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At least 667 records · Page 37Linked to original sources
PubMed2001

Real-time PCR analysis of DNA and RNA extracted from formalin-fixed and paraffin-embedded biopsies.

The archives of departments of pathology represent a unique source of morphologically defined biopsies derived from normal and pathologically altered tissues for which extensive clinical data are available. The exact quantification of nucleic acids in these biopsies offers a promising extension of current methodology to study the pathogenesis of many different diseases. The development of real-time PCR technology has greatly facilitated the realization of nucleic acid quantification. Now it is feasible to analyze large series of samples for the exact quantification of nucleic acids even if the number of target molecules is small and the amount of material available for analysis is limiting. This review focuses on our own experiences concerning the extraction of nucleic acids from fixed and embedded biopsies using both conventional approaches and laser-assisted microdissection and the subsequent application of real-time PCR methods for quantification of mRNA transcripts, gene copy number, and the methylation status. We provide a number of protocols to assist in the application of these techniques.

Biopsy↗
PubMed2002

Impact of remote protonation on 13C CPMAS NMR quantitation of charred and uncharred wood.

The impact of inefficient cross polarization (long TCH values), caused by long 13C-1H internuclear distances, on 13C CPMAS NMR spectra of charred and uncharred woods is determined by simultaneously fitting data from complementary variable spin lock and variable contact time experiments. As expected, the impact is minimal for uncharred woods, but is very significant for the charred woods. Quantification of the decrease in CPMAS signal intensity caused by both inefficient cross polarization and rapid T1rhoH relaxation is achieved using an advanced spin counting methodology, for which the term "spin accounting" is proposed. 13C CPMAS NMR observabilities determined using the spin accounting methodology were close to 100% for the uncharred samples, and 69-82% for the charred samples. This represents a large improvement on the 30-40% observabilities determined using other spin counting techniques. Furthermore, it is shown that remote protonation and rapid T1rhoH relaxation are roughly equally responsible for the low signal intensity of standard (I ms contact time) 13C CPMAS spectra of charcoal.

Carbon Isotopes↗
PubMed2025

Methodologies for Mitochondrial Omic Profiling During Spaceflight.

To be able to understand how spaceflight can affect human biology, there is a need for maximizing the amount of information that can be obtained from experiments flown to space. Recently there has been an influx of data obtained from astronauts through multi-omics approaches based on both governmental and commercial spaceflight missions. In addition to data from humans, mitochondrial specific data is gathered for other experiments from rodents and other organisms that are flown in space. This data has started to universally demonstrate that mitochondrial dysfunction is the key regulator associated with increasing health risks associated with spaceflight. This mitochondrial dysfunction can have influence downstream on immune suppression, inflammation, circadian rhythm issues, and more. Due to the space environment, standard methodologies have to be altered for performing mitochondrial specific analysis and in general sample collection for omics. To perform mitochondrial specific analysis and data collection from samples flown to space we will outline the current sample collection methods, processing of the samples, and specific analysis. Specifically we will highlight the different mitochondrial methodologies and challenges involved with research associated with spaceflight.

Space Flight↗
PubMed2005

Expression of mitochondrial genes and transcription estimation in different brain areas in Alzheimer's disease patients.

Accumulation of mitochondrial defects is hypothesised to play a role in the complex pathophysiology of the sporadic form of Alzheimer's disease (SAD). Changes in expression of mtDNA encoded genes have been reported in SAD. However no conclusive results were obtained by using different methodologies and analysing distinct transcripts in a variety of brain areas. Here, we measured the expression of five mitochondrial-encoded genes in three brain areas and in lymphocytes from patients and controls by performing reverse transcription followed by quantitative real-time PCR. The analysis of gene expression was also performed by carrying out classic dot-blot experiments to evaluate the two techniques. SAD and control samples showed similar gene expression levels. Estimation of the transcription rate was also measured. No differences were observed when comparing patients and controls. Selective differences in transcription rates were observed only when samples were pooled by tissue, lymphocytes being the tissue with the highest rates.

