A link between DNA methylation and epigenetic silencing in transgenic Volvox carteri

Epigenetic silencing of foreign genes introduced into plants poses an unsolved problem for transgenic technology. Here we have used the simple multicellular green alga Volvox carteri as a model to analyse the relation of DNA methylation to transgenic silencing. Volvox DNA contains on average 1.1% 5-methylcytosine and 0.3% N6-methyladenine, as revealed by electrospray mass spectrometry and phosphoimaging of chromatographically separated 32P-labelled nucleotides. In two nuclear transformants of V.carteri, produced in 1993 by biolistic bombardment with a foreign arylsulphatase gene (C-ars), the transgene is still expressed in one (Hill 181), but not in the other (Hill 183), after an estimated 500–1000 generations. Each transformant clone contains multiple intact copies of C-ars, most of them integrated into the genome as tandem repeats. When the bisulphite genomic sequencing protocol was applied to examine two select regions of transgenic C-ars, we found that the inactivated copies (Hill 183) exhibited a high-level methylation (40%) of CpG dinucleotides, whereas the active copies (Hill 181) displayed low-level (7%) CpG methylation. These are average values from 40 PCR clones sequenced from each DNA strand in the two portions of C-ars. The observed correlation of CpG methylation and transgene inactivation in a green alga will be discussed in the light of transcriptional silencing.

Arabidopsis nph1 and npl1: Blue light receptors that mediate both phototropism and chloroplast relocation

UV-A/blue light acts to regulate a number of physiological processes in higher plants. These include light-driven chloroplast movement and phototropism. The NPH1 gene of Arabidopsis encodes an autophosphorylating protein kinase that functions as a photoreceptor for phototropism in response to low-intensity blue light. However, nph1 mutants have been reported to exhibit normal phototropic curvature under high-intensity blue light, indicating the presence of an additional phototropic receptor. A likely candidate is the nph1 homologue, npl1, which has recently been shown to mediate the avoidance response of chloroplasts to high-intensity blue light in Arabidopsis. Here we demonstrate that npl1, like nph1, noncovalently binds the chromophore flavin mononucleotide (FMN) within two specialized PAS domains, termed LOV domains. Furthermore, when expressed in insect cells, npl1, like nph1, undergoes light-dependent autophosphorylation, indicating that npl1 also functions as a light receptor kinase. Consistent with this conclusion, we show that a nph1npl1 double mutant exhibits an impaired phototropic response under both low- and high-intensity blue light. Hence, npl1 functions as a second phototropic receptor under high fluence rate conditions and is, in part, functionally redundant to nph1. We also demonstrate that both chloroplast accumulation in response to low-intensity light and chloroplast avoidance movement in response to high-intensity light are lacking in the nph1npl1 double mutant. Our findings therefore indicate that nph1 and npl1 show partially overlapping functions in two different responses, phototropism and chloroplast relocation, in a fluence rate-dependent manner.

Improved adenovirus packaging cell lines to support the growth of replication-defective gene-delivery vectors.

Adenovirus (Ad) vectors have been extensively used to deliver recombinant genes to a great variety of cell types in vitro and in vivo. Ad-based vectors are available that replace the Ad early region 1 (E1) with recombinant foreign genes. The resultant E1-deleted vectors can then be propagated on 293 cells, a human embryonal kidney cell line that constitutively expresses the E1 genes. Unfortunately, infection of cells and tissues in vivo results in low-level expression of Ad early and late proteins (despite the absence of E1 activity) resulting in immune recognition of virally infected cells. The infected cells are subsequently eliminated, resulting in only a transient expression of foreign genes in vivo. We hypothesize that a second-generation Ad vector with a deletion of viral genes necessary for Ad genome replication should block viral DNA replication and decrease viral protein production, resulting in a diminished immune response and extended duration of foreign gene expression in vivo. As a first step toward the generation of such a modified vector, we report the construction of cell lines that not only express the E1 genes but also constitutively express the Ad serotype 2 140-kDa DNA polymerase protein, one of three virally encoded proteins essential for Ad genome replication. The Ad polymerase-expressing cell lines support the replication and growth of H5ts36, an Ad with a temperature-sensitive mutation of the Ad polymerase protein. These packaging cell lines can be used to prepare Ad vectors deleted for the E1 and polymerase functions, which should facilitate development of viral vectors for gene therapy of human diseases.

