Development and validation of a rapid multiplex ELISA for pyrrolizidine alkaloids and their N-oxides in honey and feed Rapid Detection in Food and Feed

Pyrrolizidine alkaloids (PAs) are a group of plant secondary metabolites with carcinogenic and hepatotoxic properties. When PA-producing plants contaminate crops, toxins can be transferred through the food chain and cause illness in humans and animals, most notably hepatic veno-occlusive disease. Honey has been identified as a direct risk of human exposure. The European Food Safety Authority has recently identified four groups of PAs that are of particular importance for food and feed: senecionine-type, lycopsamine-type, heliotrine-type and monocrotaline-type. Liquid or gas chromatography methods are currently used to detect PAs but there are no rapid screening assays available commercially. Therefore, the aim of this study was to develop a rapid multiplex ELISA test for the representatives of three groups of alkaloids (senecionine, lycopsamine and heliotrine types) that would be used as a risk-management tool for the screening of these toxic compounds in food and feed. The method was validated for honey and feed matrices and was demonstrated to have a detection capability less than 25 µg/kg for jacobine, lycopsamine, heliotrine and senecionine. The zinc reduction step introduced to the extraction procedure allows for the additional detection of the presence of N-oxides of PAs. This first multiplex immunoassay for PA detection with N-oxide reduction can be used for the simultaneous screening of 21 samples for >12 PA analytes. Honey samples (n?=?146) from various origins were analysed for PA determination. Six samples were determined to contain measurable PAs >25 µg/kg by ELISA which correlated to >10 µg/kg by LC-MS/MS.

An indirect bridge inspection method incorporating a wavelet-based damage indicator and pattern recognition

In recent years, there has been a move towards the development of indirect structural health monitoring (SHM)techniques for bridges; the low-cost vibration-based method presented in this paper is such an approach. It consists of the use of a moving vehicle fitted with accelerometers on its axles and incorporates wavelet analysis and statistical pattern recognition. The aim of the approach is to both detect and locate damage in bridges while reducing the need for direct instrumentation of the bridge. In theoretical simulations, a simplified vehicle-bridge interaction model is used to investigate the effectiveness of the approach in detecting damage in a bridge from vehicle accelerations. For this purpose, the accelerations are processed using a continuous wavelet transform as when the axle passes over a damaged section, any discontinuity in the signal would affect the wavelet coefficients. Based on these coefficients, a damage indicator is formulated which can distinguish between different damage levels. However, it is found to be difficult to quantify damage of varying levels when the vehicle’s transverse position is varied between bridge crossings. In a real bridge field experiment, damage was applied artificially to a steel truss bridge to test the effectiveness of the indirect approach in practice; for this purpose a two-axle van was driven across the bridge at constant speed. Both bridge and vehicle acceleration measurements were recorded. The dynamic properties of the test vehicle were identified initially via free vibration tests. It was found that the resulting damage indicators for the bridge and vehicle showed similar patterns, however, it was difficult to distinguish between different artificial damage scenarios.

Direct and indirect antimicrobial activities of neuropeptides and their therapeutic potential

As global resistance to conventional antibiotics rises we need to develop new strategies to develop future novel therapeutics. In our quest to design novel anti-infectives and antimicrobials it is of interest to investigate host-pathogen interactions and learn from the complexity of host defense strategies that have evolved over millennia. A myriad of host defense molecules are now known to play a role in protection against human infection. However, the interaction between host and pathogen is recognized to be a multifaceted one, involving countless host proteins, including several families of peptides. The regulation of infection and inflammation by multiple peptide families may represent an evolutionary failsafe in terms of functional degeneracy and emphasizes the significance of host defense in survival. One such family is the neuropeptides (NPs), which are conventionally defined as peptide neurotransmitters but have recently been shown to be pleiotropic molecules that are integral components of the nervous and immune systems. In this review we address the antimicrobial and anti-infective effects of NPs both in vitro and in vivo and discuss their potential therapeutic usefulness in overcoming infectious diseases. With improved understanding of the efficacy of NPs, these molecules could become an important part of our arsenal of weapons in the treatment of infection and inflammation. It is envisaged that targeted therapy approaches that selectively exploit the anti-infective, antimicrobial and immunomodulatory properties of NPs could become useful adjuncts to our current therapeutic modalities. © 2012 Bentham Science Publishers.

