Rapid confirmatory method for the determination of sixteen synthetic growth promoters and bisphenol A in bovine milk using dispersive solid-phase extraction and liquid chromatography-tandem mass spectrometry

A rapid liquid chromatographic-tandem mass spectrometric (LC-MS/MS) multi-residue method for the simultaneous quantitation and identification of sixteen synthetic growth promoters and bisphenol A in bovine milk has been developed and validated. Sample preparation was straightforward, efficient and economically advantageous. Milk was extracted with acetonitrile followed by phase separation with NaCl. After centrifugation, the extract was purified by dispersive solid-phase extraction with C18 sorbent material. The compounds were analysed by reversed-phase LC-MS/MS using both positive and negative ionization and operated in multiple reaction monitoring (MRM) mode, acquiring two diagnostic product ions from each of the chosen precursor ions for unambiguous confirmation. Total chromatographic run time was less than 10 min for each sample. The method was validated at a level of 1 mu g L-1. A wide variety of deuterated internal standards were used to improve method performance. The accuracy and precision of the method were satisfactory for all analytes. The confirmative quantitative liquid chromatographic tandem mass spectrometric (LC-MS/MS) method was validated according to Commission Decision 2002/657/EC. The decision limit (CC alpha) and the detection capability (CC beta) were found to be below the chosen validation level of 1 mu g L-1 for all compounds. (C) 2010 Elsevier B.V. All rights reserved.

Remote, subsurface detection of the algal toxin domoic acid onboard the Environmental Sample Processor: Assay development and field trials

The ability to detect harmful algal bloom (HAB) species and their toxins in real- or near real-time is a critical need for researchers studying HAB/toxin dynamics, as well as for coastal resource managers charged with monitoring bloom populations in order to mitigate their wide ranging impacts. The Environmental Sample Processor (ESP), a robotic electromechanical/fluidic system, was developed for the autonomous, subsurface application of molecular diagnostic tests and has successfully detected several HAB species using DNA probe arrays during field deployments. Since toxin production and thus the potential for public health and ecosystem effects varies considerably in natural phytoplankton populations, the concurrent detection of HAB species and their toxins onboard the ESP is essential. We describe herein the development of methods for extracting the algal toxin domoic acid (DA) from Pseudonitzschia cells (extraction efficiency >90%) and testing of samples using a competitive ELISA onboard the ESP. The assay detection limit is in the low ng/mL range (in extract), which corresponds to low ng/L levels of DA in seawater for a 0.5 L sample volume acquired by the ESP. We also report the first in situ detection of both a HAB organism (i.e., Pseudo-nitzschia) and its toxin, domoic acid, via the sequential (within 2-3 h) conduct of species- and toxin-specific assays during ESP deployments in Monterey Bay, CA, USA. Efforts are now underway to further refine the assay and conduct additional calibration exercises with the aim of obtaining more reliable, accurate estimates of bloom toxicity and thus their potential impacts. Published by Elsevier B.V.

Risk of Malignant Progression in Barrett’s Esophagus Patients: Results from a Large Population-Based Study

Background: Barrett's esophagus (BE) is a premalignant lesion that predisposes to esophageal adenocarcinoma. However, the reported incidence of esophageal adenocarcinoma in patients with BE varies widely. We examined the risk of malignant progression in patients with BE using data from the Northern Ireland Barrett's esophagus Register (NIBR), one of the largest population-based registries of BE worldwide, which includes every adult diagnosed with BE in Northern Ireland between 1993 and 2005.

Subjects and Methods: We followed 8522 patients with BE, defined as columnar lined epithelium of the esophagus with or without specialized intestinal metaplasia (SIM), until the end of 2008. Patients with incident adenocarcinomas of the esophagus or gastric cardia or with high-grade dysplasia of the esophagus were identified by matching the NIBR with the Northern Ireland Cancer Registry, and deaths were identified by matching with records from the Registrar General's Office. Incidence of cancer outcomes or high-grade dysplasia was calculated as events per 100 person-years (% per year) of follow-up, and Cox proportional hazard models were used to determine incidence by age, sex, length of BE segment, presence of SIM, macroscopic BE, or low-grade dysplasia. All P values were from two-sided tests.

