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Automated protein NMR structure determination using wavelet de-noised NOESY spectra.

A major time-consuming step of protein NMR structure determination is the generation of reliable NOESY cross peak lists which usually requires a significant amount of manual interaction. Here we present a new algorithm for automated peak picking involving wavelet de-noised NOESY spectra in a process where the identification of peaks is coupled to automated structure determination. The core of this method is the generation of incremental peak lists by applying different wavelet de-noising procedures which yield peak lists of a different noise content. In combination with additional filters which probe the consistency of the peak lists, good convergence of the NOESY-based automated structure determination could be achieved. These algorithms were implemented in the context of the ARIA software for automated NOE assignment and structure determination and were validated for a polysulfide-sulfur transferase protein of known structure. The procedures presented here should be commonly applicable for efficient protein NMR structure determination and automated NMR peak picking.

Algorithms↗

A microfluidic system for high-speed reproducible DNA sizing and quantitation.

Microfabrication technology was used to develop a system consisting of disposable glass chips containing etched channels, reagents including polymer matrix and size standards, computer-controlled instrumentation for performing electrophoretic separations and fluorescence detection of double-stranded DNA, and software for automated data analysis. System performance was validated for separation and quantitation reproducibility using samples varying in amount and size of DNA fragments, buffer composition, and salt concentrations. Several applications of the microfluidic system for DNA analysis have been demonstrated, such as of polymerase chain reaction (PCR) products, sizing of plasmid digests, and detection of point mutations by restriction fragment length polymorphism (RFLP) mapping.

Animals↗

Acousto-optic laser-scanning cytometer.

An instrument has been developed that uses a computer-controlled rapidly scanning laser beam to make cytometric measurements on cells or particles and which can measure low levels of fluorescence when using low-power lasers (Gershman, Hoffman, and O'Connell, "Methods and Apparatus for Analysis of Particles and Cells.") The method used is based upon acousto-optic principles of light diffraction. A vertically polarized 5-mW He-Ne laser is directed into an acousto-optic Bragg cell in which a portion of the incident light undergoes a small angular variation or deflection. Suitable optics focus the beam to a 25 microns diameter spot, at the 1/e2 point, in a sample cuvette while translating the angular variation into a linear scan. The cuvette enclosing the sample is slowly moved (approximately 1 micron/ms) via a stepper drive into the scanning beam while the forward angle light scatter sensor is monitored for the presence of valid signal events. When an event occurs, appropriate software optimizes the position of the focused laser beam onto the cell. Subsequently, scanning is stopped to allow for cell interrogation times that last for milliseconds or longer.

Acoustics↗

Permutation tests for factorially designed neuroimaging experiments.

Permutation methods for analysis of functional neuroimaging data acquired as factorially designed experiments are described and validated. The F ratio was estimated for main effects and interactions at each voxel in standard space. Critical values corresponding to probability thresholds were derived from a null distribution sampled by appropriate permutation of observations. Spatially informed, cluster-level test statistics were generated by applying a preliminary probability threshold to the voxel F maps and then computing the sum of voxel statistics in each of the resulting three-dimensional clusters, i.e., cluster "mass." Using simulations comprising two between- or within-subject factors each with two or three levels, contaminated by Gaussian and non-normal noise, the voxel-wise permutation test was compared to the standard parametric F test and to the performance of the spatially informed statistic using receiver operating characteristic (ROC) curves. Validity of the permutation-testing algorithm and software is endorsed by almost identical performance of parametric and permutation tests of the voxel-level F statistic. Permutation testing of suprathreshold voxel cluster mass, however, was found to provide consistently superior sensitivity to detect simulated signals than either of the voxel-level tests. The methods are also illustrated by application to an experimental dataset designed to investigate effects of antidepressant drug treatment on brain activation by implicit sad facial affect perception in patients with major depression. Antidepressant drug effects in left amygdala and ventral striatum were detected by this software for an interaction between time (within-subject factor) and group (between-subject factor) in a representative two-way factorial design.

