Comparative proteomic analysis of GS-NSO murine myeloma cell lines with varying recombinant monoclonal antibody production rate

We have employed an inverse engineering strategy based on quantitative proteome analysis to identify changes in intracellular protein abundance that correlate with increased specific recombinant monoclonal antibody production (qMab) by engineered murine myeloma (NSO) cells. Four homogeneous NSO cell lines differing in qMab were isolated from a pool of primary transfectants. The proteome of each stably transfected cell line was analyzed at mid-exponential growth phase by two-dimensional gel electrophoresis (2D-PAGE) and individual protein spot volume data derived from digitized gel images were compared statistically. To identify changes in protein abundance associated with qMab clatasets were screened for proteins that exhibited either a linear correlation with cell line qMab or a conserved change in abundance specific only to the cell line with highest qMab. Several proteins with altered abundance were identified by mass spectrometry. Proteins exhibiting a significant increase in abundance with increasing qMab included molecular chaperones known to interact directly with nascent immunoglobulins during their folding and assembly (e.g., BiP, endoplasmin, protein disulfide isomerase). 2D-PAGE analysis showed that in all cell lines Mab light chain was more abundant than heavy chain, indicating that this is a likely prerequisite for efficient Mab production. In summary, these data reveal both the adaptive responses and molecular mechanisms enabling mammalian cells in culture to achieve high-level recombinant monoclonal antibody production. (C) 2004 Wiley Periodicals, Inc.

Pilot study to determine the ability of health-care professionals to undertake drug dose calculations

Background: It is essential for health-care professionals to calculate drug doses accurately. Previous studies have demonstrated that many hospital doctors were unable to accurately convert dilutions (e.g. 1:1000) or percentages (e.g. percentage w/v) of drug concentrations into mass concentrations (e.g. mg/mL). Aims: The aims of the present study were to evaluate the ability of health-care professionals to perform drug dose calculations accurately and to determine their preferred concentration convention when calculating drug doses. Methods: A selection of nurses, medical students, house surgeons, registrars and pharmacists undertook a written survey to assess their ability to perform five drug dose calculations. Participants were also asked which concentration convention they preferred when calculating drug doses. The surveys were marked then analysed for health-care professionals as a whole and then by subgroup analysis to assess the performance of each health-care-professional group. Results: Overall, less than 14% of the surveyed health-care professionals could answer all five questions correctly. Subgroup analysis revealed that health-care pro-fessionals' ability to calculate drug doses were ranked in the following order: registrars approximate to pharmacists > house surgeons > medical students >> nurses. Ninety per cent of health-care professionals preferred to calculate drug doses using the mass concentration convention. Conclusions: Overall, drug dose calculations were performed poorly. Mass concentration was clearly indicated as the preferred convention for calculating drug doses.

Functional promoter analysis using an approach based on an in vitro evolution strategy

In vitro evolution imitates the natural evolution of genes and has been very successfully applied to the modification of coding sequences, but it has not yet been applied to promoter sequences. We propose an alternative method for functional promoter analysis by applying an in vitro evolution scheme consisting of rounds of error-prone PCR, followed by DNA shuffling and selection of mutant promoter activities. We modified the activity in embryogenic sugarcane cells of the promoter region of the Goldfinger isolate of banana streak virus and obtained mutant promoter sequences that showed an average mutation rate of 2.5% after applying one round of error-prone PCR and DNA shuffling. Selection and sequencing of promoter sequences with decreased or unaltered activity allowed us to rapidly map the position of one cis-acting element that influenced promoter activity in embryogenic sugarcane cells and to discover neutral mutations that did not affect promoter Junction. The selective-shotgun approach of this promoter analysis method immediately after the promoter boundaries have been defined by 5' deletion analysis dramatically reduces the labor associated with traditional linker-scanning deletion analysis to reveal the position of functional promoter domains. Furthermore, this method allows the entire promoter to be investigated at once, rather than selected domains or nucleotides, increasing the, prospect of identifying interacting promoter regions.

Multivariate genetic analysis of academic skills of the Queensland Core Skills Test and IQ highlight the importance of genetic g

This study examined the genetic and environmental relationships among 5 academic achievement skills of a standardized test of academic achievement, the Queensland Core Skills Test (QCST; Queensland Studies Authority, 2003a). QCST participants included 182 monozygotic pairs and 208 dizygotic pairs (mean 17 years +/- 0.4 standard deviation). IQ data were included in the analysis to correct for ascertainment bias. A genetic general factor explained virtually all genetic variance in the component academic skills scores, and accounted for 32% to 73% of their phenotypic variances. It also explained 56% and 42% of variation in Verbal IQ and Performance IQ respectively, suggesting that this factor is genetic g. Modest specific genetic effects were evident for achievement in mathematical problem solving and written expression. A single common factor adequately explained common environmental effects, which were also modest, and possibly due to assortative mating. The results suggest that general academic ability, derived from genetic influences and to a lesser extent common environmental influences, is the primary source of variation in component skills of the QCST.

