Development and characterization of monoclonal antibodies to spring viraemia of carp virus

Five monoclonal antibodies (mAbs) against spring viraemia of carp (SVCV0504, isolated from common carp in China) were produced from mice immunized with purified virus preparations. The virion of SVCV contains five structural proteins, representing the nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and RNA-dependent RNA polymerase (Q. Western blotting analysis revealed that three mAbs (1145, IE10, and 11-17) recognized specifically to a single protein of 47 kDa (N), the mAb 3G4 reacted with, two SVCV0504 proteins of 69 kDa (G) and 47 kDa (N), while the mAb 1A9 reacted with three SVCV0504 proteins of 69 kDa (G), 50 kDa (P), and 47 kDa (N). By indirect ELISA, two mAbs (1H5 and 11-17) showed cross-reactivity with pike fry rhabdovirus (PFRV), but no cross-reactions with the Siniperca chuatsi rhabdovirus (SCRV), Scophthalmus maximus rhabdovirus (SMRV), Paralichthys olivaceus rhabdovirus (PoRV) were demonstrated with the five mAbs. Indirect immunofluorescence showed intense fluorescence in the cytoplasm of the SVCV0504-infected epithelioma papulosum cyprini (EPC) cells in areas corresponding to the location of granular structures. The sucrose gradient-purified SVCV0504 particles could be detected successfully by these mAbs using immunodot blotting. mAb 1A9 could completely neutralize 100 TCID50 (50% tissue culture infective dose) of SVCV0504 at a dilution of 1:8. This is the first report of development of the neutralizing mAbs against SVCV. The mAb 1A9 was analyzed further and could be used to successfully detect viral antigens in the infected-EPC cell cultures or in cryosections from experimentally infected crucian carp (Carassius auratus) by immunohistochemistry assay. Furthermore, a flow cytometry procedure for the detection and quantification of cytoplasmic SVCV0504 in cell cultures was developed with mAb 1A9. At 28 h after inoculation with the virus (0.01 PFU/cell), 10.12% of infected cells could be distinguished from the uninfected cells. These mAbs will be useful in diagnostic test development and pathogenesis studies for fish rhabdovirus. (c) 2008 Elsevier B.V. All rights reserved.

HPCC在IBM刀片机群上的诊断测试与结果分析

在IBMJS21 Blade Center上进行2次HPCC测试,介绍HPCC的结果分析方法,并采用分层模型AHPCC对HPCC的测试结果进行分析。其目的是通过在高性能机群上执行HPCC测试说明HPCC测试对机群系统的评价和诊断能力。实验发现,在之前的HPL测试结果一直不理想并且无法更进一步发现和解决问题的情况下,采用HPCC测试能够较好地评价系统和诊断系统问题。通过分层模型的评价,能够得到更多关于目标系统的性能参数和发现可能的性能瓶颈,为系统设计及构建积累有价值的经验。

Self-Supervised ARTMAP

Computational models of learning typically train on labeled input patterns (supervised learning), unlabeled input patterns (unsupervised learning), or a combination of the two (semisupervised learning). In each case input patterns have a fixed number of features throughout training and testing. Human and machine learning contexts present additional opportunities for expanding incomplete knowledge from formal training, via self-directed learning that incorporates features not previously experienced. This article defines a new self-supervised learning paradigm to address these richer learning contexts, introducing a neural network called self-supervised ARTMAP. Self-supervised learning integrates knowledge from a teacher (labeled patterns with some features), knowledge from the environment (unlabeled patterns with more features), and knowledge from internal model activation (self-labeled patterns). Self-supervised ARTMAP learns about novel features from unlabeled patterns without destroying partial knowledge previously acquired from labeled patterns. A category selection function bases system predictions on known features, and distributed network activation scales unlabeled learning to prediction confidence. Slow distributed learning on unlabeled patterns focuses on novel features and confident predictions, defining classification boundaries that were ambiguous in the labeled patterns. Self-supervised ARTMAP improves test accuracy on illustrative lowdimensional problems and on high-dimensional benchmarks. Model code and benchmark data are available from: http://techlab.bu.edu/SSART/.

Cost-effectiveness analysis of the diagnosis of meniscus tears.

