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The Horizontal Gene Pool: an ESF workshop summary.

The European Science Foundation (ESF) funds a limited number of exploratory workshops each year that enable scientists to meet and develop plans for a program of integrated research which would benefit from a coordinated European effort. In summer 2003, the Standing Committee for Life and Environmental Sciences (LESC) sponsored such a workshop called The Horizontal Gene Pool: The Functional Role of Mobile Genetic Information--How Bacteria Perceive, Sample, and Utilize Genetic Elements in evolution and Local Adaptation. The workshop took place at St. Catherine's College, Oxford, UK. Its purpose was to identify how recent advances in the application of genomics and microbial ecology can be harnessed to determine the genetic mechanisms that underpin the biological role of the horizontal gene pool. Scientific excellence at the workshop was contributed by senior scientists and young investigators from research institutes located in nine European countries.

Bacteria↗

Statistical methodology to evaluate food exposure to a contaminant and influence of sanitary limits: application to Ochratoxin A.

This paper presents some statistical methodologies to evaluate the food exposure to a contaminant and quantify the outcome of a new maximum limit on a food item. Our application deals with Ochratoxin A (OTA). We focus on the quantitative evaluation of the distribution of exposure based on both consumption data and contamination data. One specific aspect of contamination data is left censorship due to the limits of detection. Three calculation procedures are proposed: [P1] a deterministic method using means of contamination; [P2] a probabilistic method using a parametric adjustment of the distributions of contamination taking into account the left censorship; and [P3] a non-parametric method which consists in randomly selecting the consumption data and the contamination values. Our main result shows that a non-parametric probabilistic approach is well adapted for the purpose of exposure assessment, when large samples are available. In the application to OTA, the probability to exceed a safe level is high, particularly for children. Simulations show that the impact of the existing standards on cereals and the currently proposed standards on wine generally do not significantly reduce the risk to be overexposed to OTA.

Adult↗

Late onset polio sequelae: disabilities and handicaps in a population-based cohort of the 1956 poliomyelitis outbreak in The Netherlands.

OBJECTIVE: To investigate the prevalence of new neuromuscular symptoms, disabilities, and handicaps in a group of polio survivors. DESIGN: A self-constructed health questionnaire about neuromuscular complaints and disability and handicap levels during the stable period after recovery from polio and at present. SUBJECTS: Three hundred fifty subjects, derived from the 1,784 polio cases registered during the 1956 polio outbreak in The Netherlands. RESULTS: Respondents totaled 260 (74%), 27 of whom denied or did not recall having had paralytic poliomyelitis. The remaining 233 subjects comprised the study group (mean age, 44yrs; range, 39 to 77; SD = 6.3). Frequency of all neuromuscular complaints at present time was significantly higher than that during the stable period after polio (range in p of .001 to .004). Fifty-eight percent of cases reported an increase in muscle weakness in comparison with muscle condition during the stable period. Fifty-six percent reported an increase in disabilities, mainly a restriction in gait functions. Fifty-three percent reported increased handicaps with regard to occupation and social integration, and there was an increased need for adaptive measures and devices. CONCLUSION: Nearly 60% of a sample of Dutch survivors of the 1956 polio outbreak experience late onset polio sequelae, resulting in increased severity of disabilities and handicaps.

Adult↗

Energy content of the evening meal alters nocturnal body temperature but not sleep.

