Evaluation of individual protein errors in silver-stained two-dimensional gels

The relationship between spot volume and variation for all protein spots observed on large format 2D gels when utilising silver stain technology and a model system based on mammalian NSO cell extracts is reported. By running multiple gels we have shown that the reproducibility of data generated in this way is dependent on individual protein spot volumes, which in turn are directly correlated with the coefficient of variation. The coefficients of variation across all observed protein spots were highest for low abundant proteins which are the primary contributors to process error, and lowest for more abundant proteins. Using the relationship between spot volume and coefficient of variation we show it is necessary to calculate variation for individual protein spot volumes. The inherent limitations of silver staining therefore mean that errors in individual protein spot volumes must be considered when assessing significant changes in protein spot volume and not global error. (C) 2003 Elsevier Science (USA). All rights reserved.

Alpha-lipoic acid does not acutely affect resistance and conduit artery function or oxidative stress in healthy men

Aims Alpha-lipoic acid (ALA) is a thiol compound with antioxidant properties used in the treatment of diabetic polyneuropathy. ALA may also improve arterial function, but there have been scant human trials examining this notion. This project aimed to investigate the effects of oral and intra-arterial ALA on changes in systemic and regional haemodynamics, respectively. Methods In study 1, 16 healthy older men aged 58 +/- 7 years (mean +/- SD) received 600 mg of ALA or placebo, on two occasions 1 week apart, in a randomized cross-over design. Repeated measures of peripheral and central haemodynamics were then obtained for 90 min. Central blood pressure and indices of arterial stiffness [augmentation index (AIx) and estimated aortic pulse wave velocity] were recorded non-invasively using pulse wave analysis. Blood samples obtained pre- and post-treatments were analysed for erythrocyte antioxidant enzyme activity, plasma nitrite and malondialdehyde. In study 2 the effects of incremental cumulative doses (0.5, 1.0, 1.5 and 2.0 mg ml(-1) min(-1)) of intra-arterial ALA on forearm blood flow (FBF) were assessed in eight healthy subjects (aged 31 +/- 5 years) by conventional venous occlusion plethysmography. Results There were no significant changes on any of the central or peripheral haemodynamic measures after either oral or direct arterial administration of ALA. Plasma ALA was detected after oral supplementation (95% confidence intervals 463, 761 ng ml(-1)), but did not alter cellular or plasma measures of oxidative stress. Conclusions Neither oral nor intra-arterial ALA had any effect on regional and systemic haemodynamics or measures of oxidative stress in healthy men.

Transient production of recombinant proteins by chinese hamster ovary cells using polyethyleneimine/DNA complexes in combination with microtubule disrupting anti-mitotic agents

We have developed a simple and robust transient expression system utilizing the 25 kDa branched cationic polymer polyethylenimine (PEI) as a vehicle to deliver plasmid DNA into suspension-adapted Chinese hamster ovary cells synchronized in G2/M phase of the cell cycle by anti-mitotic microtubule disrupting agents. The PEI-mediated transfection process was optimized with respect to PEI nitrogen to DNA phosphate molar ratio and the plasmid DNA mass to cell ratio using a reporter construct encoding firefly luciferase. Optimal production of luciferase was observed at a PEI N to DNA P ratio of 10:1 and 5 mug DNA 10(6) cells(-1). To manipulate transgene expression at mitosis, we arrested cells in G2/M phase of the cell cycle using the microtubule depolymerizing agent nocodazole. Using secreted human alkaline phosphatase (SEAP) and enhanced green fluorescent protein (eGFP) as reporters we showed that continued inclusion of nocodazole in cell culture medium significantly increased both transfection efficiency and reporter protein production. In the presence of nocodazole, greater than 90% of cells were eGFP positive 24 h post-transfection and qSEAP was increased almost fivefold, doubling total SEAP production. Under optimal conditions for PEI-mediated transfection, transient production of a recombinant chimeric IgG(4) encoded on a single vector was enhanced twofold by nocodazole, a final yield of approximately 5 mug mL(-1) achieved at an initial viable cell density of 1 x 10(6) cells mL(-1). The glycosylation of the recombinant antibody at Asn(297) was not significantly affected by nocodazole during transient production by this method. (C) 2004 Wiley Periodicals, Inc.

