Tetracycline-Inducible packaging cell line for production of Flavivirus replicon particles.

We have previously developed replicon vectors derived from the Australian flavivirus Kunjin that have a unique noncytopathic nature and have been shown to direct prolonged high-level expression of encoded heterologous genes in vitro and in vivo and to induce strong and long-lasting immune responses to encoded immunogens in mice. To facilitate further applications of these vectors in the form of virus-like particles (VLPs), we have now generated a stable BHK packaging cell line, tetKUNCprME, carrying a Kunjin structural gene cassette under the control of a tetracycline-inducible promoter. Withdrawal of tetracycline from the medium resulted in production of Kunjin structural proteins that were capable of packaging transfected and self-amplified Kunjin replicon RNA into the secreted VLPs at titers of up to 1.6 x 10(9) VLPs per ml. Furthermore, secreted KUN replicon VLPs from tetKUNCprME cells could be harvested continuously for as long as 10 days after RNA transfection, producing a total yield of more than 1010 VLPs per 106 transfected cells. Passaging of VLPs on Vero cells or intracerebral injection into 2- to 4-day-old suckling mice illustrated the complete absence of any infectious Kunjin virus. tetKUNCprME cells were also capable of packaging replicon RNA from closely and distantly related flaviviruses, West Nile virus and dengue virus type 2, respectively. The utility of high-titer KUN replicon VLPs was demonstrated by showing increasing CD8(+)-T-cell responses to encoded foreign protein with increasing doses of KUN VLPs. A single dose of 2.5 x 10(7) VLPs carrying the human respiratory syncytial virus M2 gene induced 1,400 CD8 T cells per 10(6) splenocytes in an ex vivo gamma interferon enzyme-linked immunospot assay. The packaging cell line thus represents a significant advance in the development of the noncytopathic Kunjin virus replicon-based gene expression system and may be widely applicable to the basic studies of flavivirus RNA packaging and virus assembly as well as to the development of gene expression systems based on replicons from different flaviviruses.

Risk of serious NSAID-related gastrointestinal events during long-term exposure: a systematic review

Objective: Exposure to non-steroidal anti-inflammatory drugs (NSAIDs) is associated wit increased risk of serious gastrointestinal (GI) events compared with non-exposure. We investigated whether that risk is sustained over time. Data sources: Cochrane Controlled Trials Register (to 2002); MEDLINE, EMBASE, Derwent Drug File and Current Contents (1999-2002); manual searching of reviews (1999-2002). Study selection: From 479 search results reviewed and 221 articles retrieved, seven studies of patients exposed to prescription non-selective NSAIDs for more than 6 months and reporting time-dependent serious GI event rates were selected for quantitative data synthesis. These were stratified into two groups by study design. Data extraction: Incidence of GI events and number of patients at specific time points were extracted. Data synthesis: Meta-regression analyses were performed. Change in risk was evaluated by testing whether the slope of the regression line declined over time. Four randomised controlled trials (RCTs) provided evaluable data from five NSAID arms (aspirin, naproxen, two ibuprofen arms, and diclofenac). When the RCT data were combined, a small significant decline in annualised risk was seen: -0.005% (95% Cl, -0.008% to -0.001%) per month. Sensitivity analyses were conducted because there was disparity within the RCT data. The pooled estimate from three cohort studies showed no significant decline in annualised risk over periods up to 2 years: -0.003% (95% Cl, -0.008% to 0.003%) per month. Conclusions: Small decreases in risk over time were observed; these were of negligible clinical importance. For patients who need long-term (> 6 months) treatment, precautionary measures should be considered to reduce the net probability of serious GI events over the anticipated treatment duration. The effect of intermittent versus regular daily therapy on long-term risk needs further investigation.

Rapid screening of 4000 individuals for germ-line variations in the BRAF gene

Background: The BRAF gene is frequently somatically altered in malignant melanoma. A majority of variations are at the valine 600 residue leading to a V600E substitution that constitutively activates the kinase. We screened 4000 patient and control DNAs for germ-line variations at the valine 600 residue. Methods: We developed a novel assay by adapting single-base variation assays and software for MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry to screen for all 5 reported variants at codon 600 of the BRAF gene. We screened a case-control collection comprising samples from 1082 melanoma patients and 154 of their unaffected relatives from 1278 families and from 2744 individuals from 659 unselected twin families with no history of melanoma. A panel of 66 melanoma cell lines was used for variation-positive controls. Results: All melanoma cell lines that we had found previously to carry a codon 600 variation were verified in this study. Three of the 4 possible variants (V600E n = 47, V600K n = 2, V600R n = 1) were detected, but no case of V600D was available. No germ-line variants were found in the samples from the 3980 melanoma patients or from the control individuals. Conclusions: This new assay is a high-throughput, automated alternative to standard sequencing and can be used as a rapid initial screen for somatic variants associated with melanoma. Germ-line variants at valine 600 are unlikely to exist and do not contribute to the reported role of the BRAF gene in melanoma predisposition. (c) 2006 American Association for Clinical Chemistry.