Meals of varying energy content and episodes of sleep influence body temperature. We compared the effect of an evening meal, varying from high-energy (11.91 +/- 0.86 MJ) to average (5.74 +/- 0.88 MJ) and a 10-h fast (no evening meal), on nocturnal body temperature and sleep. Seven healthy men (20-24 years, mean body mass index of 23.4 +/- 2.6 kg/m2) reported to the laboratory for an evening meal at 2000 h having consumed similar amounts of food before 1300 h. After completing the meal, subjective hunger ratings were assessed, and a venous blood sample taken. The subjects spent 4 nonconsecutive nights (an adaptation night, followed by either of the two meal conditions or the fast in random order) in the sleep laboratory when polysomnographic recordings were made from 2300 to 0700 h. Meal energy content and serum concentrations of insulin, triglyceride, and low-density lipoproteins (LDL) varied significantly. Lower rectal temperatures were measured during the fast than following the meals. Over the 8-h recording period, thermal response indices (TRI) varied with higher body temperatures following the higher energy meal. Similar rectal temperatures were attained by the end of the sleep periods. There were no significant differences in any of the subjective or objective sleep measures. The physiological responses associated with the transient dietary changes of an evening meal or a 10-h fast altered nocturnal body temperature but did not significantly affect sleep of good sleepers when sleep was initiated 2 to 3 h after finishing the meal.

Adult↗

XANES spectroscopy: a promising tool for toxicology: a tutorial.

X-ray absorption near edge structure (XANES) spectroscopy can provide information on the oxidation state of metal ions within a biological sample and also the complexes in which it is found. This type of information could be of great use to toxicologists in understanding the mechanism of action of many toxic agents. The prospect of using a sophisticated physical technique such as XANES may be somewhat intimidating for those without a strong physical background. Here, we explain the concepts necessary to understand XANES spectroscopy at a level that can be easily understood by biological scientists without a strong physics background and describe useful sample preparation and data analysis techniques which can be adapted for a variety of applications. Examples are taken from an ongoing study of manganese in brain mitochondria and neuron-like cells.

Animals↗

Energy storage and return prostheses: does patient perception correlate with biomechanical analysis?

The development and prescription of energy storage and return prosthetic feet in favor of conventional feet is largely based upon prosthetist and amputee experience. Regretfully, the comparative biomechanical analysis of energy storage and return and conventional prosthetic feet is rarely a motivation to either the technical development or clinical prescription of such devices. The development and prescription of prosthetic feet without supportive scientific evidence is likely due to the conflicting or non-significant results often presented in the scientific literature. Despite the sizeable history of comparative prosthetic literature and continued analysis of prosthetic components, the link between clinical experience and scientific evidence remains largely unexplored.A review of the comparative analysis literature evaluating energy storage and return and conventional prosthetic feet is presented to illustrate consistencies between the perceptive assessments and the objective biomechanical data. Results suggest that while experimental methodologies may limit the statistical significance of objective gait analysis results, consistent trends in temporal, kinetic, and kinematic parameters correlate well with perceptive impressions of these feet. These correlations provide insight to subtle changes in gait parameters that are deemed neither clinically nor statistically significant, yet are perceived by amputees to affect their preference for and performance of prosthetic feet during locomotion. Acknowledging and targeting areas of perceptive significance will help researchers develop more structured protocols for energy storage and return prosthesis evaluation as well as provide clinicians with information needed to enhance the appropriateness of their clinical recommendations. Expanding test environments to measure activities of perceived improvement such as high-velocity motions, stair ascent/descent, and uneven ground locomotion will provide a more appropriate assessment of the conditions for which energy storage and return prosthetic feet were designed. Concentrating research to specific test populations by age or amputation etiologies can overcome statistical limitations imposed by small study samples. Finally, directing research toward the areas of gait adaptation, heel performance, and the temporal release of energy in energy storage and return feet may reinforce the selection and utilization of advanced prosthetic components. These enhancements to current biomechanical analyses may serve to reduce the boundaries of perceptive significance and provide clinicians, designers, and researchers with the supportive data needed to prescribe, design, and evaluate energy storage and return prosthetic feet.

Amputees↗

Performance of 225 Dutch school children on Rey's Auditory Verbal Learning Test (AVLT): parallel test-retest reliabilities with an interval of 3 months and normative data.