Aged↗
PubMed2003

Internalized stigma of mental illness: psychometric properties of a new measure.

The study evaluated the Internalized Stigma of Mental Illness (ISMI) scale, designed to measure the subjective experience of stigma, with subscales measuring Alienation, Stereotype Endorsement, Perceived Discrimination, Social Withdrawal and Stigma Resistance. The ISMI was developed in collaboration with people with mental illnesses and contains 29 Likert items. The validation sample included 127 mental health outpatients. Results showed that the ISMI had high internal consistency and test-retest reliability. Construct validity was supported by comparisons against scales measuring related constructs with the same methodology. As expected, the ISMI had positive correlations with measures of stigma beliefs and depressive symptoms, and it had negative correlations with measures of self-esteem, empowerment and recovery orientation. Factor analyses of the joint set of items from the ISMI and each scale supported the distinction between constructs. Having a validated measure of internalized stigma may encourage clinicians to include stigma reduction as a verifiable treatment goal in addition to symptom reduction.

Adaptation, Psychological↗
PubMed2006

Determination of steroidal estrogens in flushed dairy manure wastewater by gas chromatography-mass spectrometry.

There is a critical need to accurately measure the concentrations of natural steroidal estrogens in flushed dairy manure wastewater (FDMW) to assess any potential risk of waterway contamination resulting from land application. Estrogens are a concern because low concentrations (10-100 ng L-1) in water can adversely affect aquatic vertebrate species such as fish, turtles, and frogs by disrupting the normal function of their endocrine systems. The objective of this study was to develop a sample preparation method that permits the quantification of four natural steroidal estrogens (17alpha-estradiol, 17beta-estradiol, estrone, and estriol) in FDMW by gas chromatography-mass spectrometry (GC-MS). Solid-phase extraction with graphitized carbon black was used for the bulk extraction of estrogens from FDMW and additional sample purification was accomplished with C-18. The sample preparation method allowed estrogens to be detected accurately by GC-MS in FDMW. Spiked recovery experiments indicated that the method is satisfactory for measuring the estrogens of interest in FDMW with average recovery of >90%. As expected in FDMW, characterization of the estrogen profile revealed a large abundance of 17alpha-estradiol relative to 17beta-estradiol and estrone. Estriol was not detected in FDMW. The methodology developed in this research helps provide an analytical foundation for the quantification of steroidal estrogens in FDMW by GC-MS.

Animals↗
PubMed1997

DNA denaturation sensitivity may invalidate bromodeoxyuridine--DNA flow cytometric analysis of potential doubling times in colorectal tumours.

BACKGROUND: Flow cytometric analysis of 5-bromo-2'-deoxyuridine (BrdU)-substituted DNA has been used to calculate tumour cell proliferation rate. In this methodology, DNA denaturation, commonly by hydrochloric acid, is essential to expose incorporated BrdU for quantification with monoclonal antibodies. This study was designed to establish the validity of this technique by examining the change in flow cytometric DNA profiles introduced by DNA denaturation procedures. METHODS: Four experiments were performed using suspensions of nuclei derived from human colorectal tumours exposed in vivo to 150 mg/m2 BrdU 8-11 h before sampling. RESULTS: After denaturation with hydrochloric acid 2 mol/l a significant decrease was observed in the DNA aneuploid G1 population (P < 0.001) with a concurrent increase in the DNA aneuploid S phase fraction (P < 0.05). These changes were independent of the washing-centrifugation step and were maximal at different hydrochloric acid concentrations for different tumours. CONCLUSION: Hydrochloric acid denaturation introduces a tumour-specific non-linear variation in the analysis of BrdU.

Aged↗
PubMed2002

An example of maximizing survey return rates. Methodological issues for health professionals.

