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Validation Experiments (validation + experiment)

Distribution by Scientific Domains

Medical Sciences	42%
Chemistry	42%

Selected Abstracts

Validation of the AMPF,STR® MiniFilerTM PCR Amplification Kit for Use in Forensic Casework,

JOURNAL OF FORENSIC SCIENCES, Issue 5 2009
Coral Luce M.S.
Abstract:, The AmpF,STR® MiniFilerTM PCR Amplification Kit is designed to genotype degraded and/or inhibited DNA samples when the AmpF,STR® IdentifilerTM PCR Amplification Kit is incapable of generating a complete genetic profile. Validation experiments, following the SWGDAM guidelines, were designed to evaluate the performance of MiniFiler. Data obtained demonstrated that MiniFiler, when used in conjunction with Identifiler, provided an increased ability to obtain genetic profiles from challenged samples. The optimum template range was found to be between 0.2 and 0.6 ng, with 0.3 ng yielding the best results. Full concordance was achieved between the MiniFiler kit and Identifiler kit except in a single case of a null allele at locus D21S11. Numerous instances of severe heterozygous peak imbalance (<50%) were observed in single source samples amplified within the optimum range of input DNA suggesting that caution be taken when attempting to deduce component genotypes in a mixture. [source]

Talairach-Based Parcellation of Neonatal Brain Magnetic Resonance Imaging Data: Validation of a New Approach

JOURNAL OF NEUROIMAGING, Issue 4 2005
Haissam Haidar PhD
ABSTRACT Background and Purpose. Talairach-based parcellation (TP) of human brain magnetic resonance imaging (MRI) data has been used increasingly in clinical research to make regional measurements of brain structures in vivo. Recently, TP has been applied to pediatric research to elucidate the changes in regional brain volumes related to several neurological disorders. However, all freely available tools have been designed to parcellate adult brain MRI data. Parcellation of neonatal MRI data is very challenging owing to the lack of strong signal contrast, variability in signal intensity within tissues, and the small size and thus difficulty in identifying small structures used as landmarks for TP. Hence the authors designed and validated a new interactive tool to parcellate brain MRI data from newborns and young infants. Methods. The authors' tool was developed as part of a postprocessing pipeline, which includes registration of multichannel MR images, segmentation, and parcellation of the segmented data. The tool employs user-friendly interactive software to visualize and assign the anatomic landmarks required for parcellation, after which the planes and parcels are generated automatically by the algorithm. The authors then performed 3 sets of validation experiments to test the precision and reliability of their tool. Results. Validation experiments of intra-and interrater reliability on data obtained from newborn and 1-year-old children showed a very high sensitivity of >95% and specificity >99.9%. The authors also showed that rotating and reformatting the original MRI data results in a statistically significant difference in parcel volumes, demonstrating the importance of using a tool such as theirs that does not require realignment of the data prior to parcellation. Conclusions. To the authors' knowledge, the presented approach is the first TP method that has been developed and validated specifically for neonatal brain MRI data. Their approach would also be valuable for the analysis of brain MRI data from older children and adults. [source]

Identification and Functional Characterization of microRNAs Involved in the Malignant Progression of Gliomas

BRAIN PATHOLOGY, Issue 3 2010
Bastian Malzkorn
Abstract Diffuse astrocytoma of World Health Organization (WHO) grade II has an inherent tendency to spontaneously progress to anaplastic astrocytoma WHO grade III or secondary glioblastoma WHO grade IV. We explored the role of microRNAs (miRNAs) in glioma progression by investigating the expression profiles of 157 miRNAs in four patients with primary WHO grade II gliomas that spontaneously progressed to WHO grade IV secondary glioblastomas. Thereby, we identified 12 miRNAs (miR-9, miR-15a, miR-16, miR-17, miR-19a, miR-20a, miR-21, miR-25, miR-28, miR-130b, miR-140 and miR-210) showing increased expression, and two miRNAs (miR-184 and miR-328) showing reduced expression upon progression. Validation experiments on independent series of primary low-grade and secondary high-grade astrocytomas confirmed miR-17 and miR-184 as promising candidates, which were selected for functional analyses. These studies revealed miRNA-specific influences on the viability, proliferation, apoptosis and invasive growth properties of A172 and T98G glioma cells in vitro. Using mRNA and protein expression profiling, we identified distinct sets of transcripts and proteins that were differentially expressed after inhibition of miR-17 or overexpression of miR-184 in glioma cells. Taken together, our results support an important role of altered miRNA expression in gliomas, and suggest miR-17 and miR-184 as interesting candidates contributing to glioma progression. [source]