Performance on Dutch adaptations of Rey's AVLT was examined in a sample of 225 6- to 12-year-old Dutch school children, selected to be representative of the general population. With an interval of 3 months, they were tested twice, using two out of three test forms which proved to be parallel. No test-retest practice effects were apparent. Age had a considerable impact on test performance. Measured imperfectly, socioeconomic background and intellectual level had some additional influence, as did the examiner who administered the tests. Boys made more errors than girls. Normative data corresponded well with those collected in a smaller Australian sample, suggesting usefulness outside The Netherlands. With a parallel test-retest reliability of.70, which was reduced to.59 when the influence of age was taken into account, the most reliable AVLT measure was the total number of words correctly recalled over the five learning trials. On the basis of reliability data, implications for the clinical use of the AVLT are discussed.

Journal Article↗

Quality of life and coping styles of HIV-positive women with children.

This descriptive study explored the quality of life and coping methods of HIV positive women (N = 52) with children. The sample consisted of women with HIV/AIDS who answered an adapted Padilla Quality of Life Index and the Jalowiec Coping Scale. Among the psychological, physical, and social quality of life subscales, social quality of life was rated highest and the psychological scale rated the lowest. Among the three styles of coping, confrontive was used most frequently, passive and emotive less frequently. Significant relationships between coping and quality of life were examined. It is essential that nurses working with women with HIV disease be aware of their common concerns and provide effective strategies to improve quality of life such as enhancing decision making and coping skills within the context of the family unit.

Adaptation, Psychological↗

[Quality control of labile blood products. Why and howto properly take a specimen? Labile Blood Products Group of the French Blood Transfusion Society].

This article reviews the various techniques of sampling used for the quality control of blood cell products. The importance of this stage for the validity of quality control results is emphasized. Three sampling methods, i.e., stripping, the sterile connection of sampling bag and the destructive method, are described in the form of operating modes and analyzed according to their advantages and drawbacks. The results of a comparative study carried out by the working group 'Blood Cell Products' of the French Society of Blood Transfusion are presented, showing that each method is valid and permits one to obtain a representative sample of the product to be controlled. Thus the diversity of the sampling methods allows us to select the one most adapted to the product to be controlled and to the analyses to be carried out afterward.

Blood Specimen Collection↗

Scalable transcriptional analysis routine--multiplexed quantitative real-time polymerase chain reaction platform for gene expression analysis and molecular diagnostics.

We report the development of a new technology for simultaneous quantitative detection of multiple targets in a single sample. Scalable transcriptional analysis routine (STAR) represents a novel integration of reverse transcriptase-polymerase chain reaction and capillary electrophoresis that allows detection of dozens of gene transcripts in a multiplexed format using amplicon size as an identifier for each target. STAR demonstrated similar or better sensitivity and precision compared to two commonly used methods, SYBR Green-based and TaqMan probe-based real-time reverse transcriptase-polymerase chain reaction. STAR can be used as a flexible platform for building a variety of applications to monitor gene expression, from single gene assays to assays analyzing the expression level of multiple genes. Using severe acute respiratory syndrome (SARS) corona virus as a model system, STAR technology detected single copies of the viral genome in a two-gene multiplex. Blinded studies using RNA extracted from various tissues of a SARS-infected individual showed that STAR correctly identified all samples containing SARS virus and yielded negative results for non-SARS control samples. Using alternate priming strategies, STAR technology can be adapted to transcriptional profiling studies without requiring a priori sequence information. Thus, STAR technology offers a flexible platform for development of highly multiplexed assays in gene expression analysis and molecular diagnostics.

Animals↗

Stochastic detection of enantiomers.

The rapid quantification of the enantiomers of small chiral molecules is very important, notably in pharmacology. Here, we show that the enantiomers of drug molecules can be distinguished by stochastic sensing, a single-molecule detection technique. The sensing element is an engineered alpha-hemolysin protein pore, fitted with a beta-cyclodextrin adapter. By using the approach, the enantiomeric composition of samples of ibuprofen and thalidomide can be determined in less than 1 s.

Hemolysin Proteins↗

A 768-lane microfabricated system for high-throughput DNA sequencing.