Obtaining a high response rate in survey research can bolster statistical power, reduce sampling error, and enhance the generalizability of the results to the population surveyed. We describe a mail survey designed to achieve a high return rate of completed questionnaires from members of the American Psychological Association who were engaged in clinical practice. We adapted the Total Design Method for survey research and were able to achieve a high response rate (68%) among health professionals. This was not an experiment in which we assessed the best method to increase survey response rates, hence we cannot empirically evaluate how each of the steps influenced our overall response rate. Future research is needed to identify the relative effectiveness of each of these principles in enhancing survey response rates. Research is also needed to distinguish general principles that apply across populations from those that must be tailored to specific subpopulations.

Child↗
PubMedDate not supplied

The experiences and health care needs of Asian coronary patients and their partners. Methodological issues and preliminary findings.

The incidence of heart disease in the Asian population in the UK is high and mortality from coronary heart disease for both Asian men and women is also higher than for non-Asians. The need to provide more information on cardiac rehabilitation programmes is highlighted. Cultural factors influence how Asian people perceive the heart, health and health care practices. A number of methodological issues are pertinent to conducting research involving ethnic groups, namely inter vs intra group differences, victim blaming, sampling, measurement, language issues and researcher effect. The aim of the study is to design, refine and validate a number of tools for use with Asian people in relation to cardiac rehabilitation. Preliminary and tentative conclusions suggest that cardiac rehabilitation services need a higher profile within the Asian community and that established approaches to data collection may need to be challenged if accurate and relevant feedback is to be achieved.

Aged↗
PubMed1995

Bulimia nervosa in a Canadian community sample: prevalence and comparison of subgroups.

OBJECTIVE: Previous epidemiological studies of bulimia nervosa have generated differing estimates of the incidence and prevalence of the disorder. These differences are attributable, in part, to varying definitions of the illness and a range of methodologies. The authors sought to define the prevalence of bulimia nervosa in a nonclinical community sample, examine the clinical significance of DSM-III-R threshold criteria, and examine comorbidity. METHOD: Subjects across Ontario (N = 8,116) were assessed with a structured interview, the World Health Organization Composite International Diagnostic Interview, with specific questions added for bulimia nervosa. Subjects who met DSM-III-R criteria for bulimia nervosa were compared with those who were missing only the frequency criterion (two or more binge-eating episodes per week for 3 months). RESULTS: In this sample, the lifetime prevalence of bulimia nervosa was 1.1% for female subjects and 0.1% for male subjects. The subjects with full- and partial-syndrome bulimia nervosa showed significant vulnerability for mood and anxiety disorders. Lifetime rates of alcohol dependence were high in the full-syndrome group. Rates of parental psychopathologies were high in both bulimic groups but tended to be higher in the subjects with full-syndrome bulimia nervosa. Both bulimic groups were significantly more likely to experience childhood sexual abuse than a normal female comparison group. CONCLUSIONS: This study confirms other prevalence estimates of bulimia nervosa and its comorbid diagnoses from studies that were based on sound methodologies. It also points to the arbitrary aspects of the frequency of binge eating as a diagnostic threshold criterion for the disorder.

Adolescent↗
PubMed1989

A methodological improvement for experimental control and blood sampling in rats.

We have developed an apparatus and techniques that permit repeated, undisturbed blood sampling and concurrent assessment of activity in rats. The apparatus also allows experimental control of conditioning stimuli and contingencies without the need to transfer the rat to a separate chamber. In the experimental apparatus, body weights remain stable at approximately 90% of the original body weight. Plasma corticosterone and prolactin levels in the apparatus are comparable to levels obtained in undisturbed free-ranging rats and remain at basal levels for the 7 day duration of this experiment. Additionally, circadian activity rhythms in the apparatus are similar to those seen in free-ranging rats. This technique has potential applications for investigators who need to be able to control shock delivery, monitor activity, and sample blood without disturbing rats.

Animals↗
PubMed1982

An antiglobulin microcytotoxicity assay to analyze non-complement fixing monoclonal antibodies to human histocompatibility antigens.

