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PubMed1981

Gas Chromatography-Mass Spectrometry of N- Heptafluorobutyryl Isobutyl Esters of Amino Acids in the Analysis of the Kinetics of [N]H(4) Assimilation in Lemna minor L.

Rapid, sensitive, and selective methods for the determination of the (15)N abundance of amino acids in isotopic tracer experiments with plant tissues are described and discussed. Methodology has been directly tested in an analysis of the kinetics of [(15)N]H(4) (+) assimilation in Lemna minor L. The techniques utilize gas chromatography-mass spectrometry selected ion monitoring of major fragments containing the N moiety of N-heptafluorobutyryl isobutyl esters of amino acids. The ratio of selected ion pairs at the characteristic retention time of each amino acid derivative can be used to calculate (15)N abundance with an accuracy of +/-1 atom% excess (15)N using samples containing as little as 30 picomoles of individual amino acids. Up to 11 individual amino acid derivatives can be selectively monitored in a single chromatogram of 30 minutes. It is suggested that these techniques will be useful in situations where the small quantities of N available for analysis have hitherto hindered the use of (15)N-labeled precursors.

Journal Article↗
PubMedDate not supplied

[The work process and occupational health risks in a cement factory].

The authors evaluate the work process and its effect on workers' health in a cement factory in the State of Rio de Janeiro. The interactive methodology consisted of different approaches to assessing the workplace through the incorporation of various institutions working in the field of Workers' Health, professionals from different backgrounds, and the trade union, valorizing the workers' experience and actively contributing to the surveillance process under the Unified National Health System (SUS). Levels of particulate matter and noise were measured. The mean level of free crystalline silica in the particulate matter was 2%, resulting in a tolerance limit as specified under Brazilian legislation (NR-15), or 2.0mg/m3. The concentration of particles both in samples collected in the workers' respiratory zone and in area samples varied from 3.59 to 52.44mg/m3. Noise varied from 83dB to 110dB. The majority of the values were higher than the maximum limits set by Brazilian legislation. These results, together with the opinions expressed by the workers themselves, showed an unhealthy workplace and work process, placing the workers' health at risk.

Brazil↗
PubMed1994

Multiple blinded reviews of the same two manuscripts. Effects of referee characteristics and publication language.

OBJECTIVE: To study the association between referee characteristics and their manuscript assessments, the influence of manuscript language on referees' judgments, and the usefulness, quality, and extent of referees' free-text comments. DESIGN: Two nonauthentic, but realistic, short manuscripts with a number of common methodological flaws were sent to 180 Scandinavian referees. Through randomization, each referee received one of the manuscripts in English and the other manuscript in the national language. A structured assessment of the manuscript quality was expressed on a 5-point scale, and the impact of referee characteristics (age, gender, experience, and so on) was analyzed by multiple linear regression. MAIN OUTCOME: Manuscript quality assessed by referees. RESULTS: A total of 312 reviews from 156 referees could be used for the study of referee characteristics and language. With increasing experience, the referees gave lower quality scores (P < .05). A tendency toward stricter assessment with younger age was seen (P < .05). No influence of referees' gender, specialty, or nationality was found. For the test manuscript of the poorest quality, the English version was assessed to be better than the national-language version (P < .05). A total of 159 of 312 reviews included free-text comments applicable for the methodological study. In 54 reviews (34%), no methodological comments accompanied the assessment, and in six reviews they were only incomplete. Wrong sampling unit was mentioned by one fourth of 80 referees. Only one referee mentioned the incorrect use of a parametric test in the analysis of data whose distribution was nonparametric. CONCLUSIONS: Experienced and young referees gave a stricter assessment of the manuscripts than their less experienced and older colleagues. An English version seemed to be accepted more easily than a national-language version of the same manuscript. Most referees spontaneously mentioned the shortcomings of the manuscripts only as part of their overall judgment.

Humans↗
PubMed2006

Methodological approaches to exploring epileptic disorders in the human brain in vitro.

Brain surgery, and in particular epilepsy surgery, offers the unique opportunity to study viable human central nervous tissue in vitro. This does not only open a window to address the basic mechanisms underlying human disease, such as epilepsy, but it allows to venture into investigating neurophysiological functions per se. In the present paper, we describe the most commonly used methods in the electrophysiological (and, at least to some extent, also histochemical and molecular) analysis of human tissue in vitro. In addition, we consider the pitfalls and limitations of such studies, in particular regarding the issue of tissue sampling procedures and control experiments.

Biopsy↗
PubMed2003

Spatial normalization of diffusion tensor fields.