A INFLUÊNCIA DAS COMPETÊNCIAS COLETIVAS SOBRE A EFICÁCIA DO ATENDIMENTO AO CLIENTE

O relacionamento de longo prazo é atualmente o elemento-chave para o sucesso das organizações. Com um mercado tão competitivo, as empresas estão desenvolvendo estratégias que melhor possam satisfazer e criar valor para seus clientes. As empresas precisam definir muito bem as suas estratégias com foco na percepção de seus clientes e do mercado. Porém a criação de valor não advém apenas da qualidade de um produto, da tecnologia e da infraestrutura. Ela também tem base em valores intrínsecos como, por exemplo, as competências. Sendo assim, as empresas precisam investir nos profissionais de linha de frente para que eles desenvolvam competências e atendam às necessidades e desejos dos clientes. Este trabalho tem o intuito de demonstrar se as competências coletivas, desenvolvidas a partir da interação social de um grupo podem trazer algum(s) benefício(s) ou não para o atendimento ao cliente em termos de satisfação e criação de valor, promovendo o marketing de relacionamento. Em seu desenvolvimento, foram utilizadas como base as teorias de competência, competências coletivas, gestão do conhecimento, satisfação e criação de valor para o cliente e marketing de relacionamento. Porém, não foi possível encontrar na literatura qualquer relação entre os temas competências coletivas e marketing de relacionamento. Neste contexto, o presente trabalho teve como objetivo geral analisar como o desenvolvimento das competências coletivas de uma equipe de vendas pode influenciar a satisfação do cliente. Especificamente buscou-se a) levantar quais os principais fatores ligados à equipe de vendas que geram satisfação nos clientes; b) analisar como as competências coletivas da equipe de vendas influenciam a satisfação do cliente; c) identificar como se formam competências coletivas em uma equipe de vendas. A partir destes objetivos, o procedimento metodológico de abordagem qualitativa foi orientado pelo método de estudo de caso, com procedimentos de análise de dados de entrevistas, observação direta e levantamento de registros. Foram entrevistadas duas equipes de vendas de uma empresa multinacional no segmento de serviços para análise e proteção de crédito, envolvendo o gerente, três vendedores e um cliente de cada equipe, atendidos por estes vendedores. As observações foram realizadas em duas reuniões de vendas envolvendo vendedores e clientes, e os registros analisados referem-se a metas e número de clientes atendidos no mês. Os resultados evidenciam que as competências coletivas podem influenciar na satisfação do cliente, embora estes só percebam as competências individuais e organizacionais. Mas ficou evidente, por parte de vendedores e gerentes de equipe, que as competências coletivas, além de promover a satisfação do cliente, auxiliam no desenvolvimento e na formação de competências individuais das pessoas na área comercial.

Monitoring long distance WDM communication lines using a high-loss loopback supervisory system

In this paper, we present experimental results for monitoring long distance WDM communication links using a line monitoring system suitable for legacy optically amplified long-haul undersea systems. This monitoring system is based on setting up a simple, passive, low cost high-loss optical loopback circuit at each repeater that provides a connection between the existing anti-directional undersea fibres, and can be used to define fault location. Fault location is achieved by transmitting a short pulse supervisory signal along with the WDM data signals where a portion of the overall signal is attenuated and returned to the transmit terminal by the loopback circuit. A special receiver is used at the terminal to extract the weakly returned supervisory signal where each supervisory signal is received at different times corresponding to different optical repeaters. Therefore, the degradation in any repeater appears on its corresponding supervisory signal level. We use a recirculating loop to simulate a 4600 km fibre link, on which a high-loss loopback supervisory system is implemented. Successful monitoring is accomplished through the production of an appropriate supervisory signal at the terminal that is detected and identified in a satisfactory time period after passing through up to 45 dB attenuation in the loopback circuit. © 2012 Elsevier B.V. All rights reserved.

Long-range translational coupling in single-stranded RNA bacteriophages: an evolutionary analysis

In coliphage MS2 RNA a long-distance interaction (LDI) between an internal segment of the upstream coat gene and the start region of the replicase gene prevents initiation of replicase synthesis in the absence of coat gene translation. Elongating ribosomes break up the repressor LDI and thus activate the hidden initiation site. Expression studies on partial MS2 cDNA clones identified base pairing between 1427-1433 and 1738-1744, the so-called Min Jou (MJ) interaction, as the molecular basis for the long-range coupling mechanism. Here, we examine the biological significance of this interaction for the control of replicase gene translation. The LDI was disrupted by mutations in the 3'-side and the evolutionary adaptation was monitored upon phage passaging. Two categories of pseudorevertants emerged. The first type had restored the MJ interaction but not necessarily the native sequence. The pseudorevertants of the second type acquired a compensatory substitution some 80 nt downstream of the MJ interaction that stabilizes an adjacent LDI. In one examined case we confirmed that the second site mutations had restored coat-replicase translational coupling. Our results show the importance of translational control for fitness of the phage. They also reveal that the structure that buries the replicase start extends to structure elements bordering the MJ interaction.

