Characterization of Vitis vinifera NPR1 homologs involved in the regulation of Pathogenesis-Related gene expression

<p>Abstract</p> <p>Background</p> <p>Grapevine protection against diseases needs alternative strategies to the use of phytochemicals, implying a thorough knowledge of innate defense mechanisms. However, signalling pathways and regulatory elements leading to induction of defense responses have yet to be characterized in this species. In order to study defense response signalling to pathogens in <it>Vitis vinifera</it>, we took advantage of its recently completed genome sequence to characterize two putative orthologs of <it>NPR1</it>, a key player in salicylic acid (SA)-mediated resistance to biotrophic pathogens in <it>Arabidopsis thaliana</it>.</p> <p>Results</p> <p>Two cDNAs named <it>VvNPR1.1 </it>and <it>VvNPR1.2 </it>were isolated from <it>Vitis vinifera </it>cv Chardonnay, encoding proteins showing 55% and 40% identity to Arabidopsis NPR1 respectively. Constitutive expression of <it>VvNPR1.1 </it>and <it>VvNPR1.2 </it>monitored in leaves of <it>V. vinifera </it>cv Chardonnay was found to be enhanced by treatment with benzothiadiazole, a SA analog. In contrast, <it>VvNPR1.1 </it>and <it>VvNPR1.2 </it>transcript levels were not affected during infection of resistant <it>Vitis riparia </it>or susceptible <it>V. vinifera </it>with <it>Plasmopara viticola</it>, the causal agent of downy mildew, suggesting regulation of VvNPR1 activity at the protein level. VvNPR1.1-GFP and VvNPR1.2-GFP fusion proteins were transiently expressed by agroinfiltration in <it>Nicotiana benthamiana </it>leaves, where they localized predominantly to the nucleus. In this system, <it>VvNPR1.1 </it>and <it>VvNPR1.2 </it>expression was sufficient to trigger the accumulation of acidic SA-dependent Pathogenesis-Related proteins PR1 and PR2, but not of basic chitinases (PR3) in the absence of pathogen infection. Interestingly, when <it>VvNPR1.1 </it>or <it>AtNPR1 </it>were transiently overexpressed in <it>Vitis vinifera </it>leaves, the induction of grapevine <it>PR1 </it>was significantly enhanced in response to <it>P. viticola</it>.</p> <p>Conclusion</p> <p>In conclusion, our data identified grapevine homologs of NPR1, and their functional analysis showed that VvNPR1.1 and VvNPR1.2 likely control the expression of SA-dependent defense genes. Overexpression of <it>VvNPR1 </it>has thus the potential to enhance grapevine defensive capabilities upon fungal infection. As a consequence, manipulating <it>VvNPR1 </it>and other signalling elements could open ways to strengthen disease resistance mechanisms in this crop species.</p

What is the efficacy of viral and bacterial vaccines in feedlot cattle to reduce bovine respiratory disease (BRD) and subsequent antibiotic use?

Bovine respiratory disease complex is the most economically significant disease of feedlot cattle[4]. Putative causal organisms include Mannheimia haemolytica, Pasteurella multocida, Histophilus somni and Mycoplasma bovis, bovine herpesvirus, bovine viral diarrhea virus, bovine respiratory syncytial virus, and parainfluenza type 3 virus[5]. Vaccination against the putative causal organisms is a frequently used approach to aid in the prevention of BRD. With a more significant concern for prudent antibiotic use in the beef industry, it is vital that decision making with regards BRDC management be based on an understanding of the efficacy of vaccination programs and management factors that might modify the efficacy of the preventive management practice[6, 7]. Systematic reviews of randomized controlled trials yield the highest level of evidence for the efficacy of treatment under field conditions, and comparative efficacy can be examined using network meta-analysis for multiple comparisons. Establishing the efficacy of monovalent and polyvalent vaccinations for the prevention of BRDC in feedlot cattle will serve to improve decision makers’ ability to engage in effective stewardship of antibiotics

What is the efficacy of metaphylaxis using antibiotics for the prevention of Bovine Respiratory Disease in beef cattle?

