Validation and reproducibility of a semi-quantitative Food Frequency Questionnaire for use in elderly Swiss women.

OBJECTIVE: The principal aim of this study was to develop a Swiss Food Frequency Questionnaire (FFQ) for the elderly population for use in a study to investigate the influence of nutritional factors on bone health. The secondary aim was to assess its validity and both short-term and long-term reproducibility. DESIGN: A 4-day weighed record (4 d WR) was applied to 51 randomly selected women of a mean age of 80.3 years. Subsequently, a detailed FFQ was developed, cross-validated against a further 44 4-d WR, and the short- (1 month, n = 15) and long-term (12 months, n = 14) reproducibility examined. SETTING: French speaking part of Switzerland. SUBJECTS: The subjects were randomly selected women recruited from the Swiss Evaluation of the Methods of Measurement of Osteoporotic Fracture cohort study. RESULTS: Mean energy intakes by 4-d WR and FFQ showed no significant difference [1564.9 kcal (SD 351.1); 1641.3 kcal (SD 523.2) respectively]. Mean crude nutrient intakes were also similar (with nonsignifcant P-values examining the differences in intake) and ranged from 0.13 (potassium) to 0.48 (magnesium). Similar results were found in the reproducibility studies. CONCLUSION: These findings provide evidence that this FFQ adequately estimates nutrient intakes and can be used to rank individuals within distributions of intake in specific populations.

Construction and Quantitative Evaluation of a Dual Specific Promoter System for Monitoring the Expression Status of Stra8 and c-kit Genes.

Applications of genetic constructs with multiple promoters, which are fused with reporter genes and simultaneous monitoring of various events in cells, have gained special attention in recent years. Lentiviral vectors, with their distinctive characteristics, have been considered to monitor the developmental changes of cells in vitro. In this study, we constructed a novel lentiviral vector (FUM-M), containing two germ cell-specific promoters (Stra8 and c-kit), fused with ZsGreen and DsRed2 reporter genes, and evaluated its efficiency in different cells following treatments with retinoic acid and DMSO. Several cell lines (P19, GC-1 spg and HEK293T) were transduced with this vector, and functional capabilities of the promoters were verified by flow cytometry and quantitative RT-PCR. Our results indicate that FUM-M shows dynamic behavior in the presence and absence of extrinsic factors. A correlation was also observed between the function of promoters, present in the lentiviral construct and the endogenous level of the Stra8 and c-kit mRNAs in the cells. In conclusion, we recommend this strategy, which needs further optimization of the constructs, as a beneficial and practical way to screen chemical inducers involved in cellular differentiation toward germ-like cells.

A quantitative genetic signature of senescence in a short-lived perennial plant.

The evolution of senescence (the physiological decline of organisms with age) poses an apparent paradox because it represents a failure of natural selection to increase the survival and reproductive performance of organisms. The paradox can be resolved if natural selection becomes less effective with age, because the death of postreproductive individuals should have diminished effects on Darwinian fitness [1, 2]. A substantial body of empirical work is consistent with this prediction for animals, which transmit their genes to progeny via an immortal germline. However, such evidence is still lacking in plants, which lack a germline and whose reproduction is diffuse and modular across the soma. Here, we provide experimental evidence for a genetic basis of senescence in the short-lived perennial plant Silene latifolia. Our pedigree-based analysis revealed a marked increase with age in the additive genetic variance of traits closely associated with fitness. This result thus extends to plants the quantitative genetic support for the evolutionary theory of senescence.

Detection of live and antibiotic-killed bacteria by quantitative real-time PCR of specific fragments of ribosomal RNA