Algorithms↗

Patient based 'burden-of-illness' in Swedish primary health care. Applying the Johns Hopkins ACG case-mix system in a retrospective study of electronic patient records.

Patients from one municipality in Sweden utilizing primary health care (PHC) during 1998 and 1999 have been categorized into 81 groups. The groups show each patient's own case-mix in terms of illness. Grouping was carried out using the case-mix instrument adjusted clinical groups (ACG), developed by the School of Hygiene and Public Health at Johns Hopkins University in Baltimore, USA. The resulting pattern provided a more adequate reflection of the scope of primary care's task than that yielded by diagnoses alone. Changes over time in terms of illness patterns for a population could be described, analysed and assessed from medical and health economic perspectives. One of the conclusions from this study was that the ACG instrument is a relevant tool in describing the outcome of work by the primary health care centre. The ACG is of interest in the improvement of the quality of primary care in Sweden. The ACG should be a driving force in the development of health indices in both national and international comparisons, as a result of its focus on the health status of patients and populations instead of on diagnoses and diseases.

Acute Disease↗

Evaluation of mucosal eosinophils in the pediatric colon.

To evaluate the clinical significance of colonic eosinophilia, we conducted a retrospective study of all children older than 1 year evaluated at Children's Hospital of Pittsburgh from January 1999 to June 2001 with a description of colonic eosinophilia in the pathology report. Medical records were reviewed. Diagnoses were confirmed by contacting the patients. Histological slides (H&E) were reviewed by an investigator blind to the patients' data. Biopsies were grouped according to the site they were taken from and then screened at low power for areas of maximal eosinophilia for further quantitative analysis. Results of manual counts were validated by image analysis using Metaphor Image Analysis Software. Sixty-nine children with colonic eosinophilia were identified (36 male; mean age, 135.2 +/- 55.4 months). Their final diagnoses were inflammatory bowel disease in 32% (group A), irritable bowel syndrome in 33% (group B), food allergies in 10% (group C), and other diagnoses in 25% (group D). The maximal eosinophil count per crypt area was significantly (P < 0.05) higher in group A vs groups B, C, and D (34.8 +/- 17.1 vs 21.3 +/- 8.8, 25.4 +/- 16.7, and 24.2 +/- 9.7, respectively). The total cellularity of the lamina propria was considered high only in group A (P < 0.05 vs groups B and C). A mostly equal vertical distribution of eosinophils throughout the lamina propria was found significantly more frequently in group A vs groups C (P = 0.04) and D (P = 0.007). We conclude that children with inflammatory bowel disease have an equal distribution of eosinophils throughout the lamina propria, with intraepithelial and intracryptal eosinophils and with a higher overall total cellularity. In irritable bowel syndrome and patients with a variety of other diagnoses, including allergies, the distribution is mostly superficial, with a lower total cellularity.

Adolescent↗

Development of a simplex polymerase chain reaction-based assay for the detection of Neisseria meningitidis.

BACKGROUND: This study was undertaken to develop a sensitive and specific polymerase chain reaction (PCR)-based assay for Neisseria meningitidis and Neisseria gonorrhoeae that could ultimately be incorporated into a multiplex assay designed to screen cerebrospinal fluid (CSF) for a panel of pyogenic bacterial species associated with bacterial meningitis. METHODS: N.meningitidis-specific primers were designed from porA gene sequences with the aid of a commercial software program and were used to develop and validate a clinical assay using a liquid hybridization-gel retardation detection system. The analytic sensitivity of the assay was determined using CSF spiked with N. meningitidis and comparing the PCR-based results with culture. RESULTS: Analytic sensitivity experiments showed the assay's limit of detection to be 100 fg purified input target DNA and 0.0125 colony-forming units of N. meningitidis spiked into CSF. Specificity experiments showed the assay could detect all strains of N. meningitidis and N. gonorrhoeae tested, but did not support amplification of the commensal neisserial species or a panel of other human bacterial pathogens. CONCLUSIONS: This PCR-based assay for pathogenic neisserial species is sensitive and specific and suitable for incorporation into a multiplex assay for the clinical differentiation of aseptic and septic meningitis.

