Developing an international Pseudomonas aeruginosa reference panel

Pseudomonas aeruginosa is a major opportunistic pathogen in cystic fibrosis (CF) patients and causes a wide range of infections among other susceptible populations. Its inherent resistance to many antimicrobials also makes it difficult to treat infections with this pathogen. Recent evidence has highlighted the diversity of this species, yet despite this, the majority of studies on virulence and pathogenesis focus on a small number of strains. There is a pressing need for a P. aeruginosa reference panel to harmonize and coordinate the collective efforts of the P. aeruginosa research community. We have collated a panel of 43 P. aeruginosa strains that reflects the organism's diversity. In addition to the commonly studied clones, this panel includes transmissible strains, sequential CF isolates, strains with specific virulence characteristics, and strains that represent serotype, genotype or geographic diversity. This focussed panel of P. aeruginosa isolates will help accelerate and consolidate the discovery of virulence determinants, improve our understanding of the pathogenesis of infections caused by this pathogen, and provide the community with a valuable resource for the testing of novel therapeutic agents.

Bioinformatic evaluation of transcriptional regulation of WNT pathway genes with reference to diabetic nephropathy

Objective: Diabetic nephropathy (DN) is a microvascular complication of diabetes. Members of the WNT/ β-catenin pathways have been implicated in interstitial fibrosis and glomerular sclerosis, characteristic hallmarks of DN. These processes are controlled, in part, by transcription factors (TFs), proteins which bind to gene promoter regions attenuating their regulation. We sought to identify predicted cis-acting transcription factor binding sites (TFBS) over-represented within the promoter regions of WNT pathway members compared to genes across the genome.Methods: We assessed the frequency of 62 TFBS motifs from the JASPAR databases on 65 WNT pathway genes. P-values were estimated on the hypergeometric distribution for each TF. Gene expression profiles of enriched motifs were examined from DN-related datasets to assess clinical significance.Results: TFBS motifs transcription factor AP-2 alpha (TFAP2A), myeloid zinc finger 1 (MZF1), and specificity protein 1 (SP1) were significantly enriched within WNT pathway genes (P-values<6.83x10-29, 1.34x10-11 and 3.01x10-6 respectively). MZF1 gene expression was significantly increased in DN in a whole kidney dataset (fold change = 1.16; 16% increase; P = 0.03). TFAP2A gene expression was decreased in an independent dataset (fold change = -1.02; P = 0.03). SP1 was not differentially expressed in any datasets examined.Conclusions: Three TFBS profiles are significantly enriched within the WNT pathway genes examined highlighting the use of in silico analyses for identifying key regulators of this pathway. Modification of TF binding to gene promoter regions involved in DN pathology may limit progression, making refinement of targeted therapeutic strategies possible through clearer delineation of their role.

Pilot tone reference-less phase conjugator for phase modulated retrodirective antenna applications

This letter presents a simple tracking phased locked loop (PLL) that can be used to track phase-modulated signals and provide a phase-conjugated signal for retrodirective retransmission. The configuration allows the retrodirective antenna to directly track phase-modulated signals with no requirement for a separate continuous wave (CW) pilot tone. The ability to directly track phase-modulated signals is carried out using a 4× multiplier on the tracking PLL reference signal. Practical phase conjugation results are presented for a five-element retrodirective array simultaneously sending and receiving phase-modulated (QPSK) signals. Signals with levels as low as -122 dBm can be phase-conjugated and retransmitted with 30 dBm EIRP.