RÉSUMÉ Le but d'un traitement antimicrobien est d'éradiquer une infection bactérienne. Cependant, il est souvent difficile d'en évaluer rapidement l'efficacité en utilisant les techniques standard. L'estimation de la viabilité bactérienne par marqueurs moléculaires permettrait d'accélérer le processus. Ce travail étudie donc la possibilité d'utiliser le RNA ribosomal (rRNA) à cet effet. Des cultures de Streptococcus gordonii sensibles (parent Wt) et tolérants (mutant Tol 1) à l'action bactéricide de la pénicilline ont été exposées à différents antibiotiques. La survie bactérienne au cours du temps a été déterminée en comparant deux méthodes. La méthode de référence par compte viable a été comparée à une méthode moléculaire consistant à amplifier par PCR quantitative en temps réel une partie du génome bactérien. La cible choisie devait refléter la viabilité cellulaire et par conséquent être synthétisée de manière constitutive lors de la vie de la bactérie et être détruite rapidement lors de la mort cellulaire. Le choix s'est porté sur un fragment du gène 16S-rRNA. Ce travail a permis de valider ce choix en corrélant ce marqueur moléculaire à la viabilité bactérienne au cours d'un traitement antibiotique bactéricide. De manière attendue, les S. gordonii sensibles à la pénicilline ont perdu ≥ 4 log10 CFU/ml après 48 heures de traitement par pénicilline alors que le mutant tolérant Tol1 en a perdu ≥ 1 log10 CFU/ml. De manière intéressant, la quantité de marqueur a augmenté proportionnellement au compte viable durant la phase de croissance bactérienne. Après administration du traitement antibiotique, l'évolution du marqueur dépendait de la capacité de la bactérie à survivre à l'action de l'antibiotique. Stable lors du traitement des souches tolérantes, la quantité de marqueur détectée diminuait de manière proportionnelle au compte viable lors du traitement des souches sensibles. Cette corrélation s'est confirmée lors de l'utilisation d'autres antibiotiques bactéricides. En conclusion, l'amplification par PCR du RNA ribosomal 16S permet d'évaluer rapidement la viabilité bactérienne au cours d'un traitement antibiotique en évitant le recours à la mise en culture dont les résultats ne sont obtenus qu'après plus de 24 heures. Cette méthode offre donc au clinicien une évaluation rapide de l'efficacité du traitement, particulièrement dans les situations, comme le choc septique, où l'initiation sans délai d'un traitement efficace est une des conditions essentielles du succès thérapeutique. ABSTRACT Assessing bacterial viability by molecular markers might help accelerate the measurement of antibiotic-induced killing. This study investigated whether ribosomal RNA (rRNA) could be suitable for this purpose. Cultures of penicillin-susceptible and penicillin-tolerant (Tol1 mutant) Streptococcus gordonii were exposed to mechanistically different penicillin and levofloxacin. Bacterial survival was assessed by viable counts, and compared to quantitative real-time PCR amplification of either the 16S-rRNA genes (rDNA) or the 16S rRNA, following reverse transcription. Penicillin-susceptible S. gordonii lost ≥ 4 log10 CFU/ml of viability over 48 h of penicillin treatment. In comparison, the Toll mutant lost ≤ 1 log10 CFU/ml. Amplification of a 427-base fragment of 16S rDNA yielded amplicons that increased proportionally to viable counts during bacterial growth, but did not decrease during drug-induced killing. In contrast, the same 427-base fragment amplified from 16S rDNA paralleled both bacterial growth and drug-induced killing. It also differentiated between penicillin-induced killing of the parent and the Toll mutant (≥4 log10 CFU/ml and ≤1 lo10 CFU/ml, respectively), and detected killing by mechanistically unrelated levofloxacin. Since large fragments of polynucleotides might be degraded faster than smaller fragments the experiments were repeated by amplifying a 119-base region internal to the origina1 427-base fragment. The amount of 119-base amplicons increased proportionally to viability during growth, but remained stable during drug treatment. Thus, 16S rRNA was a marker of antibiotic-induced killing, but the size of the amplified fragment was critical to differentiate between live and dead bacteria.

Identification of reference genes for quantitative RT-PCR in ascending aortic aneurysms.

Hypertension and congenital aortic valve malformations are frequent causes of ascending aortic aneurysms. The molecular mechanisms of aneurysm formation under these circumstances are not well understood. Reference genes for gene activity studies in aortic tissue that are not influenced by aortic valve morphology and its hemodynamic consequences, aortic dilatation, hypertension, or antihypertensive medication are not available so far. This study determines genes in ascending aortic tissue that are independent of these parameters. Tissue specimens from dilated and undilated ascending aortas were obtained from 60 patients (age ≤70 years) with different morphologies of the aortic valve (tricuspid undilated n = 24, dilated n = 11; bicuspid undilated n = 6, dilated n = 15; unicuspid dilated n = 4). Of the studied individuals, 36 had hypertension, and 31 received ACE inhibitors or AT1 receptor antagonists. The specimens were obtained intraoperatively from the wall of the ascending aorta. We analyzed the expression levels of 32 candidate reference genes by quantitative RT-PCR (RT-qPCR). Differential expression levels were assessed by parametric statistics. The expression analysis of these 32 genes by RT-qPCR showed that EIF2B1, ELF1, and PPIA remained constant in their expression levels in the different specimen groups, thus being insensitive to aortic valve morphology, aortic dilatation, hypertension, and medication with ACE inhibitors or AT1 receptor antagonists. Unlike many other commonly used reference genes, the genes EIF2B1, ELF1, and PPIA are neither confounded by aortic comorbidities nor by antihypertensive medication and therefore are most suitable for gene expression analysis of ascending aortic tissue.