Bacterial Proteins↗

Fully automated flexible docking of ligands into flexible synthetic receptors using forward and inverse docking strategies.

The prediction of the structure of host-guest complexes is one of the most challenging problems in supramolecular chemistry. Usual procedures for docking of ligands into receptors do not take full conformational freedom of the host molecule into account. We describe and apply a new docking approach which performs a conformational sampling of the host and then sequentially docks the ligand into all receptor conformers using the incremental construction technique of the FlexX software platform. The applicability of this approach is validated on a set of host-guest complexes with known crystal structure. Moreover, we demonstrate that due to the interchangeability of the roles of host and guest, the docking process can be inverted. In this inverse docking mode, the receptor molecule is docked around its ligand. For all investigated test cases, the predicted structures are in good agreement with the experiment for both normal (forward) and inverse docking. Since the ligand is often smaller than the receptor and, thus, its conformational space is more restricted, the inverse docking approach leads in most cases to considerable speed-up. By having the choice between two alternative docking directions, the application range of the method is significantly extended. Finally, an important result of this study is the suitability of the simple energy function used here for structure prediction of complexes in organic media.

Algorithms↗

Comparison of ligand-based and structure-based 3D-QSAR approaches: a case study on (aryl-)bridged 2-aminobenzonitriles inhibiting HIV-1 reverse transcriptase.

Ligand- (GRIND) and structure-based (GLUE/GRIND) 3D-QSAR approaches were compared for 55 (aryl-)bridged 2-aminobenzonitriles inhibiting HIV-1 reverse transcriptase (HIV-1 RT). The ligand-based model was built from conformers selected by in vacuo minimization. The available X-ray structure of 3v in complex with HIV-1 RT allowed comparative structure-based calculations using the new docking software GLUE for conformer selection. Both models were validated via statistics and via virtual receptor sites (VRS) considering pharmacophoric regions and mutual distances, which were also compared with experimental evidence. The statistics show slight superiority of the structure-based approach in terms of fitting and prediction. By encoding relevant molecular interaction fields (MIF) into pharmacophoric regions, 10 such regions were derived from both models; they all fit the real receptor except HBD2. Also mutual distances highly agree between the real site and both VRS. Although distances from the structure-based approach are closer to the real receptor, present data prove the validity of the ligand-based GRIND approach.

Binding Sites↗

Epigenesis and dynamic similarity in two regulatory networks in Pseudomonas aeruginosa.

Mucoidy and cytotoxicity arise from two independent modifications of the phenotype of the bacterium Pseudomonas aeruginosa that contribute to the mortality and morbidity of cystic fibrosis. We show that, even though the transcriptional regulatory networks controlling both processes are quite different from a molecular or mechanistic point of view, they may be identical from a dynamic point of view: epigenesis may in both cases be the cause of the acquisition of these new phenotypes. This was highlighted by the identity of formal graphs modelling these networks. A mathematical framework based on formal methods from computer science was defined and implemented with a software environment. It allows an easy and rigorous validation and certification of these models and of the experimental methods that can be proposed to falsify or validate the underlying hypothesis.

Epigenesis, Genetic↗

Use of clinical audit for revalidation: is it sufficiently accurate?

In order to provide better patient care, clinicians will be subject to revalidation and re-certification. This may be partially based on existing and ongoing data collection, yet many units fail to incorporate mechanisms that validate the data that may be used. The accuracy of audit data was evaluated in a unit that has been using commercially available audit software for over 10 years. A total of 655 consecutive surgical admissions were documented over a 6-month period and errors in data collection and entry were gathered and analyzed. An overall accuracy of 90.5% was confirmed but examination of the data found them to be open to misinterpretation. Moreover, 13% of errors were made during a single week when locum staff were involved. The study highlights the fallibility of data collection during audit, and urges caution if using such data when judging performance-related issues as part of the process of appraisal.

