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PubMed2006

Controlled loading of oligodeoxyribonucleotide monolayers onto unoxidized crystalline silicon; fluorescence-based determination of the surface coverage and of the hybridization efficiency; parallel imaging of the process by Atomic Force Microscopy.

Unoxidized crystalline silicon, characterized by high purity, high homogeneity, sturdiness and an atomically flat surface, offers many advantages for the construction of electronic miniaturized biosensor arrays upon attachment of biomolecules (DNA, proteins or small organic compounds). This allows to study the incidence of molecular interactions through the simultaneous analysis, within a single experiment, of a number of samples containing small quantities of potential targets, in the presence of thousands of variables. A simple, accurate and robust methodology was established and is here presented, for the assembling of DNA sensors on the unoxidized, crystalline Si(100) surface, by loading controlled amounts of a monolayer DNA-probe through a two-step procedure. At first a monolayer of a spacer molecule, such as 10-undecynoic acid, was deposited, under optimized conditions, via controlled cathodic electrografting, then a synthetic DNA-probe was anchored to it, through amidation in aqueous solution. The surface coverage of several DNA-probes and the control of their efficiency in recognizing a complementary target-DNA upon hybridization were evaluated by fluorescence measurements. The whole process was also monitored in parallel by Atomic Force Microscopy (AFM).

Biosensing Techniques↗

PubMed1984

[Statistical standards for clinical long-term studies].

Interest in the implementation of long-term clinical studies has grown considerably in Austria in recent years, partly encouraged by studies of this nature performed in West Germany, Great Britain and the United States. However, such studies have to be based on biometric standards that must be adhered to in the planning, implementation and reporting phases of long-term studies if they are to be regarded as authoritative and to find international acceptance. The present paper deals with such standards, based on the current level of methodological development as well as the authors' experience gathered over many years of participation in such projects. Special emphasis is placed on planning, random sampling and analysis, randomization, documentation, organization, termination of studies, reporting, multicentre investigations and cooperation between doctors and statisticians. The authors present several alternatives with and without the use of computers, attempt to explain to clinicians the role of the biostatistician in the supervision of long-term studies and offer their services to those interested in their cooperation.

Austria↗

PubMed1975

Fluorescent antibody method for the detection of Salmonella: development, evaluation, and collaborative study.

In preliminary studies, several commercial polyvalent fluorescent antibody (FA) preparations were evaluated for specificity and crossreactivity and an FA method was developed for the screening of Salmonella in products. Approximately 4000 product samples were tested by the FA method and the results were compared to those from the official final action AOAC method, 46.013-46.026. Only 4 FA false-negatives were found for a total of 619 confirmed positive Salmonella samples. The FA false-positive rate was 7%. The method was then subjected to a 2-phase collaborative study. In Phase I, 22 analysts tested 5 inoculated and 5 uninoculated samples of dried milk. In Phase II, 5 naturally contaminated and 5 presumably uncontaminated foods were analyzed. The study was designed to compare results from 11 analysts experienced in FA methodology with those from 11 analysts with little or no experience. Selenite cystine (SC) and tetrathionate (TT) broths were used for enrichment and both were inoculated into SC for post-enrichment. All 4 combinations (SC, TT, SC-SC, and TT-SC) were used with the FA method to determine the best technique. Results were compared to the analysis with TT and SC by the AOAC culture method. In all studies, FA analysis with SC-SC gave the highest correlation with the AOAC method. In a total of 200 samples, the experienced group found 125 AOAC positives and 127 FA positives; no FA false-negatives and only 2 false-positives were reported. The inexperienced group reported 9 FA false-negatives and 5 FA false-positives. All false-negatives occurred in only 3 of the inexperienced laboratories. These studies showed that enrichment and post-enrichment in SC gave the best FA results and that training in FA methodology is required for correlation with existing AOAC methodology. The FA method for the detection of Salmonella has been adopted as official first action.

Evaluation Studies as Topic↗

PubMed1999

Waveform interactions and the segregation of concurrent vowels.

