Evaluation of the dao1 gene as a selectable marker in transformation of apple rootstock M26 as well as transformation of a vector containing GA20 oxidase gene into Agrobacterium

The aim of this project was to evaluate the possibility to use D-alanine as a selective marker instead of commonly used antibiotics and herbicides in transformation of apple rootstock M26. The transformation was carried out using Agrobacterium tumefaciens strain C58C1(pGV3101) containing the vector pPCV70:dao1. The vector contained the dao1 gene resistant to D-amino acids and the nptII gene resistant to antibiotic, kanamycin. The dao1 encodes for the enzyme D-amino-acid oxidase (DAAO). The enzyme detoxifies D-amino acids, such as, D-alanine, which are harmful to plants. The transformed explants were grown on medium containing increased concentrations of D-alanine, with a starting concentration of 1.2 mM. Eight putative transgenic clones were obtained from the selection medium. However, further molecular analyses have to be carried out to confirm the result. A second experiment was performed to transform the GA20 oxidase gene into A. tumefaciens to get a ready-to-use transformation vector. The vector containing the GA20 oxidase gene was transformed into the A. tumefaciens strain C58C1(pGV3850). DNA from the strain was isolated and digested. The result was visualized on an agarose gel under UV light. The conclusion of the experiment is that the vector containing GA20 oxidase gene has been transformed into the strain C58C1.Syftet med detta projekt var att undersöka möjligheten att vid transformering av äpplegrundstammen M26 genomföra selektion med hjälp av D-aminosyra (D-alanin) istället för andra vanligt förekommande selektionsämnen, så som antibiotika och herbicider. Transformeringar med D-alanin som selektionsämne genomfördes, med Agrobacterium tumefaciens stammen C58C1(pGV3101) innehållande vektorn pPCV70:dao1. Vektorn innehöll även genen nptII, som ger de transformerade explantaten resistans mot antibiotika, kanamycin. dao1 genen kodar för enzymet D-amino-acid oxidase (DAAO). Enzymet bryter ner det giftiga ämnet D-alanin till ofarliga ämnen för växten. De transformerade explantaten odlades på medium innehållande ökande koncentration av D-alanin, med utgångskoncentrationen 1.2 mM. Åtta putativa transformerade kloner selekterades på selektionsmediet. Ytterligare molekylära analyser måste dock genomföras för att bekräfta resultaten. Försök genomfördes även för att transformera en vektor innehållande GA20 oxidas genen i A. tumefaciens, för att få fram en vektor färdig för användning vid transformering av växter. Vektorn innehållande GA20 oxidas genen och nptII genen transformerades in i A. tumefaciens stammen C58C1(pGV3850). DNA från A. tumefaciens var sedan isolerad och fragmenterad. Resultatet visualiserades på en agarose gel under UV ljus. Slutsatsen av försöket är att vektorn transformerats in i A. tumefaciens stammen C58C1

Differential regulation of Knotted1-like genes during establishment of the shoot apical meristem in Norway spruce (Picea abies)

Establishment of the shoot apical meristem (SAM) in Arabidopsis embryos requires the KNOXI transcription factor SHOOT MERISTEMLESS. In Norway spruce (Picea abies), four KNOXI family members (HBK1, HBK2, HBK3 and HBK4) have been identified, but a corresponding role in SAM development has not been demonstrated. As a first step to differentiate between the functions of the four Norway spruce HBK genes, we have here analyzed their expression profiles during the process of somatic embryo development. This was made both under normal embryo development and under conditions of reduced SAM formation by treatment with the polar auxin transport inhibitor NPA. Concomitantly with the formation of an embryonic SAM, the HBK2 and HBK4 genes displayed a significant up-regulation that was delayed by NPA treatment. In contrast, HBK1 and HBK3 were up-regulated prior to SAM formation, and their temporal expression was not affected by NPA. Ectopic expression of the four HBK genes in transgenic Arabidopsis plants further supported similar functions of HBK2 and HBK4, distinct from those of HBK1 and HBK3. Together, the results suggest that HBK2 and HBK4 exert similar functions related to the SAM differentiation and somatic embryo development in Norway spruce, while HBK1 and HBK3 have more general functions during embryo development

Genetics of callous-unemotional behavior in children

Callous-unemotional behavior (CU) is currently under consideration as a subtyping index for conduct disorder diagnosis. Twin studies routinely estimate the heritability of CU as greater than 50%. It is now possible to estimate genetic influence using DNA alone from samples of unrelated individuals, not relying on the assumptions of the twin method. Here we use this new DNA method (implemented in a software package called Genome-wide Complex Trait Analysis, GCTA) for the first time to estimate genetic influence on CU. We also report the first genome-wide association (GWA) study of CU as a quantitative trait. We compare these DNA results to those from twin analyses using the same measure and the same community sample of 2,930 children rated by their teachers at ages 7, 9 and 12. GCTA estimates of heritability were near zero, even though twin analysis of CU in this sample confirmed the high heritability of CU reported in the literature, and even though GCTA estimates of heritability were substantial for cognitive and anthropological traits in this sample. No significant associations were found in GWA analysis, which, like GCTA, only detects additive effects of common DNA variants. The phrase ‘missing heritability’ was coined to refer to the gap between variance associated with DNA variants identified in GWA studies versus twin study heritability. However, GCTA heritability, not twin study heritability, is the ceiling for GWA studies because both GCTA and GWA are limited to the overall additive effects of common DNA variants, whereas twin studies are not. This GCTA ceiling is very low for CU in our study, despite its high twin study heritability estimate. The gap between GCTA and twin study heritabilities will make it challenging to identify genes responsible for the heritability of CU

