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81.
Christine Le Signor Vincent Savois Grégoire Aubert Jérôme Verdier Marie Nicolas Gaelle Pagny Françoise Moussy Myriam Sanchez Dave Baker Jonathan Clarke Richard Thompson 《Plant biotechnology journal》2009,7(5):430-441
Medicago truncatula has been widely adopted as a model plant for crop legume species of the Vicieae. Despite the availability of transformation and regeneration protocols, there are currently limited tools available in this species for the systematic investigation of gene function. Within the framework of the European Grain Legumes Integrated Project ( http://www.eugrainlegumes.org ), chemical mutagenesis was applied to M. truncatula to create two mutant populations that were used to establish a TILLING (targeting induced local lesions in genomes) platform and a phenotypic database, allowing both reverse and forward genetics screens. Both populations had the same M2 line number, but differed in their M1 population size: population 1 was derived from a small M1 population (one-tenth the size of the M2 generation), whereas population 2 was generated by single seed descent and therefore has M1 and M2 generations of equal size. Fifty-six targets were screened, 10 on both populations, and 546 point mutations were identified. Population 2 had a mutation frequency of 1/485 kb, twice that of population 1. The strategy used to generate population 2 is more efficient than that used to generate population 1, with regard to mutagenesis density and mutation recovery. However, the design of population 1 allowed us to estimate the genetically effective cell number to be three in M. truncatula . Phenotyping data to help forward screenings are publicly available, as well as a web tool for ordering seeds at http://www.inra.fr/legumbase 相似文献
82.
A relatively simple method for the determination of the diffusion coefficient of a substance that has been injected into tissue is described. We illustrate this method using [3]dexamethasone injected into the subcutaneous tissue of rats. Digital autoradiography was used to measure the distribution of the [3H] dexamethasone within the subcutaneous tissue at 2.5 and 20 min after injection. Measured concentration profiles of the injection were compared to a mathematical model of drug diffusion from an injection. There was good agreement between the experimental data and the mathematical model. The diffusion coefficient found using this simple injection method was (4.01 +/- 2.01) x 10(-10) m2/s. This D value was very close to the value of D = (4.11 +/- 1.77) x 10(-10) m2/s found previously using different mathematical and experimental techniques with osmotic pumps implanted for 6, 24, and 60 h in rats (1). The simple method given here for the determination of the diffusion coefficient is general enough to be applied to other substances and tissues as well. 相似文献
83.
Melissa C. Brouwers Michelle E. Kho George P. Browman Jako S. Burgers Francoise Cluzeau Gene Feder Béatrice Fervers Ian D. Graham Steven E. Hanna Julie Makarski 《CMAJ》2010,182(10):1045-1052
Background
We undertook research to improve the AGREE instrument, a tool used to evaluate guidelines. We tested a new seven-point scale, evaluated the usefulness of the original items in the instrument, investigated evidence to support shorter, tailored versions of the tool, and identified areas for improvement.Method
We report on one component of a larger study that used a mixed design with four factors (user type, clinical topic, guideline and condition). For the analysis reported in this article, we asked participants to read a guideline and use the AGREE items to evaluate it based on a seven-point scale, to complete three outcome measures related to adoption of the guideline, and to provide feedback on the instrument’s usefulness and how to improve it.Results
Guideline developers gave lower-quality ratings than did clinicians or policy-makers. Five of six domains were significant predictors of participants’ outcome measures (p < 0.05). All domains and items were rated as useful by stakeholders (mean scores > 4.0) with no significant differences by user type (p > 0.05). Internal consistency ranged between 0.64 and 0.89. Inter-rater reliability was satisfactory. We received feedback on how to improve the instrument.Interpretation
Quality ratings of the AGREE domains were significant predictors of outcome measures associated with guideline adoption: guideline endorsements, overall intentions to use guidelines, and overall quality of guidelines. All AGREE items were assessed as useful in determining whether a participant would use a guideline. No clusters of items were found more useful by some users than others. The measurement properties of the seven-point scale were promising. These data contributed to the refinements and release of the AGREE II.Evidence-based guidelines are systematically developed statements aimed at assisting clinicians and patients in decisions about appropriate health care for specific clinical circumstances. 1 Guidelines assist decision-makers in solving system-level and population-level challenges. 