Results: After a mean of 7.0 years of follow-up, 79 patients were diagnosed with esophageal cancer, 16 with cancer of the gastric cardia, and 36 with high-grade dysplasia. In the entire cohort, incidence of esophageal or gastric cardia cancer or high-grade dysplasia combined was 0.22% per year (95% confidence interval [CI] = 0.19% to 0.26%). SIM was found in 46.0% of patients. In patients with SIM, the combined incidence was 0.38% per year (95% CI = 0.31 to 0.46%). The risk of cancer was statistically significantly elevated in patients with vs without SIM at index biopsy (0.38% per year vs 0.07% per year; hazard ratio [HR] = 3.54, 95% CI = 2.09 to 6.00, P <. 001), in men compared with women (0.28% per year vs 0.13% per year; HR = 2.11, 95% CI = 1.41 to 3.16, P <. 001), and in patients with low-grade dysplasia compared with no dysplasia (1.40% per year vs 0.17% per year; HR = 5.67, 95% CI = 3.77 to 8.53, P <. 001).

Conclusion: We found the risk of malignant progression among patients with BE to be lower than previously reported, suggesting that currently recommended surveillance strategies may not be cost-effective. © The Author 2011. Published by Oxford University Press. All rights reserved.

In vivo and in vitro function of human UDP-galactose 4 '-epimerase variants

Type III galactosemia results from reduced activity of the enzyme UDP-galactose 4'-epimerase. Five disease-associated alleles (G90E, V94M, D103G, N34S and L183P) and three artificial alleles (Y105C, N268D, and M284K) were tested for their ability to alleviate galactose-induced growth arrest in a Saccharomyces cerevisiae strain which lacks endogenous UDP-galactose 4'-epimerase. For all of these alleles, except M284K, the ability to alleviate galactose sensitivity was correlated with the UDP-galactose 4'-epimerase activity detected in cell extracts. The M284K allele, however, was able to substantially alleviate galactose sensitivity, but demonstrated near-zero activity in cell extracts. Recombinant expression of the corresponding protein in Escherichia coil resulted in a protein with reduced enzymatic activity and reduced stability towards denaturants in vitro. This lack of stability may result from the introduction of an unpaired positive charge into a bundle of three alpha-helices near the surface of the protein. The disparities between the in vivo and in vitro data for M284K-hGALE further suggest that there are additional, stabilising factors present in the cell. Taken together, these results reinforce the need for care in the interpretation of in vitro, enzymatic diagnostic tests for type III galactosemia. (C) 2011 Elsevier Masson SAS. All rights reserved.

Reducing the future threat from (liver) fluke: realistic prospect or quixotic fantasy?

The liver fluke remains an economically significant parasite of livestock and is emerging as an important zoonotic infection of humans. The incidence of the disease has increased in the last few years, as a possible consequence of changes to the World's climate. Future predictions suggest that this trend is likely to continue. Allied to the changing pattern of disease, reports of resistance to triclabendazole (TCBZ) have appeared in the literature, although they do not all represent genuine cases of resistance. Nevertheless, any reports of resistance are a concern, because triclabendazole is the only drug that has high activity against the migratory and damaging juvenile stages of infection. How to deal with the twin problems (of increasing incidence and drug resistance) is the overall theme of the session on “Trematodes: Fasciola hepatica epidemiology and control” and of this review to introduce the session.

