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PubMed2004

Membrane protein structure determination using solid-state NMR.

Solid-state NMR is emerging as a method for resolving structural information for large biomolecular complexes, such as membrane-embedded proteins. In principle, there is no molecular weight limit to the use of the approach, although the complexity and volume of data is still outside complete assignment and structural determinations for any large (Mr > approx 30,000) complex unless specific methods to reduce the information content to a manageable amount are employed. Such methods include specific residue-type labeling, labeling of putative segments of a protein, or examination of complexes made up of smaller, manageable units, such as oligomeric ion channels. Labeling possibilities are usually limited to recombinant or synthesized proteins, and labeling strategies often follow models from a bioinformatics approach. In all cases, and in common with most membrane studies, sample preparation is vital, and this activity alone can take considerable effort before NMR can be applied--peptide or protein production (synthesis or expression) followed by reconstitution into bilayers and resolution of suitable sample geometry is still technically challenging. As experience is gained in the field, this development time should decrease. Here, the practical aspects of the use of solid-state NMR for membrane protein structural determinations are presented, as well as how the methodology can be applied. Some successes to date are discussed, with an indication of how the area might develop.

Membrane Proteins↗

PubMed1997

Dissociation between categorical and coordinate spatial computations: modulation by cerebral hemispheres, task properties, mode of response, and age.

Kosslyn suggested a dissociation of two kinds of spatial representations: categorical and coordinate, the former computed by the left hemisphere and the latter by the right hemisphere. In addition, with practice, a "categorization" of the coordinate computation would appear. These suggestions resulted largely from an experiment, replicated by others, in which the subjects had to estimate the relative position of a dot and a line by giving an oral response, and feedback was provided. The present series of five experiments was an attempt to test whether this finding could be generalized, under several methodological manipulations, some of which have already been used by researchers in separate studies. In the five experiments, accuracy was a more contributive dependent variable than correct latencies, and practice effects on the task X field interaction were not verified. Experiments 1-3 concerned the kind of response. When a manual instead of an oral response was required (Experiment 1; n = 32 Ss), the expected dissociation was observed (as well as when the latency of responses made by the right hand were studied). When the number of oral responses was increased from two (a binary choice) to eight, the dissociation was still observed for accuracy but disappeared when a more liberal criterion of accuracy was used to reduce the considerable task effect (Experiment 2; n = 32 Ss), or when response requirements were equated for both tasks (Experiment 3; n = 32 Ss). In Experiment 4 (n = 32 Ss), a manual response was called but the feedback was removed and the task dissociation disappeared. Finally, the task dissociation observed in Experiment 1 was not verified when a sample of elderly subjects was enrolled (Experiment 5; n = 32 Ss). However, age per se was the source of an interesting additional dissociation since only the coordinate computation was age sensitive. Our results suggest that the dissociation proposed by Kosslyn between the computation of categorical vs coordinate spatial relationships is highly unstable and sensitive to subtle methodological factors (vocal vs manual response, presence vs absence of feedback, binary vs "continuous" response, age) which could preclude its general application.

Adolescent↗

PubMed1997

A lognormal distribution-based exposure assessment method for unbalanced data.

We present a generalization of existing statistical methodology for assessing occupational exposures while explicitly accounting for between- and within-worker sources of variability. The approach relies upon an intuitively reasonable model for shift-long exposures, and requires repeated exposure measurements on at least some members of a random sample of workers from a job group. We make the methodology more readily applicable by providing the necessary details for its use when the exposure data are unbalanced (that is, when there are varying numbers of measurements per worker). The hypothesis testing strategy focuses on the probability that an arbitrary worker in a job group experiences a long-term mean exposure above the occupational exposure limit (OEL). We also provide a statistical approach to aid in the determination of an appropriate intervention strategy in the event that exposure levels are deemed unacceptable for a group of workers. We discuss important practical considerations associated with the methodology, and we provide several examples using unbalanced sets of shift-long exposure data-taken on workers in various sectors of the nickel-producing industry. We conclude that the statistical methods discussed afford sizable practical advantages, while maintaining similar overall performance to that of existing methods appropriate for balanced data only.

Air Pollutants, Occupational↗

PubMed2000

Physician-assisted suicide in Connecticut: physicians' attitudes and experiences.