2,3 The potential benefits of guidelines, however, are only as good as the quality of the guidelines themselves. Rigorous development and strategies for reporting are important precursors to successful implementation of the resulting recommendations. 4The quality of guidelines is variable, often falling short of basic standards.5–7 To address this variability, an international team of guideline developers and researchers, the AGREE Collaboration (Appraisal of Guidelines, Research and Evaluation), created a generic instrument to assess the process of guideline development and reporting. The result of this work was the AGREE instrument, a 23-item tool targeting six quality-related domains.8,9 It became accepted by many as the standard for guideline evaluation.10As with any new assessment tool, ongoing development of the instrument is required. The AGREE Next Steps Consortium was established to conduct a program of research aimed at improving the AGREE and advancing the overall guideline enterprise. We report on the first of two studies designed to achieve these goals. This study focused on four key issues related to methodology and implementation (Figure 1).Open in a separate windowFigure 1Program of research.First, the original four-point response scale was not in keeping with standards for test construction that are intended to maximize the reliability and discriminability of an instrument and minimize the number of appraisers required to evaluate a guideline.11 To address this issue, we introduced a seven-point response scale, tested its performance and conducted a preliminary analysis of some of its measurement properties.Second, to be of value, the AGREE instrument needs to be easy to apply and needs to generate information that is useful. To generate a reliable estimate of guideline quality, it is recommended that the 23 items of the AGREE instrument be applied by four independent reviewers.8,9 This process can be cumbersome and resource-intensive. However, no systematic analysis has been undertaken previously to determine if all items of the AGREE instrument generate information that is equally useful across different groups. This fact opens the possibility that fewer than 23 items, and varying combinations of items unique to different users, may be sufficient for evaluation purposes. Therefore, we explored whether evidence exists to inform the development of abridged versions of the AGREE that could be tailored to the unique priorities of different user groups.Third, to be useful as well, the AGREE ratings should be associated with outcomes that are relevant to guideline usage. In keeping with previous findings,4 guidelines of higher quality should be more attractive, endorsed or used than those of lower quality. We therefore explored whether these relationships existed and whether they were consistent across different types of users.Finally, given that the AGREE had been in the field for some time, we systematically collected feedback from users on how the items and domains might be improved, updated and refined.In combination with the results of the second study,12 the data were used by the consortium to craft the AGREE II,13 the next version of the AGREE instrument. 相似文献84.
Creswell C Hentges F Parkinson M Sheffield P Willetts L Cooper P 《Mental health in family medicine》2010,7(1):49-57
Anxiety disorders in childhood are common, disabling and run a chronic course. Cognitive behaviour therapy (CBT) is effective but expensive and trained therapists are scarce. Guided self-help treatments may be a means of widening access to treatment. This study aimed to examine the feasibility of guided CBT self-help in primary care for childhood anxiety disorders, specifically in terms of therapist adherence, patient and therapist satisfaction and clinical gain.Participants were children aged between five and 12 years referred to two primary child and adolescent mental health services (PCAMHSs) in Oxfordshire, UK, who met diagnostic criteria for a primary anxiety disorder. Of the 52 eligible children, 41 anxious children were assessed for anxiety severity and interference before and after receiving CBT self-help delivered via a parent (total therapy time = five hours) by primary mental health workers (PMHWs). Therapy sessions were rated for treatment adherence and parents and PMHWs completed satisfaction questionnaires after treatment completion. Over 80% of therapy sessions were rated at a high level of treatment adherence. Parents and PMHWs reported high satisfaction with the treatment. Sixty-one percent of the children assessed no longer met the criteria for their primary anxiety disorder diagnosis following treatment, and 76% were rated as 'much'/'very much' improved on the Clinical Global Impression-Improvement (CGI-I) scale. There were significant reductions on parent and child report measures of anxiety symptoms, interference and depression. Preliminary exploration indicated that parental anxiety was associated with child treatment outcome. The findings suggest that guided CBT self-help represents a promising treatment for childhood anxiety in primary care. 相似文献
85.