Wheat grain cooking process as investigated by modulated temperature differential scanning calorimetry

The thermal properties of soft and hard wheat grains, cooked in a steam pressure cooker, as a function of cooking temperature and time were investigated by modulated temperature differential scanning calorimetry (MTDSC). Four cooking temperatures (110, 120, 130 and 140 degrees C) and six cooking times (20, 40, 60, 80, 100 and 120 min) for each temperature were studied. It was found that typical non-reversible heat flow thermograms of cooked and uncooked wheat grains consisted of two endothermic baseline shifts localised around 40-50 degrees C and then 60-70 degrees C. The second peaks of non-reversible heat flow thermograms (60-70 degrees C) were associated with starch gelatinisation. The degree of gelatinisation was quantified based on these peaks. In this study, starch was completely gelatinised within 60-80 min for cooking temperatures at 110-120 degrees C and within 20 min for cooking temperatures at 130-140 degrees C. MTDSC detected reversible endothermic baseline shifts in most samples, localised broadly around 48-67 degrees C with changes in heat capacity ranging from 0.02 to 0.06 J/g per degrees C. These reversible endothermic baseline shifts are related to the glass transition, which occurs during starch gelatinisation. Data on the specific heat capacity of the cooked wheat samples are provided. (C) 2005 Elsevier Ltd. All rights reserved.

On the optimal ratio of heavy to light chain genes for efficient recombinant antibody production by CHO cells

Monoclonal antibodies (Mab) are heterotetramers consisting of an equimolar ratio of heavy chain (HC) and light chain (LC) polypeptides. Accordingly, most recombinant Mab expression systems utilize an equimolar ratio of heavy chain (he) to light chain (lc) genes encoded on either one or two plasmids. However, there is no evidence to suggest that this gene ratio is optimal for stable or transient production of recombinant Mab. In this study we have determined the optimal ratio of hc:lc genes for production of a recombinant IgG(4) Mab, cB72.3, by Chinese hamster ovary (CHO) cells using both empirical and mathematical modeling approaches. Polyethyleneimine-mediated transient expression of cB72.3 at varying ratios of hc:lc genes encoded on separate plasmids yielded an optimal Mab titer at a hc:lc gene ratio of 3:2; a conclusion confirmed by separate mathematical modeling of the Mab folding and assembly process using transient expression data. On the basis of this information, we hypothesized that utilization of he genes at low hc:lc gene ratios is more efficient. To confirm this, cB72.3 Mab was transiently produced by CHO cells at constant he and varying lc gene dose. Under these conditions, Mab yield was increased with a concomitant increase in lc gene dose. To determine if the above findings also apply to stably transfected CHO cells producing recombinant Mab, we compared the intra- and extracellular ratios of HC and LC polypeptides for three GS-CHO cells lines transfected with a 1:1 ratio of hc:lc genes and selected for stable expression of the same recombinant Mab, cB72.3. Intra- and extracellular HC:LC polypeptide ratios ranged from 1:2 to 1:5, less than that observed on transient expression of the same Mab in parental CHO cells using the same vector. In conclusion, our data suggest that the optimal ratio of hc:lc genes used for transient and stable expression of Mab differ. In the case of the latter, we infer that optimal Mab production by stably transfected cells represents a compromise between HC abundance limiting productivity and the requirement for excess LC to render Mab folding and assembly more efficient.

Mentoring for population health in general practice divisions

This paper describes the implementation and evaluation of a three-way model of service development mentoring. This population health mentoring program was funded by the Commonwealth Department of Health and Ageing to enable staff from eight Divisions of General Practice in South Australia to gain a sound understanding of population health concepts relevant to their workplace. The distinguishing features of service development mentoring were that the learning was grounded within an individual's work setting and experience; there was an identified population health problem or issue confronting the Division of General Practice; and there was an expectation of enhanced organisational performance. A formal evaluation found a consensus among all learners that mentoring was a positive and worthwhile experience, where they had achieved what they had set out to do. Mentors found the model of learning agreeable and effective. Division executive officers recognised enhanced skills among their "learner" colleagues, and commented positively on the benefits to their organisations through the development of well researched and relevant projects, with the potential to improve the efficiency of their population health activities.

Distribution of 19 major virulence genes in Legionella pneumophila serogroup 1 isolates from patients and water in Queensland, Australia

The distribution of 19 major virulence genes and the presence of plasmids were surveyed in 141 Legionella pneumophila serogroup (SG) 1 isolates from patients and water in Queensland, Australia. The results showed that 16 of the virulence genes examined were present in all isolates, suggesting that they are life-essential genes for isolates in the environment and host cells. The 65 kb pathogenicity island identified originally in strain Philadelphia-1(T) was detected more frequently in isolates from water (44.2 %) than in those from patients (2.7 %), indicating that the 65 kb DNA fragment may aid the survival of L. pneumophila in the sampled environment. However, the low frequency of the 65 kb fragment in isolates from patients suggests that the pathogenicity island may not be necessary for L. pneumophila to cause disease. Plasmids were not detected in the L. pneumophila SG1 isolates from patients or water studied. There was an association of both lvh and rtxA with the virulent and predominant genotype detected by amplified fragment length polymorphism, termed AF1, whereas the avirulent common isolate from water termed AF16 did not have lvh or rtxA genes, with the exception of one isolate with rtxA. It was found that a PCR detection test strategy with lvh and rtxA as pathogenesis markers would be useful for determining the infection potential of an isolate.