Neurofibromatosis of von Recklinghausen: a quantitative study of the epidermal keratinocyte and melanocyte populations.

The numerical keratinocyte to melanocyte relation was studied in café au lait spots and adjacent normally pigmented skin of 9 patients with classical neurofibromatosis. Compared to normal skin of healthy individuals, the keratinocyte:melanocyte ratio distributions obtained in neurofibromatosis indicated a shift to lower values in the biopsies of café au lait spots and normally pigmented skin. These results are evidence in favor of an impaired tissue organization of the epidermis in neurofibromatosis with regard to the keratinocyte-melanocyte interrelation.

Automated quantitative histology reveals vascular morphodynamics during Arabidopsis hypocotyl secondary growth.

Among various advantages, their small size makes model organisms preferred subjects of investigation. Yet, even in model systems detailed analysis of numerous developmental processes at cellular level is severely hampered by their scale. For instance, secondary growth of Arabidopsis hypocotyls creates a radial pattern of highly specialized tissues that comprises several thousand cells starting from a few dozen. This dynamic process is difficult to follow because of its scale and because it can only be investigated invasively, precluding comprehensive understanding of the cell proliferation, differentiation, and patterning events involved. To overcome such limitation, we established an automated quantitative histology approach. We acquired hypocotyl cross-sections from tiled high-resolution images and extracted their information content using custom high-throughput image processing and segmentation. Coupled with automated cell type recognition through machine learning, we could establish a cellular resolution atlas that reveals vascular morphodynamics during secondary growth, for example equidistant phloem pole formation. DOI: http://dx.doi.org/10.7554/eLife.01567.001.

Plasmid-based controls to detect rpoB mutations in Mycobacterium tuberculosis by quantitative polymerase chain reaction-high-resolution melting

Quantitative polymerase chain reaction-high-resolution melting (qPCR-HRM) analysis was used to screen for mutations related to drug resistance in Mycobacterium tuberculosis. We detected the C526T and C531T mutations in the rifampicin resistance-determining region (RRDR) of the rpoB gene with qPCR-HRM using plasmid-based controls. A segment of the RRDR region from M. tuberculosis H37Rv and from strains carrying C531T or C526T mutations in the rpoB were cloned into pGEM-T vector and these vectors were used as controls in the qPCR-HRM analysis of 54 M. tuberculosis strains. The results were confirmed by DNA sequencing and showed that recombinant plasmids can replace genomic DNA as controls in the qPCR-HRM assay. Plasmids can be handled outside of biosafety level 3 facilities, reducing the risk of contamination and the cost of the assay. Plasmids have a high stability, are normally maintained in Escherichia coli and can be extracted in large amounts.

Inference about the number of contributors to a DNA mixture: Comparative analyses of a Bayesian network approach and the maximum allele count method.

In the forensic examination of DNA mixtures, the question of how to set the total number of contributors (N) presents a topic of ongoing interest. Part of the discussion gravitates around issues of bias, in particular when assessments of the number of contributors are not made prior to considering the genotypic configuration of potential donors. Further complication may stem from the observation that, in some cases, there may be numbers of contributors that are incompatible with the set of alleles seen in the profile of a mixed crime stain, given the genotype of a potential contributor. In such situations, procedures that take a single and fixed number contributors as their output can lead to inferential impasses. Assessing the number of contributors within a probabilistic framework can help avoiding such complication. Using elements of decision theory, this paper analyses two strategies for inference on the number of contributors. One procedure is deterministic and focuses on the minimum number of contributors required to 'explain' an observed set of alleles. The other procedure is probabilistic using Bayes' theorem and provides a probability distribution for a set of numbers of contributors, based on the set of observed alleles as well as their respective rates of occurrence. The discussion concentrates on mixed stains of varying quality (i.e., different numbers of loci for which genotyping information is available). A so-called qualitative interpretation is pursued since quantitative information such as peak area and height data are not taken into account. The competing procedures are compared using a standard scoring rule that penalizes the degree of divergence between a given agreed value for N, that is the number of contributors, and the actual value taken by N. Using only modest assumptions and a discussion with reference to a casework example, this paper reports on analyses using simulation techniques and graphical models (i.e., Bayesian networks) to point out that setting the number of contributors to a mixed crime stain in probabilistic terms is, for the conditions assumed in this study, preferable to a decision policy that uses categoric assumptions about N.