BACKGROUND: Diagnostic imaging represents the fastest growing segment of costs in the US health system. This study investigated the cost-effectiveness of alternative diagnostic approaches to meniscus tears of the knee, a highly prevalent disease that traditionally relies on MRI as part of the diagnostic strategy. PURPOSE: To identify the most efficient strategy for the diagnosis of meniscus tears. STUDY DESIGN: Economic and decision analysis; Level of evidence, 1. METHODS: A simple-decision model run as a cost-utility analysis was constructed to assess the value added by MRI in various combinations with patient history and physical examination (H&P). The model examined traumatic and degenerative tears in 2 distinct settings: primary care and orthopaedic sports medicine clinic. Strategies were compared using the incremental cost-effectiveness ratio (ICER). RESULTS: In both practice settings, H&P alone was widely preferred for degenerative meniscus tears. Performing MRI to confirm a positive H&P was preferred for traumatic tears in both practice settings, with a willingness to pay of less than US$50,000 per quality-adjusted life-year. Performing an MRI for all patients was not preferred in any reasonable clinical scenario. The prevalence of a meniscus tear in a clinician's patient population was influential. For traumatic tears, MRI to confirm a positive H&P was preferred when prevalence was less than 46.7%, with H&P preferred above that. For degenerative tears, H&P was preferred until the prevalence reaches 74.2%, and then MRI to confirm a negative was the preferred strategy. In both settings, MRI to confirm positive physical examination led to more than a 10-fold lower rate of unnecessary surgeries than did any other strategy, while MRI to confirm negative physical examination led to a 2.08 and 2.26 higher rate than H&P alone in primary care and orthopaedic clinics, respectively. CONCLUSION: For all practitioners, H&P is the preferred strategy for the suspected degenerative meniscus tear. An MRI to confirm a positive H&P is preferred for traumatic tears for all practitioners. Consideration should be given to implementing alternative diagnostic strategies as well as enhancing provider education in physical examination skills to improve the reliability of H&P as a diagnostic test. CLINICAL RELEVANCE: Alternative diagnostic strategies that do not include the use of MRI may result in decreased health care costs without harm to the patient and could possibly reduce unnecessary procedures.

A prospective clinical trial of a real-time polymerase chain reaction assay for the diagnosis of candidemia in nonneutropenic, critically ill adults

Background. Invasive Candida infection among nonneutropenic, critically ill adults is a clinical problem that has received increasing attention in recent years. Poor performance of extant diagnostic modalities has promoted risk-based, preemptive prescribing in view of the poor outcomes associated with inadequate or delayed antifungal therapy; this risks unnecessary overtreatment. A rapid, reliable diagnostic test could have a substantial impact on therapeutic practice in this patient population.

Methods. Three TaqMan-based real-time polymerase chain reaction assays were developed that are capable of detecting the main medically important Candida species, categorized according to the likelihood of fluconazole susceptibility. Assay 1 detected Candida albicans, Candida parapsilosis, Candida tropicalis, and Candida dubliniensis. Assays 2 and 3 detected Candida glabrata and Candida krusei, respectively. The clinical performance of these assays, applied to serum, was evaluated in a prospective trial of nonneutropenic adults in a single intensive care unit.

Results. In all, 527 specimens were obtained from 157 participants. All 3 assays were run in parallel for each specimen; they could be completed within 1 working day. Of these, 23 specimens were obtained from 23 participants categorized as having proven Candida infection at the time of sampling. If a single episode of Candida famata candidemia was excluded, the estimated clinical sensitivity, specificity, and positive and negative predictive values of the assays in this trial were 90.9%, 100%, 100% and 99.8%, respectively.

Conclusions. These data suggest that the described assays perform well in this population for enhancing the diagnosis of candidemia. The extent to which they may affect clinical outcomes, prescribing practice, and cost-effectiveness of care remains to be ascertained.