A method for the spatial normalization and reorientation of diffusion tensor (DT) fields is presented. Spatial normalization of tensor fields requires an appropriate reorientation of the tensor on each voxel, in addition to its relocation into the standardized space. This appropriate tensor reorientation is determined from the spatial normalization transformation and from an estimate of the underlying fiber direction. The latter is obtained by treating the principal eigenvectors of the tensor field around each voxel as random samples drawn from the probability distribution that represents the direction of the underlying fiber. This approach was applied to DT images from nine normal volunteers, and the results show a significant improvement in signal-to-noise ratio (SNR) after spatial normalization and averaging of tensor fields across individuals. The statistics of the spatially normalized tensor field, which represents the tensor characteristics of normal individuals, may be useful for quantitatively characterizing individual variations of white matter structures revealed by DT imaging (DTI) and deviations caused by pathology. Simulated experiments using this methodology are also described.

Algorithms↗
PubMed2004

Characterizing high-affinity antigen/antibody complexes by kinetic- and equilibrium-based methods.

Two biophysical methods, Biacore and KinExA, were used to kinetically and thermodynamically characterize high-affinity antigen/antibody complexes. Three to five independent experiments were performed on each platform with three different antigen/antibody complexes possessing nanomolar to picomolar equilibrium dissociation constants. By monitoring the dissociation phase on Biacore for 4 h, we were able to measure dissociation rate constants (kd) on the order of 1 x 10(-5)s(-1). To characterize high-affinity interactions by KinExA, samples needed to be equilibrated for up to 35 h to reach equilibrium. In the end, we show that similar kinetic rate constants and affinities were determined with both solution-phase and solid-phase methodologies. These results help further validate both interaction technologies and illustrate their suitability for characterizing extremely high-affinity interactions.

Animals↗
PubMed2000

Rapid identification of local T cell expansion in inflammatory organ diseases by flow cytometric T cell receptor Vbeta analysis.

Oligoclonal expansion of antigen-specific T cells occurs frequently during inflammatory diseases. These cells may persist for a long time at high frequency in the body and be enriched in the affected tissues. As a screening test for expanded cell T cell populations at sites of inflammation, we developed an optimized methodology for flow-cytometry-based quantification of T cell receptor Vbeta (TCRBV) expression. We first validated the specificity of a TCRBV-specific monoclonal antibody set by direct comparison with PCR-based analysis of mono- and polyclonal T cell samples. This monoclonal antibody (mAb) panel recognized approximately two thirds of the T cell receptor alpha/beta repertoire in a group of 64 healthy donors and allowed defining TCR usage in the CD4+ and CD8+ subsets. The reliable detection of expanded Vbeta gene families in T cell populations was confirmed in experiments on superantigen-stimulated T cells. Through differential TCR analysis on T cell subpopulations in cerebrospinal fluid and blood in patients with acute encephalitis, we were able to identify locally expanded CD8+ T cells. The power of this approach affords not only high-throughput comparative TCR analysis for immunological studies in vitro, but also rapid ex vivo identification of cell populations enriched in organ compartments during inflammatory diseases.

Acute Disease↗
PubMed1999

Radiotherapy for bone metastases: a critical appraisal of outcome measures.

Pain from bone metastases is a common problem in patients with advanced cancer, and radiotherapy plays an important role in its palliation. Single fraction treatments are often prescribed, but there is no clear consensus on this issue and clinical practice shows significant variability. This situation is unsatisfactory for all patients--the patient, the clinician, and the health care administrator. Randomized trials may use poor outcome measures and this contributes to practice variability. The credibility of outcome studies is often reduced due to poor study design, small sample sizes, and the use of endpoints that are both unreliable and unsuitable. The endpoints used have been narrowly defined, the patient's perspective has generally been overlooked, and quality of life has only once been used as an endpoint. A review of the current literature suggests that instruments specific to bone metastases are required. These must be based on patient experience, and rely on self-report. In addition, there is a need to understand the relative priority that patients attribute to treatment outcomes. The use of better instruments and methodologies in future trials will enhance the credibility of results and reduce practice variations.

Bone Neoplasms↗
PubMed2001

Health-related quality of life in childhood disorders: a modified focus group technique to involve children.

OBJECTIVES: Qualitative methodology has been under-utilized in child health research due to lack of a specific set of instruments. The objective of this study was to develop a child-centred qualitative research methodology to facilitate direct exploration of health-related quality of life (HRQL) issues and to identify the quality of life elements in pre-adolescent children with a chronic medical condition. STUDY DESIGN: Purposeful stratified sampling of children, ages 6-12, who function in a regular school class, with active epilepsy who were assembled in small focus groups. The groups met in four phases and were led by moderators who probed preset open questions and activities. RESULTS: The study demonstrated that our modified focus groups process was a powerful exploratory experience eliciting meaningful and important issues in quality of life beyond what parents and health professionals expected, and helped identify HRQL elements in childhood epilepsy. CONCLUSION: Modified focus groups are appropriate and suitable to explore quality of life issues in pre-adolescent children with a chronic medical condition. The process is feasible and trustworthy.