Orbital phase-resolved spectroscopy of 4U 1538−52 with MAXI

Context. 4U 1538−52, an absorbed high mass X-ray binary with an orbital period of ~3.73 days, shows moderate orbital intensity modulations with a low level of counts during the eclipse. Several models have been proposed to explain the accretion at different orbital phases by a spherically symmetric stellar wind from the companion. Aims. The aim of this work is to study both the light curve and orbital phase spectroscopy of this source in the long term. In particular, we study the folded light curve and the changes in the spectral parameters with orbital phase to analyse the stellar wind of QV Nor, the mass donor of this binary system. Methods. We used all the observations made from the Gas Slit Camera on board MAXI of 4U 1538−52 covering many orbits continuously. We obtained the good interval times for all orbital phase ranges, which were the input for extracting our data. We estimated the orbital period of the system and then folded the light curves, and we fitted the X-ray spectra with the same model for every orbital phase spectrum. We also extracted the averaged spectrum of all the MAXI data available. Results. The MAXI spectra in the 2–20 keV energy range were fitted with an absorbed Comptonisation of cool photons on hot electrons. We found a strong orbital dependence of the absorption column density but neither the fluorescence iron emission line nor low energy excess were needed to fit the MAXI spectra. The variation in the spectral parameters over the binary orbit were used to examine the mode of accretion onto the neutron star in 4U 1538−52. We deduce a best value of Ṁ/v∞ = 0.65 × 10-9M⊙ yr-1/ (km s-1) for QV Nor.

The assessment of the level of knowledge about dietary fibre among the portuguese population

The aim of this work was to assess the degree of knowledge about dietary fibre (DF), as influenced by factors such as age, gender, level of education and living environment. For this, a descriptive cross- sectional study was undertaken by means of a questionnaire and the total number of respondents was 382. The results revealed that the participants had a not satisfying global level of knowledge (3.59 ± 0.64, on a scale from 1 to 5). The overall knowledge was found lower for young adults aged 19 to 30 years, when compared to adults aged over 31 and up to 65 years, being this difference statistically significant (p=0.030). However, no statistical significant differences were found between genders, levels of education or living environments. The low level of knowledge demonstrated by the enquired alerts for the need to plan some additional actions to further inform the Portuguese population about dietary fibre.

Lichtheimia Infection in a Lymphoma Patient: Case Report and a Brief Review of the Available Diagnostic Tools

We describe the case of a patient with a T-lymphoblastic lymphoma whose disseminated mucormycosis was diagnosed with delay, and we address the diagnostic and therapeutic decision-making process and review the diagnostic workup of patients with potential IFD. The diagnosis was delayed despite a suggestive radiological presentation of the patient's pulmonary lesion. The uncommon risk profile (T-lymphoblastic lymphoma, short neutropenic phases) wrongly led to a low level of suspicion. The diagnosis was also hampered by the lack of indirect markers for infections caused by Mucorales, the low sensitivity of both fungal culture and panfungal PCR, and the limited availability of species-specific PCR. A high level of suspicion of IFD is needed, and aggressive diagnostic procedures should be promptly initiated even in apparently low-risk patients with uncommon presentations. The extent of the analytical workup should be decided on a case-by-case base. Diagnostic tests such as the galactomannan and β-D-glucan test and/or PCR on biological material followed by sequencing should be chosen according to their availability and after evaluation of their specificity and sensitivity. In high-risk patients, preemptive therapy with a broad-spectrum mould-active antifungal agent should be started before definitive diagnostic findings become available.

Why is Coccidoxenoides perminutus, a mealybug parasitoid, ineffective as a biocontrol agent - Inaccurate measures of parasitism or low adult survival?