Comparison of drug survival rates for tumor necrosis factor antagonists in rheumatoid arthritis.

BACKGROUND Persistence of anti-tumor necrosis factor (TNF) therapy in rheumatoid arthritis (RA) is an overall marker of treatment success. OBJECTIVE To assess the survival of anti-TNF treatment and to define the potential predictors of drug discontinuation in RA, in order to verify the adequacy of current practices. DESIGN An observational, descriptive, longitudinal, retrospective study. SETTING The Hospital Clínico Universitario de Valladolid, Valladolid, Spain. PATIENTS RA patients treated with anti-TNF therapy between January 2011 and January 2012. MEASUREMENTS Demographic information and therapy assessments were gathered from medical and pharmaceutical records. Data is expressed as means (standard deviations) for quantitative variables and frequency distribution for qualitative variables. Kaplan-Meier survival analysis was used to assess persistence, and Cox multivariate regression models were used to assess potential predictors of treatment discontinuation. RESULTS In total, 126 treatment series with infliximab (n = 53), etanercept (n = 51) or adalimumab (n = 22) were administered to 91 patients. Infliximab has mostly been used as a first-line treatment, but it was the drug with the shortest time until a change of treatment. Significant predictors of drug survival were: age; the anti-TNF agent; and the previous response to an anti-TNF drug. LIMITATION The small sample size. CONCLUSION The overall efficacy of anti-TNF drugs diminishes with time, with infliximab having the shortest time until a change of treatment. The management of biologic therapy in patients with RA should be reconsidered in order to achieve disease control with a reduction in costs.

Quantitative ultrasound of bone in institutionalized elderly women: a cross-sectional and longitudinal study.

Quantitative ultrasound of bone is a promising method for bone assessment: radiation-free, portable and predictive of hip fracture. Its portability allowed us to study the relationships between ultrasonic parameters of bone with age and with non-vertebral fractures in elderly women living in 19 nursing homes. Broadband ultrasound attenuation (BUA) and speed of sound (SOS) of the calcaneus were measured (and the stiffness index calculated) in a sample of 270 institutionalized women, aged 85 +/- 7 years, using an Achilles bone densitometer (Lunar). The effects of age, history of non-vertebral and non-traumatic fractures, body mass index, triceps skinfold and arm circumference were assessed on BUA, SOS and stiffness index. Furthermore, to evaluate longitudinally the influence of aging on the ultrasound parameters of bone, 60 subjects from the same group had a second ultrasound measurement after 1 year. The cross-sectional analysis of the data on all 270 women showed a significant decrease (p < 0.001) with age in BUA, SOS and stiffness index (-0.47%, -0.06%, and -1.01% respectively per year). In the 94 women, (35%) with a history of previous non-vertebral fractures, ultrasound parameters were significantly lower (p < 0.0001) than in the 176 women with no history of fracture (-8.3% for BUA, -1.3% for SOS, -18.9% for stiffness index). In contrast, there was no significant difference in anthropometric measurements between the groups with and without previous non-vertebral fractures, although the measurements decreased significantly with age. In the longitudinal study, repeated quantitative ultrasound after 11.4 +/- 0.8 months showed no significant decrease in BUA (-1%) but a significant decrease in SOS (-0.3%, p < 0.0001) and in stiffness index (-3.6%, p < 0.0002). In conclusion, quantitative ultrasound of the calcaneus measures properties of bone which continue to decline in institutionalized elderly women, and is able to discriminate women with previous non-vertebral fractures.

Bilateral sequential nonarteritic anterior ischemic optic neuropathy: a comparison of visual outcomes in fellow eyes using quantitative analysis of goldmann visual fields.