A Review of Indirect Bridge Monitoring Using Passing Vehicles

Indirect bridge monitoring methods, using the responses measured from vehicles passing over bridges, are under development for about a decade. A major advantage of these methods is that they use sensors mounted on the vehicle, no sensors or data acquisition system needs to be installed on the bridge. Most of the proposed methods are based on the identification of dynamic characteristics of the bridge from responses measured on the vehicle, such as natural frequency, mode shapes, and damping. In addition, some of the methods seek to directly detect bridge damage based on the interaction between the vehicle and bridge. This paper presents a critical review of indirect methods for bridge monitoring and provides discussion and recommendations on the challenges to be overcome for successful implementation in practice.

LL-37 quantification in gingival crevicular fluid (GCF) from periodontitis patients

Background: LL-37, an anti-microbial peptide belonging to the cathelicidin family, derives its name from two N-terminal leucine residues and the 37 amino acids comprising the peptide. LL-37 is the only known cathelicidin to exist in humans. It exhibits both anti-bacterial and immunomodulatory properties. Objectives: In the current study, LL-37 was quantified in GCF from periodontitis patients. Previous studies have relied on qualitative results from Western blotting to detect LL-37 in GCF. This study aims to quantitatively determine LL-37 levels in GCF. Methods: GCF and bacterial plaque samples, pre- and post non-surgical periodontal treatment, were collected from 4 sites in 12 patients presenting with advanced periodontitis. Plaque samples were analysed by QPCR for the presence or absence of the periodontopathic bacterium Porphyromonas gingivalis (P. gingivalis). The concentrations of LL-37 in patient samples pre- and post-treatment were deduced by indirect enzyme linked immunosorbent assay (ELISA). Results: Concentrations of LL-37 in samples varied between a minimum and maximum of 1 and 40 ng/ml. LL-37 levels were shown, pre-treatment, to be higher in deep pockets (6-9 mm) compared with shallower pockets (3-5 mm) and highest in those sites which were positive for P. gingivalis. Non-surgical therapy resulted in a significant improvement in clinical indices while expression levels of P. gingivalis were reduced. Following treatment, LL-37 levels in GCF decreased from an average of 6.5 ± 1 - 5.8 ± 1.2 ng/ml. The most interesting observation however was the reduction in LL-37 levels, from an average of 7 ± 1.3 – 2.5 ± 1.1 ng/ml in those sites where P. gingivalis infection was eradicated post-treatment. Conclusions: LL-37 levels are increased at sites showing advanced periodontal disease, reduce following treatment and appear to be linked to the presence of P. gingivalis. This study will further our knowledge of host defence in chronic diseases such as periodontitis.

Production of extended-spectrum β-lactamases and the potential indirect pathogenic role of Prevotella isolates from the cystic fibrosis respiratory microbiota

Extended-spectrum β-lactamase (ESBL) production and the prevalence of the β-lactamase-encoding gene blaTEM were determined in Prevotella isolates (n=50) cultured from the respiratory tract of adults and young people with cystic fibrosis (CF). Time-kill studies were used to investigate the concept of passive antibiotic resistance and to ascertain whether a β-lactamase-positive Prevotella isolate can protect a recognised CF pathogen from the action of ceftazidime in vitro. The results indicated that approximately three-quarters (38/50; 76%) of Prevotella isolates produced ESBLs. Isolates positive for ESBL production had higher minimum inhibitory concentrations (MICs) of β-lactam antibiotics compared with isolates negative for production of ESBLs (P<0.001). The blaTEM gene was detected more frequently in CF Prevotella isolates from paediatric patients compared with isolates from adults (P=0.002), with sequence analysis demonstrating that 21/22 (95%) partial blaTEM genes detected were identical to blaTEM-116. Furthermore, a β-lactamase-positive Prevotella isolate protected Pseudomonas aeruginosa from the antimicrobial effects of ceftazidime (P=0.03). Prevotella isolated from the CF respiratory microbiota produce ESBLs and may influence the pathogenesis of chronic lung infection via indirect methods, including shielding recognised pathogens from the action of ceftazidime.