Greater knowledge of fluke epidemiology and population genetics will highlight those regions where surveillance is most required and indicate how quickly resistant populations of fluke may arise. Models of disease risk are becoming increasingly sophisticated and precise, with more refined data analysis programmes and Geographic Information Systems (GIS) data. Recent improvements have been made in our understanding of the action of triclabendazole and the ways in which flukes have become resistant to it. While microtubules are the most likely target for drug action, tubulin mutations do not seem to be involved in the resistance mechanism. Rather, upregulation of drug uptake and metabolism processes appear to be more important and the data relating to them will be discussed. The information may help in the design of new treatment strategies or pinpoint potential molecular markers for monitoring fluke populations. Advances in the identification of novel targets for drugs and vaccines will be made by the various “-omics” technologies that are now being applied to Fasciola. A major area of concern in the current control of fasciolosis is the lack of reliable tests for the diagnosis of drug (TCBZ) resistance. This has led to inaccurate reports of resistance, which is hindering successful disease management, as farmers may be encouraged to switch to less effective drugs. Progress with the development of a number of new diagnostic tests will be reviewed.

Molecular detection of CF lung pathogens:current status and future potential

Molecular diagnostic tests, based on the detection and identification of nucleic acids in human biological samples, are increasingly employed in the diagnosis of infectious diseases and may be of future benefit to CF microbiology services. Our growing understanding of the complex polymicrobial nature of CF airway infection has highlighted current and likely future shortcomings in standard diagnostic practices. Failure to detect fastidious or slow growing microbes and misidentification of newly emerging pathogens could potentially be addressed using culture-independent molecular technologies with high target specificity. This review considers existing molecular diagnostic tests in the context of the key requirements for an envisaged CF microbiology focussed assay. The issues of assay speed, throughput, detection of multiple pathogens, data interpretation and antimicrobial susceptibility testing are discussed.

Accuracy of LightCycler SeptiFast for the detection and identification of pathogens in the blood of patients with suspected sepsis: a systematic review and meta-analysis

Purpose: There is an urgent need to develop diagnostic tests to improve the detection of pathogens causing life-threatening infection (sepsis). SeptiFast is a CE-marked multi-pathogen real-time PCR system capable of detecting DNA sequences of bacteria and fungi present in blood samples within a few hours. We report here a systematic review and meta-analysis of diagnostic accuracy studies of SeptiFast in the setting of suspected sepsis.

Methods: A comprehensive search strategy was developed to identify studies that compared SeptiFast with blood culture in suspected sepsis. Methodological quality was assessed using QUADAS. Heterogeneity of studies was investigated using a coupled forest plot of sensitivity and specificity and a scatter plot in receiver operator characteristic space. Bivariate model method was used to estimate summary sensitivity and specificity.

Results: From 41 phase III diagnostic accuracy studies, summary sensitivity and specificity for SeptiFast compared with blood culture were 0.68 (95 % CI 0.63–0.73) and 0.86 (95 % CI 0.84–0.89) respectively. Study quality was judged to be variable with important deficiencies overall in design and reporting that could impact on derived diagnostic accuracy metrics.

Conclusions: SeptiFast appears to have higher specificity than sensitivity, but deficiencies in study quality are likely to render this body of work unreliable. Based on the evidence presented here, it remains difficult to make firm recommendations about the likely clinical utility of SeptiFast in the setting of suspected sepsis.

Electron-Impact Uncertainty Analysis and its Impact on Certain Temperature Diagnostics

In this study we calculate the electron-impact uncertainties in atomic data for direct ionization and recombination and investigate the role of these uncertainties on spectral diagnostics. We outline a systematic approach to assigning meaningful uncertainties that vary with electron temperature. Once these uncertainty parameters have been evaluated, we can then calculate the uncertainties on key diagnostics through a Monte Carlo routine, using the Astrophysical Emission Code (APEC) [Smith et al. 2001]. We incorporate these uncertainties into well known temperature diagnostics, such as the Lyman alpha versus resonance line ratio and the G ratio. We compare these calculations to a study performed by [Testa et al. 2004], where significant discrepancies in the two diagnostic ratios were observed. We conclude that while the atomic physics uncertainties play a noticeable role in the discrepancies observed by Testa, they do not explain all of them. This indicates that there is another physical process occurring in the system that is not being taken into account. This work is supported in part by the National Science Foundation REU and Department of Defense ASSURE programs under NSF Grant no. 1262851 and by the Smithsonian Institution.