A 768-lane DNA sequencing system based on microfluidic plates has been designed as a near-term successor to 96-lane capillary arrays. Electrophoretic separations are implemented for the first time in large-format (25 cm x 50 cm) microdevices, with the objective of proving realistic read length, parallelism, and the scaled sample requirements for long-read de novo sequencing. Two 384-lane plates are alternatively cycled between electrophoresis and regeneration via a robotic pipettor. A total of greater than 172000 bases, 99% accuracy (corresponding to quality score 20) is achieved for each iteration of a 384 lane plate. At current operating conditions, this implies a system throughput exceeding 4 megabases of raw sequence (Phred 20) per day on the new platform. Standard operation is at "1/32x" Sanger chemistry, equal to typical genome center operation on mature capillary array machines, and a 16-fold improvement in scaling relative to previous microfabricated devices. Experiments provide evidence that sample concentration can be further reduced to 1/256x Sanger chemistry in the microdevice. Life-testing indicates a usable life of >150 hours (more than 50 runs) for the 384 lane plates. The combined advances, particularly those in read length and sample requirement, directly address the cost model requirements for adaptation of the new technology as the next step beyond capillary array instruments.

DNA↗

Evaluation of two new integrated adapters for blood drawing in closed blood bag systems: influence on different molecular coagulation markers.

We compared a conventional blood donation bag system (A) with two newly developed ones (B, C) with integrated Y-shaped adapters. The new systems facilitate the withdrawal of blood samples in a closed system under aseptic conditions. The purpose of the study was to determine whether disturbances during blood donation in the different tubings influence the quality of the donated plasma due to activation processes of the coagulation system. In all bag samples from 34 probands for each system, we found higher levels of the global tests (aPTT, TT) compared to intraindividual controls. This was due to a greater amount of anticoagulant in the bag samples. An increase of procoagulant activity was demonstrated for bags A and C. Concerning the fibrinolytic system, values were reduced in all three systems compared to controls. In summary, the quality of the plasma in the three different bag devices was comparable to controls concerning factor activities measured as global tests. The activation of the coagulation system was only slightly higher in the new system B and C than in the conventional one (A). Therefore, the new closed systems are suitable for blood donation and reveal a high coagulation quality of the plasma.

Adolescent↗

Psychoeducation for the families of patients with eating disorders and changes in expressed emotion: A preliminary study.

Psychosocial variables such as expressed emotion (EE) have prognostic significance, and family psychoeducation has been developed to aid in the treatment of various psychiatric disorders. This study reports relationships among EE, family factors, and symptoms observed while conducting multifamily psychoeducation for eating disorders. Group sessions were held once a month for the relatives of patients with DSM-IV eating disorders, and the group met for five sessions that included both education and problem-solving. Thirty-seven relatives volunteered to participate in our program, and of these, 28 completed the program. EE (as measured by the Five-Minute Speech Sample [FMSS]), family function (as measured by the Family Adaptability and Cohesion Evaluation Scales [FACES]), the family's mental state (as measured by the Profile of Mood States [POMS]), and patient's symptoms (as measured by the Eating Disorder Evaluation Scales [EDES] and Global Assessment of Functioning [GAF] on clinician evaluations, and by the Anorexic Behavior Observation Scale [ABOS] assessment of the family) were administered at both the first and final sessions. The rates of high-EE relatives tended to decrease (especially high emotional overinvolvement [EOI]), and families' assessment of symptoms was also significantly improved. Twice-repeated multivariate analysis of variance (MANCOVA) showed that EOI, ABOS, and POMS scores were changed significantly during the sessions. Psychoeducation for the family members of patients with eating disorders might help lower distress and encourage positive interactions within the family. EE is an important measure in evaluations of psychoeducation. However, a randomized, controlled trial is needed to clarify the efficacy of this treatment.

Adaptation, Psychological↗

Base orientation of second DNA in RecA.DNA filaments. Analysis by combination of linear dichroism and small angle neutron scattering in flow-oriented solution.