Bovine respiratory disease complex is the most economically significant disease of feedlot cattle. Putative causal organisms include Mannheimia haemolytica, Pasteurella multocida, Histophilus somni and Mycoplasma bovis, bovine herpesvirus, bovine viral diarrhea virus, bovine respiratory syncytial virus, and parainfluenza type 3 virus. Although vaccination against the putative causal organisms is a frequently used approach to aid in the prevention of BRD, it is also common and legal for antibiotics to be used for metaphylaxis at the arrival of beef cattle at feedlots. With a more significant concern for prudent antibiotic use in the beef industry, it is essential for decision making with regards BRDC management to understand the efficacy of metaphylaxis as a preventive management practice for BRDC. Systematic reviews of randomized controlled trials yield the highest level of evidence for the efficacy of treatment under field conditions, and comparative efficacy can be examined using network meta-analysis for multiple comparisons. Establishing the efficacy of metaphylaxis for the prevention of BRDC will serve to improve decision makers’ ability to engage in effective stewardship of antibiotics

Photoresist as a sacrificial layer by dissolution in acetone

We report here a detailed study of sacrificial layer dissolution of photoresist by acetone in microchannels. The effects of channel geometry as well as photoresist characteristics such as thermal cycles and UV exposure are considered and tested. Test channels were designed and fabricated ranging in height from 2 µm to 6 µm, widths from 10 µm to 80 µm, and lengths up to 2 mm. Channels were formed by encapsulating a sacrificial photoresist layer between two layers of parylene. Sacrificial layer dissolution in acetone was monitored using time lapse digital photography through a microscope and the captured data plotted and analyzed. The data support a diffusion limited model for photoresist dissolution in acetone. Individual parameters in the diffusion limited model are tested and validated through a number of controlled experiments. These results and the final model are important for the design and fabrication of micro-fluidic systems based on the parylene-photoresist sacrificial system

Role of the enterocyte in fructose-induced hypertriglyceridaemia

Dietary fructose has been linked to an increased post-prandial triglyceride (TG) level; which is an established independent risk factor for cardiovascular disease. Although much research has focused on the effects of fructose consumption on liver-derived very-low density lipoprotein (VLDL); emerging evidence also suggests that fructose may raise post-prandial TG levels by affecting the metabolism of enterocytes of the small intestine. Enterocytes have become well recognised for their ability to transiently store lipids following a meal and to thus control post-prandial TG levels according to the rate of chylomicron (CM) lipoprotein synthesis and secretion. The influence of fructose consumption on several aspects of enterocyte lipid metabolism are discussed; including de novo lipogenesis; apolipoprotein B48 and CM-TG production; based on the findings of animal and human isotopic tracer studies. Methodological issues affecting the interpretation of fructose studies conducted to date are highlighted; including the accurate separation of CM and VLDL. Although the available evidence to date is limited; disruption of enterocyte lipid metabolism may make a meaningful contribution to the hypertriglyceridaemia often associated with fructose consumption

Phytotoxic metabolites from Neofusicoccum parvum, a pathogen of Botryosphaeria dieback of grapevine

Liquid chromatography-diode array screening of the organic extract of the cultures of 13 isolates of the fungus Neofusicoccum parvum, the main causal agent of botryosphaeria dieback of grapevine, showed similar metabolites. One strain was selected for further chemical studies and led to the isolation and characterisation of 13 metabolites. Structures were elucidated through spectroscopic analyses, including one- and two-dimensional NMR and mass spectrometry, and through comparison to literature data. The isolated compounds belong to four different chemical families: five metabolites, namely, ( )-terremutin (1), (+)-terremutin hydrate (2), (+)-epi-sphaeropsidone (3) ( )-4-chloro-terremutin hydrate (4) and(+)-4- hydroxysuccinate-terremutin hydrate (5), belong to the family of dihydrotoluquinones; two metabolites, namely, (6S,7R) asperlin (6) and (6R,7S)-dia-asperlin (7), belong to the family of epoxylactones; four metabolites, namely, (R)-( )-mellein (8), (3R,4R)-4-hydroxymellein (9), (3R,4S)-4-hydroxymellein (10) (R)( )-3-hydroxymellein (11), belong to the family of dihydroisocoumarins; and two of the metabolites, namely, 6-methyl-salicylic acid (12) and 2-hydroxypropyl salicylic acid (13), belong to the family of hydroxybenzoic acids. We determined the phytotoxic activity of the isolated metabolites through a leaf disc assay and the expression of defence-related genes in Vitis vinifera cells cv. Chardonnay cultured with ( )-terremutin (1), the most abundant metabolite. Finally, analysis of the brown stripes of grapevine wood from plants showing botryosphaeria dieback symptoms revealed the presence of two of the isolated phytotoxinsinfo:eu-repo/semantics/publishedVersio

A qualitative examination of apathy and physical activity in Huntington's and Parkinson's disease