Effects of Prochlorperazine on Normal Vestibular Ocular and Perceptual Responses: A Randomised, Double-Blind, Crossover, Placebo-Controlled Study

Background: The present study investigated whether prochlorperazine affects vestibular-ocular reflex (VOR) and vestibulo-perceptual function. Methods: We studied 12 healthy naïve subjects 3 hours after a single dose of oral prochlorperazine 5mg in a randomised, placebo-controlled, double-blind, cross-over study in healthy young subjects. Two rotational tests in yaw were used: 1) a Threshold task investigating perceptual motion detection and nystagmic thresholds (acceleration steps of 0.5deg/s/s) and 2) Suprathreshold responses to velocity steps of 90deg/s in which vestibulo-ocular (VO) and vestibulo-perceptual (VP) time constants of decay, as well as VOR gain, were measured. Results: Prochlorperazine had no effect upon any measure of nystagmic or perceptual vestibular function compared to placebo. This lack of effects on vestibular-mediated motion perception suggests that the drug is likely to act more as an antiemetic than as an anti-vertiginous agent.

Aprendizagem dos números reais : um estudo com alunos do 9.º ano

Relatório da Prática de Ensino Supervisionada, Ensino de Matemática, Universidade de Lisboa, 2013

Metodologia bayesiana e adequação de modelos

Tese de doutoramento, Estatística e Investigação Operacional (Probabilidades e Estatística), Universidade de Lisboa, Faculdade de Ciências, 2014

Descrição de uma população narcoléptica portuguesa

Tese de mestrado, Ciências do Sono, Faculdade de Medicina, Universidade de Lisboa, 2015

Análise do investimento direto estrangeiro em Portugal utilizando a metodologia de box-jenkins

Dissertação para obtenção do Grau de Mestre em Contabilidade e Finanças Orientadora: Professora Doutora Patrícia Ramos

Modelo de data mining para detecção de tumores em exames de rastreio

Dissertação para obtenção do grau de Mestre em Engenharia Informática

Sonication of removed breast implants for improved detection of subclinical infection.

BACKGROUND: Capsular fibrosis is a severe complication after breast implantation with an uncertain etiology. Microbial colonization of the prosthesis is hypothesized as a possible reason for the low-grade infection and subsequent capsular fibrosis. Current diagnostic tests consist of intraoperative swabs and tissue biopsies. Sonication of removed implants may improve the diagnosis of implant infection by detachment of biofilms from the implant surface. METHODS: Breast implants removed from patients with Baker grades 3 and 4 capsular contracture were analyzed by sonication, and the resulting sonication fluid was quantitatively cultured. RESULTS: This study investigated 22 breast implants (6 implants with Baker 3 and 16 implants with Baker 4 capsular fibrosis) from 13 patients. The mean age of the patients was 49 years (range, 31-76 years). The mean implant indwelling time was 10.4 years (range, 3 months to 30 years). Of the 22 implants, 12 were used for breast reconstruction and 10 for aesthetic procedures. The implants were located subglandularly (n = 12), submuscularly (n = 6), and subcutaneously (n = 4). Coagulase-negative staphylococci, Propionibacterium acnes, or both were detected in the sonication fluid cultures of nine implants (41%), eight of which grew significant numbers of microorganisms (>100 colonies/ml of sonication fluid). CONCLUSIONS: Sonication detected bacteria in 41% of removed breast implants. The identified bacteria belonged to normal skin flora. Further investigation is needed to determine any causal relation between biofilms and capsular fibrosis.