Child↗
PubMed2000

Inactivation of local root canal medicaments by dentine: an in vitro study.

AIMS: The aim of the study was to investigate the inactivation by dentine of the antibacterial activity of various commonly used local root canal medicaments. METHODOLOGY: The medicaments tested were saturated calcium hydroxide solution, 1% sodium hypochlorite, 0.5% and 0.05% chlorhexidine acetate, and 2/4% and 0.2/0.4% iodine potassium iodide. Dentine was sterilized by autoclaving and crushed into powder with a particle size of 0.2-20 microns. Aliquots of dentine suspension were incubated with the medicaments in sealed test tubes at 37 degrees C for 24 h or 1 h before adding the bacteria. In some experiments bacteria were added simultaneously with dentine powder and the medicament. Enterococcus faecalis A197A was used as a test organism. Samples for bacterial culturing were taken from the suspensions at 5 min, 1 h and 24 h after adding the bacteria. RESULTS: Dentine powder had an inhibitory effect on all medicaments tested. The effect was dependent on the concentration of the medicament as well as on the length of the time the medicament was preincubated with dentine powder before adding the bacteria. The effect of calcium hydroxide on E. faecalis was totally abolished by the presence of dentine powder. Similarly, 0.2/0.4% iodine potassium iodide lost its effect after preincubation for 1 h with dentine before adding the bacteria. The effect of 0.05% chlorhexidine and 1% sodium hypochlorite on E. faecalis was reduced but not totally eliminated by the presence of dentine. No inhibition could be measured when full strength solutions of chlorhexidine and iodine potassium iodide were used in killing E. faecalis. CONCLUSIONS: The dentine powder model appears to be an efficient tool for the study of interactions between local endodontic medicaments, dentine, and microbes.

Anti-Infective Agents, Local↗
PubMed2003

Evaluating innovations in maternity care: methodological approaches to a baseline postal survey.

BACKGROUND: A network of four public maternity hospitals in Melbourne, Australia, has implemented a range of initiatives aimed at improving maternity care. Comprehensive evaluation aims to determine whether or not the maternity service enhancement strategies have led to improvements in women's views and experiences of care. This paper discusses the baseline survey study design and methodological issues associated with the recruitment and reminder processes. METHOD: All women who gave birth over a 14-week period in 1999 at one of the four maternity units, except those who had a stillbirth or neonatal death, were invited to participate in the baseline postal survey. Questionnaires were sent to women at 3 months postpartum. A system of written and telephone reminders was instituted. RESULTS: The overall response fraction to the baseline survey was 65.2 percent (1256/1922). The sample was representative in terms of maternal age, method of birth, and infant birthweight. Women born overseas of non-English speaking background, single women, and women having their second or subsequent baby were underrepresented. CONCLUSIONS: The pleasing response fraction in part may be attributed to the method of recruitment and the reminder processes. Rigorous and careful approaches to methodology enhance study integrity and provide context to the interpretation of study findings.

Continuity of Patient Care↗
PubMed1977

Factors influencing contraceptive behavior of single college students.

This study investigated the premarital contraceptive behavior of 222 male and female college students. Contraceptive practice was examined in relation to dating patterns, level of emotional involvement with the sex partner was heightened, the intercourse was planned, and the individual and prior sexual experience.

Adolescent↗
PubMed2001

Safety testing for replication-competent retrovirus associated with gibbon ape leukemia virus-pseudotyped retroviral vectors.

The potential pathogenicity of replication-competent retroviruses (RCR) requires vigilant testing to exclude inadvertent contamination of clinical gene therapy vector products with RCR. Pseudotyped vectors using the gibbon ape leukemia virus (GALV) envelope have entered into clinical trials but specific recommendations regarding methods for screening of vector product and analysis of clinical samples have not been set forth. Unfortunately, current screening assays used for detecting amphotropic RCR are not suitable for GALV-pseudotyped RCR. We modified the extended S+/L- assay for RCR detection by using human 293 cells for virus amplification. Of five cell lines tested, 293 cells were selected because they combined a high transduction efficiency and an ability to generate RCR at high titer. After optimizing the amplification assay, a dilution of GALV virus could consistently be detected at a dilution of 10(-6). In coculture experiments, one GALV-infected cell could be consistently detected in 10(6) uninfected cells. A PCR-based assay was developed that was capable of detecting 100 copies of a GALV envelope containing plasmid diluted in 1 microg of DNA obtained from uninfected cells. PCR was also able to detect one GALV-infected cell in 10(6) uninfected cells. These assays will be suitable for testing of vector preparations and for monitoring of clinical samples from patients treated in clinical gene therapy protocols. The assays developed are similar in methodology and sensitivity to those currently used for certification of amphotropic retroviral vectors.