Coccidoxenoides perminutus achieves only low levels of parasitism of its host Planococcus citri in southeast Queensland citrus. Two possible causes were investigated. Adult survival under natural conditions was assessed to determine whether providing adult food sources could enhance survival. Behavioural changes of hosts, induced by C perminutus parasitism, was also investigated to establish if parasitised P. citri move from their feeding site to seek protected shelters some distance away and are thus not accounted for in field assessments of parasitism rates. Unparasitised mealybugs placed in the field for two periods were retrieved before the effects of parasitism were manifested and parasitism rates were still low (0.3% at 5 days and 1.2% at 10 days). Levels of locomotion of P. citri exposed to C perminutus were compared with those of unexposed ones. Parasitised mealybugs, regardless of instar, undergo behavioural changes. In comparison to unparasitised controls, the mealybugs become highly active 7-14 days after exposure to wasps. All parasitised mealybugs undergo physical changes, their body becomes cylindrical, their legs go so rigid that the mealybugs become immobile, and this signifies the typical mummy appearance. All mealybugs that became mummies eventually fell from the host lemon fruit because of impaired locomotion and were caught on sticky traps that had been placed beneath the lemons. Consequently, their final site of mummification was not established. C perminutus adults provided with nectar or honey survived longer (about 5 days) in the field than those without food (about a day). Nectar from two plant species, Alpinia zerumbet and Datura candida, proved to be good sources of food for the adult wasps, and were comparable in quality to honey. The low level of parasitism achieved by C perminutus in southeast Queensland citrus thus appears to be a consequence of the short adult life and the negative effects of a harsh environment. Provision of a suitable food source (e.g., nectar) may well enhance levels of parasitism in the field. (c) 2005 Elsevier Inc. All rights reserved.

A systematic review and meta-analysis of clinical trials on physical interventions for lateral epicondylalgia

A systematic review of the literature on the effectiveness of physical interventions for lateral epicondylalgia ( tennis elbow) was carried out. Seventy six randomised controlled trials were identified, 28 of which satisfied the minimum criteria for meta-analysis. The evidence suggests that extracorporeal shock wave therapy is not beneficial in the treatment of tennis elbow. There is a lack of evidence for the long term benefit of physical interventions in general. However, further research with long term follow up into manipulation and exercise as treatments is indicated.

A Comparison of Paediatric Occupational Therapy University Program Curricula in New Zealand, Australia, and Canada

Comparisons were made of the paediatric content of professional entry-level occupational therapy university program curricula in Australia, New Zealand, and Canada using an ex post facto surveymethodology. The findings indicated that in Australia/New Zealand, paediatrics made up 20% of the total curriculum, but only 13% in Canada. Canadian reference materials were utilized less often in Canadian universities than in Australia/New Zealand. Theories taught most often in Australia/New Zealand were: Sensory Integration, Neurodevelopmental Therapy, Client-Centered Practice, Playfulness, and the Model of Human Occupation. In Canada, the most frequent theories were: Piaget’s Stages ofCognitive/Intellectual Development, Neurodevelopmental Therapy, Erikson’s Eight Stages of Psychosocial Development and Sensory Integration. The most frequently taught paediatric assessment tools in both regions were the Bruininks-Oseretsky Test of Motor Proficiency and Miller Assessment for Preschoolers. Paediatric interventionmethods taught to students in all three countries focused on activities of daily living/self-care, motor skills, perceptual and visual motor integration, and infant and child development. [Article copies available for a fee from The Haworth Document Delivery Service: 1-800-HAWORTH. E-mail address: Website: ©2006 by The Haworth Press, Inc. All rights reserved.]

The feasibility and outcome of clinic plus Internet delivery of cognitive-behavior therapy for childhood anxiety

Seventy-two clinically anxious children, aged 7 to 14 years, were randomly allocated to clinic-based, cognitive-behavior therapy, the same treatment partially delivered Via the Internet. or a wait-list control (WL). Children in the clinic and clinic-plus-Internet conditions showed significantly greater reductions in anxiety from pre- to posttreatment and were more likely to be free of their anxiety diagnoses, compared with the WL group. Improvements were maintained at 12-month follow-up for both therapy conditions', with minimal difference in outcomes between interventions. The Internet treatment content was highly acceptable to families, with minimal dropout and a high level of therapy compliance.