Acute and long-term effects of metformin on the function and insulin secretory responsiveness of clonal β-cells

Functional effects of acute and prolonged (48 h) exposure to the biguanide drug metformin were examined in the clonal pancreatic ß-cell line, BRIN-BD11. Effects of metformin on prolonged exposure to excessive increased concentrations of glucose and palmitic acid were also assessed. In acute 20-min incubations, 12.5-50 µm metformin did not alter basal (1.1 mm glucose) or glucose-stimulated (16.7 mm glucose) insulin secretion. However, higher concentrations of metformin (100-1000 µm) increased (1.3-1.5-fold; p

The interrogation and multiplexing of long period grating curvature sensors using a Bragg grating based, derivative spectroscopy technique

A long period grating is interrogated with a fibre Bragg grating using a derivative spectroscopy technique. A quasi-linear relationship between the output of the sensing scheme and the curvature experienced by the long period grating is demonstrated, with a sensitivity of 5.05 m and with an average curvature resolution of 2.9 × 10-2 m-1. In addition, the feasibility of multiplexing an in-line series of long period gratings with this interrogation scheme is demonstrated with two pairs of fibre Bragg gratings and long period gratings. With this arrangement the cross-talk error between channels was less than ± 2.4 × 10-3 m-1.

Low jitter, long distance pulse transmission near net fibre dispersion zero wavelength

The authors study experimentally ~10 ps return-to-zero pulse propagation near the net dispersion zero of an optical fibre transmission line. Stable near-jitter-free propagation was observed over 70 Mm. Pulse stabilisation and ASE suppression were achieved through the saturable aborber mechanism of nonlinear polarisation rotation.

Evaluating the capacity of long haul soliton transmission

We review recent developments in the use of optical solitons for communication systems spanning transoceanic distances. The implementation of "soliton control" to alleviate the detrimental impact of effects such as amplifier noise is shown to be critical for obtaining advantages over competing technologies. The potential performance of two control strategies, namely straight line filtering and synchronous phase modulation, is examined in detail. Design diagrams are used to determine the maximum permissible amplifier spacing, which is a key determinant of system economics. To focus the enquiry, two example system spans are taken, representing transatlantic and transpacific distances. It is concluded that straight line filtering provides very little improvement over a basic design without control. However synchronous phase modulation, which may be implemented using a handful of actively driven components, provides very substantial benefits. These may be used either to extend the overall bit-rate-distance product of the system or to increase the amplifier spacing at more moderate capacities.

A high sensitivity refractometer based upon a long period grating Mach-Zehnder interferometer

A low cost interrogation scheme is demonstrated for a refractometer based on an in-line fiber long period grating (LPG) Mach–Zehnder interferometer. Using this interrogation scheme the minimum detectable change in refractive index of ?n ~ 1.8×10-6 is obtained, which is the highest resolution achieved using a fiber LPG device, and is comparable to precision techniques used in the industry including high performance liquid chromatography and ultraviolet spectroscopy.

Long distance dispersion managed soliton transmission experiments

This thesis presents results of transmission experiments using optical solitons in a dispersion managed optical fibre recirculating loop. The basic concepts of pulse propagation in optical fibre are introduced before optical solitons and their use in optically amplified fibre systems are discussed. The role of dispersion management in such systems is then considered. The design, operation and limitations of the recirculating loop and soliton sources which were used and the experimental techniques are described before the experimental work is presented. The experimental work covers a number of areas all of which used dispersion management of the transmission line. A novel ultra-long distance propagation scheme which achieved low timing jitter by suppression of the amplifier noise and by working close to the zero dispersion wavelength has been discovered. The use of fibre Bragg gratings as wavelength filters to suppress noise and reduce timing jitter has been investigated. The performance of the fibre grating cornpared favourably with that of a bulk device and was in good agreement with theoretical predictions. The upgrade of existing standard fibre systems to higher bit rates is currently an important issue. The possibility of using solitons with dispersion compensation to allow an increase in data rate of existing standard fibre systems to 10Gbit/s over 5000km has been demonstrated. The applicability of this technique to longer distances, higher bit rates or longer amplifier spans is also investigated by optimisation of the dispersion management scheme. The use of fibre Bragg gratings as the dispersion compensating elements in such standard fibre transmission experiments has been examined and the main problem that these devices currently have, high polarisation mode dispersion, is discussed. The likely future direction of optical communications and what part solitons and dispersion management will play in this development is discussed in the thesis conclusions