Assessing colonoscopic inspection skill using a virtual withdrawal simulation: a preliminary validation of performance metrics.

BACKGROUND: The effectiveness of colonoscopy for diagnosing and preventing colon cancer is largely dependent on the ability of endoscopists to fully inspect the colonic mucosa, which they achieve primarily through skilled manipulation of the colonoscope during withdrawal. Performance assessment during live procedures is problematic. However, a virtual withdrawal simulation can help identify and parameterise actions linked to successful inspection, and offer standardised assessments for trainees. METHODS: Eleven experienced endoscopists and 18 endoscopy novices (medical students) completed a mucosal inspection task during three simulated colonoscopic withdrawals. The two groups were compared on 10 performance metrics to preliminarily assess the validity of these measures to describe inspection quality. Four metrics were related to aspects of polyp detection: percentage of polyp markers found; number of polyp markers found per minute; percentage of the mucosal surface illuminated by the colonoscope (≥0.5 s); and percentage of polyp markers illuminated (≥2.5 s) but not identified. A further six metrics described the movement of the colonoscope: withdrawal time; linear distance travelled by the colonoscope tip; total distance travelled by the colonoscope tip; and distance travelled by the colonoscope tip due to movement of the up/down angulation control, movement of the left/right angulation control, and axial shaft rotation. RESULTS: Statistically significant experienced-novice differences were found for 8 of the 10 performance metrics (p's < .005). Compared with novices, experienced endoscopists inspected more of the mucosa and detected more polyp markers, at a faster rate. Despite completing the withdrawals more quickly than the novices, the experienced endoscopists also moved the colonoscope more in terms of linear distance travelled and overall tip movement, with greater use of both the up/down angulation control and axial shaft rotation. However, the groups did not differ in the number of polyp markers visible on the monitor but not identified, or movement of the left/right angulation control. All metrics that yielded significant group differences had adequate to excellent internal consistency reliability (α = .79 to .90). CONCLUSIONS: These systematic differences confirm the potential of the simulated withdrawal task for evaluating inspection skills and strategies. It may be useful for training, and assessment of trainee competence.

Fungal protein production: design and production of chimeric proteins.

For more than a century, filamentous fungi have been used for the production of a wide variety of endogenous enzymes of industrial interest. More recently, with the use of genetic engineering tools developed for these organisms, this use has expanded for the production of nonnative heterologous proteins. In this review, an overview is given of examples describing the production of a special class of these proteins, namely chimeric proteins. The production of two types of chimeric proteins have been explored: (a) proteins grafted for a specific substrate-binding domain and (b) fusion proteins containing two separate enzymatic activities. Various application areas for the use of these chimeric proteins are described.

Scientists' views about attribution of global warming.

Results are presented from a survey held among 1868 scientists studying various aspects of climate change, including physical climate, climate impacts, and mitigation. The survey was unique in its size, broadness and level of detail. Consistent with other research, we found that, as the level of expertise in climate science grew, so too did the level of agreement on anthropogenic causation. 90% of respondents with more than 10 climate-related peer-reviewed publications (about half of all respondents), explicitly agreed with anthropogenic greenhouse gases (GHGs) being the dominant driver of recent global warming. The respondents' quantitative estimate of the GHG contribution appeared to strongly depend on their judgment or knowledge of the cooling effect of aerosols. The phrasing of the IPCC attribution statement in its fourth assessment report (AR4)-providing a lower limit for the isolated GHG contribution-may have led to an underestimation of the GHG influence on recent warming. The phrasing was improved in AR5. We also report on the respondents' views on other factors contributing to global warming; of these Land Use and Land Cover Change (LULCC) was considered the most important. Respondents who characterized human influence on climate as insignificant, reported having had the most frequent media coverage regarding their views on climate change.

What drives the ecological quality of surface waters? A review of 11 predictive modeling tools.

What policy is needed to ensure that good-quality water is available for both people's needs and the environment? The EU Water Framework Directive (WFD), which came into force in 2000, established a framework for the assessment, management, protection and improvement of the status of water bodies across the European Union. However, recent reviews show that the ecological status of the majority of surface waters in the EU does not meet the requirement of good status. Thus, it is an important question what measures water management authorities should take to improve the ecological status of their water bodies. To find concrete answers, several institutes in the Netherlands cooperated to develop a software tool, the WFD Explorer, to assist water managers in selecting efficient measures. This article deals with the development of prediction tools that allow one to calculate the effect of restoration and mitigation measures on the biological quality, expressed in terms of Ecological Quality Ratios (EQRs). To find the ideal modeling tool we give a review of 11 predictive models: 10 models from the field of Machine Learning and, additionally, the Multiple Regression model. We present our results in terms of a 'prediction-interpretation competition'. All these models were tested in a multiple-stressor setting: the values of 15 stressors (or steering factors) are available to predict the EQR values of four biological quality elements (phytoplankton, other aquatic flora, benthic invertebrates and fish). Analyses are based on 29 data sets from various water clusters (streams, ditches, lakes, channels). All 11 models were ranked by their predictive performance and their level of model transparency. Our review shows a trade-off between these two aspects. Models that have the best EQR prediction performance show non-transparent model structures. These are Random Forest and Boosting. However, models with low prediction accuracies show transparent response relationships between EQRs on the one hand and individual steering factors on the other hand. These models are Multiple Regression, Regression Trees and Product Unit Neural Networks. To acknowledge both aspects of model quality - predictive power and transparency - we recommend that models from both groups are implemented in the WFD Explorer software.