On the effects of triangulated terrain resolution on distributed hydrologic model response

HYDROLOGICAL PROCESSES, Issue 11 2005
Enrique R. Vivoni
Abstract Distributed hydrologic models based on triangulated irregular networks (TIN) provide a means for computational efficiency in small to large-scale watershed modelling through an adaptive, multiple resolution representation of complex basin topography. Despite previous research with TIN-based hydrology models, the effect of triangulated terrain resolution on basin hydrologic response has received surprisingly little attention. Evaluating the impact of adaptive gridding on hydrologic response is important for determining the level of detail required in a terrain model. In this study, we address the spatial sensitivity of the TIN-based Real-time Integrated Basin Simulator (tRIBS) in order to assess the variability in the basin-averaged and distributed hydrologic response (water balance, runoff mechanisms, surface saturation, groundwater dynamics) with respect to changes in topographic resolution. Prior to hydrologic simulations, we describe the generation of TIN models that effectively capture topographic and hydrographic variability from grid digital elevation models. In addition, we discuss the sampling methods and performance metrics utilized in the spatial aggregation of triangulated terrain models. For a 64 km2 catchment in northeastern Oklahoma, we conduct a multiple resolution validation experiment by utilizing the tRIBS model over a wide range of spatial aggregation levels. Hydrologic performance is assessed as a function of the terrain resolution, with the variability in basin response attributed to variations in the coupled surface,subsurface dynamics. In particular, resolving the near-stream, variable source area is found to be a key determinant of model behaviour as it controls the dynamic saturation pattern and its effect on rainfall partitioning. A relationship between the hydrologic sensitivity to resolution and the spatial aggregation of terrain attributes is presented as an effective means for selecting the model resolution. Finally, the study highlights the important effects of terrain resolution on distributed hydrologic model response and provides insight into the multiple resolution calibration and validation of TIN-based hydrology models. Copyright © 2005 John Wiley & Sons, Ltd. [source]

A novel 2D-based approach to the discovery of candidate substrates for the metalloendopeptidase meprin

FEBS JOURNAL, Issue 18 2008
Daniel Ambort
In the past, protease-substrate finding proved to be rather haphazard and was executed by in vitro cleavage assays using singly selected targets. In the present study, we report the first protease proteomic approach applied to meprin, an astacin-like metalloendopeptidase, to determine physiological substrates in a cell-based system of Madin,Darby canine kidney epithelial cells. A simple 2D IEF/SDS/PAGE-based image analysis procedure was designed to find candidate substrates in conditioned media of Madin,Darby canine kidney cells expressing meprin in zymogen or in active form. The method enabled the discovery of hitherto unkown meprin substrates with shortened (non-trypsin-generated) N- and C-terminally truncated cleavage products in peptide fragments upon LC-MS/MS analysis. Of 22 (17 nonredundant) candidate substrates identified, the proteolytic processing of vinculin, lysyl oxidase, collagen type V and annexin A1 was analysed by means of immunoblotting validation experiments. The classification of substrates into functional groups may propose new functions for meprins in the regulation of cell homeostasis and the extracellular environment, and in innate immunity, respectively. [source]