An examination of the dynamic relationship between self-efficacy and performance across levels of analysis and levels of specificity

This research used resource allocation theory to generate predictions regarding dynamic relationships between self-efficacy and task performance from 2 levels of analysis and specificity. Participants were given multiple trials of practice on an air traffic control task. Measures of task-specific self-efficacy and performance were taken at repeated intervals. The authors used multilevel analysis to demonstrate differential and dynamic effects. As predicted, task-specific self-efficacy was negatively associated with task performance at the within-person level. On the other hand, average levels of task-specific self-efficacy were positively related to performance at the between-persons level and mediated the effect of general self-efficacy. The key findings from this research relate to dynamic effects - these results show that self-efficacy effects can change over time, but it depends on the level of analysis and specificity at which self-efficacy is conceptualized. These novel findings emphasize the importance of conceptualizing self-efficacy within a multilevel and multispecificity framework and make a significant contribution to understanding the way this construct relates to task performance.

Coupled thermal, structural and vibrational analysis of a hypersonic engine for flight test

This paper describes a relatively simple and quick method for implementing aerodynamic heating models into a finite element code for non-linear transient thermal-structural and thermal-structural-vibrational analyses of a Mach 10 generic HyShot scramjet engine. The thermal-structural-vibrational response of the engine was studied for the descent trajectory from 60 to 26 km. Aerodynamic heating fluxes, as a function of spatial position and time for varying trajectory points, were implemented in the transient heat analysis. Additionally, the combined effect of varying dynamic pressure and thermal loads with altitude was considered. This aero-thermal-structural analysis capability was used to assess the temperature distribution, engine geometry distortion and yielding of the structural material due to aerodynamic heating during the descent trajectory, and for optimising the wall thickness, nose radius of leading edge, etc. of the engine intake. A structural vibration analysis was also performed following the aero-thermal-structural analysis to determine the changes in natural frequencies of the structural vibration modes that occur at the various temperatures associated with the descent trajectory. This analysis provides a unique and relatively simple design strategy for predicting and mitigating the thermal-structural-vibrational response of hypersonic engines. (C) 2006 Elsevier SAS. All rights reserved.

Minimising cold damage during reproductive development among temperate rice genotypes. II. Genotypic variation and flowering traits related to cold tolerance screening

Low temperature during microspore development increases spikelet sterility and reduces grain yield in rice (Oryza sativa L.). The objectives of this study were to determine genotypic variation in spikelet sterility in the field in response to low-temperature and then to examine the use of physio-morphological traits at flowering to screen for cold tolerance. Multiple-sown field experiments were conducted over 4 consecutive years in the rice-growing region of Australia to increase the likelihood of encountering low-temperature during microspore development. More than 50 cultivars of various origins were evaluated, with 7 cultivars common to all 4 years. The average minimum temperature for 9 days during microspore development was used as a covariate in the analysis to compare cultivars at a similar temperature. The low-temperature conditions in Year 4 identified cold-tolerant cultivars such as Hayayuki and HSC55 and susceptible cultivars such as Sasanishiki and Doongara. After low temperature conditions, spikelet sterility was negatively correlated with the number of engorged pollen grains, anther length, anther area, anther width, and stigma area. The number of engorged pollen grains and anther length were found to be facultative traits as their relationships with spikelet sterility were identified only after cold water exposure and did not exist under non-stressed conditions.

Identification and analysis of novel genes expressed in the mouse embryonic facial primordia

Craniofacial anomalies are a common feature of human congenital dysmorphology syndromes, suggesting that genes expressed in the developing face are likely to play a wider role in embryonic development. To facilitate the identification of genes involved in embryogenesis, we previously constructed an enriched cDNA library by subtracting adult mouse liver cDNA from that of embryonic day (E)10.5 mouse pharyngeal arch cDNA. From this library, 273 unique clones were sequenced and known proteins binned into functional categories in order to assess enrichment of the library (1). We have now selected 31 novel and poorly characterised genes from this library and present bioinformatic analysis to predict proteins encoded by these genes, and to detect evolutionary conservation. Of these genes 61% (19/31) showed restricted expression in the developing embryo, and a subset of these was chosen for further in silico characterisation as well as experimental determination of subcellular localisation based on transient transfection of predicted full-length coding sequences into mammalian cell lines. Where a human orthologue of these genes was detected, chromosomal localisation was determined relative to known loci for human congenital disease.