OBJECTIVE: This study examined aspects of physician attitudes toward physician-assisted suicide (PAS) not fully examined to date: evaluation of risks related to PAS, particularly the presence of depression, and the influence of religious and professionally-based values. DESIGN: Anonymous, self-administered mailed questionnaire using Dillman methodology. PARTICIPANTS: Targeted sample of physicians licensed by the Connecticut Department of Public Health in 1997 (n = 2,805 completed surveys; 40% response rate). DATA INCLUDE: Physician and patient characteristics, attitudes toward physician assisted suicide, and confidence in treating depression. RESULTS: Religious affiliation, religiosity, ethnicity and medical specialty were strongly associated with views on PAS. Seven percent of respondents had been asked to write a lethal prescription during the past year, 15% of whom (n = 24) had complied with at least one request. CONCLUSIONS: Most respondents expressed concern regarding certain risks associated with PAS, including movement toward involuntary euthanasia and the influence of undetected depression. Findings raise practical issues to be addressed through statutory or professional safeguards if PAS were to be legalized.

Adult↗

PubMed2005

Identification of impurities in acarbose by using an integrated liquid chromatography-nuclear magnetic resonance and liquid chromatography-mass spectrometry approach.

The usefulness of applying an integrated LC-NMR and LC-MS approach to acarbose bulk drug impurity profiling is demonstrated. LC-MS and LC-NMR methodologies were employed for the online separation and structural elucidation of a final drug product. Combining data provided by the stop-flow LC-NMR and LC-MS experiments made it possible to identify the main components present in the acarbose sample. Spectral analysis revealed that A and B were known impurities while C was an unknown compound. LC-MS and LC-NMR analyses revealed that C was a pentasaccharide differing from the acarbose in number and nature of sugar subunits in the molecule. It was subsequently isolated and its structure was confirmed by the offline 1- and 2-D NMR experiments, and atom assignment was made.

Acarbose↗

PubMed2005

A method for generating reproducible evidence in fMRI studies.

Insights into cognitive neuroscience from neuroimaging techniques are now required to go beyond the localisation of well-known cognitive functions. Fundamental to this is the notion of reproducibility of experimental outcomes. This paper addresses the central issue that functional magnetic resonance imaging (fMRI) experiments will produce more desirable information if researchers begin to search for reproducible evidence rather than only p value significance. The study proposes a methodology for investigating reproducible evidence without conducting separate fMRI experiments. The reproducible evidence is gathered from the separate runs within the study. The associated empirical Bayes and ROC extensions of the linear model provide parameter estimates to determine reproducibility. Empirical applications of the methodology suggest that reproducible evidence is robust to small sample sizes and sensitive to both the magnitude and persistency of brain activation. It is demonstrated that research findings in fMRI studies would be more compelling with supporting reproducible evidence in addition to standard hypothesis testing evidence.

Algorithms↗

PubMed2006

Reexamining psychokinesis: comment on Bösch, Steinkamp, and Boller (2006).

H. Bösch, F. Steinkamp, and E. Boller's review of the evidence for psychokinesis confirms many of the authors' earlier findings. The authors agree with Bösch et al. that existing studies provide statistical evidence for psychokinesis, that the evidence is generally of high methodological quality, and that effect sizes are distributed heterogeneously. Bösch et al. postulated the heterogeneity is attributable to selective reporting and thus that psychokinesis is "not proven." However, Bösch et al. assumed that effect size is entirely independent of sample size. For these experiments, this assumption is incorrect; it also guarantees heterogeneity. The authors maintain that selective reporting is an implausible explanation for the observed data and hence that these studies provide evidence for a genuine psychokinetic effect.

Cognition↗

PubMed1991

Importance of locally derived birth weight nomograms.

Significant differences exist between the published nomograms of birth weight for gestational age. Differences in demographics, altitude above sea level and study methodology have all been implicated. A three-year experience of uncomplicated singleton gestations (N = 14,766) was analyzed for birth weight and gestational age. The mean, 10th percentile and their 95% confidence intervals were compared to Brenner's data and a pooled sample from other studies in this country. The mean birth weights were significantly higher than Brenner's after 32 weeks' gestation and higher than the pooled mean after 35. The 10th percentiles were significantly greater than Brenner's after 36 weeks' gestation. Approximately 66% of neonates at risk, below our 10th percentile, would not be identified with Brenner's criteria. We agree with Goldenberg that uniform criteria for the definition of the normal population are important in identifying aberrant growth. However, birth weight standards should be developed for the specific population in question.

Altitude↗

PubMed2000

Venepuncture versus heel lance for blood sampling in term neonates.