Two experiments investigated the effects of small values of fundamental frequency difference (delta F0) on the identification of concurrent vowels. As delta F0's get smaller, mechanisms that exploit them must necessarily fail, and the pattern of breakdown may tell which mechanisms are used by the auditory system. Small delta F0's also present a methodological difficulty. If the stimulus is shorter than the beat period, its spectrum depends on which part of the beat pattern is sampled. A different starting phase might produce a different experimental outcome, and the experiment may lack generality. The first experiment explored the effects of delta F0's as small as 0.4%. The smallest delta F0 conditions were synthesized with several starting phases obtained by gating successive segments of the beat pattern. An improvement in identification was demonstrated for delta F0's as small as 0.4% for all segments. Differences between segments (or starting phase) were also observed, but when averaged over vowel pairs they were of small magnitude compared to delta F0 effects. The nature of delta F0-induced waveform interactions and the factors that affect them are discussed in detail in a tutorial section, and the hypothesis that the improvement in identification is the result of such interactions (beat hypothesis) is examined. It is unlikely that this hypothesis can account for the effects observed. The reduced benefit of delta F0 for identification at smaller delta F0's more likely reflects the breakdown of the same F0-guided segregation mechanism that operates at larger delta F0's.

Biomechanical Phenomena↗

PubMed1997

Women's experiences of non-invasive breast cancer: literature review and study report.

A review of the post-1990 literature on breast cancer has identified a large body of psychosocial research focusing on coping styles, quality of life and women's participation in choice of treatment. However, numerous methodological problems were found, including a variety of different diagnoses included in samples both within studies and between studies, making comparisons difficult. In particular, no article was traced which focused on non-invasive breast cancer, or ductal carcinoma in situ (DCIS). Therefore a small exploratory study was conducted using qualitative methods to explore women's experience of DCIS, in an attempt to begin exploration of possible similarities or differences in the way women experience invasive and non-invasive breast cancer. A convenience sample of 10 women treated at two hospitals in an Australian state capital city was interviewed, and three themes which emerged from these interviews are discussed in this article. These are discovering the problem, reaction to the diagnosis, and information. Most women unexpectedly reacted in a calm and accepting way to receiving the diagnosis of DCIS, and this may be because they had had no signs of disease but been recalled for further tests after routine screening. They felt well and had no cause to assume a poor prognosis. It is possible also that their reactions were affected by a television health education campaign at the time, which gave a very optimistic picture of the benefits of early detection of breast cancer. Information-giving, as reported by the women, seemed deficient. None of them knew that they had had a non-invasive condition, one thought her disease had been benign, and another that hers was "evasive'. Findings are discussed in relation to the literature on information-giving and on the role of the nurse in this area, and suggestions are made for further research to systematically compare women's reactions to having invasive and non-invasive breast cancer in order that nurses may be aware of their possibly different needs and respond to them appropriately.

Adaptation, Psychological↗

PubMed2003

Meanings and practical knowledge of people with long-term urinary catheters.

The lived experience of people with long-term indwelling urethral catheters is described and interpreted. This hermeneutic (interpretive) phenomenologic study used Merleau-Ponty's philosophy and van Manen's methodology. Fourteen adults aged 35 to 95 years who had worn a catheter more than 4 months (range = 6 months to 18 years) comprised the purposive sample. Data were obtained through audiotaped and transcribed interviews conducted in the homes of participants. The study provided both meanings and practical knowledge related to the experience of catheter use. Despite its drawbacks, participants recognized the device's value in their daily lives and weighed its positive features against concerns associated with long-term use. Individual meanings included making aesthetic harmony with a new catheter, the convenience of the catheter compared with the inconvenience of incontinence, and the catheter as a symbol and reminder of one's mortality. Practical knowledge related to participants' awareness of their catheter needs and practices, such as features of supplies, intervals for changes, catheter insertions, emptying the urine bag, and changes associated with sex. Knowledge of the lived experience with a urinary catheter can assist nurses in sensitive decision making about care and help them become better patient advocates.