Epidemiology, patterns of care, and mortality for patients with acute respiratory distress syndrome in intensive care units in 50 countries

IMPORTANCE: Limited information exists about the epidemiology, recognition, management, and outcomes of patients with the acute respiratory distress syndrome (ARDS). OBJECTIVES: To evaluate intensive care unit (ICU) incidence and outcome of ARDS and to assess clinician recognition, ventilation management, and use of adjuncts-for example prone positioning-in routine clinical practice for patients fulfilling the ARDS Berlin Definition. DESIGN, SETTING, AND PARTICIPANTS:The Large Observational Study to Understand the Global Impact of Severe Acute Respiratory Failure (LUNG SAFE) was an international, multicenter, prospective cohort study of patients undergoing invasive or noninvasive ventilation, conducted during 4 consecutive weeks in the winter of 2014 in a convenience sample of 459 ICUs from 50 countries across 5 continents. EXPOSURES:Acute respiratory distress syndrome. MAIN OUTCOMES AND MEASURES: The primary outcome was ICU incidence of ARDS. Secondary outcomes included assessment of clinician recognition of ARDS, the application of ventilatory management, the use of adjunctive interventions in routine clinical practice, and clinical outcomes from ARDS. RESULTS: Of 29,144 patients admitted to participating ICUs, 3022 (10.4%) fulfilled ARDS criteria. Of these, 2377 patients developed ARDS in the first 48 hours and whose respiratory failure was managed with invasive mechanical ventilation. The period prevalence of mild ARDS was 30.0% (95% CI, 28.2%-31.9%); of moderate ARDS, 46.6% (95% CI, 44.5%-48.6%); and of severe ARDS, 23.4% (95% CI, 21.7%-25.2%). ARDS represented 0.42 cases per ICU bed over 4 weeks and represented 10.4% (95% CI, 10.0%-10.7%) of ICU admissions and 23.4% of patients requiring mechanical ventilation. Clinical recognition of ARDS ranged from 51.3% (95% CI, 47.5%-55.0%) in mild to 78.5% (95% CI, 74.8%-81.8%) in severe ARDS. Less than two-thirds of patients with ARDS received a tidal volume 8 of mL/kg or less of predicted body weight. Plateau pressure was measured in 40.1% (95% CI, 38.2-42.1), whereas 82.6% (95% CI, 81.0%-84.1%) received a positive end-expository pressure (PEEP) of less than 12 cm H2O. Prone positioning was used in 16.3% (95% CI, 13.7%-19.2%) of patients with severe ARDS. Clinician recognition of ARDS was associated with higher PEEP, greater use of neuromuscular blockade, and prone positioning. Hospital mortality was 34.9% (95% CI, 31.4%-38.5%) for those with mild, 40.3% (95% CI, 37.4%-43.3%) for those with moderate, and 46.1% (95% CI, 41.9%-50.4%) for those with severe ARDS. CONCLUSIONS AND RELEVANCE: Among ICUs in 50 countries, the period prevalence of ARDS was 10.4% of ICU admissions. This syndrome appeared to be underrecognized and undertreated and associated with a high mortality rate. These findings indicate the potential for improvement in the management of patients with ARDS

Rangewide genetic analysis of Lesser Prairie-Chicken reveals population structure, range expansion, and possible introgression

The distribution of the Lesser Prairie-Chicken (Tympanuchus pallidicinctus) has been markedly reduced due to loss and fragmentation of habitat. Portions of the historical range, however, have been recolonized and even expanded due to planting of conservation reserve program (CRP) fields that provide favorable vegetation structure for Lesser Prairie-Chickens. The source population(s) feeding the range expansion is unknown, yet has resulted in overlap between Lesser and Greater Prairie-Chickens (T. cupido) increasing the potential for hybridization. Our objectives were to characterize connectivity and genetic diversity among populations, identify source population(s) of recent range expansion, and examine hybridization with the Greater Prairie-Chicken. We analyzed 640 samples from across the range using 13 microsatellites. We identified three to four populations corresponding largely to ecoregions. The Shinnery Oak Prairie and Sand Sagebrush Prairie represented genetically distinct populations (F (ST) > 0.034 and F (ST) > 0.023 respectively). The Shortgrass/CRP Mosaic and Mixed Grass ecoregions appeared admixed (F (ST) = 0.009). Genetic diversity was similar among ecoregions and N (e) ranged from 142 (95 % CI 99-236) for the Shortgrass/CRP Mosaic to 296 (95 % CI 233-396) in the Mixed Grass Prairie. No recent migration was detected among ecoregions, except asymmetric dispersal from both the Mixed Grass Prairie and to a lesser extent the Sand Sagebrush Prairie north into adjacent Shortgrass/CRP Mosaic (m = 0.207, 95 % CI 0.116-0.298, m = 0.097, 95 % CI 0.010-0.183, respectively). Indices investigating potential hybridization in the Shortgrass/CRP Mosaic revealed that six of the 13 individuals with hybrid phenotypes were significantly admixed suggesting hybridization. Continued monitoring of diversity within and among ecoregions is warranted as are actions promoting genetic connectivity and range expansion.Keywords: Spatial genetic population structure, Genetic diversity, Tympanuchus pallidicinctus, Gene flow, Hybridizatio