Biological Assay↗
PubMed2006

Evaluation of the effect of EDTA, EDTAC and citric acid on the microhardness of root dentine.

AIM: To evaluate the effect of citric acid, ethylenediaminetetraacetic acid (EDTA) and ethylenediaminetetraacetic acid plus Cetavlon (EDTAC) solutions on the microhardness of human root canal dentine. METHODOLOGY: Sixteen maxillary human canines were sectioned transversely at the cemento-enamel junction and the crowns were discarded. Subsequently, each root was embedded in an epoxy resin cylinder and their middle third sectioned horizontally into 4 mm thick slices. The samples were randomly divided into three groups according to the chelating agent employed, as follows (n = 6): group 1: EDTA 17%, group 2: EDTAC 17% and group 3: citric acid 10%. Dentine microhardness was then measured with a load of 50 g for 15 s. At the beginning of the experiment, reference microhardness values were obtained for samples without any etching (t = 0 min). The same samples were then exposed to 50 microL of the chelator solution for 1, 3 and 5 min. The Student's t-test (P < 0.05) was used to compare results for different times for each chelator and different chelators for each time. RESULTS: Microhardness decreased with increasing time of application of chelating solutions. There were no significant (P > 0.05) differences between initial microhardness for the three groups as well as after 1 min of application of the substances. After 3 min, EDTA produced a significantly greater reduction in microhardness. However, there was no significant difference between EDTA and EDTAC after 5 min. Citric acid caused significantly less reduction in microhardness. CONCLUSIONS: Overall, citric acid was least effective in reducing dentine hardness whilst EDTA had the strongest effect.

Cetrimonium↗
PubMed2005

Characterization of the microbial community in indoor environments by chemical marker analysis: an update and critical evaluation.

UNLABELLED: We published recently an integrated procedure for applying chemical marker analysis to characterize the microbiology of indoor environments comprising a scheme for extraction and analysis of markers of endotoxin, peptidoglycan/bacterial biomass, and fungal biomass. In the present paper, we report some significant improvements and also new possibilities of the described approach. We found that while 3-hydroxy fatty acids (3-OH FAs) of 10-14 carbon chain lengths are useful endotoxin markers, longer 3-OH FAs (i.e. with 16 carbon atoms and more) may rather serve as markers of Actinobacteria. We introduced 13C-labeled 3-hydroxytridecanoic acid, from labeled Pectinatus cerevisiiphilus, as an internal standard to improve quantification of the 3-OH FAs in the gas chromatography-mass spectrometry analysis. Finally, in experiments aiming to identify a suitable method for collection of house dust for chemical marker analysis, we found that the marker compositions of dusts sedimented on plexiglass plates that were spatially well-distributed in a studied room at different heights above floor level, were undistinguishable. This type of sampling thus appears to be well suited for use, e.g. in epidemiological studies. In summary, the presented work describes important new capabilities of chemical marker analysis in defining human exposure to microorganisms in indoor environments. PRACTICAL IMPLICATIONS: We are developing an integrated methodology for characterizing the microbiology of indoor environments where specific microbial monomeric constituents in building materials and inhalable house dust particles are determined by using mass spectrometry-based methods. The methodology should represent a firm basis in research aiming to relate microbial exposure indoors to well-being and health.

Air Microbiology↗
PubMed2001

Recruiting older women to research studies: the san diego cooperative mammography project.

BACKGROUND: Despite the growing number of older people in the population, this age group continues to be under represented in clinical trials. As a result, physicians must base treatment decisions for older patients on data from studies involving primarily younger, and presumably healthier, adults. Little experience is available to guide the development of study methodologies that will enhance the recruitment of older patients to clinical studies. METHODS: This pilot study compared two methods of recruiting women 75 years and older to a clinical research study related to their most recent screening mammogram. The effectiveness of a single, "in-person" invitation to participate made during the screening mammogram appointment was compared with the effectiveness of a single invitation to participate sent "by-mail" following a screening mammogram. RESULTS: Both methods succeeded in recruiting a sizable sample (N=2,394). The "in-person" invitation to participate was more labor-intense and less likely to be inclusive of all eligible women, but secured a significantly greater proportion of the women to participate. However, once recruited, women in the "by-mail" method were significantly more likely to comply with the optional elements of the study and to express a willingness to continue with follow-up studies than those recruited by the "in-person" method. CONCLUSIONS: Lack of participation of older women in clinical research may be more a reflection of not being asked, rather than their lack of willingness to participate, thus reinforcing the key role health care providers can play in recruiting older women to clinical studies.

Journal Article↗