Magne D Palmer G Barton JL Mézin F Talabot-Ayer D Bas S Duffy T Noger M Guerne PA Nicklin MJ Gabay C 《Arthritis research & therapy》2006,8(3):R80-11
Six novel members of the IL-1 family of cytokines were recently identified, primarily through the use of DNA database searches for IL-1 homologues, and were named IL-1F5 to IL-1F10. In the present study, we investigated the effect of IL-1F8 on primary human joint cells, and examined the expression of the new IL-1 family members in human and mouse joints. Human synovial fibroblasts (hSFs) and human articular chondrocytes (hACs) expressed the IL-1F8 receptor (IL-1Rrp2) and produced pro-inflammatory mediators in response to recombinant IL-1F8. IL-1F8 mRNA expression was increased in hSFs upon stimulation with proinflammatory cytokines, whereas in hACs IL-1F8 mRNA expression was constitutive. However, IL-1F8 protein was undetectable in hSF and hAC culture supernatants. Furthermore, although IL-1beta protein levels were increased in inflamed human and mouse joint tissue, IL-1F8 protein levels were not. IL-1F8 levels in synovial fluids were similar to or lower than those in matched serum samples, suggesting that the joint itself is not a major source of IL-1F8. Serum levels of IL-1F8 were similar in healthy donors, and patients with rheumatoid arthritis, osteoarthritis and septic shock, and did not correlate with inflammatory status. Interestingly however, we observed high IL-1F8 levels in several serum samples in all groups. In conclusion, IL-1F8 exerts proinflammatory effects in primary human joint cells. Joint and serum IL-1F8 protein levels did not correlate with inflammation, but they were high in some human serum samples tested, including samples from patients with rheumatoid arthritis. It remains to be determined whether circulating IL-1F8 can contribute to joint inflammation in rheumatoid arthritis. 相似文献
86.
Reybier K Boyer J Farines V Camus F Souchard JP Monje MC Bernardes-Genisson V Goldstein S Nepveu F 《Free radical research》2006,40(1):11-20
The ability of ten imidazolyl nitrones to directly scavenge free radicals (R(*)) generated in polar ((*)OH, O(*)(2)(-), SO(*)(3)(-) cysteinyl, (*)CH(3)) or in apolar (CH(3)-(*)CH-CH(3)) media has been studied. When oxygen or sulfur-centered radicals are generated in polar media, EPR spectra are not or weakly observed with simple spectral features. Strong line intensities and more complicated spectra are observed with the isopropyl radical generated in an apolar medium. Intermediate results are obtained with (*)CH(3) generated in a polar medium. EPR demonstrates the ability of these nitrones to trap radicals to the nitrone C(alpha) atom (alpha radical adduct) and to the imidazol C(5) atom (5-radical adduct). Beside the nucleophilic addition of the radical to the C(alpha) atom, the EPR studies suggest a two-step mechanism for the overall reaction of R(*) attacking the imidazol core. The two steps seem to occur very fast with the (*)OH radical obtained in a polar medium and slower with the isopropyl radical prepared in benzene. In conclusion, imidazolyl nitrones present a high capacity to trap and stabilize carbon-centered radicals. 相似文献
87.
The MicroArray Quality Control (MAQC) project shows inter- and intraplatform reproducibility of gene expression measurements 总被引:2,自引:0,他引:2
MAQC Consortium Shi L Reid LH Jones WD Shippy R Warrington JA Baker SC Collins PJ de Longueville F Kawasaki ES Lee KY Luo Y Sun YA Willey JC Setterquist RA Fischer GM Tong W Dragan YP Dix DJ Frueh FW Goodsaid FM Herman D Jensen RV Johnson CD Lobenhofer EK Puri RK Schrf U Thierry-Mieg J Wang C Wilson M Wolber PK Zhang L Amur S Bao W Barbacioru CC Lucas AB Bertholet V Boysen C Bromley B Brown D Brunner A Canales R Cao XM Cebula TA Chen JJ Cheng J Chu TM Chudin E Corson J Corton JC Croner LJ 《Nature biotechnology》2006,24(9):1151-1161
Over the last decade, the introduction of microarray technology has had a profound impact on gene expression research. The publication of studies with dissimilar or altogether contradictory results, obtained using different microarray platforms to analyze identical RNA samples, has raised concerns about the reliability of this technology. The MicroArray Quality Control (MAQC) project was initiated to address these concerns, as well as other performance and data analysis issues. Expression data on four titration pools from two distinct reference RNA samples were generated at multiple test sites using a variety of microarray-based and alternative technology platforms. Here we describe the experimental design and probe mapping efforts behind the MAQC project. We show intraplatform consistency across test sites as well as a high level of interplatform concordance in terms of genes identified as differentially expressed. This study provides a resource that represents an important first step toward establishing a framework for the use of microarrays in clinical and regulatory settings. 相似文献
88.