Certification↗

The role of computer-aided assessment in health professional education: a comparison of student performance in computer-based and paper-and-pen multiple-choice tests.

There is a lack of empirical research in the use of computers in assessment. A study was conducted to compare student performance in computerized and paper-and-pen multiple-choice tests and assess the extent and effect of 'computer anxiety'. The LXR.TEST 5.1 software was used for the computerized test. A validated rating scale was used to assess computer anxiety. Most students had little computer experience other than in word processing. Only about a third of the students had moderate to mild computer anxiety. Neither computer experience nor anxiety correlated significantly with performance in the computer test. Whereas students performed significantly better in the paper test than in the computer test, there was no statistical difference in ranking in the two assessments formats. This suggests that computer-based assessment could be used with confidence for the purposes of ranking students, but care should be taken in using it for grading.

Journal Article↗

A web-based database for diagnosis of haematologic neoplasms using immunophenotyping by flow cytometry.

The interpretation of immunophenotyping results by flow cytometry involves pattern recognition of different haematologic neoplasms that may have similar immunologic marker patterns. The numerous markers available in the flow cytometry laboratory make these patterns difficult to remember, especially for those of uncommon neoplasms. We describe the design and implementation of a Web-based database for diagnosis of haematologic neoplasms using results of immunophenotyping by flow cytometry. This database aims to assist pathology and haematology residents in interpreting flow cytometry data, and is designed to reach a wide base of users who use a variety of browsers on different computer platforms. Five modules are developed in this comprehensive program: (a) differential diagnosis: to generate a list of differential diagnoses that closely match the marker results in a given case; (b) display of disorders: typical results of markers for each disorder; (c) display of markers: relevant information of each immunologic marker; (d) display of archived cases for a disorder: marker results of cases previously diagnosed for a disorder; and (e) display of summary for archived cases: summary of marker results of all the archived cases for each disorder. Our experience with this Web-based database in teaching pathology residents has been very encouraging. Since the World Wide Web is increasingly more accessible to computer users, it has become an ideal medium for distribution of clinical decision-support software.

Antigens, CD↗

Computer-assisted sleep staging.

To address the subjectivity in manual scoring of polysomnograms, a computer-assisted sleep staging method is presented in this paper. The method uses the principles of segmentation and self-organization (clustering) based on primitive sleep-related features to find the pseudonatural stages present in the record. Sample epochs of these natural stages are presented to the user, who can classify them according to the Rechtschaffen and Kales (RK) or any other standard. The method then learns from these samples to complete the classification. This step allows the active participation of the operator in order to customize the staging to his/her preferences. The method was developed and tested using 12 records of varying types (normal, abnormal, male, female, varying age groups). Results showed an overall concurrence of 80.6% with manual scoring of 20-s epochs according to RK standard. The greatest amount of errors occurred in the identification of the highly transitional Stage 1, 54% of which was misclassified into neighboring stages 2 or Wake.

Adolescent↗

Assessment of nutritional status in rhesus monkeys: comparison of dual-energy X-ray absorptiometry and stable isotope dilution.

Body composition estimates from dual-energy X-ray absorptiometry and stable isotope dilution ((2)H and (18)O) were compared in 61 rhesus monkeys (Macaca mulatta) from the ongoing long-term energy restriction study at the University of Wisconsin. Their average age was 18.9 +/- 2.5 y/o. Of the animals, 51% were in the energy restricted group and 38% were females. Although the correlation between methods was highly significant for fat mass (R(2) = 0.97, SEE = 0.25 kg or 7.5%, P < 0.0001) and fat-free mass (R(2) = 0.98, SEE = 0.29 kg or 3.6%, P < 0.0001), we observed that dual-energy X-ray absorptiometry underestimated fat mass by 0.67 +/- 0.26 kg (7.5%, P < 0.0001) and overestimated fat-free mass by 0.57 +/- 0.29 kg (20%, P < 0.0001) when compared with isotope dilution. Taken together with data from the literature, the present results emphasize the usefulness of dual-energy X-ray absorptiometry to derive body composition and thus nutritional status in monkeys, but demonstrate the importance of validation experiments for a given DXA model and software.