Evidence for heritable predisposition to epigenetic silencing of MLH1

INTERNATIONAL JOURNAL OF CANCER, Issue 8 2007
Huiping Chen
Abstract Epigenetic silencing of MLH1 is the most common cause of defective DNA mismatch repair in endometrial and colorectal cancers. We hypothesized that variation in the MLH1 gene might contribute to the risk for MLH1 methylation and epigenetic silencing. We undertook a case-control study to test for the association between MLH1 variants and abnormal MLH1 methylation. Eight MLH1 SNPs were typed in the normal DNA from women with endometrial carcinoma. For these studies, the cases were women whose cancers exhibited MLH1 methylation (N = 98) and the controls were women whose cancers had no MLH1 methylation (N = 219). One MLH1 SNP, rs1800734, located in the MLH1 CpG island at ,93 from the translation start site, was significantly associated with MLH1 methylation as were age at diagnosis and patient body mass index. In validation experiments, a similar-sized cohort of colorectal carcinoma patients (N = 387) showed a similar degree of association with the ,93 SNP; a smaller cohort of endometrial carcinomas (N = 181) showed no association. Combining all 3 cohorts showed an odds ratio of 1.61 (95% CI: 1.20,2.16) for the AA or AG vs. GG genotype at the ,93 SNP. Identification of risk alleles for MLH1 methylation could shed light on mechanisms of epigenetic silencing and may ultimately lead to new approaches to the prevention or treatment of malignancies associated with MLH1 inactivation. © 2007 Wiley-Liss, Inc. [source]

Optimisation of the medium composition for production of protease and soybean peptides by Bacillus subtilis SHZ using response surface methodology

INTERNATIONAL JOURNAL OF FOOD SCIENCE & TECHNOLOGY, Issue 7 2008
Bo Yu
Summary Responses surface methodology was employed to enhance the production of protease and soybean peptides by Bacillus subtilis SHZ. For screening of medium composition significantly influencing protease and soybean peptides yield, the two-level Plackett,Burman design was used. Among thirteen variables tested; KH2PO4, glucose and defatted soybean flour (DSF) were selected based on their high significant effect on both protease activity and soybean peptides yield. Then, a three-level Box,Behnken design was employed to optimise the medium composition for the production of the protease and soybean peptides in submerged fermentation. Mathematical models were then developed to show the effect of each medium composition and their interactions on the production of protease and soybean peptides. The model estimated that, the maximal protease activity (320 ± 1 U mL,1) could be obtained when the concentrations of glucose, KH2PO4, DSF were set at 8,9 g L,1, 2,3 g L,1, 55,65 g L,1, respectively; while a maximal yield of soybean peptides (8.5 ± 0.1 g L,1) could be achieved when the concentrations of glucose, KH2PO4, DSF were set at 7,9 g L,1, 3,4 g L,1 and 55,58 g L,1, respectively. These predicted values were also verified by validation experiments. [source]

Assessment of nutritional status in rhesus monkeys: comparison of dual-energy X-ray absorptiometry and stable isotope dilution

JOURNAL OF MEDICAL PRIMATOLOGY, Issue 3 2005
Stéphane Blanc
Abstract:, Body composition estimates from dual-energy X-ray absorptiometry and stable isotope dilution (2H and 18O) were compared in 61 rhesus monkeys (Macaca mulatta) from the ongoing long-term energy restriction study at the University of Wisconsin. Their average age was 18.9 ± 2.5 y/o. Of the animals, 51% were in the energy restricted group and 38% were females. Although the correlation between methods was highly significant for fat mass (R2 = 0.97, SEE = 0.25 kg or 7.5%, P < 0.0001) and fat-free mass (R2 = 0.98, SEE = 0.29 kg or 3.6%, P < 0.0001), we observed that dual-energy X-ray absorptiometry underestimated fat mass by 0.67 ± 0.26 kg (7.5%, P < 0.0001) and overestimated fat-free mass by 0.57 ± 0.29 kg (20%, P < 0.0001) when compared with isotope dilution. Taken together with data from the literature, the present results emphasize the usefulness of dual-energy X-ray absorptiometry to derive body composition and thus nutritional status in monkeys, but demonstrate the importance of validation experiments for a given DXA model and software. [source]