Progress in virtual reality simulators for surgical training and certification.

There is increasing evidence that educating trainee surgeons by simulation is preferable to traditional operating-room training methods with actual patients. Apart from reducing costs and risks to patients, training by simulation can provide some unique benefits, such as greater control over the training procedure and more easily defined metrics for assessing proficiency. Virtual reality (VR) simulators are now playing an increasing role in surgical training. However, currently available VR simulators lack the fidelity to teach trainees past the novice-to-intermediate skills level. Recent technological developments in other industries using simulation, such as the games and entertainment and aviation industries, suggest that the next generation of VR simulators should be suitable for training, maintenance and certification of advanced surgical skills. To be effective as an advanced surgical training and assessment tool, VR simulation needs to provide adequate and relevant levels of physical realism, case complexity and performance assessment. Proper validation of VR simulators and an increased appreciation of their value by the medical profession are crucial for them to be accepted into surgical training curricula.

Engineering of geranylgeranyl pyrophosphate synthase levels and physiological conditions for enhanced carotenoid and astaxanthin synthesis in Xanthophyllomyces dendrorhous.

The basidiomycetous yeast, Xanthophyllomyces dendrorhous, is one of the very few organisms which can be used for biological production of the carotenoid astaxanthin. crtE cDNA has been cloned from this fungus for engineering of the terpenoid pathway. The function of its gene product as a geranylgeranyl pyrophosphate synthase was established. X. dendrorhous was transformed with the crtE cDNA to divert metabolite flow from the sterol pathway towards carotenoid biosynthesis. Transformants were obtained with increased levels of geranylgeranyl pyrophosphate synthase leading to higher carotenoid levels including astaxanthin. Physiological conditions for maximum carotenoid synthesis for wild type and the CrtE transformant were dim light and extra air supply of the shaking culture. These conditions and the transformation with crtE had additive effects and resulted in an 8-fold higher astaxanthin formation as compared to the initial wild type culture without illumination and extra air supply yielding 451 µg/g dry wt within 4 days of growth.

Post-genomic insights into the plant polysaccharide degradation potential of Aspergillus nidulans and comparison to Aspergillus niger and Aspergillus oryzae.

The plant polysaccharide degradative potential of Aspergillus nidulans was analysed in detail and compared to that of Aspergillus niger and Aspergillus oryzae using a combination of bioinformatics, physiology and transcriptomics. Manual verification indicated that 28.4% of the A. nidulans ORFs analysed in this study do not contain a secretion signal, of which 40% may be secreted through a non-classical method.While significant differences were found between the species in the numbers of ORFs assigned to the relevant CAZy families, no significant difference was observed in growth on polysaccharides. Growth differences were observed between the Aspergilli and Podospora anserina, which has a more different genomic potential for polysaccharide degradation, suggesting that large genomic differences are required to cause growth differences on polysaccharides. Differences were also detected between the Aspergilli in the presence of putative regulatory sequences in the promoters of the ORFs of this study and correlation of the presence of putative XlnR binding sites to induction by xylose was detected for A. niger. These data demonstrate differences at genome content, substrate specificity of the enzymes and gene regulation in these three Aspergilli, which likely reflect their individual adaptation to their natural biotope.

The role of quadratus lumborum asymmetry in the occurrence of lesions in the lumbar vertebrae of cricket fast bowlers.

In cricket fast bowlers an increased incidence of stress fractures or lesions in the L4 pars interarticularis is observed, which shows a strong statistical correlation with the presence of hypertrophy in the contralateral Quadratus Lumborum (QL) muscle. This study aims to find a physical explanation for this correlation. A mathematical model was used to estimate the forces and moments on the L3 and L4 vertebrae in six postures attained during fast bowling. These forces and moments were used in finite element models to estimate the stresses in the pars interarticularis. Two scenarios were examined per posture: symmetric QL muscles, and right QL muscle volume 30% enlarged. Influence of muscle activation was also investigated. QL asymmetry only correlates with significant stress increases when stress levels are relatively low. When stress levels are high, due to extreme posture or muscle activation, asymmetry only causes small stress changes, suggesting that asymmetry is not the cause of stress fractures in the pars. There are even indications that asymmetry might help to reduce stresses, but more detailed knowledge of the size and activation of the lumbar muscles is needed to confirm this.