Activities of Daily Living↗

PubMedDate not supplied

Methodologic challenges in developing and implementing measures of quality for child health care.

OBJECTIVE: To review the major building blocks in measurement of quality for child health care, with recommendations for future research. METHODS: We describe a framework of building blocks for quality measurement and discuss how an investigator's choices for each component are constrained by the special features of child health care. RESULTS: Methodologic challenges for children's health care include developmental change and dependency on others, fragmentary care and inadequate health care data, unusual care settings, potential for long-term consequences, proxy reporting of outcomes and patient experience, small sample sizes, and lack of evidence that links processes and outcomes of care and of methods for risk adjustment. We cite examples of child-specific measures of quality that illustrate solutions to these challenges. CONCLUSIONS: Children are different from adults, and measures of health care quality for children must differ from those for adults. We suggest future research on measures of quality directed toward overcoming the methodologic problems specific to child health care.

Adolescent↗

PubMed2006

Separation of a BMS drug candidate and acyl glucuronide from seven glucuronide positional isomers in rat plasma via high-performance liquid chromatography with tandem mass spectrometric detection.

A high-performance liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed and validated for the determination of a BMS drug candidate and its acyl glucuronide (1-O-beta glucuronide) in rat plasma. A 50-microL aliquot of each plasma sample was fortified with acetonitrile containing the internal standard to precipitate proteins and extract the analytes of interest. After mixing and centrifugation, the supernatant from each sample was transferred to a 96-well plate and injected into an LC/MS/MS system. Chromatographic separation was achieved isocratically on a Phenomenex Luna C(18), 3 mm x 150 mm, 3 microm column. The mobile phase contained 0.075% formic acid in 70:30 (v/v) acetonitrile/water. Under the optimized chromatographic conditions, the BMS drug candidate and its acyl glucuronide were separated from its seven glucuronide positional isomers within 10 min. Resolution of the parent from all glucuronides and acyl glucuronide from its positional isomers was critical to avoid their interference with quantitation of parent or acyl glucuronide. Detection was by positive ion electrospray MS/MS on a Sciex API 4000. The standard curve, which ranged from 5 to 5000 ng/mL, was fitted to a 1/x(2) weighted quadratic regression model for both the BMS drug candidate and its acyl glucuronide. Whole blood and plasma stability experiments were conducted to establish the sample collection, storage, and processing conditions. The validation results demonstrated that this method was rugged and repeatable. The same methodology has also been used in mouse and human plasma for the determination of the BMS drug candidate and its acyl glucuronide.

Animals↗

PubMed2007

Evaluation of an on-target sample preparation system for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in conjunction with normal-flow peptide high-performance liquid chromatography for peptide mass fingerprint analyses.

Large-scale mass spectrometry (MS)-based proteomic analyses require high-throughput sample preparation techniques due to the increasing numbers of samples that make up a typical proteomics experiment. Moreover, extensive sample pre-treatment steps are necessary prior to MS acquisition for even the most rapid and robust MS-based proteomics methodology, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS followed by peptide mass fingerprinting (PMF) analysis. These include sample purification and fractionation, removal of digestion buffers or solvents, and spotting of sample with matrix onto the MALDI target. These multiple steps of time-consuming sample handling can result in high overall analysis costs and the likelihood of sample contamination and loss. In order to overcome some of these limitations in sample processing, we have investigated the use of a novel, simple, inexpensive 96-well elastomeric array that affixes to a MALDI target to create an on-target 96-well plate that accommodates a high solution volume (ca. 200 microL), thereby enabling the on-target processing of samples for MALDI-TOFMS. We explored several factors that influence MALDI sample preparation: type of matrix, solution volume, solution organic composition, solution drying rates and matrix/analyte co-crystallization methods. We also investigated the use of the 96-well elastomeric device for coupling MALDI-TOFMS analysis directly to high flow rate (1 mL/min) reversed-phase (rp)-HPLC. By developing an optimized, robust sample preparation protocol, we were able to obtain mass spectra with a high signal-to-noise ratio from peptide standards present at the 50-fmol level in large starting volumes of solution. PMF analyses were possible from 1-pmol and 500-fmol protein-digest standards. Coupling the device to high-flow HPLC (750 microL/min) yielded a robust and semi-automated means to obtain enhanced MALDI-TOFMS data at 500 ng of protein digest. These methodologies developed for this simple, on-target, elastomeric device show promise for streamlining the sample preparation process from HPLC to MALDI-MS.

