Semliki Forest virus as an expression vector in insect cell lines

Studies were undertaken to determine if replication-deficient Semliki Forest virus expression vectors could be successfully used to express foreign gene constructs in insect cell lines. Using green fluorescent protein (GFP) as a marker we recorded infection levels of nearly 100% in the Aedes albopictus cell lines C6/36 and Aa23T, as well as in the Ae. aegypti cell line MOS20. The virus was capable of infecting an Anopheles gambiae cell line MOS55. The amount of GFP protein produced in each cell line was quantified. Northern analysis of viral transcription revealed the presence of novel transcripts in Aa23T, C6/36, and MOS55 cell lines, but not in the BHK or MOS20. The initial characterization of these transcripts is described.

Expression of the hepatitis C virus structural proteins in mammalian cells induces morphology similar to that in natural infection

Like many positive-strand RNA viruses, replication of the hepatitis C virus (HCV) is associated with cytoplasmic membrane rearrangements. However, it is unclear which HCV Proteins induce these ultrastructural features. This work examined the morphological changes induced by expression of the HCV structural proteins, core, E1 and E2, expressed from a Semliki Forest Virus (SFV) recombinant RNA replicon. Electron microscopy of cells expressing these proteins showed cytoplasmic vacuoles containing membranous and electron-dense material that were distinct from the type I cytoplasmic vacuoles induced during SFV replicon replication. Immunogold labelling showed that the core and E2 proteins localized to the external and internal membranes of these vacuoles. At times were also associated with some of the internal amorphous material. Dual immunogold labelling with antibodies raised against the core protein and against an endoplasmic reticulum (ER)-resident protein (protein disulphide isomerase) showed that the HCV-induced vacuoles were associated with ER-labelled membranes. This report has identified an association between the HCV core and E2 proteins with induced cytoplasmic vacuoles which are morphologically similar to those observed in HCV-infected liver tissue, suggesting that the HCV structural proteins may be responsible for the induction of these vacuoles during HCV replication in vivo.

Expression of the alpha 1b-adrenergic receptor and G protein subunits in mammalian cell lines using the Semliki Forest virus expression system.

Semliki Forest virus (SFV) vectors have been efficiently used for rapid high level expression of several G protein-coupled receptors. Here we describe the use of SFV vectors to express the alpha 1b-adrenergic receptor (AR) alone or in the presence of the G protein alpha q and/or beta 2 and gamma 2 subunits. Infection of baby hamster kidney (BHK) cells with recombinant SFV-alpha 1b-AR particles resulted in high specific binding activity of the alpha 1b-AR (24 pmol receptor/mg protein). Time-course studies indicated that the highest level of receptor expression was obtained 30 hours post-infection. The stimulation of BHK cells, with epinephrine led to a 5-fold increase in inositol phosphate (IP) accumulation, confirming the functional coupling of the receptor to G protein-mediated activation of phospholipase C. The SFV expression system represents a rapid and reproducible system to study the pharmacological properties and interactions of G protein coupled receptors and of G protein subunits.

Evaluation de la qualité de vie après prélèvement de la veine fémorale superficielle lors de reconstructions infra-inguinales [Quality of life after superficial femoral vein harvest for infra-inguinal reconstructions]

BACKGROUND: The superficial femoral vein (SFV) is a well-established alternative conduit for infra-inguinal reconstructivenous hypertension after SFV harvest may however result in significant morbidity. This study reports the efficiency of SFV as conduit for infra-inguinal reconstructions and characterize the anatomic and physiologic changes in harvest limbs and their relationship to the development of venous complications. METHODS: From May 1999 through November 2003, 23 SFV were harvested from 21 patients undergoing infra-inguinal reconstructions. Bypasses were controlled by regular duplex-ultrasound. The venous morbidity was assessed by measurements of leg circumferences, strain-gauge plethysmography and quality of life, investigated by the VEINES-QOL scale. RESULTS: At a mean follow-up of 10.4 months (range 1-56), primary, secondary patency and limb salvage rates of infra-inguinal bypasses using SFV are 71.4%, 76.2% and 85.7% respectively. No patient had major venous claudication. Oedema was significantly present in nine patients. Strain-gauge plethysmography showed outflow obstruction in all patients. The VEINES-QOL assessment showed no limitation in social and domestic activity, moderate complain about leg heaviness despite presence of oedema. CONCLUSION: The SFV harvest is a reliable conduit for infra-inguinal reconstructions and results in moderate venous morbidity in terms of functional consequences and quality of life.

Superficial femoral vein as substitute for pulmonary artery reconstruction after resection for bronchovascular fistula

Post-lobectomy bronchovascular fistula (BVF) associated with massive hemoptysis is a rare but life-threatening complication. Surgical options include completion pneumonectomy or BVF resection with end-to-end anastomosis of the airways and reconstruction of the pulmonary artery (PA) by interposition of an appropriate substitute. We report PA resection and successful reconstruction by interposition of an autologous reversed superficial femoral vein (SFV) segment for this purpose.