Mirror mirror on the wall... an unobtrusive intelligent multisensory mirror for well-being status self-assessment and visualization

A person’s well-being status is reflected by their face through a combination of facial expressions and physical signs. The SEMEOTICONS project translates the semeiotic code of the human face into measurements and computational descriptors that are automatically extracted from images, videos and 3D scans of the face. SEMEOTICONS developed a multisensory platform in the form of a smart mirror to identify signs related to cardio-metabolic risk. The aim was to enable users to self-monitor their well-being status over time and guide them to improve their lifestyle. Significant scientific and technological challenges have been addressed to build the multisensory mirror, from touchless data acquisition, to real-time processing and integration of multimodal data

Genetic risk and a primary role for cell-mediated immune mechanisms in multiple sclerosis.

Multiple sclerosis is a common disease of the central nervous system in which the interplay between inflammatory and neurodegenerative processes typically results in intermittent neurological disturbance followed by progressive accumulation of disability. Epidemiological studies have shown that genetic factors are primarily responsible for the substantially increased frequency of the disease seen in the relatives of affected individuals, and systematic attempts to identify linkage in multiplex families have confirmed that variation within the major histocompatibility complex (MHC) exerts the greatest individual effect on risk. Modestly powered genome-wide association studies (GWAS) have enabled more than 20 additional risk loci to be identified and have shown that multiple variants exerting modest individual effects have a key role in disease susceptibility. Most of the genetic architecture underlying susceptibility to the disease remains to be defined and is anticipated to require the analysis of sample sizes that are beyond the numbers currently available to individual research groups. In a collaborative GWAS involving 9,772 cases of European descent collected by 23 research groups working in 15 different countries, we have replicated almost all of the previously suggested associations and identified at least a further 29 novel susceptibility loci. Within the MHC we have refined the identity of the HLA-DRB1 risk alleles and confirmed that variation in the HLA-A gene underlies the independent protective effect attributable to the class I region. Immunologically relevant genes are significantly overrepresented among those mapping close to the identified loci and particularly implicate T-helper-cell differentiation in the pathogenesis of multiple sclerosis

Mitochondrial mosaics in the liver of 3 infants with mtDNA defects

<p>Abstract</p> <p>Background</p> <p>In muscle cytochrome oxidase (COX) negative fibers (mitochondrial mosaics) have often been visualized.</p> <p>Methods</p> <p>COX activity staining of liver for light and electron microscopy, muscle stains, blue native gel electrophoresis and activity assays of respiratory chain proteins, their immunolocalisation, mitochondrial and nuclear DNA analysis.</p> <p>Results</p> <p>Three unrelated infants showed a mitochondrial mosaic in the liver after staining for COX activity, i.e. hepatocytes with strongly reactive mitochondria were found adjacent to cells with many negative, or barely reactive, mitochondria. Deficiency was most severe in the patient diagnosed with Pearson syndrome. Ragged-red fibers were absent in muscle biopsies of all patients. Enzyme biochemistry was not diagnostic in muscle, fibroblasts and lymphocytes. Blue native gel electrophoresis of liver tissue, but not of muscle, demonstrated a decreased activity of complex IV; in both muscle and liver subcomplexes of complex V were seen. Immunocytochemistry of complex IV confirmed the mosaic pattern in two livers, but not in fibroblasts. MRI of the brain revealed severe white matter cavitation in the Pearson case, but only slight cortical atrophy in the Alpers-Huttenlocher patient, and a normal image in the 3rd. MtDNA in leucocytes showed a common deletion in 50% of the mtDNA molecules of the Pearson patient. In the patient diagnosed with Alpers-Huttenlocher syndrome, mtDNA was depleted for 60% in muscle. In the 3rd patient muscular and hepatic mtDNA was depleted for more than 70%. Mutations in the nuclear encoded gene of <it>POLG </it>were subsequently found in both the 2nd and 3rd patients.</p> <p>Conclusion</p> <p>Histoenzymatic COX staining of a liver biopsy is fast and yields crucial data about the pathogenesis; it indicates whether mtDNA should be assayed. Each time a mitochondrial disorder is suspected and muscle data are non-diagnostic, a liver biopsy should be recommended. Mosaics are probably more frequent than observed until now. A novel pathogenic mutation in <it>POLG </it>is reported.</p> <p>Tentative explanations for the mitochondrial mosaics are, in one patient, unequal partition of mutated mitochondria during mitoses, and in two others, an interaction between products of several genes required for mtDNA maintenance.</p