A DHA14 drug efflux gene from Xanthomonas albilineans confers high-level albicidin antibiotic resistance in Escherichia coli

Aims: Identification of a gene for self-protection from the antibiotic-producing plant pathogen Xanthomonas albilineans, and functional testing by heterologous expression. Methods and Results: Albicidin antibiotics and phytotoxins are potent inhibitors of prokaryote DNA replication. A resistance gene (albF) isolated by shotgun cloning from the X. albilineans albicidin-biosynthesis region encodes a protein with typical features of DHA14 drug efflux pumps. Low-level expression of albF in Escherichia coli increased the MIC of albicidin 3000-fold, without affecting tsx-mediated albicidin uptake into the periplasm or resistance to other tested antibiotics. Bioinformatic analysis indicates more similarity to proteins involved in self-protection in polyketide-antibiotic-producing actinomycetes than to multi-drug resistance pumps in other Gram-negative bacteria. A complex promoter region may co-regulate albF with genes for hydrolases likely to be involved in albicidin activation or self-protection. Conclusions: AlbF is the first apparent single-component antibiotic-specific efflux pump from a Gram-negative antibiotic producer. It shows extraordinary efficiency as measured by resistance level conferred upon heterologous expression. Significance and Impact of the Study: Development of the clinical potential of albicidins as potent bactericidial antibiotics against diverse bacteria has been limited because of low yields in culture. Expression of albF with recently described albicidin-biosynthesis genes may enable large-scale production. Because albicidins are X. albilineans pathogenicity factors, interference with AlbF function is also an opportunity for control of the associated plant disease.

INFLUÊNCIA DO LASER DE BAIXA INTENSIDADE NA VELOCIDADE DA MOVIMENTAÇÃO ORTODÔNTICA

Este estudo investigou os efeitos do laser de baixa intensidade na velocidade da movimentação ortodôntica de caninos submetidos à retração inicial. A amostra constou de 26 caninos superiores e inferiores, submetidos à retração inicial realizada com mola Niti, com força de 150g. Um dos caninos foi irradiado com laser de diodo, seguindo o protocolo de aplicação: 780nm/20mW/5Jcm2/0,2J por ponto/Et=2J, nos dias 0, 3 e 7 pós-ativação, sendo que o contralateral foi considerado placebo. A retração durou em média 4 meses, num total de 9 aplicações de laser. Os modelos de cada mês foram escaneados com scanner 3D (3Shape) e as imagens tridimensionais foram analisadas por meio do Software Geomagic Studio 5, para a mensuração da quantidade de movimentação dos caninos retraídos. Foi empregada a Análise de Variância a três critérios, seguida pelo teste de Tukey (p<0,05). Para verificação da integridade tecidual, foram efetuadas radiografias periapicais iniciais e finais dos caninos retraídos e dos molares, nas quais foram avaliados uma possível reabsorção na crista alveolar, por meio da distância da crista óssea alveolar até a junção cemento-esmalte e os níveis de reabsorção radicular, por meio do índice de Levander e Malmgreen, sendo este último avaliado somente nos caninos retraídos. Para isto, foi empregado o teste não paramétrico de Wilcoxon (p<0,05). Os resultados indicaram que houve um aumento estatisticamente significante na velocidade da movimentação dos caninos irradiados comparados ao seu contralateral, em todos os tempos avaliados, como também a preservação da integridade tecidual. Com isso, concluiu-se que o laser de diodo pode acelerar a movimentação ortodôntica, podendo contribuir para a diminuição do tempo de tratamento.(AU)

Efeito do laser de baixa intensidade no procedimento de expansão rápida da maxila