Phenotypic characterization of an international Pseudomonas aeruginosa reference panel: strains of cystic fibrosis (CF) origin show less in vivo virulence than non-CF strains

Pseudomonas aeruginosa causes chronic lung infections in people with cystic fibrosis (CF) and acute opportunistic infections in people without CF. Forty two P. aeruginosa strains from a range of clinical and environmental sources were collated into a single reference strain panel to harmonise research on this diverse opportunistic pathogen. To facilitate further harmonized and comparable research on P. aeruginosa, we characterised the panel strains for growth rates, motility, virulence in the Galleria mellonella infection model, pyocyanin and alginate production, mucoid phenotype, lipopolysaccharide (LPS) pattern, biofilm formation, urease activity, antimicrobial and phage susceptibilities. Phenotypic diversity across the P. aeruginosa panel was apparent for all phenotypes examined agreeing with the marked variability seen in this species. However, except for growth rate, the phenotypic diversity among strains from CF versus non-CF sources was comparable. CF strains were less virulent in the G. mellonella model than non-CF strains (p=0.037). Transmissible CF strains generally lacked O antigen, produced less pyocyanin, and had low virulence in G. mellonella. Further, in the three sets of sequential CF strains, virulence, O-antigen expression and pyocyanin production were higher in the earlier isolate compared to the isolate obtained later in infection. Overall, full phenotypic characterization of the defined panel of P. aeruginosa strains increases our understanding of the virulence and pathogenesis of P. aeruginosa and may provide a valuable resource for the testing of novel therapies against this problematic pathogen.

Characterization of a reference material for BCR-ABL (M-BCR) mRNA quantitation by real-time amplification assays:towards new standards for gene expression measurements

Monitoring of BCR-ABL transcripts has become established practice in the management of chronic myeloid leukemia. However, nucleic acid amplification techniques are prone to variations which limit the reliability of real-time quantitative PCR (RQ-PCR) for clinical decision making, highlighting the need for standardization of assays and reporting of minimal residual disease (MRD) data. We evaluated a lyophilized preparation of a leukemic cell line (K562) as a potential quality control reagent. This was found to be relatively stable, yielding comparable respective levels of ABL, GUS and BCR-ABL transcripts as determined by RQ-PCR before and after accelerated degradation experiments as well as following 5 years storage at -20 degrees C. Vials of freeze-dried cells were sent at ambient temperature to 22 laboratories on four continents, with RQ-PCR analyses detecting BCR-ABL transcripts at levels comparable to those observed in primary patient samples. Our results suggest that freeze-dried cells can be used as quality control reagents with a range of analytical instrumentations and could enable the development of urgently needed international standards simulating clinically relevant levels of MRD.

Reference gene selection in human periodontal ligament cells

Background: Real-time quantitative PCR (qPCR) is a highly sensitive and specific method which is used extensively for determining gene expression profiles in a variety of cell and tissue types. In order to obtain accurate and reliable gene expression quantification, qPCR data are generally normalised against so-called reference or housekeeping genes. Ideally, reference genes should have abundant and stable RNA transcriptomes under the experimental conditions employed. However, reference genes are often selected rather arbitrarily and indeed some have been shown to have variable expression in a variety of in vitro experimental conditions.
Objective: The objective of the current study was to investigate reference gene expression in human periodontal ligament (PDL) cells in response to treatment with lipopolysaccharide (LPS).
Method: Primary human PDL cells were grown in Dulbecco’s Modified Eagle Medium with L-glutamine supplemented with 10% fetal bovine serum, 100UI/ml penicillin and 100µg/ml streptomycin. RNA was isolated using the RNeasy Mini Kit (Qiagen) and reverse transcribed using the QuantiTect Reverse Transcription Kit (Qiagen). The expression of a total of 19 reference genes was studied in the presence and absence of LPS treatment using the Roche Reference Gene Panel. Data were analysed using NormFinder and Bestkeeper validation programs.
Results: Treatment of human PDL cells with LPS resulted in changes in expression of several commonly used reference genes, including GAPDH. On the other hand the reference genes β-actin, G6PDH and 18S were identified as stable genes following LPS treatment.
Conclusion: Many of the reference genes studied were robust to LPS treatment (up to 100 ng/ml). However several commonly employed reference genes, including GAPDH varied with LPS treatment, suggesting they would not be ideal candidates for normalisation in qPCR gene expression studies.

Pathogens in Milk: Enterobacter Species. Reference Module in Food Sciences.