Aim: In Huntington's disease (HD) and Parkinson's disease (PD), apathy is a frequently cited barrier to participation in physical activity. Current diagnostic criteria emphasize dissociable variants of apathy that differentially affect goal-directed behavior. How these dimensions present and affect physical activity in HD and PD is unknown. Methods: Using a qualitative approach, we examined the experience of apathy and its impact on physical activity in 20 people with early-manifest HD or idiopathic PD. Results: Two major themes emerged: the multidimensionality of apathy, including initiation or goal-identification difficulties, and the interplay of apathy and fatigue; and facilitators of physical activity, including routines, safe environments and education. Conclusion: Physical activity interventions tailored to apathy phenotypes may maximize participant engagement

The clinical application of genome-wide sequencing for monogenic diseases in Canada: Position statement of the Canadian College of medical geneticists

Purpose and scope: The aim of this Position Statement is to provide recommendations for Canadian medical geneticists, clinical laboratory geneticists, genetic counsellors and other physicians regarding the use of genome-wide sequencing of germline DNA in the context of clinical genetic diagnosis. This statement has been developed to facilitate the clinical translation and development of best practices for clinical genome-wide sequencing for genetic diagnosis of monogenic diseases in Canada; it does not address the clinical application of this technology in other fields such as molecular investigation of cancer or for population screening of healthy individuals. Methods of statement development: Two multidisciplinary groups consisting of medical geneticists, clinical laboratory geneticists, genetic counsellors, ethicists, lawyers and genetic researchers were assembled to review existing literature and guidelines on genome-wide sequencing for clinical genetic diagnosis in the context of monogenic diseases, and to make recommendations relevant to the Canadian context. The statement was circulated for comment to the Canadian College of Medical Geneticists (CCMG) membership-at-large and, following incorporation of feedback, approved by the CCMG Board of Directors. The CCMG is a Canadian organisation responsible for certifying medical geneticists and clinical laboratory geneticists, and for establishing professional and ethical standards for clinical genetics services in Canada. Results and conclusions: Recommendations include (1) clinical genome-wide sequencing is an appropriate approach in the diagnostic assessment of a patient for whom there is suspicion of a significant monogenic disease that is associated with a high degree of genetic heterogeneity, or where specific genetic tests have failed to provide a diagnosis; (2) until the benefits of reporting incidental findings are established, we do not endorse the intentional clinical analysis of disease-associated genes other than those linked to the primary indication; and (3) clinicians should provide genetic counselling and obtain informed consent prior to undertaking clinical genome-wide sequencing. Counselling should include discussion of the limitations of testing, likelihood and implications of diagnosis and incidental findings, and the potential need for further analysis to facilitate clinical interpretation, including studies performed in a research setting. These recommendations will be routinely reevaluated as knowledge of diagnostic and clinical utility of clinical genome-wide sequencing improves. While the document was developed to direct practice in Canada, the applicability of the statement is broader and will be of interest to clinicians and health jurisdictions internationally

Steric Clash in the SET Domain of Histone Methyltransferase NSD1 as a Cause of Sotos Syndrome and Its Genetic Heterogeneity in a Brazilian Cohort

Most histone methyltransferases (HMTase) harbor a predicted Su(var)3–9, Enhancer-of-zeste, Trithorax (SET) domain, which transfers a methyl group to a lysine residue in their substrates. Mutations of the SET domains were reported to cause intellectual disability syndromes such as Sotos, Weaver, or Kabuki syndromes. Sotos syndrome is an overgrowth syndrome with intellectual disability caused by haploinsufficiency of the nuclear receptor binding SET domain protein 1 (NSD1) gene, an HMTase at 5q35.2–35.3. Here, we analyzed NSD1 in 34 Brazilian Sotos patients and identified three novel and eight known mutations. Using protein modeling and bioinformatic approaches, we evaluated the effects of one novel (I2007F) and 21 previously reported missense mutations in the SET domain. For the I2007F mutation, we observed conformational change and loss of structural stability in Molecular Dynamics (MD) simulations which may lead to loss-of-function of the SET domain. For six mutations near the ligand-binding site we observed in simulations steric clashes with neighboring side chains near the substrate S-Adenosyl methionine (SAM) binding site, which may disrupt the enzymatic activity of NSD1. These results point to a structural mechanism underlying the pathology of the NSD1 missense mutations in the SET domain in Sotos syndrome. NSD1 mutations were identified in only 32% of the Brazilian Sotos patients in our study cohort suggesting other genes (including unknown disease genes) underlie the molecular etiology for the majority of these patients. Our studies also found NSD1 expression to be profound in human fetal brain and cerebellum, accounting for prenatal onset and hypoplasia of cerebellar vermis seen in Sotos syndrome