A proposta do presente estudo foi avaliar os efeitos do laser de baixa intensidade na regeneração óssea no procedimento de expansão rápida da maxila. Utilizou-se 27 indivíduos com média de idade de 10,2 anos, divididos em dois grupos: grupo laser (n=14), no qual se realizou a expansão rápida da maxila, associada ao laser e grupo sem laser (n=13), que realizou somente a expansão rápida da maxila. O protocolo de ativação do parafuso expansor foi de 1 volta completa no primeiro dia e ½ volta diária até a sobrecorreção. O laser utilizado foi o de diodo (TWIN Laser MMOptics®, São Carlos), seguindo o protocolo de aplicação: comprimento de onda de 780nm, potência de 40mW, densidade de 10J/cm2, em 10 pontos localizados ao redor da sutura palatina mediana. Os estágios de aplicação foram: L1 (do primeiro ao quinto dia de aplicação), L2 (travamento do parafuso e 3 dias seguidos), L3, L4 e L5 (após 7, 14 e 21 dias do L2, respectivamente). Radiografias oclusais da maxila foram realizadas com auxílio de uma escala de alumínio, para referencial densitométrico, em diferentes tempos: T1 (inicial), T2 (dia de travamento do parafuso), T3 (3 a 5 dias do T2), T4 (30 dias do T3), T5 (60 dias do T4). As radiografias foram digitalizadas e submetidas a um programa de imagem (Image Tool - UTHSCSA, Texas, USA), para mensuração da densidade óptica das áreas previamente selecionadas. Para realização do teste estatístico, utilizou-se a Análise de Covariância usando como covariável o tempo para a fase avaliada. Em todos os testes foi adotado nível de significância de 5% (p<0,05).Para o Grupo Laser, os dados mostram que houve uma queda significante de densidade durante a abertura do parafuso (T2-T1), um aumento significante da mesma no período final de avaliação (T5-T4), e um aumento também da densidade no período de regeneração propriamente dito (T5-T2), ou seja, a partir do momento em que finalizou a fase de abertura do parafuso expansor. Enquanto que no Grupo Sem Laser, a densidade não mostrou diferença estatisticamente significantemente em nenhum período analisado. Os resultados mostraram que o laser propiciou consideravelmente uma melhor abertura da sutura palatina mediana, além de influenciar no processo de regeneração óssea da sutura, acelerando seus processos de reparo.(AU)

A co-ordinated business object approach for supporting tactical level management decisions

The proliferation of data throughout the strategic, tactical and operational areas within many organisations, has provided a need for the decision maker to be presented with structured information that is appropriate for achieving allocated tasks. However, despite this abundance of data, managers at all levels in the organisation commonly encounter a condition of ‘information overload’, that results in a paucity of the correct information. Specifically, this thesis will focus upon the tactical domain within the organisation and the information needs of management who reside at this level. In doing so, it will argue that the link between decision making at the tactical level in the organisation, and low-level transaction processing data, should be through a common object model that used a framework based upon knowledge leveraged from co-ordination theory. In order to achieve this, the Co-ordinated Business Object Model (CBOM) was created. Detailing a two-tier framework, the first tier models data based upon four interactive object models, namely, processes, activities, resources and actors. The second tier analyses the data captured by the four object models, and returns information that can be used to support tactical decision making. In addition, the Co-ordinated Business Object Support System (CBOSS), is a prototype tool that has been developed in order to both support the CBOM implementation, and to also demonstrate the functionality of the CBOM as a modelling approach for supporting tactical management decision making. Containing a graphical user interface, the system’s functionality allows the user to create and explore alternative implementations of an identified tactical level process. In order to validate the CBOM, three verification tests have been completed. The results provide evidence that the CBOM framework helps bridge the gap between low level transaction data, and the information that is used to support tactical level decision making.

Studies on bacterial lung infections in cystic fibrosis

The major cause of death in CF is a continuous inflammation of the lungs colonised with Pseudomonas aeruginosa and occasionally also with Burkholderia cepacia. A combination of serum IgG to LPS and serum PCT levels were found to be good markers for detection of early colonisation with P. aeruginosa. Colomycin sulphomethate (colistin E) is one of the antibiotics used to treat P. aeruginosa infections in CF. Electrophoretic methods were developed to monitor the rate of conversion of colomycin sulphomethate to the active form of the drug. Antimicrobial activity towards P. aeruginosa was generated as the sulphomethate substituents were released. Clinical resistance of P. aeruginosa to colomycin is rare, but a number of isolates have been isolated. Twelve colomycin-resistant clinical isolates were investigated to determine the mechanism of resistance. It was found that the low level of resistance was due to over expression of outer membrane protein H (OprH) in 5 isolates. A novel mechanism of resistance involving modification of the phosphate groups in LPS was identified in one of the isolates. Drugs which reduce inflammation in infected CF lungs would be of great advantage for therapy. Reducing inflammation would preserve the lung function and increase the quality of life for CF patients. Antibiotics like tetracyclines, macrolides and polymyxins were tested for their potential anti-inflammatory effects using cultured human monocytic (U937) cells which secrete the pro-inflammatory cytokines IL1- and TNF- in response to LPS from P. aeruginosa and B. cepacia. It was found that tetracyclines, and especially doxycycline, are good inhibitors of cytokine release by U937 cells and therefore could reduce the inflammatory cascade.