Environmental occurrence and fate of psychiatric pharmaceuticals

Os fármacos são importantes contaminantes ambientais. Nas últimas duas décadas, o número de estudos sobre a ocorrência destes poluentes emergentes em matrizes ambientais aumentou significativamente. Esta ocorrência generalizada preocupa a comunidade científica devido a evidências que comprovam a sua capacidade de interferir nos ecossistemas, mesmo em concentrações muito baixas. No caso particular dos fármacos psiquiátricos é expectável que constituam um risco ecológico significativo. Para uma melhor compreensão do impacto real destes poluentes é essencial que se proceda a uma avaliação extensiva da sua persistência e destino em matrizes ambientais. Os estudos apresentados nesta tese pretendem contribuir para melhorar o conhecimento acerca da ocorrência, persistência e destino ambiental de fármacos psiquiátricos. Para este efeito, foram seleccionados, como objecto de estudo, dois grupos de fármacos: anti-epilépticos (carbamazepina) e fármacos com efeitos ansiolíticos e sedativos (as benzodiazepinas diazepam, oxazepam, lorazepam e alprazolam). A fotodegradação é o principal processo que afecta a persistência de poluentes orgânicos em ambientes aquáticos. Consequentemente, a persistência dos cinco fármacos seleccionados foi avaliada através de estudos de fotodegradação directa e indirecta, tendo em consideração a influência de parâmetros relevantes tais como pH, nível de oxigenação e matéria orgânica dissolvida. Os estudos de fotodegradação aqui descritos foram seguidos por cromatografia micelar electrocinética com a aplicação de um capilar com revestimento dinâmico. Adicionalmente, os fotoprodutos resultantes de fotodegradação directa foram identificados por espectrometria de massa. O estudo da carbamazepina no ambiente é particularmente relevante uma vez que esta foi proposta como um potencial marcador de poluição antropogénica. A sua ocorrência em água superficiais, de sub-solo e residuais foi investigada através da implementação de um ensaio imunológico (ELISA), optimizado para a aplicação a triagens ambientais e amostras com matrizes complexas. O destino deste fármaco na interface água/solo foi também investigado usando solos agrícolas submetidos a fertilizações de longo prazo; este estudo permitiu tirar conclusões acerca da contaminação de águas adjacentes por solos contaminados. O trabalho aqui descrito constitui uma abordagem multidisplinar à problemática da ocorrência de fármacos psiquiátricos no ambiente, contribuindo de forma relevante para esta área de estudo.

Occurrence and fate of estrogens and antibiotics in the environment evaluated by low-cost analytical methodologies

Endocrine disruptors and pharmaceuticals are considered to be concerning environmental contaminants. During the last two decades, studies dealing with the occurrence and fate of these emerging contaminants in the aquatic environment have raised attention and its number is constantly increasing. The presence of these contaminants in the environment is particularly important since they are known to induce adverse effects in the ecosystems even at extremely low concentrations. Estrogens and antibiotics, in particular, are identified as capable of induce endocrine disruption and contribute for the appearance of multi-resistant bacteria, respectively. A better assessment and understanding of the real impact of these contaminants in the aquatic environment implies the evaluation of their occurrence and fate, which is the main aim of this Thesis. Two estrogens (17-estradiol and 17-ethinylestradiol) and an antibiotic (sulfamethoxazole) were the contaminants under study and their occurrence in surface and waste waters was assessed by the implementation of enzyme linked immunosorbent assays (ELISAs). The assays were optimized in order to accomplish two important aspects: to analyze complex water samples, giving special attention to matrix effects, and to increase the sensitivity. Since the levels of these contaminants in the environment are extremely low, a pre-concentration methodology was also object of study in this Thesis. Dispersive liquid-liquid microextraction (DLLME) was developed for the preconcentration of E2 and EE2, subsequently quantified by either highperformance liquid chromatography (HPLC) and the previously optimized ELISAs. Moreover, the use of anthropogenic markers, i.e. indicators of human presence or activity, has been discussed as a tool to track the origin and type of contamination. An ELISA for the quantification of caffeine, as an anthropogenic marker, was also developed in order to assess the occurrence of human domestic pollution in Portuguese surface waters. Finally, photodegradation is considered to be one of the most important pathways contributing for the mitigation of pollutants’ presence in the aquatic environment. Both direct and indirect photodegradation of E2 and EE2 were evaluated. Since the presence of humic substances (HS) is known to have a noticeable influence on the photodegradation of pollutants and in order to mimic the real aquatic environment, special attention was given to the influence of the presence and concentration of different fractions of HS on the photodegradation of both hormones.

Protection of wind energy systems against the indirect effects of lightning

This paper is concerned with the protection of wind energy systems against the indirect effects of lightning. As wind energy is gaining increasing importance throughout the world, lightning damages involving wind energy systems have come to be regarded with more attention. Nevertheless, there are still very few studies in Portugal regarding lightning protection of wind energy systems using models of the Electro-Magnetic Transients Program (EMTP). Hence, a new case study is presented in this paper, based on a wind turbine with an interconnecting transformer, considering that lightning strikes the soil near the tower at a distance such that galvanic coupling occurs through the grounding electrode. Computer simulations obtained by using EMTP-RV are presented and conclusions are duly drawn. (C) 2011 Elsevier Ltd. All rights reserved.