Automated visibility map of the internal colon surface from colonoscopy video.

PURPOSE: Optical colonoscopy is a prominent procedure by which clinicians examine the surface of the colon for cancerous polyps using a flexible colonoscope. One of the main concerns regarding the quality of the colonoscopy is to ensure that the whole colonic surface has been inspected for abnormalities. In this paper, we aim at estimating areas that have not been covered thoroughly by providing a map from the internal colon surface. METHODS: Camera parameters were estimated using optical flow between consecutive colonoscopy frames. A cylinder model was fitted to the colon structure using 3D pseudo stereo vision and projected into each frame. A circumferential band from the cylinder was extracted to unroll the internal colon surface (band image). By registering these band images, drift in estimating camera motion could be reduced, and a visibility map of the colon surface could be generated, revealing uncovered areas by the colonoscope. Hidden areas behind haustral folds were ignored in this study. The method was validated on simulated and actual colonoscopy videos. The realistic simulated videos were generated using a colonoscopy simulator with known ground truth, and the actual colonoscopy videos were manually assessed by a clinical expert. RESULTS: The proposed method obtained a sensitivity and precision of 98 and 96 % for detecting the number of uncovered areas on simulated data, whereas validation on real videos showed a sensitivity and precision of 96 and 78 %, respectively. Error in camera motion drift could be reduced by almost 50 % using results from band image registration. CONCLUSION: Using a simple cylindrical model for the colon and reducing drift by registering band images allows for the generation of visibility maps. The current results also suggest that the provided feedback through the visibility map could enhance clinicians' awareness of uncovered areas, which in return could reduce the probability of missing polyps.

Genes involved in carotene synthesis and mating in Blakeslea trispora.

Mating of Blakeslea trispora and other molds of the order Mucorales requires the interaction of mycelia of opposite sex, (+) and (-), leading to the development of specialized structures and to an enhanced accumulation of beta-carotene. Industry obtains beta-carotene by co-cultivating appropriate strains of Blakeslea ("mated cultures"). Gene transcription in single and mated cultures was assayed by cDNA-AFLP, a technique to observe the differential expression of subsets of mRNA fragments. Overexpression in mated cultures is about ten times more frequent than underexpression. We obtained and sequenced fragments of 97 candidate genes that appeared to be overexpressed during mating and confirmed four of them by reverse transcription and real-time PCR. Comparisons with gene sequences from other organisms suggest functions in carotene biosynthesis (4 genes), energy metabolism (8), cell wall synthesis (1), transfer of acetyl groups (1), and regulatory processes (10). Sodium acetate inhibited sexual overexpression in about two-thirds of the candidate genes and acted as a signal with broad effects on the metabolism and the morphology of mated cultures. Our work offers new materials for the study of carotene biosynthesis and its regulation and for the improvement of carotene production with Mucorales.

High-level production of beta-carotene in Saccharomyces cerevisiae by successive transformation with carotenogenic genes from Xanthophyllomyces dendrorhous.

To determine whether Saccharomyces cerevisiae can serve as a host for efficient carotenoid and especially beta-carotene production, carotenogenic genes from the carotenoid-producing yeast Xanthophyllomyces dendrorhous were introduced and overexpressed in S. cerevisiae. Because overexpression of these genes from an episomal expression vector resulted in unstable strains, the genes were integrated into genomic DNA to yield stable, carotenoid-producing S. cerevisiae cells. Furthermore, carotenoid production levels were higher in strains containing integrated carotenogenic genes. Overexpression of crtYB (which encodes a bifunctional phytoene synthase and lycopene cyclase) and crtI (phytoene desaturase) from X. dendrorhous was sufficient to enable carotenoid production. Carotenoid production levels were increased by additional overexpression of a homologous geranylgeranyl diphosphate (GGPP) synthase from S. cerevisiae that is encoded by BTS1. Combined overexpression of crtE (heterologous GGPP synthase) from X. dendrorhous with crtYB and crtI and introduction of an additional copy of a truncated 3-hydroxy-3-methylglutaryl-coenzyme A reductase gene (tHMG1) into carotenoid-producing cells resulted in a successive increase in carotenoid production levels. The strains mentioned produced high levels of intermediates of the carotenogenic pathway and comparable low levels of the preferred end product beta-carotene, as determined by high-performance liquid chromatography. We finally succeeded in constructing an S. cerevisiae strain capable of producing high levels of beta-carotene, up to 5.9 mg/g (dry weight), which was accomplished by the introduction of an additional copy of crtI and tHMG1 into carotenoid-producing yeast cells. This transformant is promising for further development toward the biotechnological production of beta-carotene by S. cerevisiae.