Chromatography, High Pressure Liquid↗

PubMed1999

Reverse transcription polymerase chain reaction method for the detection of glycopeptide resistance in enterococci.

In this work we have developed reverse transcription polymerase chain reaction (RT-PCR) methods for detecting specific mRNA from enterococci, particularly vanA and vanB genes, responsible for glycopeptide resistance in this genus. mRNA from the two genes was detected immediately after RNA extraction of a midlog phase culture, determined by growth rate analysis. Because of the short half-life associated with many bacterial RNA species (1.5-2 min), time is an important factor in obtaining RNA of good yield and high purity. Our results showed that: (i) the transcription of mRNA related to vanA ligase in enterococci showing Van A phenotype happens only after induction with both vancomycin and teicoplanin; (ii) the transcription of mRNA related to vanB ligase happens only in the presence of vancomycin and (iii) there was no transcription of mRNA in the two strains positive to vanA gene after PCR experiments. RT-PCR methodology can have numerous applications in microbiology for studying gene expression in isolated bacteria and also in nonculturable cells in environmental samples, for studies of mechanisms and/or as an indicator of viability in bacterial communities.

Anti-Bacterial Agents↗

PubMed2002

Human blood and environmental media screening method for pesticides and polychlorinated biphenyl compounds using liquid extraction and gas chromatography-mass spectrometry analysis.

Screening assessment methods have been developed for semi- and non-volatile persistent organic pollutants (POPs) for human blood and solid environmental media. The specific methodology is developed for measuring the presence of "native" compounds, specifically, a variety of organochlorine pesticides (OCPs), organophosphate pesticides (OPPs), and for polychlorinated biphenyls (PCBs). The method is demonstrated on anonymous Red Cross blood samples as well as two potential environmental sources, tracked in soil and dog hair. This work is based on previously developed methods for semi-volatile hydrocarbon exposure from fuels usage and similarly employs liquid solvent extraction, evaporative volume reduction. and subsequent specialized gas chromatography-mass spectrometry analysis (GC-MS). Standard curves, estimates of recovery efficiency, and specific GC-MS SIM quantification methods were developed for common pesticides including diazinon. aldrin, chlorpyrifos, malathion, dieldrin, DDT, permethrin, cyhalothrin, and cypermethrin, and for seven selected PCBs. Trace levels of certain PCBs and pesticides such as permethrin, dieldrin, malathion, lindane, diazinon, and chlorpyrifos were tentatively identified in anonymous blood samples as well as in two potential environmental sources. tracked in soil and dog hair. The method provides a simple screening procedure for various media and a variety of common organic pollutants without extensive sample preparation. It is meant to complement and augment data from more specific or complex methodology, to provide initial broad spectrum guidance for designing targeted experiments, and to provide confirmatory evidence for the usual metabolic biomarker measurements made to assess human exposure.

Environmental Monitoring↗

PubMed2005

Use of within-array replicate spots for assessing differential expression in microarray experiments.

MOTIVATION: Spotted arrays are often printed with probes in duplicate or triplicate, but current methods for assessing differential expression are not able to make full use of the resulting information. The usual practice is to average the duplicate or triplicate results for each probe before assessing differential expression. This results in the loss of valuable information about genewise variability. RESULTS: A method is proposed for extracting more information from within-array replicate spots in microarray experiments by estimating the strength of the correlation between them. The method involves fitting separate linear models to the expression data for each gene but with a common value for the between-replicate correlation. The method greatly improves the precision with which the genewise variances are estimated and thereby improves inference methods designed to identify differentially expressed genes. The method may be combined with empirical Bayes methods for moderating the genewise variances between genes. The method is validated using data from a microarray experiment involving calibration and ratio control spots in conjunction with spiked-in RNA. Comparing results for calibration and ratio control spots shows that the common correlation method results in substantially better discrimination of differentially expressed genes from those which are not. The spike-in experiment also confirms that the results may be further improved by empirical Bayes smoothing of the variances when the sample size is small. AVAILABILITY: The methodology is implemented in the limma software package for R, available from the CRAN repository http://www.r-project.org

Algorithms↗

PubMed1999

Maximally selected chi2 statistics for k x 2 tables.