In situ aortobiiliac reconstruction of infected aneurysm using a single superficial femoral vein.

Objective: To demonstrate successful in situ aortoiliac reconstruction of an infected infrarenal aneurysm using one single superficial femoral vein (SFV). Methods: In situ reconstruction using the right SFV sutured in end-to-end anastomosis with the aorta and distally with the right common iliac artery and in end-to-side anastomosis with the left common iliac artery. Results: The operating time was less than reported for aortic in situ reconstruction with bilateral SFV harvesting. The duplex scan 3 months postoperatively showed permeability of the bypass without any anastomotic stenosis or pseudoaneurysm. The right common femoral, popliteal, and greater saphenous veins were patent without thrombus, and the patient did not complain about peripheral edema. Conclusions: The use of only one instead of both the SFVs for aortobiiliac in situ reconstruction might be a way to reduce operating time and allow autogenous venous reconstruction even in patients with limited availability of venous material.

Effect of different tumbling marination methods and time on the quality characteristics of prepared pork chops

AbstractThe combined effects of tumbling marination methods (Vacuum continuous tumbling marination, CT; Vacuum intermittent tumbling marination, IT) and effective tumbling time (4, 6, 8 and 10 h) on quality characteristics of prepared boneless pork chops were investigated. The results showed that regardless of tumbling time, CT method significantly increased the pH, product yield, cohesiveness, resilience, sensory tenderness and overall flavor (p<0.05) compared with IT method, and CT method also significantly decreased the pressing loss, cooking loss, shear force value (SFV), hardness and chewiness (p<0.05) compared with IT method. With the effective tumbling time increasing from 4 h to 10 h, the product yield and sensory attributes of prepared pork chops increased at first and then decreased, whereas the pressing loss, cooking loss, SFV, hardness and chewiness decreased at first and then increased. Additionally, an interaction between CT method and effective tumbling time was also observed. These results suggested that CT method of 8 h obtained the best quality characteristics of prepared pork chops, which should be adopted.

Modelling the hydraulics of the Carlisle 2005 flood event

The performance of a 2D numerical model of flood hydraulics is tested for a major event in Carlisle, UK, in 2005. This event is associated with a unique data set, with GPS surveyed wrack lines and flood extent surveyed 3 weeks after the flood. The Simple Finite Volume (SFV) model is used to solve the 2D Saint-Venant equations over an unstructured mesh of 30000 elements representing channel and floodplain, and allowing detailed hydraulics of flow around bridge piers and other influential features to be represented. The SFV model is also used to corroborate flows recorded for the event at two gauging stations. Calibration of Manning's n is performed with a two stage strategy, with channel values determined by calibration of the gauging station models, and floodplain values determined by optimising the fit between model results and observed water levels and flood extent for the 2005 event. RMS error for the calibrated model compared with surveyed water levels is ~±0.4m, the same order of magnitude as the estimated error in the survey data. The study demonstrates the ability of unstructured mesh hydraulic models to represent important hydraulic processes across a range of scales, with potential applications to flood risk management.

Effect of fluoride solutions on the shear bond strength of orthodontic brackets

The aim of this in vitro study was to evaluate the shear bond strength of brackets after pre-treatment with different fluoride solutions. This study used 48 freshly extracted sound bovine incisors that were randomly assigned to 4 experimental groups (n=12). CG: (control) without treatment; NF: 4 min application of neutral fluoride; APF: application of 1.23% acidulated phosphate fluoride (APF) for 4 min; and SFV: application of 5% sodium fluoride varnish for 6 h. For each group, after surface treatment, prophylaxis of enamel and bracket bonding with Transbond XT composite resin (3M) were performed following the manufacturer's specifications. The shear bond strength was performed with a universal testing machine 24 h after fixing the brackets. The tooth surfaces were analyzed to verify the adhesive remnant index (ARI). Data were analyzed statistically by ANOVA and Tukey's test (α=0.05). There was statistically significant difference among the groups (p<0.0001). CG and NF groups presented significantly higher bond strength than APF and SFV. There was no significant difference between CG and NF or between APF and SFV (p>0.05). The analysis of ARI scores revealed that most failures occurred at the enamel-resin interface. It may be concluded that the pre-treatment of enamel with 1.23% APF and 5% SFV prior to fixing orthodontic brackets reduces shear bond strength values.

Semliki Forest Virus as a Vector: Pros and Cons for Its Use in Biopharmaceuticals Production

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Immunogenicity and safety of seasonal and 2009 pandemic A/H1N1 influenza vaccines for patients with autoimmune diseases: a prospective, monocentre trial on 199 patients

The 2009 pandemic A/H1N1 influenza outbreak represented a theoretical risk for patients with autoimmune diseases (AID), especially those immunosuppressed. This study was undertaken to evaluate immunogenicity and tolerance of seasonal (SFV) and A/H1N1 flu vaccines (HFV) in AID patients.