A practical miniature long-term bone marrow culture system for investigating early myelodysplasia

A method was devised to grow haemopoietic cells in long-term bone marrow culture (LTBMC) which requires only 1 x 10(6) cells/culture. Such miniature cultures were used to study growth patterns of marrow from patients with myelodysplastic syndromes (MDS). Consistent differences in LTBMC cellularity and cellular composition were noted between MDS and normal marrow. These differences were accentuated by rGM-CSF. The criteria which distinguished between and MDS marrows were: cell count at weeks 1 and 4, % neutrophils and % blasts. In 10 patients with unexplained macrocytosis or pancytopenia miniature LTBMC results clearly segregated into either 'normal' or 'MDS' growth patterns. Miniature LTBMC with rGM-CSF may therefore be a useful diagnostic test for early MDS.

The clinical effectiveness and cost-effectiveness of screening for open angle glaucoma:A systematic review and economic evaluation

Objectives: To assess whether open angle glaucoma (OAG) screening meets the UK National Screening Committee criteria, to compare screening strategies with case finding, to estimate test parameters, to model estimates of cost and cost-effectiveness, and to identify areas for future research. Data sources: Major electronic databases were searched up to December 2005. Review methods: Screening strategies were developed by wide consultation. Markov submodels were developed to represent screening strategies. Parameter estimates were determined by systematic reviews of epidemiology, economic evaluations of screening, and effectiveness (test accuracy, screening and treatment). Tailored highly sensitive electronic searches were undertaken. Results: Most potential screening tests reviewed had an estimated specificity of 85% or higher. No test was clearly most accurate, with only a few, heterogeneous studies for each test. No randomised controlled trials (RCTs) of screening were identified. Based on two treatment RCTs, early treatment reduces the risk of progression. Extrapolating from this, and assuming accelerated progression with advancing disease severity, without treatment the mean time to blindness in at least one eye was approximately 23 years, compared to 35 years with treatment. Prevalence would have to be about 3-4% in 40 year olds with a screening interval of 10 years to approach cost-effectiveness. It is predicted that screening might be cost-effective in a 50-year-old cohort at a prevalence of 4% with a 10-year screening interval. General population screening at any age, thus, appears not to be cost-effective. Selective screening of groups with higher prevalence (family history, black ethnicity) might be worthwhile, although this would only cover 6% of the population. Extension to include other at-risk cohorts (e.g. myopia and diabetes) would include 37% of the general population, but the prevalence is then too low for screening to be considered cost-effective. Screening using a test with initial automated classification followed by assessment by a specialised optometrist, for test positives, was more cost-effective than initial specialised optometric assessment. The cost-effectiveness of the screening programme was highly sensitive to the perspective on costs (NHS or societal). In the base-case model, the NHS costs of visual impairment were estimated as £669. If annual societal costs were £8800, then screening might be considered cost-effective for a 40-year-old cohort with 1% OAG prevalence assuming a willingness to pay of £30,000 per quality-adjusted life-year. Of lesser importance were changes to estimates of attendance for sight tests, incidence of OAG, rate of progression and utility values for each stage of OAG severity. Cost-effectiveness was not particularly sensitive to the accuracy of screening tests within the ranges observed. However, a highly specific test is required to reduce large numbers of false-positive referrals. The findings that population screening is unlikely to be cost-effective are based on an economic model whose parameter estimates have considerable uncertainty, in particular, if rate of progression and/or costs of visual impairment are higher than estimated then screening could be cost-effective. Conclusions: While population screening is not cost-effective, the targeted screening of high-risk groups may be. Procedures for identifying those at risk, for quality assuring the programme, as well as adequate service provision for those screened positive would all be needed. Glaucoma detection can be improved by increasing attendance for eye examination, and improving the performance of current testing by either refining practice or adding in a technology-based first assessment, the latter being the more cost-effective option. This has implications for any future organisational changes in community eye-care services. Further research should aim to develop and provide quality data to populate the economic model, by conducting a feasibility study of interventions to improve detection, by obtaining further data on costs of blindness, risk of progression and health outcomes, and by conducting an RCT of interventions to improve the uptake of glaucoma testing. © Queen's Printer and Controller of HMSO 2007. All rights reserved.

A PCR-based method to distinguish fungi of the rice sheath-blight complex, Rhizoctonia solani, R. oryzae and R. oryzae-sativae

Identification of Rhizoctonia solani, R. oryzae and R. oryzae-sativae, components of the rice sheath disease complex, is extremely difficult and often inaccurate and as a result may hinder the success of extensive breeding programmes throughout Asia. In this study, primers designed from unique regions within the rDNA internal transcribed spacers have been used to develop a rapid PCR-based diagnostic test to provide an accurate identification of the species on rice. Tests on the specificity of the primers concerned showed that they provide the means for accurate identification of the Rhizoctonia species responsible for sheath diseases in rice.