OBJECTIVE To better define the concordance of visual loss in patients with nonarteritic anterior ischemic optic neuropathy (NAION). METHODS The medical records of 86 patients with bilateral sequential NAION were reviewed retrospectively, and visual function was assessed using visual acuity, Goldmann visual fields, color vision, and relative afferent papillary defect. A quantitative total visual field score and score per quadrant were analyzed for each eye using the numerical Goldmann visual field scoring method. RESULTS Outcome measures were visual acuity, visual field, color vision, and relative afferent papillary defect. A statistically significant correlation was found between fellow eyes for multiple parameters, including logMAR visual acuity (P = .01), global visual field (P < .001), superior visual field (P < .001), and inferior visual field (P < .001). The mean deviation of total (P < .001) and pattern (P < .001) deviation analyses was significantly less between fellow eyes than between first and second eyes of different patients. CONCLUSIONS Visual function between fellow eyes showed a fair to moderate correlation that was statistically significant. The pattern of vision loss was also more similar in fellow eyes than between eyes of different patients. These results may help allow better prediction of visual outcome for the second eye in patients with NAION.

An electronic portfolio for quantitative assessment of surgical skills in undergraduate medical education.

BACKGROUND We evaluated a newly designed electronic portfolio (e-Portfolio) that provided quantitative evaluation of surgical skills. Medical students at the University of Seville used the e-Portfolio on a voluntary basis for evaluation of their performance in undergraduate surgical subjects. METHODS Our new web-based e-Portfolio was designed to evaluate surgical practical knowledge and skills targets. Students recorded each activity on a form, attached evidence, and added their reflections. Students self-assessed their practical knowledge using qualitative criteria (yes/no), and graded their skills according to complexity (basic/advanced) and participation (observer/assistant/independent). A numerical value was assigned to each activity, and the values of all activities were summated to obtain the total score. The application automatically displayed quantitative feedback. We performed qualitative evaluation of the perceived usefulness of the e-Portfolio and quantitative evaluation of the targets achieved. RESULTS Thirty-seven of 112 students (33%) used the e-Portfolio, of which 87% reported that they understood the methodology of the portfolio. All students reported an improved understanding of their learning objectives resulting from the numerical visualization of progress, all students reported that the quantitative feedback encouraged their learning, and 79% of students felt that their teachers were more available because they were using the e-Portfolio. Only 51.3% of students reported that the reflective aspects of learning were useful. Individual students achieved a maximum of 65% of the total targets and 87% of the skills targets. The mean total score was 345 ± 38 points. For basic skills, 92% of students achieved the maximum score for participation as an independent operator, and all achieved the maximum scores for participation as an observer and assistant. For complex skills, 62% of students achieved the maximum score for participation as an independent operator, and 98% achieved the maximum scores for participation as an observer or assistant. CONCLUSIONS Medical students reported that use of an electronic portfolio that provided quantitative feedback on their progress was useful when the number and complexity of targets were appropriate, but not when the portfolio offered only formative evaluations based on reflection. Students felt that use of the e-Portfolio guided their learning process by indicating knowledge gaps to themselves and teachers.

Quantitative analysis of postnatal neurogenesis and neuron number in the macaque monkey dentate gyrus

The dentate gyrus is one of only two regions of the mammalian brain where substantial neurogenesis occurs postnatally. However, detailed quantitative information about the postnatal structural maturation of the primate dentate gyrus is meager. We performed design-based, stereological studies of neuron number and size, and volume of the dentate gyrus layers in rhesus macaque monkeys (Macaca mulatta) of different postnatal ages. We found that about 40% of the total number of granule cells observed in mature 5-10-year-old macaque monkeys are added to the granule cell layer postnatally; 25% of these neurons are added within the first three postnatal months. Accordingly, cell proliferation and neurogenesis within the dentate gyrus peak within the first 3 months after birth and remain at an intermediate level between 3 months and at least 1 year of age. Although granule cell bodies undergo their largest increase in size during the first year of life, cell size and the volume of the three layers of the dentate gyrus (i.e. the molecular, granule cell and polymorphic layers) continue to increase beyond 1 year of age. Moreover, the different layers of the dentate gyrus exhibit distinct volumetric changes during postnatal development. Finally, we observe significant levels of cell proliferation, neurogenesis and cell death in the context of an overall stable number of granule cells in mature 5-10-year-old monkeys. These data identify an extended developmental period during which neurogenesis might be modulated to significantly impact the structure and function of the dentate gyrus in adulthood.