modulation pathways. Our goal is to investigates the role of a tomato glutathione S-transferase (GST - Solyc07g056420) gene in controlling plant stress response. Tobacco lines overexpressing the Solyc07g056420 coding sequence (OE) accumulated significantly higher levels of hydrogen peroxide in leaves and decreased leaf levels of flavonoids, chlorophyll A and antioxidant capacity compared with control plants. OE 40 days old plants underwent differential watering treatments, i.e. full reintegration of water lost by evapo transpiration (FWR) and restitution of 50% of lost water (HWR). Under HWR conditions, OE plants showed a reduced occurrence of leaf injuries and responded to drought with a significantly higher increase in leaf chlorophyll A, chlorophyll B, hydrogen peroxide and antioxidant capacity compared to control plants. These results suggested that the hyper-accumulation of H2O2 induced in leaves by the overexpression of the Solyc07g056420 coding sequence and compensated by the adjusting the antioxidant capacity might lead to an enhancement of plant responsiveness to drought. Ongoing experiments will further investigate the functional role of the tomato Solyc07g056420 within the stress signaling network and its possible involvement in the modulation of plant response to other environmental stresses

Functional pattern of Brain FDG-PET in Amyotrophic Lateral Sclerosis

Objective: We investigated a large sample of patients with amyotrophic lateral sclerosis (ALS) at rest in order to assess the value of 18F-2-fluoro-2-deoxy-D-glucose (18F-FDG) PET as a biomarker to discriminate patients from controls. Methods: A total of 195 patients with ALS and 40 controls underwent brain 18F-FDG-PET, most within 5 months of diagnosis. Spinal and bulbar subgroups of ALS were also investigated. Twenty-five bilateral cortical and subcortical volumes of interest and cerebellum were taken into account, and 18F-FDG uptakes were individually normalized by whole-brain values. Group analyses investigated the ALS-related metabolic changes. Discriminant analysis investigating sensitivity and specificity was performed using the 51 volumes of interest as well as age and sex. Metabolic connectivity was explored by voxel-wise interregional correlation analysis. Results: Hypometabolismwas found in frontal, motor, and occipital cortex and hypermetabolismin midbrain, temporal pole, and hippocampus in patients with ALS compared to controls. A similar metabolic pattern was also found in the 2 subgroups. Discriminant analysis showed a sensitivity of 95% and a specificity of 83% in separating patients from controls. Connectivity analysis found a highly significant positive correlation between midbrain and white matter in corticospinal tracts in patients with ALS. Conclusions: 18F-FDG distribution changes in ALS showed a clear pattern of hypometabolism in frontal and occipital cortex and hypermetabolism in midbrain. The latter might be interpreted as the neurobiological correlate of diffuse subcortical gliosis. Discriminant analysis resulted in high sensitivity and specificity in differentiating patients with ALS from controls. Once validated by diseased-control studies, the present methodology might represent a potentially useful biomarker for ALS diagnosis. Classificaton of evidence: This study provides Class III evidence that 18F-FDG-PET accurately distinguishes patients with ALS from normal controls (sensitivity 95.4%, specificity 82.5%)

Lifetime Sport Practice and Brain Metabolism in Amyotrophic Lateral Sclerosis

Objective: To evaluate the metabolic correlates of lifetime sport practice in ALS through brain 18F-FDG-PET. Methods: 131 patients completed a questionnaire about lifetime exposures, including physical activity related to sports, hobbies and occupations, and underwent brain 18F-FDG-PET. Exposure to sports was expressed as MET (Metabolic Equivalent of Task). We considered only regular practice (at least 2 h/week, for at least three months). We compared brain metabolism between two groups: subjects who did not report regular sport practice during life (N-group) and patients who did (Y-group). The resulting significant clusters were used in each group as seed regions in an interregional correlation analysis (IRCA) to evaluate the impact of lifetime sport practice on brain networks typically involved by the neurodegenerative process of ALS. Each group was compared to healthy controls (HC, n = 40). Results: We found a significant, relative cerebellar hypermetabolism in the N-group compared to the Y-group. The metabolism of such cerebellar cluster resulted correlated to more significant and widespread metabolic changes in areas known to be affected by ALS (i.e. frontotemporal regions and corticospinal tracts) in the N-group as compared to the Y-group, despite the same level of disability as expressed by the ALS FRS-R. Such findings resulted independent of age, sex, site of onset (bulbar/spinal), presence/absence of C9ORF72 expansion, cognitive status and physical activity related to hobbies and occupations. When compared to HC, the N-group showed more widespread metabolic changes than the Y-group in cortical regions known to be relatively hypometabolic in ALS patients as compared to HC. Conclusions: We hypothesize that patients of the N-group might cope better with the neurodegenerative process, since they show more widespread metabolic changes as compared to the Y-group, despite the same level of disability. Nevertheless, further studies are necessary to corroborate this hypothesis