A PCR-based method to characterise and identify benzimidazole resistance in Helminthosporium solani

Control of Helminthosporium solani, the cause of silver scurf in potato tubers, has been impaired by selection of benzimidazole-resistant strains as a result of repeated use of the fungicide thiabendazole. Identification of thiabendazole-resistant strains of H. solani by conventional techniques takes several weeks. Primers designed from conserved regions of the fungal beta-tubulin gene were used to PCR amplify and sequence a portion of the gene. A point mutation was detected at codon 198 in thiabendazole-resistant isolates causing a change in the amino acid sequence from glutamic acid to alanine or glutamine. Species-specific PCR primers designed to amplify this region were used in conjunction with a restriction endonuclease to cause cleavage in sensitive isolates only and thus provide a rapid diagnostic test to differentiate field isolates.

Identification of benzimidazole resistance in Cladobotryum dendroides using a PCR-based method

Cladobotryutn dendroides, causal agent of cobweb disease of Agaricus bisporus, has become increasingly resistant to methylbenzimidazole carbamate (MBC) fungicides following the extensive use of MBC in cultivated mushroom production in Ireland. Of 38 isolates of C. dendroides obtained from Irish mushroom units, 34 were resistant to carbendazim. Primers based on conserved regions of the -tubulin gene were used to amplify and sequence a portion of the -tubulin gene in C. dendroides. A point mutation was detected at codon 50 in isolates resistant to benzimidazole fungicides, causing an amino acid substitution from tyrosine to cysteine. Species-specific PCR primers were designed to amplify the region of the -tubulin gene containing this substitution. The point mutation removed an Ace I restriction site in the -tubulin gene sequence of resistant isolates. Digestion of the PCR product with Ace I thus provides a rapid diagnostic test to differentiate sensitive and resistant isolates of this fungus. EMBL accession number: YI2256.

A ética como criação de qualidade de vida : o apreço pela vida como pilar de sustentação da acção humana

Relatório da Prática de Ensino Supervisionada, Mestrado em Ensino da Filosofia, Universidade de Lisboa, 2011

O ensino da multimédia com recurso ao Project Based Learning : realização de vídeos promocionais de um estabelecimento de ensino

Relatório da prática de ensino supervisionada, Mestrado em Ensino de Informática, Universidade de Lisboa, 2014

Pullulation bactérienne de l'intestin grêle [Small intestine bacterial overgrowth]

Small intestine bacterial overgrowth (SIBO) is a condition characterised by nutrient malabsorption and excessive bacteria in the small intestine. It typically presents with diarrhea, flatulence and a syndrome of malabsorption (steatorrhea, macrocytic anemia). However, it may be asymptomatic in the eldery. A high index of suspicion is necessary in order to differentiate SIBO from other similar presenting disorders such as coeliac disease, lactose intolerance or the irritable bowel syndrome. A search for predisposing factor is thus necessary. These factors may be anatomical (stenosis, blind loop), or functional (intestinal hypomotility, achlorydria). The hydrogen breath test is the most frequently used diagnostic test although it lacks standardisation. The treatment of SIBO consists of eliminating predisposing factors and broad-spectrum antibiotic therapy.

Exact Skewness-Kurtosis Tests for Multivariate Normality and Goodness-of-fit in Multivariate Regressions with Application to Asset Pricing Models

We study the problem of testing the error distribution in a multivariate linear regression (MLR) model. The tests are functions of appropriately standardized multivariate least squares residuals whose distribution is invariant to the unknown cross-equation error covariance matrix. Empirical multivariate skewness and kurtosis criteria are then compared to simulation-based estimate of their expected value under the hypothesized distribution. Special cases considered include testing multivariate normal, Student t; normal mixtures and stable error models. In the Gaussian case, finite-sample versions of the standard multivariate skewness and kurtosis tests are derived. To do this, we exploit simple, double and multi-stage Monte Carlo test methods. For non-Gaussian distribution families involving nuisance parameters, confidence sets are derived for the the nuisance parameters and the error distribution. The procedures considered are evaluated in a small simulation experi-ment. Finally, the tests are applied to an asset pricing model with observable risk-free rates, using monthly returns on New York Stock Exchange (NYSE) portfolios over five-year subperiods from 1926-1995.