Physical activity and strength of the femoral neck during the adolescent growth spurt: A longitudinal analysis

Loading of the femoral neck (FN) is dominated by bending and compressive stresses. We hypothesize that adaptation of the FN to physical activity would be manifested in the cross-sectional area (CSA) and section modulus (Z) of bone, indices of axial and bending strength, respectively. We investigated the influence of physical activity on bone strength during adolescence using 7 years of longitudinal data from 109 boys and 121 girls from the Saskatchewan Paediatric Bone and Mineral Accrual Study (PBMAS). Physical activity data (PAC-Q physical activity inventory) and anthropometric measurements were taken every 6 months and DXA bone scans were measured annually (Hologic QDR2000, array mode). We applied hip structural analysis to derive strength and geometric indices of the femoral neck using DXA scans. To control for maturation, we determined a biological maturity age defined as years from age at peak height velocity (APHV). To account for the repeated measures within individual nature of longitudinal data, multilevel random effects regression analyses were used to analyze the data. When biological maturity age and body size (height and weight) were controlled, in both boys and girls, physical activity was a significant positive independent predictor of CSA and Z of the narrow region of the femoral neck (P < 0.05). There was no independent effect of physical activity on the subperiosteal width of the femoral neck. When leg length and leg lean mass were introduced into the random effects models to control for size and muscle mass of the leg (instead of height and weight), all significant effects of physical activity disappeared. Even among adolescents engaged in normal levels of physical activity, the statistically significant relationship between physical activity and indices of bone strength demonstrate that modifiable lifestyle factors like exercise play an important role in optimizing bone strength during the growing years. Physical activity differences were explained by the interdependence between activity and lean mass considerations. Physical activity is important for optimal development of bone strength. (c) 2005 Elsevier Inc. All rights reserved.

Putative glycogen-accumulating organisms belonging to the Alphaproteobacteria identified through rRNA-based stable isotope probing

Deterioration of enhanced biological phosphorus removal (EBPR) has been linked to the proliferation of glycogen-accumulating organisms (GAOs), but few organisms possessing the GAO metabolic phenotype have been identified. An unidentified GAO was highly enriched in a laboratory-scale bioreactor and attempts to identify this organism using conventional 16S rRNA gene cloning had failed. Therefore, rRNA-based stable isotope probing followed by full-cycle rRNA analysis was used to specifically identify the putative GAOs based on their characteristic metabolic phenotype. The study obtained sequences from a group of Alphaproteobacteria not previously shown to possess the GAO phenotype, but 90% identical by 16S rRNA gene analysis to a phylogenetic clade containing cloned sequences from putative GAOs and the isolate Defluvicoccus vanus. Fluorescence in situ hybridization (FISH) probes (DF988 and DF1020) were designed to target the new group and post-FISH chemical staining demonstrated anaerobic-aerobic cycling of polyhydroxyalkanoates, as per the GAO phenotype. The successful use of probes DF988 and DF1020 required the use of unlabelled helper probes which increased probe signal intensity up to 6.6-fold, thus highlighting the utility of helper probes in FISH. The new group constituted 33% of all Bacteria in the lab-scale bioreactor from which they were identified and were also abundant (51 and 55% of Bacteria) in two other similar bioreactors in which phosphorus removal had deteriorated. Unlike the previously identified Defluvicoccus-related organisms, the group identified in this study were also found in two full-scale treatment plants performing EBPR, suggesting that this group may be industrially relevant.

Reconstruction and in silico analysis of the MAPK signaling pathways in the human blood fluke, Schistosoma japonicum

At present, little is known about signal transduction mechanisms in schistosomes, which cause the disease of schistosomiasis. The mitogen-activated protein kinase (MAPK) signaling pathways, which are evolutionarily conserved from yeast to Homo sapiens, play key roles in multiple cellular processes. Here, we reconstructed the hypothetical MAPK signaling pathways in Schistosoma japonicum and compared the schistosome pathways with those of model eukaryote species. We identified 60 homologous components in the S. japoncium MAPK signaling pathways. Among these, 27 were predicted to be full-length sequences. Phylogenetic analysis of these proteins confirmed the evolutionary conservation of the MAPK signaling pathways. Remarkably, we identified S. japonicum homologues of GTP-binding protein beta and alpha-I subunits in the yeast mating pathway, which might be involved in the regulation of different life stages and female sexual maturation processes as well in schistosomes. In addition, several pathway member genes, including ERK, JNK, Sja-DSP, MRAS and RAS, were determined through quantitative PCR analysis to be expressed in a stage-specific manner, with ERK, JNK and their inhibitor Sja-DSP markedly upregulated in adult female schistosomes. (c) 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