BACKGROUND: Heel lance has been the conventional method of blood sampling in neonates for screening tests. Neonates undergoing this procedure experience pain. Despite various studies evaluating the role of pharmacological and non-pharmacological interventions to date, there are no effective and practical methods to alleviate pain from heel lance. OBJECTIVES: To determine whether venepuncture or heel lance is less painful and more effective for blood sampling in term neonates. SEARCH STRATEGY: Systematic search in accordance with Cochrane Neonatal Collaborative Review Group. All randomized controlled trials which compared pain response to venepuncture vs. heel lance were searched using MEDLINE, Embase, CINAHL, the Cochrane Library, reference lists of identified trials and personal files. No language restrictions were applied. SELECTION CRITERIA: Randomized controlled trials which compared pain response to venepuncture vs. heel lance were selected for this review. DATA COLLECTION AND ANALYSIS: Data regarding the primary outcome of pain response to venepuncture vs. heel lance as assessed by validated pain measures were abstracted. Secondary outcomes including the need of repeat blood sampling, bruising/hematoma at local site, and parental perception of their own anxiety and infant's pain were also abstracted. All data were analysed using Revman 3.1. When possible, meta-analysis was done using relative risk (RR) and event rate difference (ERD), along with their 95% confidence intervals (CI). If ERD was significant, number needed to treat (NNT) was calculated. MAIN RESULTS: Two trials were eligible for inclusion in the review. One study using Neonatal Infant Pain Scale (NIPS) and one study using Neonatal Facial Action Coding System (NFCS) and cry duration showed statistically significantly lower pain scores for venepuncture as compared to heel lance. In one study, maternal anxiety was noted to be higher in the venepuncture group as compared to heel lance group prior to the procedure; however, after observing the procedure, mothers rated their infant's pain to be lower in the venepuncture group. A meta-analysis was performed on the need for at least one additional skin puncture to obtain the required amount of blood using venepuncture as compared to heel lance. The relative risk for requiring more than one skin puncture for venepuncture versus heel lance was 0.19 [95% confidence interval (CI) 0.10, 0.38]. The risk difference was -53% (95% CI -66%, -40%). The number needed to treat (NNT) to avoid one repeat skin puncture was 1.9 (95% CI 1.5, 2.5). REVIEWER'S CONCLUSIONS: Venepuncture, when performed by a trained phlebotomist, appears to be the method of choice for blood sampling in term neonates. For each two venepunctures instead of heel lance, the need for one additional skin puncture can be avoided. In view of the promising results derived from small studies with some methodological limitations, further well designed randomized controlled trials need to be conducted. The interventions should be compared in settings where several individuals perform the venepuncture and/or the heel lance.

Blood Specimen Collection↗

PubMed1988

Microsphere estimates of blood flow: methodological considerations.

The microsphere technique is a standard method for measuring blood flow in experimental animals. Sporadic reports have appeared outlining the limitations of this method. In this study we have systematically assessed the effect of blood withdrawals for reference sampling, microsphere numbers, and anesthesia on blood flow estimates using radioactive microspheres in dogs. Experiments were performed on 18 conscious and 12 anesthetized dogs. Four blood flow estimates were performed over 120 min using 1 X 10(6) microspheres (15 microns) each time. The effects of excessive numbers of microspheres (13 million), pentobarbital sodium anesthesia (30 mg/kg), and replacement of volume loss for reference samples with dextran 70 were assessed. In both conscious and anesthetized dogs a progressive decrease in gastric mucosal blood flow and cardiac output was observed over 120 min. This was also observed in the pancreas in conscious dogs. The major factor responsible for these changes was the volume loss due to reference sample withdrawals. Replacement of the withdrawn blood with dextran 70 led to stable blood flows to all organs. The injection of excessive numbers of microspheres did not modify hemodynamics to a greater extent than did the injection of 4 million microspheres. Anesthesia exerted no influence on blood flow other than raising coronary flow. We conclude that although blood flow to the gastric mucosa and the pancreas is sensitive to the minor hemodynamic changes associated with the microsphere technique, replacement of volume loss for reference samples ensures stable blood flow to all organs over a 120-min period.

Animals↗

PubMed2006

Statistical analysis of an RNA titration series evaluates microarray precision and sensitivity on a whole-array basis.