Étiopathogenèse de la scoliose idiopathique de l'adolescent : implication de la mélatonine et de l'ostéopontine

La scoliose idiopathique de l’adolescent (SIA) est une maladie dont la cause est encore inconnue, et qui génère des déformations complexes du rachis, du thorax et du bassin. La prévalence est de 4% dans la population adolescente au Québec. Cette pathologie affecte surtout les filles durant leur poussée de croissance pubertaire. Parmi plusieurs hypothèses émises, l’hypothèse neuroendocrinienne, impliquant une déficience en mélatonine comme agent étiologique de la SIA a suscité beaucoup d’intérêt. Cette hypothèse découle du fait que l’ablation de la glande pinéale chez le poulet produit une scoliose ressemblant sous plusieurs aspects à la pathologie humaine. La pertinence biologique de la mélatonine dans la scoliose est controversée, étant donné que la majorité des études chez l’homme n’ont pu mettre en évidence une diminution significative des niveaux de mélatonine circulante chez les patients scoliotiques. Nous avons démontré un dysfonctionnement dans la signalisation de la mélatonine au niveau des tissus musculo-squelettiques chez une série de patients atteints de SIA (Moreau & coll. 2004). Nous avons confirmé ce défaut chez un plus grand nombre de patients ainsi qu’en utilisant une nouvelle technologie (spectroscopie cellulaire diélectrique) n’ayant pas recours à un prétraitement des cellules donnant ainsi des résultats plus précis. Cette technique a montré la présence des mêmes groupes fonctionnels identifiés auparavant par la technique d’AMPc. Le dysfonctionnement de la signalisation de la mélatonine est dû à une phosphorylation accrue des protéines G inhibitrices. Ce défaut pourrait être causé par un déséquilibre de l’activité des kinases et phosphatases capables de réguler la phosphorylation des protéines Gi. Parmi ces kinases, PKCd a suscité initialement notre intérêt vu qu’elle peut phosphoryler les protéines Gi. Nous avons démontré que cette kinase interagit avec le récepteur de la mélatonine MT2 et que cette interaction varie selon le groupe fonctionnel auquel un patient SIA appartient. Par la suite nos travaux se sont dirigés vers la découverte d’effecteurs cellulaires régulés par la mélatonine et plus spécifiquement l’ostéopontine (OPN), compte tenu de son rôle présumé comme mécanorécepteur et dans certaines structures jouant un rôle dans la proprioception, le contrôle postural et la fonction vestibulaire. L’OPN a été identifiée initialement par sa surexpression au niveau protéique et de l’ARNm dans la musculature paraspinale uniquement chez les poulets scoliotiques. Nous avons également utilisé un autre modèle animal, la souris C57Bl/6 naturellement déficiente en mélatonine. Nous avons généré des souris bipèdes en amputant les membres antérieurs de souris OPN KO, des souris CD44 KO ainsi que des souris contrôles C57Bl/6. Nos résultats ont montré qu’aucune souris OPN KO (n=50) ou CD44 KO (n=60) ne développe la maladie, contrairement aux souris contrôles C57Bl/6 (n=50) dont 45% deviennent scoliotiques. Ces résultats nous ont poussés à investiguer le rôle de cette protéine dans l’étiopathogenèse de la maladie chez l’humain. Nos résultats ont montré une augmentation des niveaux circulants d’OPN chez les patients atteints de la SIA et que l’élevation en OPN corrélait avec la sévérité de la maladie. Nos études chez les enfants asymptomatiques nés de parents scoliotiques et qui sont plus à risque de développer la maladie ont aussi démontré des différences significatives au niveau des concentrations en OPN en comparaison avec les sujets sains. En effet, plusieurs enfants à risque présentaient des niveaux d’OPN supérieurs à 800ng/ml suggérant un plus grand risque de développer une scoliose indiquant aussi que l’augmentation des niveaux en OPN précède le début de la maladie.