Endo-(1,4)-beta-xylanases of plant and fungal origin play an important role in the degradation of arabinoxylans. Two distinct classes of proteinaceous endoxylanase inhibitors, the Triticum aestivum xylanase inhibitor (TAXI) and the xylanase inhibitor protein (XIP), have been identified in cereals. Engineering of proteins in conjunction with enzyme kinetics, thermodynamic, real-time interaction, and X-ray crystallographic studies has provided knowledge on the mechanism of inhibition of XIP-I towards endoxylanases. XIP-I is a 30 kDa protein which belongs to glycoside hydrolase family 18, and folds as a typical (beta/alpha)8 barrel. Although the inhibitor shows highest homology with plant chitinases, XIP-I does not hydrolyse chitin; probably due to structural differences in the XIP-I binding cleft. The inhibitor is specific for fungal xylanases from glycoside hydrolases families 10 and 11, but does not inhibit bacterial enzymes. The inhibition is competitive and, depending on the xylanase, the Ki value can be as low as 3.4 nM. Site-directed mutagenesis of a xylanase from Aspergillus niger suggested that the XIP-I binding site was the conserved hairpin loop "thumb" region of family 11 xylanases. Furthermore, XIP-I shows the ability to inhibit barley alpha-amylases of glycoside hydrolase family 13, providing the first example of a protein able to inhibit members of different glycoside hydrolase families (10, 11, and 13), and additionally a novel function for a protein of glycoside hydrolase family 18. 相似文献
89.
Bès C Briant-Longuet L Cerutti M Heitz F Troadec S Pugnière M Roquet F Molina F Casset F Bresson D Péraldi-Roux S Devauchelle G Devaux C Granier C Chardes T 《The Journal of biological chemistry》2003,278(16):14265-14273
We analyzed antigen-binding residues from the variable domains of anti-CD4 antibody 13B8.2 using the Spot method of parallel peptide synthesis. Sixteen amino acids, defined as Spot critical residues (SCR), were identified on the basis of a 50% decrease in CD4 binding to alanine analogs of reactive peptides. Recombinant Fab 13B8.2 mutants were constructed with alanine residues in place of each of the 16 SCR, expressed in the baculovirus cell system, and purified. CD measurements indicated that the mutated proteins were conformationally intact, with a beta-sheet secondary structure similar to that of wild-type Fab. Compared with the CD4-binding capacity of wild-type Fab 13B8.2, 11 light (Y32-L, W35-L, Y36-L, H91-L, and Y92-L) and heavy chain (H35-H, R38-H, W52-H, R53-H, F100K-H, and W103-H) Fab single mutants showed a decrease in CD4 recognition as demonstrated by enzyme-linked immunosorbent assay, BIAcore, and flow cytometry analyses. The five remaining Fab mutants showed antigen-binding properties similar to those of wild-type Fab. Recombinant Fab mutants that showed decreased CD4 binding also lost their capacity to inhibit human immunodeficiency virus promoter activation and the antigen-presenting ability that wild-type Fab displays. Molecular modeling of the 13B8.2 antibody paratope indicated that most of these critical residues are appropriately positioned inside the putative CD4-binding pocket, whereas the five SCR that were not confirmed by mutagenesis show an unfavorable positioning. Taken together, these results indicate that most of the residues defined by the Spot method as critical matched with important residues defined by mutagenesis in the whole protein context. The identification of critical residues for CD4 binding in the paratope of anti-CD4 recombinant Fab 13B8.2 provides the opportunity for the generation of improved anti-CD4 molecules with more efficient pharmacological properties. 相似文献
90.
Schanstra JP Duchene J Praddaude F Bruneval P Tack I Chevalier J Girolami JP Bascands JL 《American journal of physiology. Heart and circulatory physiology》2003,284(6):H1904-H1908
Bradykinin B(2) receptor knockout mice (B(2)-/-) have been useful to study the role of bradykinin under pathological conditions. With the use of these mice, it was shown that bradykinin plays an important role in angiogenesis, heart failure, salt-induced hypertension, and kidney fibrosis. Data on the role of the bradykinin B(2) receptor under physiological conditions using these mice are controversial and scarce, because these mice have no typical phenotype. For this reason, we have studied, under physiological conditions, renal hemodynamics as well as a number of morphometric glomerular parameters of B(2)-/- mice on a homogenized genetic background and on mice bred in a pathogen-free environment. Backcrossed B(2)-/- mice had normal blood pressure and normal apparent renal hemodynamics and morphology. However, reduced renal nitrite excretion and glomerular cGMP content were found, which was associated with a reduced glomerular capillary surface area. These differences had, however, no detectable effects on renal hemodynamics. These differences between B(2)-/- and wild-type mice might become important under pathological conditions as shown by a number of studies using these bradykinin B(2) receptor knockout mice. 相似文献