BACKGROUND: Concerns are often raised about the accuracy of microarray technologies and the degree of cross-platform agreement, but there are yet no methods which can unambiguously evaluate precision and sensitivity for these technologies on a whole-array basis. RESULTS: A methodology is described for evaluating the precision and sensitivity of whole-genome gene expression technologies such as microarrays. The method consists of an easy-to-construct titration series of RNA samples and an associated statistical analysis using non-linear regression. The method evaluates the precision and responsiveness of each microarray platform on a whole-array basis, i.e., using all the probes, without the need to match probes across platforms. An experiment is conducted to assess and compare four widely used microarray platforms. All four platforms are shown to have satisfactory precision but the commercial platforms are superior for resolving differential expression for genes at lower expression levels. The effective precision of the two-color platforms is improved by allowing for probe-specific dye-effects in the statistical model. The methodology is used to compare three data extraction algorithms for the Affymetrix platforms, demonstrating poor performance for the commonly used proprietary algorithm relative to the other algorithms. For probes which can be matched across platforms, the cross-platform variability is decomposed into within-platform and between-platform components, showing that platform disagreement is almost entirely systematic rather than due to measurement variability. CONCLUSION: The results demonstrate good precision and sensitivity for all the platforms, but highlight the need for improved probe annotation. They quantify the extent to which cross-platform measures can be expected to be less accurate than within-platform comparisons for predicting disease progression or outcome.

Algorithms↗

PubMed2005

Oxytocin is not a valid biomarker when measured in saliva by immunoassay.

The integration of oxytocin (OT) into behavioral science seems to hold considerable promise for advancing our understanding of human health and development but methodological issues restrict the measurement of OT in large studies, in everyday social settings, or when repeated sampling is required. Measuring OT in saliva could overcome many of these limitations. In this paper, we rigorously evaluate the feasibility of doing so. A series of experiments leads to the conclusion that saliva does not contain oxytocin in measurable amounts, and that OT is not a valid salivary biomarker when measured by currently available immunological methods. Levels of immuno-reactive OT in saliva are primarily due to non-specific interference with antibody-antigen binding. We can state with a high degree of certainty that measurement of OT in saliva does not yield meaningful indices of individual differences or intra-individual change.

Biomarkers↗

PubMed2004

Traumatic events and posttraumatic stress disorder in patients with psychogenic nonepileptic seizures: a critical review.

Although video-EEG monitoring has revolutionized the diagnosis of psychogenic nonepileptic seizure (PNES), the etiology of this condition remains poorly understood. This article is a critical review of studies on the prevalence of traumatic events, abuse, and/or posttraumatic stress disorder (PTSD) in patients with PNES. Searches carried out on MEDLINE (1966-2004) and Web of Science (1945-2004) identified 17 relevant studies. PNES samples showed very high rates of trauma (44-100%) and abuse (23-77%), which were 15-40% higher than those found in control groups. This suggests that traumatic experiences may be a potential risk factor for PNES. PNES samples also showed a higher prevalence of PTSD than control groups, raising the possibility that PNES may arise as a clinical expression of a hypothetical PTSD subtype the core symptoms of which are dissociative. Methodological limitations do not permit the confirmation of these hypotheses. Stronger research designs are needed, such as prospective and case-control studies in both hospital and community settings.

Adult↗

PubMed2004

Genome wide identification and classification of alternative splicing based on EST data.

MOTIVATION: Alternative splicing is currently seen to explain the vast disparity between the number of predicted genes in the human genome and the highly diverse proteome. The mapping of expressed sequences tag (EST) consensus sequences derived from the GeneNest database onto the genome provides an efficient way of predicting exon-intron boundaries, gene structure and alternative splicing events. However, the alternative splicing events are obscured by a large number of putatively artificial exon boundaries arising due to genomic contamination or alignment errors. The current work describes a methodology to associate quality values to the predicted exon-intron boundaries. High quality exon-intron boundaries are used to predict constitutive and alternative splicing ranked by confidence values, aiming to facilitate large-scale analysis of alternative splicing and splicing in general. RESULTS: Applying the current methodology, constitutive splicing is observed in 33,270 EST clusters, out of which 45% are alternatively spliced. The classification derived from the computed confidence values for 17 of these splice events frequently correlate (15/17) with RT-PCR experiments performed for 40 different tissue samples. As an application of the confidence measure, an evaluation of distribution of alternative splicing revealed that majority of variants correspond to the coding regions of the genes. However, still a significant fraction maps to non-coding regions, thereby indicating a functional relevance of alternative splicing in untranslated regions. AVAILABILITY: The predicted alternative splice variants are visualized in the SpliceNest database at http://splicenest.molgen.mpg.de

Algorithms↗

PubMed2001

Approach to high-resolution ex situ NMR spectroscopy.

Nuclear magnetic resonance (NMR) experiments are typically performed with samples immersed in a magnet shimmed to high homogeneity. However, there are many circumstances in which it is impractical or undesirable to insert objects or subjects into the bore of a high-field magnet. Here we present a methodology based on an adaptation of nutation echoes that provides resolved spectra in the presence of matched inhomogeneous static and radiofrequency fields, thereby opening the way to high-resolution ex situ NMR. The observation of chemical shifts is regained through the use of multiple-pulse sequences of correlated, composite z-rotation pulses, producing resolved NMR spectra of liquid samples.