To gain insight into the mechanism of pairing two complementary DNA strands by the RecA protein, we have determined the nucleobase orientation of the first and the second bound DNA strands in the RecA.DNA filament by combined measurements of linear dichroism and small angle neutron scattering on flow-oriented samples. An etheno-modified DNA, poly(depsilonA) was adapted as the first DNA and an oligo(dT) as the second DNA, making it possible to distinguish between the linear dichroism signals of the two DNA strands. The results indicate that binding of the second DNA does not alter the nucleobase orientation of the first bound strand and that the bases of the second DNA are almost coplanar to the bases of the first strand although somewhat more tilted (60 degrees relative to the fiber axis compared with 70 degrees for the first DNA strand). Similar results were obtained for the RecA.DNA complex formed with unmodified poly(dA) and oligo(dT). An almost coplanar orientation of nucleobases of two DNA strands in a RecA-DNA filament would facilitate scanning for, and recognition of, complementary base sequences. The slight deviation from co-planarity could increase the free energy of the duplex to facilitate dissociation in case of mismatching base sequences.

DNA↗

Factors affecting accuracy of perception on a task requiring the ability to identify viewpoints.

A study was undertaken to determine the relative contribution of age, sex, and three stimulus features (board shape, block arrangement, and block shape) to perceptual accuracy on 39 board/block adaptations of Piaget's three-mountain task. The sample consisted of 830 children (420 1st graders and 410 5th graders) and 130 college students. Stimulus features of the board/block task accounted for between 22% and 26% of the explained variability among accuracy scores; however, interactions of other factors with age were nonsignificant. When data from college students were combined with children, a curvilinear developmental relationship between age and perceptual accuracy was obtained. The board/block task has promise as an instrument for longitudinal studies.

Adolescent↗

Toward the application of proteomics to human thyroid tissue.

CONTEXT: In this paper we describe for the first time a systematic approach to proteome analysis of human thyroid tissue. OBJECTIVE AND DESIGN: We report different methods to decrease the complexity of the human thyroid tissue proteome by applying different solubilization strategies and correcting for thyroglobulin protein abundance; to increase the protein resolution by prefractionation and by the use of narrow-range pH gradients; to detect proteins using sensitive and quantitative stains; and to identify soluble and membrane-bound thyroid tissue proteins by mass spectrometry analysis. MAIN OUTCOME/RESULTS: We found that buffers containing high contents of urea and detergents allow the best solubilization of human thyroid tissue proteins; highly variable abundance of thyroglobulin is a major pitfall of human thyroid proteome analysis, which in contrast to centrifugal ultrafiltration, size-exclusion chromatography and microdissection, can be countered best by adapting the protein amount to the thyroglobulin content per sample; prefractionation leads to a significant enrichment of proteins and allows subcellar localization of thyroid proteins; application of narrow-range immobilized pH gradient (IPG) strips allows further improvement of spot detection and separation; and protein detection with the fluorescent stain ruthenium II Tris bathophenanthroline disulfonate (RuBPs) is a highly sensitive and reliable tool for quantitative proteome analysis. Finally, in a pilot study of four patients with benign nodular thyroid disease we found that the described procedures allow a highly reproducible detection and identification of alterations in protein expression between nodular and corresponding normal thyroid tissues. CONCLUSIONS: Application of the described methods provides the basis for a highly sensitive and reproducible proteome analysis of the human thyroid, providing an additional novel tool to elucidate complex proteins changes in human thyroid biology as well as pathophysiology of human thyroid disease.

Cell Fractionation↗

The use of a radiometric technic for the rapid detection of contaminated tissue specimens from burned patients.

The 14CO2-dependent BACTEC system for the detection of bacteria in fluid specimens was adapted for the semiquantitative bacteriologic screening of solid tissue samples from 33 burned patients. The radiometric system detected 87% of all specimens harboring > 10(5) organisms per gram of tissue and 100% of all heavily contaminated specimens (> 10(8) organisms per gram) within eight hours of incubation. The BACTEC radiometric system correlated well (98.1%) with conventional culture methods in the detection of all contaminated samples. No false-positive result was obtained from sterile tissue specimens.

Bacteria↗