Talairach-Based Parcellation of Neonatal Brain Magnetic Resonance Imaging Data: Validation of a New Approach

FPRL-1 induces modifications of migration-associated proteins in human neutrophils

PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 17 2006
Karsten Boldt
Abstract Human polymorphonuclear neutrophils (PMNs) are an important cell population of the innate immune system, which migrates following concentration gradients of chemokines or chemoattractants to locations of infection and inflammation in order to eliminate invading microorganisms and cell debris. For both migration and adhesion of PMNs to various tissues, the dynamic remodeling of the cytoskeleton is key prerequisite. In this context, the formyl peptide receptor-like,1 (FPRL-1) is an important chemoattractant receptor expressed on PMNs. In this study, we show that a short stimulation of FPRL-1 with either a synthetic peptide ligand (W-peptide) or a natural ligand (sCK,8-1) changes the protein pattern of PMNs as assessed by 2-D-DIGE. MS analysis of selected deregulated protein species resulted in the identification of proteins that are involved in the remodeling process of the actin- and tubulin-based cytoskeleton, such as L -plastin, moesin, cofilin, and stathmin. Subsequent validation experiments performed either by Western blotting or phosphoprotein-specific gel staining (Pro-Q Diamond) revealed that L -plastin is phosphorylated, whereas moesin, cofilin, and stathmin are dephosphorylated in PMNs upon FPRL-1 stimulation. These findings suggest that FPRL-1 signaling targets proteins that regulate the motility of PMNs and moreover show that 2-D-DIGE is a technique capable of detecting and quantifying differently modified (e.g., phosphorylated) protein variants. [source]

Development of a validated liquid chromatography/tandem mass spectrometry method for the distinction of thyronine and thyronamine constitutional isomers and for the identification of new deiodinase substrates

RAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 20 2008
Susanne Piehl
Thyronines (THs) and thyronamines (TAMs) are two groups of endogenous iodine-containing signaling molecules whose representatives differ from each other only regarding the number and/or the position of the iodine atoms. Both groups of compounds are substrates of three deiodinase isozymes, which catalyze the sequential reductive removal of iodine from the respective precursor molecule. In this study, a novel analytical method applying liquid chromatography/tandem mass spectrometry (LC-MS/MS) was developed. This method permitted the unequivocal, simultaneous identification and quantification of all THs and TAMs in the same biological sample. Furthermore, a liquid-liquid extraction procedure permitting the concurrent isolation of all THs and TAMs from biological matrices, namely deiodinase (Dio) reaction mixtures, was established. Method validation experiments with extracted TH and TAM analytes demonstrated that the method was selective, devoid of matrix effects, sensitive, linear over a wide range of analyte concentrations and robust in terms of reproducible recoveries, process efficiencies as well as intra-assay and inter-assay stability parameters. The method was applied to study the deiodination reactions of iodinated THs catalyzed by the three deiodinase isozymes. With the HPLC protocol developed herein, sufficient chromatographic separation of all constitutional TH and TAM isomers was achieved. Accordingly, the position of each iodine atom removed from a TH substrate in a Dio-catalyzed reaction was backtracked unequivocally. While several established deiodination reactions were verified, two as yet unknown reactions, namely the phenolic ring deiodination of 3,,5,-diiodothyronine (3,,5,-T2) by Dio2 and the tyrosyl ring deiodination of 3-monoiodothyronine (3-T1) by Dio3, were newly identified. Copyright © 2008 John Wiley & Sons, Ltd. [source]

Analysis of neuroactive amino acids from microdialysate samples by fluorescence detection using a modification of the 6-aminoquinolyl- N -hydroxysuccinimidyl carbamate method