An antiglobulin microcytotoxicity assay has been used to analyze non-complement fixing monoclonal antibodies to human histocompatibility antigens. The assay utilizes methodology similar to that of the widely used microcytotoxicity assay for HLA typing, requires low numbers of target cells and is suitable to test large numbers of samples. The sensitivity of the assay is influenced by the anti-mouse Ig antiserum used, by the sequence of addition of the various reagents and by the incubation time. The assay is suitable to screen supernatants of clones derived from hybridization experiments and to characterize the serological specificity of anti-HLA monoclonal antibodies.

Animals↗
PubMed1984

Psychological approaches to the management of arthritis pain.

The present review examines the literature regarding the efficacy of cognitive-behavioral and other self-control interventions in helping arthritis patients reduce their pain and functional disabilities. The evidence indicates that self-control interventions have produced significant and positive changes in the pain and functional disabilities of patients with rheumatoid arthritis and arthritis secondary to hemophilia. However, the literature suffers from deficiencies with regard to the use of small subject samples; inadequate control procedures and follow-up assessments; failure to demonstrate that positive outcomes are related to changes in subjects' covert experiences or control of physiological variables; and reliance upon self-report measures of outcome. The review is followed by a description of a multidisciplinary study of the efficacy of a biofeedback-assisted, cognitive-behavioral group therapy program for rheumatoid arthritis patients that features several methodological improvements relative to previous investigations. The preliminary outcome data show that the cognitive-behavioral intervention is associated with reductions in pain behavior and self-reports of pain and disability. It is concluded that, although the self-control interventions have shown promising results, psychologists must demonstrate positive and reliable outcomes among large numbers of arthritis patients over extended periods of time if the interventions are to be viewed as credible by rheumatologists.

Arthritis↗
PubMed1991

RIA-linked microdialysis sampling in the awake rat: application to free-drug pharmacokinetics of hydrocortisone.

The purpose of this research was to combine microdialysis sampling techniques with a highly sensitive radioimmunoassay (RIA) to study the in vivo kinetic response of pharmacologically important substances. This technique allowed for a dense sampling regimen from an awake, free-roaming experimental animal with no loss of blood and with rapid analysis of the dialysate. An important methodological criterion for accurate quantitation of a test drug in the extracellular space was knowledge of the relative recovery of the sampling system at the time of experimentation. Accordingly, the factors which influenced the recovery of drug during dense in vivo microdialysis sampling were examined and an analytical technique was developed to measure the instantaneous recovery of drug from the extracellular space. This information was applied to in vivo (iv) sampling experiments on anaesthetized and awake, free-roaming rats following bolus and multiple long-term iv administrations of the highly protein bound steroid (i.e. greater than 90%), hydrocortisone-21-phosphate. These studies indicated that unbound hydrocortisone levels as determined by the RIA-linked microdialysis (RIALM) technique fluctuated rapidly between each 2-min sampling interval, but nevertheless decreased to predose endogenous concentrations in a first-order fashion (t1/2 = 17-29 min). The rapid fluctuations of unbound hydrocortisone may reflect real pharmacokinetic or pharmacodynamic phenomena, attributed, perhaps, to reequilibration of the unbound drug pool with proteins and tissues in the blood.

Animals↗
PubMed2002

[Pilot study of The National Child and Adolescent Health Survey: sampling design].

The concept of a nation-wide Health Survey for Children and Adolescents has been tested over one year in a pilot study with 1,630 participating children and adolescents. A major methodological aspect of this pilot phase was to compare different variants of study sample recruitment regarding their feasibility. There was a choice between a sample originating from the population registry and a cluster sample of schools and school classes for the age group from 11 to 15 years. As a result, after weighing the advantages and disadvantages of both sampling designs, a continuous population registry sample has been recommended. The acquired knowledge and experience will be translated into reality in 2003 when the main course of the survey, which will include about 18,000 children and adolescents, will start.