Double-heater-wire circuits and heat-and-moisture exchangers and the risk of ventilator-associated pneumonia

Objective: To compare the incidence of ventilator-associated pneumonia (VAP) in patients ventilated in intensive care by means of circuits humidified with a hygroscopic heat-and-moisture exchanger with a bacterial viral filter (HME) or hot-water humidification with a heater wire in both inspiratory and expiratory circuit limbs (DHW) or the inspiratory limb only (SHW). Design: A prospective, randomized trial. Setting: A metropolitan teaching hospital's general intensive care unit. Patients: Three hundred eighty-one patients requiring a minimum period of mechanical ventilation of 48 hrs. Interventions: Patients were randomized to humidification with use of an HME (n = 190), SHW (n = 94), or DHW (n = 97). Measurements and Main Results. Study end points were VAP diagnosed on the basis of Clinical Pulmonary Infection Score (CPIS) (1), HME resistance after 24 hrs of use, endotracheal tube resistance, and HME use per patient. VAP occurred with similar frequency in all groups (13%, HME; 14%, DHW; 10%, SHW; p = 0.61) and was predicted only by current smoking (adjusted odds ratio [AOR], 2.1; 95% confidence interval [CI], 1.1-3.9; p =.03) and ventilation days (AOR, 1.05; 95% Cl, 1.0-1.2; p =.001); VAP was less likely for patients with an admission diagnosis of pneumonia (AOR, 0.40; 95% Cl, 0.4-0.2; p =.04). HME resistance after 24 hrs of use measured at a gas flow of 50 L/min was 0.9 cm H2O (0.4-2.9). Endotracheal tube resistance was similar for all three groups (16-19 cm H2O min/L; p =.2), as were suction frequency, secretion thickness, and blood on suctioning (p =.32, p =.06, and p =.34, respectively). The HME use per patient per day was 1.13. Conclusions: Humidification technique does not influence either VAP incidence or secretion characteristics, but HMEs may have air-flow resistance higher than manufacturer specifications after 24 hrs of use.

A prospective assessment of SRS-24 scores after endoscopic anterior instrumentation for scoliosis

Study Design. Prospective clinical case series. Objective. To evaluate the clinical outcome of anterior endoscopic instrumention for scoliosis using the SRS-24 questionnaire and to examine how these scores change over a 2-year follow-up period. Summary of Background Data. Anterior endoscopic instrumentation correction has several advantages compared with open procedures. However, the clinical results of this technique using a validated outcome measure have rarely been reported in the literature. Methods. A total of 83 consecutive patients underwent endoscopic anterior instrumentation performed at a single unit. Patients completed the SRS-24 questionnaire before surgery and at 3, 6, 12, and 24 months after surgery. The SRS-24 scores were compared between each of the follow-up intervals. Results. The pain, general self-image, and function from back condition domains improved after surgery (P < 0.05). Activity level significantly improved between 3 and 6 months, and both function domains improved between 6 and 12 months (P < 0.05). None of the domains increased significantly after 1 year. Conclusions. Endoscopic anterior instrumentation for scoliosis significantly improved pain, self-image, and function. The greatest improvement in function occurred between 6 and 12 months after surgery. The SRS-24 scores at 1 year from surgery may provide a good indicator of patient outcome in the long-term.

Distance measurement using the change in junction voltage across a laser diode due to the self-mixing effect

Conventional detection scheme for self-mixing sensors uses an integrated photodiode within the laser package to monitor the self mixing signal. This arrangement can be simplified by directly obtaining the self-mixing signals across the laser diode itself and omitting the photodiode. This work reports on a Vertical-Cavity Surface-Emitting Laser (VCSEL) based selfmixing sensor using the laser junction voltage to obtain the selfmixing signal. We show that the same information can be obtained with only minor changes to the extraction circuitry leading to potential cost saving with reductions in component costs and complexity and significant increase in bandwidth favoring high speed modulation. Experiments using both photo current and voltage detection were carried out and the results obtained show good agreement with the theory.