The importance of forest area and configuration relative to local habitat factors for conserving forest mammals: A case study of koalas in Queensland, Australia

The loss and fragmentation of forest habitats by human land use are recognised as important factors influencing the decline of forest-dependent fauna. Mammal species that are dependent upon forest habitats are particularly sensitive to habitat loss and fragmentation because they have highly specific habitat requirements, and in many cases have limited ability to move through and utilise the land use matrix. We addressed this problem using a case study of the koala (Phascolarctos cinereus) surveyed in a fragmented rural-urban landscape in southeast Queensland, Australia. We applied a logistic modelling and hierarchical partitioning analysis to determine the importance of forest area and its configuration relative to site (local) and patch-level habitat variables. After taking into account spatial auto-correlation and the year of survey, we found koala occurrence increased with the area of all forest habitats, habitat patch size and the proportion of primary Eucalyptus tree species; and decreased with mean nearest neighbour distance between forest patches, the density of forest patches, and the density of sealed roads. The difference between the effect of habitat area and configuration was not as strong as theory predicts, with the configuration of remnant forest becoming increasingly important as the area of forest habitat declines. We conclude that the area of forest, its configuration across the landscape, as well as the land use matrix, are important determinants of koala occurrence, and that habitat configuration should not be overlooked in the conservation of forest-dependent mammals, such as the koala. We highlight the implications of these findings for koala conservation. (c) 2006 Elsevier Ltd. All rights reserved.

Reply to Paparazzo

We would like to reply to the comments made by Paparazzo on our recent paper [1] on the “effect of curve fitting parameters on quantitative analysis of Fe0.94O and Fe2O3 using XPS”. There have been many studies on the characterisation of the properties of iron oxide surfaces. The main purpose of writing the paper was to demonstrate the extent to which the selection of input parameters for curve fitting can affect the results of the quantitative analysis, and to use this analysis to develop more consistent, reproducible and quantitative methods of analysis of these data.

Stage is not a reliable indicator of tumor volume in non-small cell lung cancer: a preliminary analysis of the trans-tasman radiation oncology group 99-05 database

Background: Tumor volume has been shown to be a prognostic factor for the response of some tumors to radiotherapy. TNM stage has prognostic value for patients treated surgically for non-small cell lung cancer (NSCLC), but its value is less clear for patients treated by nonsurgical means. This may be because tumor size is not a consistent determinant of T stage or stage group. As part of the preliminary analyses for the Trans-Tasman Radiation Oncology Group 99-05 study, the authors performed this analysis to determine to what extent stage reflects tumor volume. Methods: In this prospective multicenter observational study, patients had to have histologically proven NSCLC, no evidence of disease beyond the primary site or thoracic lymph nodes, and been planned for radical radiotherapy with or without chemotherapy. Tumor volume measurements were based on computed tomography-based treatment planning images. Results: Four hundred four patients were available for analysis. There was a strong correlation between (log) maximum tumor diameter and (log) tumor volume (r = 0.93, p < 0.001). Although there was a highly significant trend of increasing volume with increasing T stage and stage group, when tumors were categorized into four groups according to increasing volume, there was only 55% concordance with T stage and 67% concordance with stage group. Conclusions: There is limited correlation between tumor size and disease stage in patients with NSCLC. This justifies documentation and investigation of size as a potential prognostic factor independent of stage. Maximum tumor diameter may be an adequate substitute for volume as a measurement of size.

Self-efficacy and task performance: An investigation from two levels of analysis and specificity

This research adopts a resource allocation theoretical framework to generate predictions regarding the relationship between self-efficacy and task performance from two levels of analysis and specificity. Participants were given multiple trials of practice on an air traffic control task. Measures of task-specific self-efficacy and performance were taken at repeated intervals. The authors used multilevel analysis to demonstrate dynamic main effects, dynamic mediation and dynamic moderation. As predicted, the positive effects of overall task specific self-efficacy and general self-efficacy on task performance strengthened throughout practice. In line with these dynamic main effects, the effect of general self-efficacy was mediated by overall task specific self-efficacy; however this pattern emerged over time. Finally, changes in task specific self-efficacy were negatively associated with changes in performance at the within-person level; however this effect only emerged towards the end of practice for individuals with high levels of overall task specific self-efficacy. These novel findings emphasise the importance of conceptualising self-efficacy within a multi-level and multi-specificity framework and make a significant contribution to understanding the way this construct relates to task performance.