Enterobacter species commonly occur in the environment and are recognized as opportunistic human pathogens in clinical settings. However, with the exception of Enterobacter sakazakii (Cronobacter), Enterobacter species are not normally considered foodborne pathogens. Cronobacter are particularly associated with illness in infants, particularly within the first 3 months after birth. Therefore, although Cronobacter are found in a wide range of fresh and dried food materials, it is their contamination of the infant formula production chain that is the major cause for concern. Cronobacter are noted for their ability to survive during desiccation and their persistence in dried infant food for at least 2 years.

Consciência cultural crítica numa comunidade virtual educativa de línguas

A difusão das tecnologias da informação e comunicação fomenta mudanças qualitativas nas práticas pedagógicas, proporcionando a criação de comunidades de aprendizagem entre aprendentes de diferentes pontos do mundo. Tendo como referência a pedagogia crítica para a emancipação (Freire, 1997; Giroux, 1997), este estudo analisou de que forma aprendentes de diferentes proveniências linguístico-culturais desenvolvem a sua consciência cultural crítica (Byram, 1997), quando colocados em situação de trabalho colaborativo on-line, formando uma comunidade de aprendizagem, através do recurso a uma plataforma especialmente concebida para o efeito, a 2ndschool.eu, na qual foram levados a desenvolver um trabalho de natureza interdisciplinar. Pretendíamos que esta plataforma fomentasse questionamentos por parte dos seus membros. Como tal, integrámos diferentes instrumentos de comunicação eletrónica (chat, fóruns e e-mail), através dos quais se promoveu a interação entre os participantes no projeto, alunos e professores (de diversas áreas disciplinares) do Ensino Secundário belga, búlgaro, grego, polaco, português e sueco, com vista à realização de uma tarefa comum: a edição de um trabalho de projeto de análise crítica de reportagens, artigos de opinião e fotos de jornais acerca de tópicos da atualidade nacional e/ou internacional. Tivemos em conta uma metodologia de investigação mais orientada para o estudo de caso e análise do discurso. Para tal, recorremos a dois tipos de instrumentos de recolha de dados: as impressões das discussões estabelecidas através de chat, fóruns, blogs e wikis e os resultados de três questionários sobre o perfil sociolinguístico e cultural dos participantes, a avaliação da plataforma virtual e o inventário de estratégias mais eficazes na negociação de saberes estabelecida. Concluímos que os alunos (re)constroem saberes, pois revelam representações que têm acerca de situações-problema, refletem acerca das mesmas e, posteriormente, disseminam ativamente pontos de vista críticos através de ferramentas Web 2.0, como forma de as resolver. Enquanto verdadeiros pronetários, foram capazes de recorrer a estratégias de comunicação que fomentam a busca de entendimento com o Outro, num caminho oscilante entre o concordar e o discordar, entre o ajudar e o solicitar ajuda, entre o opinar e o escutar, entre o avaliar e o ser avaliado e entre o corrigir e o ser corrigido. Identificámos como principais limitações do nosso estudo a dificuldade de análise das práticas interdisciplinares dos interlocutores internacionais, a desmotivação de alguns aprendentes nas tarefas e ainda o reduzido recurso ao videochat, pelo desconforto no seu uso. Por isso, consideramos que futuras investigações deverão debruçar-se nestas questões.

Parameters determination for the coupled electromagnetic transformer model, with particular reference to winding faults studies

In the past few years, a considerable research effort has been devoted to the development of transformer digital models in order to simulate its behaviour under transient and abnormal operating conditions. Although many three-phase transformer models have been presented in the literature, there is a surprisingly lack of studies regarding the incorporation of winding faults. This paper presents a coupled electromagnetic transformer model for the study of winding inter-turn short-circuits. Particular attention will be given to the model parameters determination, for both healthy and faulty operating conditions. Experimental and simulation test results are presented in the paper, demonstrating the adequacy of the model as well as the methodologies for the parameters determination.

Identification of reliable reference genes for quantitative gene expression in the Mozambique tilapia, Oreochromis mossambicus

Dissertação de mest., Biologia Marinha (Aquacultura), Faculdade de Ciências e Tecnologia, Univ. do Algarve, 2010