It is common in epidemiologic analyses to summarize continuous outcomes as falling above or below a threshold. With such a dichotomized outcome, the usual chi2 statistics for association or trend can be used to test for equality of proportions across strata of the study population. However, if the threshold is chosen to maximize the test statistic, the nominal chi2 reference distributions are incorrect. In this paper, the asymptotic distributions of maximally selected chi2 statistics for association and for trend for the k x 2 table are derived. The methodology is illustrated with data from an AIDS clinical trial. The results of simulation experiments that assess the accuracy of the asymptotic distributions in moderate sample sizes are also reported.

Anti-HIV Agents↗

PubMed2006

Palatine mucosa of aging and experimentally induced diabetic aging rabbits: light-microscopic, scanning electron microscopic and transmission electron microscopic qualitative study.

BACKGROUND: Data associating palatine mucosa, microvascular architecture, aging and diabetes mellitus are few, so the aim of the present study was to examine these conditions structurally and ultrastructurally. METHODS: We used 18 female rabbits, aged about 2 years at the beginning of the experiment, distributed into 2 groups: aging diabetic and aging animals, prepared by three different methodologies: light microscopy, scanning electron microscopy and transmission electron microscopy. The samples were stained with hematoxylin-eosin for light microscopy, and for scanning electron microscopy, we used Mercox Cl-2B vascular corrosion casts. For transmission electron microscopy, the specimens were fixed and embedded in Epon 812 resin. RESULTS: Under light microscopy, we noted small amounts of underdeveloped connective papillae and significant flattened areas through the epithelium-connective tissue interface in the aging diabetic group. Larger blood vessels were deeply located in the palatine mucosa, branching off in the direction of the epithelium-connective tissue interface. Capillaries followed the interface contour. In both groups, vascular corrosion casts revealed capillaries sprouting out in disorganized rows but parallel between themselves. 'Hair-pin' capillary loops and convoluted capillary loops were noted. CONCLUSION: The aging diabetic group showed rarefying microvasculature areas with complex tortuous capillary loops. Transmission electron microscopy results showed that the aging diabetic group presented small cytoplasmatic projections directed to the vessel lumen and micropinocytic vesicle, i.e. caveolas. Epithelium-connective tissue interface, connective papillae, microangioarchitecture, and information about endothelial cells alterations were observed in the aging diabetic and aging animals.

Age Factors↗

PubMed1992

[The biological specimen bank: methodological and organizational problems].

Little experience is available to solve methodological and organizational problems when setting up a biological specimen bank as a support to prospective studies on chronic diseases. Sample aliquots preparation, processing procedures and storage system are discussed in the present paper. Problems related to the quality of biological material stored, as to the influence of the very low temperature freezing physico-chemical effects, are discussed also in the appendix.

Blood Preservation↗

PubMed1993

Advances in chromatography for clinical drug analysis: supercritical fluid chromatography, capillary electrophoresis, and selected high-performance liquid chromatography techniques.