Magnetic Resonance Spectroscopy↗

PubMed1999

Association of herpesvirus with fibropapillomatosis of the green turtle Chelonia mydas and the loggerhead turtle Caretta caretta in Florida.

Sea turtle fibropapillomatosis (FP) is a disease marked by proliferation of benign but debilitating cutaneous fibropapillomas and occasional visceral fibromas. Transmission experiments have implicated a chloroform-sensitive transforming agent present in filtered cell-free tumor homogenates in the etiology of FP. In this study, consensus primer PCR methodology was used to test the association of a chelonian herpesvirus with fibropapillomatosis. Fibropapilloma and skin samples were obtained from 17 green and 2 loggerhead turtles affected with FP stranded along the Florida coastline. Ninety-three cutaneous and visceral tumors from the 19 turtles, and 33 skin samples from 16 of the turtles, were tested. All turtles affected with FP had herpesvirus associated with their tumors as detected by PCR. Ninety-six percent (89/93) of the tumors, but only 9% (3/33) of the skin samples, from affected turtles contained detectable herpesvirus. The skin samples that contained herpesvirus were all within 2 cm of a fibropapilloma. Also, 1 of 11 scar tissue samples from sites where fibropapillomas had been removed 2 to 51 wk earlier from 5 green turtles contained detectable herpesvirus. None of 18 normal skin samples from 2 green and 2 loggerhead turtles stranded without FP contained herpesvirus. The data indicated that herpesvirus was detectable only within or close to tumors. To determine if the same virus infected both turtle species, partial nucleotide sequences of the herpesvirus DNA polymerase gene were determined from 6 loggerhead and 2 green turtle samples. The sequences predicted that herpesvirus of loggerhead turtles differed from those of green turtles by only 1 of 60 amino acids in the sequence examined, indicating that a chelonian herpesvirus exhibiting minor intratypic variation was the only herpesvirus present in tumors of both green and loggerhead turtles. The FP-associated herpesvirus resisted cultivation on chelonian cell lines which support the replication of other chelonian herpesviruses. These results lead to the conclusion that a chelonian herpesvirus is regularly associated with fibropapillomatosis and is not merely an incidental finding in affected turtles.

Amino Acid Sequence↗

PubMed2004

Genesis of self-identity as disother: life histories of people who stutter.

This paper explores the processes shaping self-identity formation as DisOther and the actions of participants who stutter. It illuminates the experiences of adults who stutter using a biographical, narrative, life history methodology. The participants were seven South African adults of diverse racial, social and economic backgrounds from KwaZulu Natal, South Africa. Five males and two females were invited to participate via purposive and convenience sampling processes. Their stories of living with stuttering in their life worlds over time were constructed via biographical interviews using personal, social and temporal lenses typical of life history methodology. The interviews were audio-recorded and transcribed. The data were analysed at two levels using a combination of strategies. The first level entailed a narrative analysis that was represented as research stories for each participant. The cross-case and thematic analysis of research stories constituted the second level analysis of narratives. The findings explain the complex and interrelated personal and social processes over time which contribute to the genesis of self-identity formation as DisOther Social inscriptions of difference occurred in immediate home, school and work contexts over time via multiple processes such as labelling, norming, judging and teasing. Personal processes included discoveries of difference via critical events, repeated reinforcement of difference, self-judgement and temporal burdening. Furthermore, the actions participants took in negotiating stuttering were examined. The implications of the findings and limitations of the study are presented.

Adaptation, Psychological↗

PubMed1993

[Situational effects in reporting life change events? A comparison of two assessment techniques].

Studies on the role of life events in the development of malignant diseases (particularly breast cancer) often rely on so-called limited prospective designs: Women with a suspicious breast lump are interviewed prior to surgery. After having learnt the final diagnosis, the sample is divided into a group having cancer and a control group. Research using this approach was criticised for producing artifacts: knowledge of diagnosis should cause reevaluations of past events and lead to overreporting of stressful experiences. Two limited prospective studies using different assessment methodologies are compared for possible artifacts due to knowledge of diagnosis. When considering less severe events, the application of a checklist technique leads to marked memory bias as assumed above. When leaving the respondents time to reconstruct their events at length, no artifacts are present. Reporting of the severest event categories remains unaffected under both methods.

Adult↗