Absorptiometry, Photon↗

Modelling of cardiovascular system: development of a hybrid (numerical-physical) model.

Physical models of the circulation are used for research, training and for testing of implantable active and passive circulatory prosthetic and assistance devices. However, in comparison with numerical models, they are rigid and expensive. To overcome these limitations, we have developed a model of the circulation based on the merging of a lumped parameter physical model into a numerical one (producing therefore a hybrid). The physical model is limited to the barest essentials and, in this application, developed to test the principle, it is a windkessel representing the systemic arterial tree. The lumped parameters numerical model was developed in LabVIEW environment and represents pulmonary and systemic circulation (except the systemic arterial tree). Based on the equivalence between hydraulic and electrical circuits, this prototype was developed connecting the numerical model to an electrical circuit--the physical model. This specific solution is valid mainly educationally but permits the development of software and the verification of preliminary results without using cumbersome hydraulic circuits. The interfaces between numerical and electrical circuits are set up by a voltage controlled current generator and a voltage controlled voltage generator. The behavior of the model is analyzed based on the ventricular pressure-volume loops and on the time course of arterial and ventricular pressures and flow in different circulatory conditions. The model can represent hemodynamic relationships in different ventricular and circulatory conditions.

Coronary Circulation↗

Beverage intake among preschool children and its effect on weight status.