Adolescent↗
PubMed2005

What have we learned from observational studies on neonatal sepsis?

OBJECTIVE: To assess how observational studies on neonatal sepsis can help define the knowledge base required for neonatal randomized, clinical trials. DESIGN: Methodologic review of past observational studies and critical reviews. RESULTS: Observational studies on neonatal sepsis have suffered from important limitations: failure to employ multivariate analyses, considering infection in isolation (ignoring coexisting respiratory distress), failure to provide likelihood ratios for predictors and combinations of predictors, and ignoring the phenomenologic dimension of clinicians' experience. CONCLUSION: Future observational studies must address three key issues. They should begin with a clear analytic and sampling plan that pays careful attention to the proper use and reporting of multivariate analyses. Second, they must explicitly address two subpopulations: critically ill newborns with negative cultures and asymptomatic newborns with positive cultures. Finally, they should be theory driven and provide empirical physiologic data that permit situating their results in the context of the evolving systemic inflammatory response syndrome and PIRO (predisposition, infection, host response, organ dysfunction) models.

Bacteremia↗
PubMed2005

Major depressive disorder in children and adolescents: clinical trial design and antidepressant efficacy.

Major depressive disorder is a common, chronic, recurring, and disabling illness in children and adolescents. Epidemiologic data suggest that the mean age at onset for depression is becoming younger. Antidepressants are widely used in pediatric patients despite a relatively small evidence base from randomized, controlled studies. Compared with the adult population, there are very few controlled studies of antidepressants in children and adolescents, and the studies that have been conducted do not unambiguously demonstrate efficacy. These findings in general are contrary to clinicians' experiences with antidepressants in this population. The different factors associated with the outcome of antidepressant studies in children and adolescents are reviewed. Developmental issues in pediatric patients, such as age of puberty and maturity of neural circuitry, are considered in the context of failed clinical trials. Review of existing controlled trials demonstrates a wide variety in study methodology. Factors such as the study location, methods of patient recruitment, small sample sizes, inclusion/exclusion criteria, study design, and choice of outcome measures all influence the ability of a study to detect differences between an antidepressant and placebo. In the current atmosphere of increased concern about antidepressant side effects, including suicidality, it is increasingly important that clinicians who treat depressed children and adolescents make informed therapeutic decisions based on data from well-controlled clinical trials.

Adolescent↗
PubMed1993

McGill Pain Questionnaire translated into Danish: experimental and clinical findings.

OBJECTIVE: To develop a methodology for translating the McGill Pain Questionnaire (MPQ) into a Danish version, and to make comparisons to studies of patients speaking other languages. DESIGN: Finding suitable Danish adjectives using the same methodology as that in the original MPQ. Comparison of Danish descriptors to the words in the English version of MPQ. Survey in healthy subjects and patients with rheumatoid arthritis (RA) and fibromyalgia (F). SETTING: The general public and hospital outpatients. PATIENTS: A random sample of 186 healthy volunteers, 20 patients with rheumatoid arthritis and 41 patients with fibromyalgia. MAIN OUTCOME MEASURES: Danish words translated as closely as possible to the descriptors in the original McGill Pain Questionnaire. A pain-assessment instrument making international pain description possible. RESULTS: A Danish version of the McGill Pain Questionnaire was developed with scale values of Danish descriptors not differing more than 5 x SEM from the 'patient' words in the English version. The subdivision into classes and subclasses was respected. In the reliability experiment, the same rank values were found in 85% of subclasses. In a study using two experimental pain stimulus intensities, seven of 10 subjects obtained higher MPQ scores following the high-intensity stimulus. In the clinical study, the pain profiles of patients with RA and F in English, Italian, and Danish patients were almost the same. CONCLUSION: The present methodology of translating the McGill Pain Questionnaire permits comparison of studies from English-speaking and non-English-speaking populations, thus facilitating international research exchange.

Adult↗