Cloning of the astaxanthin synthase gene from Xanthophyllomyces dendrorhous (Phaffia rhodozyma) and its assignment as a beta-carotene 3-hydroxylase/4-ketolase.

A gene has been cloned from Xanthophyllomyces dendrorhous by complementation of astaxanthin formation in a beta-carotene accumulating mutant. It consists of 3,166 bp and contains 17 introns. For the beta-carotene mutant ATCC 96815, a single point mutation in the splicing sequence of intron 8 was found. The resulting improper splicing of the mRNA results in an inactive protein. The cDNA of this beta-carotene oxygenase encodes a cytochrome P450 monooxygenase belonging to the 3A subfamily. P450-specific domains were identified including a cytochrome P450 and an oxygen binding motif. Electrons are provided by a cytochrome P450 reductase. Functional characterization of the enzyme by genetic modification of X. dendrorhous demonstrated that this P450 monooxygenase is multifunctional catalyzing all steps from beta-carotene to astaxanthin formation by oxygenation of carbon 3 and 4. The reaction sequence is first 4-ketolation of beta-carotene followed by 3-hydroxylation. A hydroxylation mechanism at allylic carbon atoms has been proposed for the generation of 4-keto and 3-hydroxy groups at both beta-ionone ends.

Metabolic engineering of the astaxanthin-biosynthetic pathway of Xanthophyllomyces dendrorhous.

This review describes the different approaches that have been used to manipulate and improve carotenoid production in Xanthophyllomyces dendrorhous. The red yeast X. dendrorhous (formerly known as Phaffia rhodozyma) is one of the microbiological production systems for natural astaxanthin. Astaxanthin is applied in food and feed industry and can be used as a nutraceutical because of its strong antioxidant properties. However, the production levels of astaxanthin in wild-type isolates are rather low. To increase the astaxanthin content in X. dendrorhous, cultivation protocols have been optimized and astaxanthin-hyperproducing mutants have been obtained by screening of classically mutagenized X. dendrorhous strains. The knowledge about the regulation of carotenogenesis in X. dendrorhous is still limited in comparison to that in other carotenogenic fungi. The X. dendrorhous carotenogenic genes have been cloned and a X. dendrorhous transformation system has been developed. These tools allowed the directed genetic modification of the astaxanthin pathway in X. dendrorhous. The crtYB gene, encoding the bifunctional enzyme phytoene synthase/lycopene cyclase, was inactivated by insertion of a vector by single and double cross-over events, indicating that it is possible to generate specific carotenoid-biosynthetic mutants. Additionally, overexpression of crtYB resulted in the accumulation of beta-carotene and echinone, which indicates that the oxygenation reactions are rate-limiting in these recombinant strains. Furthermore, overexpression of the phytoene desaturase-encoding gene (crtI) showed an increase in monocyclic carotenoids such as torulene and HDCO (3-hydroxy-3',4'-didehydro-beta,-psi-carotene-4-one) and a decrease in bicyclic carotenoids such as echinone, beta-carotene and astaxanthin.

Metabolic engineering of the carotenoid biosynthetic pathway in the yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma).

The crtYB locus was used as an integrative platform for the construction of specific carotenoid biosynthetic mutants in the astaxanthin-producing yeast Xanthophyllomyces dendrorhous. The crtYB gene of X. dendrorhous, encoding a chimeric carotenoid biosynthetic enzyme, could be inactivated by both single and double crossover events, resulting in non-carotenoid-producing transformants. In addition, the crtYB gene, linked to either its homologous or a glyceraldehyde-3-phosphate dehydrogenase promoter, was overexpressed in the wild type and a beta-carotene-accumulating mutant of X. dendrorhous. In several transformants containing multiple copies of the crtYB gene, the total carotenoid content was higher than in the control strain. This increase was mainly due to an increase of the beta-carotene and echinone content, whereas the total content of astaxanthin was unaffected or even lower. Overexpression of the phytoene synthase-encoding gene (crtI) had a large impact on the ratio between mono- and bicyclic carotenoids. Furthermore, we showed that in metabolic engineered X. dendrorhous strains, the competition between the enzymes phytoene desaturase and lycopene cyclase for lycopene governs the metabolic flux either via beta-carotene to astaxanthin or via 3,4-didehydrolycopene to 3-hydroxy-3'-4'-didehydro-beta-psi-caroten-4-one (HDCO). The monocylic carotenoid torulene and HDCO, normally produced as minority carotenoids, were the main carotenoids produced in these strains.