OBJECTIVE: The obesity epidemic in the United States continues to increase. Because obesity tends to track over time, the increase in overweight among young children is of significant concern. A number of eating patterns have been associated with overweight among preschool-aged children. Recently, 100% fruit juice and sweetened fruit drinks have received considerable attention as potential sources of high-energy beverages that could be related to the prevalence of obesity among young children. Our aim was to evaluate the beverage intake among preschool children who participated in the National Health and Nutrition Examination Survey 1999-2002 and investigate associations between types and amounts of beverages consumed and weight status in preschool-aged children. METHODS: We performed a secondary analysis of the data from the National Health and Nutrition Examination Survey 1999-2002, which is a continuous, cross-sectional survey of a nationally representative sample of the noninstitutionalized population of the United States. It included the collection of parent reported demographic descriptors, a 24-hour dietary recall, a measure of physical activity, and a standardized physical examination. The 24-hour dietary recall was obtained in person by a trained interviewer and reflected the foods and beverages that were consumed by the participant the previous day. The National Health and Nutrition Examination Survey food groups were classified on the basis of the US Department of Agriculture's Food and Nutrient Database for Dietary Studies. We reviewed the main food descriptors used and classified all beverages listed. One hundred percent fruit juice was classified as only beverages that contained 100% fruit juice, without sweetener. Fruit drinks included any sweetened fruit juice, fruit-flavored drink (natural or artificial), or drink that contained fruit juice in part. Milk included any type of cow milk and then was subcategorized by percentage of milk fat. Any sweetened soft drink, caffeinated or uncaffeinated, was categorized as soda. Diet drinks included any fruit drink, tea, or soda that was sweetened by low-calorie sweetener. Several beverages were removed from the analysis because of low frequency of consumption among the sample. Water was not included in the analysis because it is not part of the US Department of Agriculture's Food and Nutrient Database categories. For the purposes of this analysis, the beverages were converted and reported as ounces, rather than grams, as reported by the National Health and Nutrition Examination Survey, to make it more clinically relevant. The child's BMI percentile for age and gender were calculated on the basis of Centers for Disease Control and Prevention criteria and used to identify children's weight status as underweight (< 5%), normal weight (5% to < 85%), at risk for overweight (85% to < 95%), or overweight (> or = 95%). Because of the small number of children in the underweight category, they were included in the normal-weight category for this analysis. Data were analyzed using SUDAAN 9.0.1 statistical software programs. SUDAAN allows for improved accuracy and validity of results by calculating test statistics for the stratified, multistage probability design of the National Health and Nutrition Examination Survey. Sample weights were applied to all analyses to account for unequal probability of selection from oversampling low-income children and black and Mexican American children. Descriptive and chi2 analyses and analysis of covariance, adjusting for age, gender, ethnicity, household income, energy intake, and physical activity, were conducted. RESULTS: All children who were aged 2 to 5 years were identified (N = 1572). Those with missing data were removed from additional analysis, resulting in a final sample of 1160 preschool children. Of the 1160 children analyzed, 579 (49.9%) were male. White children represented 35%, black children represented 28.3%, and Hispanic children represented 36.7% of the sample. Twenty-four percent of the children were overweight or at risk for overweight (BMI > or = 85%), and 10.7% were overweight (BMI > or = 95%). There were no statistically significant differences in BMI between boys and girls or among the ethnicities. Overweight children tended to be older (mean age: 3.83 years) compared with the normal-weight children (mean age: 3.48 years). Eighty-three percent of children drank milk, 48% drank 100% fruit juice, 44% drank fruit drink, and 39% drank soda. Whole milk was consumed by 46.5% of the children, and 3.1% and 5.5% of the children consumed skim milk and 1% milk, respectively. Preschool children consumed a mean total beverage volume of 26.93 oz/day, which included 12.32 oz of milk, 4.70 oz of 100% fruit juice, 4.98 oz of fruit drinks, and 3.25 oz of soda. Weight status of the child had no association with the amount of total beverages, milk, 100% fruit juice, fruit drink, or soda consumed. There was no clinically significant association between the types of milk (percentage of fat) consumed and weight status. In analysis of covariance, daily total energy intake increased with increased consumption of milk, 100% fruit juice, fruit drinks, and soda. However, there was not a statistically significant increase in BMI on the basis of quantity of milk, 100% fruit juice, fruit drink, or soda consumed. CONCLUSIONS: On average, preschool children drank less milk than the 2005 Dietary Guidelines for Americans recommendation of 16 oz/day. Only 8.6% drank low-fat or skim milk, as recommended for children who are older than 2 years. On average, preschool children drank < 6 oz/day 100% fruit juice. Increased beverage consumption was associated with an increase in the total energy intake of the children but not with their BMI. Prospectively studying preschool children beyond 2 to 5 years of age, through their adiposity rebound (approximately 5.5-6 years) to determine whether there is a trajectory increase in their BMI, may help to clarify the role of beverage consumption in total energy intake and weight status.

Beverages↗

Comparative study of methods for the quantification of biofilm on complete dentures.

This study compared the application and reliability of 4 methods for biofilm quantification (computerized, paper-weighing, point-counting, and planimetric) in complete dentures, verifying the correlation between them. The internal surfaces of 62 complete dentures were stained (5% erythrosine) and photographed. The slides were projected on paper, and the areas (total and biofilm-covered) were outlined with a pencil. These areas were measured with an equidistant point grid (point-counting method), a digital planimeter (planimetric method), and for the paper weighing method they were cut and weighed with a precision scale. For the computerized method, ImageTool software was used. In order to perform a validation test of the methods, all of them were applied to slide projections of geometric figures with known dimensions. The correlation tests showed high correlation values (r = 0.82 to 0.99) among the methods. The validation test (ANOVA) showed no statistically significant differences among the values obtained from the measurement of figures using all four quantitative methods and the real dimensions of these geometric figures. Quantitative methods were efficient and reliable for measuring quantity of biofilm in complete dentures, and may be useful in experimental studies on the efficacy of hygiene products. The computerized method was fast and easy to perform.

Aged↗