Advances have been made in chromatography complement immunoassay for clinical drug analysis. Chromatographic theory shows that the minimum detectable mass is directly proportional to the square of column radius. Small internal diameter columns are capable of analyzing lower analyte concentrations, and high resolution is achievable in open capillary columns, concomitant with greatly reduced mobile phase consumption and waste. This review, based on the author's experience and literature, focuses on supercritical fluid chromatography (SFC), capillary electrophoresis (CE), and selected high-performance liquid chromatography (HPLC) methodologies--microbore and direct-sample analysis (DSA) using commercially available Restricted Access Media (RAM) and REMEDi. In investigating the feasibility of SFC, the "normal-phase-like" selectivity of carbon dioxide was established, affecting the design of the extraction protocol and the elution order of drugs and metabolites. For example, the "more polar" tautomer eluted after FK-506, opposite to the order in the reversed-phase HPLC analysis. CE was investigated by Shihabi et al. for the analysis of pentobarbital and iohexol, while Evenson and Wiktorowicz performed preliminary evaluation of several therapeutic drug monitoring (TDM) drug groups. Innovation in HPLC column technology and hardware have greatly enhanced clinical drug analysis. Microbore column, offering enhanced mass sensitivity and high resolution, utilizes small sample size of 5 microliters of serum for the analysis of chloramphenicol. Commercially available RAM include internal surface reversed phase, shielded hydrophobic phase, and dual zone media, readily applicable for serum drug analysis without any sample preparation. Recently, an automated HPLC REMEDi offers urine and serum drug screening for toxicology.

Animals↗

PubMed2004

One breath at a time: living with cystic fibrosis.

The purpose of this qualitative investigation was to describe the lived experiences of families caring for a child with cystic fibrosis at the time of initial diagnosis. Phenomenological research methodology as described by Colaizzi (1978) was used to guide the investigation. A purposive sample of 9 family members voluntarily participated in the study. Data were gathered through focus groups and written narratives. Data analysis yielded 3 essential theme clusters with subthemes: Falling Apart, Pulling Together, and Moving Beyond. Within the theme of Falling Apart, the subthemes of Devastation of Diagnosis, An All-Encompassing Sense of Fear and Isolation, and An Overwhelming Sense of Guilt and Powerlessness are described. The theme of Pulling Together included the subthemes of Perpetual Vigilance and Returning to Normalcy, and the third theme of Moving Beyond included the subtheme of An Optimal Unfolding of a New Kind of Consciousness. This article describes in detail the themes and subthemes identified during data analysis and the fluid nature of the relationship that exists within the essential structure of caring for a family member with cystic fibrosis. The diagnosis of cystic fibrosis most often comes as a life-shattering experience to families. Lifestyle readjustments are made in an attempt to return to some sense of family normalcy. In order to achieve stability in their daily lives, families are vigilant in the care and monitoring of the health of a child with cystic fibrosis. Ongoing support from health care professionals that is grounded in the realities of living with cystic fibrosis is critical. This study describes how families develop their own unique way of controlling the experience of living with cystic fibrosis, one day and one breath at a time.

Adaptation, Psychological↗

PubMed1985

Drug absorption from inhalation aerosols administered by positive-pressure ventilation. I: Administration of a characterized, solid disodium fluorescein aerosol under a controlled respiratory regime to the beagle dog.

An apparatus and novel method is described for administration of well-characterized inhalation aerosols, under strictly controlled respiratory regimes, direct to the respiratory tract (RT) of the beagle dog by positive-pressure ventilation. The method enables the study of systemic absorption kinetics of compounds delivered as inhalation aerosols as a function of the aerosol particle size and respiratory variables provided their intrinsic pharmacokinetics are linear. Aerosol characteristics are determined by sampling the aerosol at a point close to its entry to the endotracheally intubated animal. The chosen positive-pressure ventilatory regime, which is monitored as airway pressure and exhaled volume versus time, can be held constant for the aerosol administration period. The methodology is illustrated by administration of a solid polydispersed aerosol of disodium fluorescein. Resultant plasma concentrations (C) were determined as a function of time by sampling from an indwelling venous cannula. The pharmacokinetic analysis of resultant C versus time data, together with that from an intravenous control experiment, is described to determine the amount absorbed as a function of time. Following aerosol administration according to the chosen respiratory regime, fluorescein was rapidly absorbed from the RT. The methodology will enable systematic variation of the particle size and positive-pressure respiratory regime in order to determine effects on drug absorption kinetics.

Absorption↗