BIOMEDICAL CHROMATOGRAPHY, Issue 10 2005
M. Teresa Oreiro-García
Abstract A sensitive and rapid reversed-phase high-performance liquid chromatographic method using pre-column derivatization with 6-aminoquinolyl- N -hydroxysuccinimidyl carbamate (AQC) and fluorescence detection is reported. By directly derivatizing microdialysate samples with AQC, an automatic and rapid simultaneous measurement of aspartate, serine, glutamate, glycine and histidine was developed. Excellent linearity (r2 , 0.998) was achieved for the standard mixture used for the validation experiments. Within-day and between-day precision was less than 6.2%, and the accuracy ranged from 95 to 105.2% in standards. This method is suitable for single run analysis of a high number of small volume microdialysate samples from rat hippocampus. Amino acids from microdialysate samples were quantified with RSD for reproducibility below 2%, and at approximately 0.1% for retention time. Copyright © 2005 John Wiley & Sons, Ltd. [source]

Optimization of cultivation conditions in spin tubes for Chinese hamster ovary cells producing erythropoietin and the comparison of glycosylation patterns in different cultivation vessels

BIOTECHNOLOGY PROGRESS, Issue 3 2010
Jure Strnad
Abstract This article describes the optimization of cultivation factor settings, that is the shaking rate and working volume in 50 mL spin tubes for a Chinese hamster ovary cell line expressing recombinant human ,-erythropoietin, using a response D-optimal surface method. The main objectives of the research were, firstly, to determine a setting in which the product titer and product quality attributes in spin tubes are equivalent to those in 250 mL shake flasks in a seven day batch and, secondly, to find a setting in which the product titer is maximal. The model for product titer prediction as a function of shaking rate and working volume in the defined design space was successfully applied to the optimization of cultivation conditions in spin tubes for the tested cell line. Subsequently, validation experiments were carried out simultaneously in spin tubes, shake flasks and bench scale bioreactors to compare cell culture performance parameters such as growth, productivity and product quality attributes in the form of isoform profiles and glycan antennarity structures. The results of the experiments showed that similar cell culture performance and product quality could be achieved in spin tubes when compared to shake flasks. Additionally, bioreactor titers could be reproduced in spin tubes at high shaking rates and low working volumes, but with differing product quality. Cultivation at lower shaking rates in spin tubes and shake flasks produced a glycoprotein with a product quality slightly comparable to that from bioreactors, but with titers being only two thirds. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source]

The use of small molecule high-throughput screening to identify inhibitors of the proteinase 3-NB1 interaction

CLINICAL & EXPERIMENTAL IMMUNOLOGY, Issue 2 2010
M. Choi
Summary Anti-neutrophil cytoplasmic antibodies (ANCA) to proteinase 3 (PR3) are found in patients with small-vessel vasculitis. PR3-ANCA bind strongly to membrane PR3 (mPR3) that is presented by the NB1 receptor. We performed high-throughput screening using a small molecule library to identify compounds that inhibit PR3-NB1 binding. We established a human embryonic kidney (HEK293) cell-based system, where approximately 95 ± 2% of the NB1-transfected cells expressed the NB1 receptor on the cell surface. Addition of 0·1 µg/ml human PR3 to 104 NB1-expressing HEK293 cells resulted in PR3 binding that was detected by immunofluorescence using a fluorescence plate reader assay. We identified 13 of 20 000 molecules that inhibited PR3 binding by >70%. Seven of 13 substances showed reproducible inhibition in four additional validation experiments. Two selected compounds (27519 and 27549) demonstrated a dose-dependent inhibition over a range from 6·25 to 100 µM as measured by the plate reader assay. We used flow cytometry as a second assay, and found that both compounds reproducibly inhibited PR3 binding to NB1-transfected HEK293 cells at 50 µM (inhibition to 42 ± 4% with compound 27519 and to 47 ± 6% with compound 27549 compared to the dimethylsulphoxide control). Furthermore, compounds 27519 and 27549 also inhibited binding of exogenous PR3 to human neutrophils. In contrast, the compounds did not decrease mPR3 expression on resting neutrophils, but reduced the tumour necrosis factor-,-mediated mPR3 increase on NB1pos neutrophils when present continuously during the assay. The findings suggest that small inhibitory compounds provide a potential therapeutic tool to reduce mPR3 by preventing its binding to NB1. [source]