bluuebunny/crossref_metadata_2025_split huggingface dataset:
Anomalous Structure of Urinary Glycosaminoglycans in Patients with Pseudoxanthoma Elasticum
Authors: Maccari Francesca , Gheduzzi Dealba , Volpi Nicola 
Published: February 2004

Abstract
Background: Pseudoxanthoma elasticum (PXE) is a hereditary connective tissue disease in which proteoglycans have altered properties. We investigated whether altered proteoglycan metabolism occurs in vivo and may be reflected in the urine of PXE individuals by analyzing the excreted polysaccharides.
Methods: We measured sulfated glycosaminoglycans in the urine of 10 PXE-affected patients, 12 healthy carriers, and 20 healthy controls by agarose gel electrophoresis. Chondroitin sulfate and heparan sulfate disaccharides were also quantified by treatment with specific lyases and separation of products by chromatography.
Results: Total polysaccharides were 34% lower in the urine of PXE-affected patients and 17% lower in healthy carriers than in the control group. Chondroitin sulfate was significantly (P &lt;0.01) decreased, and heparan sulfate was significantly increased. The ratio of chondroitin sulfate to heparan sulfate was 2.7 for PXE-affected patients, 2.3 for healthy carriers, and 10.7 for controls. In PXE-affected individuals and carriers, chondroitin sulfate contained more 4-sulfated disaccharide, less 6-sulfated disaccharide, and decreased nonsulfated disaccharide. Heparan sulfate from PXE-affected individuals and healthy carriers produced significantly less N-sulfated disaccharide and more disaccharide sulfated at the C-6 position with no significant abnormality of the nonsulfated disaccharide percentage and sulfates:disaccharide ratio.
Conclusions: The urinary data support the concept that the inherited defect of the ABCC6/MRP6 transporter in PXE alters metabolism of key polysaccharides. Structural analysis of urinary sulfated polyanions may be useful in the diagnosis of PXE.
Amplicon Melting Analysis with Labeled Primers: A Closed-Tube Method for Differentiating Homozygotes and Heterozygotes
Authors: Gundry Cameron N , Vandersteen Joshua G , Reed Gudrun H , Pryor Robert J , Chen Jian , Wittwer Carl T 
Published: February 2004

AbstractBackground: Common methods for identification of DNA sequence variants use gel electrophoresis or column separation after PCR.Methods: We developed a method for sequence variant analysis requiring only PCR and amplicon melting analysis. One of the PCR primers was fluorescently labeled. After PCR, the melting transition of the amplicon was monitored by high-resolution melting analysis. Different homozygotes were distinguished by amplicon melting temperature (Tm). Heterozygotes were identified by low-temperature melting of heteroduplexes, which broadened the overall melting transition. In both cases, melting analysis required ∼1 min and no sample processing was needed after PCR.Results: Polymorphisms in the HTR2A (T102C), β-globin [hemoglobin (Hb) S, C, and E], and cystic fibrosis (F508del, F508C, I507del, I506V) genes were analyzed. Heteroduplexes produced by amplification of heterozygous DNA were best detected by rapid cooling (&gt;2 °C/s) of denatured products, followed by rapid heating during melting analysis (0.2–0.4 °C/s). Heterozygotes were distinguished from homozygotes by a broader melting transition, and each heterozygote had a uniquely shaped fluorescent melting curve. All homozygotes tested were distinguished from each other, including Hb AA and Hb SS, which differed in Tm by &lt;0.2 °C. The amplicons varied in length from 44 to 304 bp. In place of one labeled and one unlabeled primer, a generic fluorescent oligonucleotide could be used if a 5′ tail of identical sequence was added to one of the two unlabeled primers.Conclusion: High-resolution melting analysis of PCR products amplified with labeled primers can identify both heterozygous and homozygous sequence variants.
Exploiting the Enzymatic Recognition of an Unnatural Base Pair to Develop a Universal Genetic Analysis System
Authors: Moser Michael J , Marshall David J , Grenier Jennifer K , Kieffer Collin D , Killeen Anthony A , Ptacin Jerod L , Richmond Craig S , Roesch Eric B , Scherrer Christopher W , Sherrill Christopher B , Van Hout Cris V , Zanton Sara J , Prudent James R 
Published: February 2004

AbstractBackground: With the invention of the DNA chip, genome-wide analysis is now a reality. Unfortunately, solid-phase detection systems such as the DNA chip suffer from a narrow range in quantification and sensitivity. Today the best methodology for sensitive, wide dynamic range quantification and genotyping of nucleic acids is real-time PCR. However, multiplexed real-time PCR technologies require complicated and costly design and manufacturing of separate detection probes for each new target.Methods: We developed a novel real-time PCR technology that uses universal energy transfer probes constructed from An Expanded Genetic Information System (AEGIS) for both quantification and genotyping analyses.Results: RNA quantification by reverse transcription-PCR was linear over four orders of magnitude for the simultaneous analysis of β-actin messenger RNA and 18S ribosomal RNA. A single trial validation study of 176 previously genotyped clinical specimens was performed by endpoint analysis for factor V Leiden and prothrombin 20210A mutation detection. There was concordance for 173 samples between the genotyping results from Invader® tests and the AEGIS universal energy transfer probe system for both factor V Leiden and prothrombin G20210A. Two prothrombin and one factor V sample gave indeterminate results (no calls).Conclusion: The AEGIS universal probe system allows for rapid development of PCR assays for nucleic acid quantification and genotyping.
Application of Optical Trapping for Cells Grown on Plates: Optimization of PCR and Fidelity of DNA Sequencing of p53 Gene from a Single Cell
Authors: Gale James M , Romero Christopher P , Tafoya Gregory B , Conia Jérôme 
Published: February 2004

Abstract
Background: Optical trapping has traditionally been used to visually select and isolate nonadherent cells grown in suspension because cells grown in monolayers will rapidly reattach to surfaces if suspended in solution. We explored methods to slow cell reattachment that are also compatible with high-fidelity PCR.
Methods: Using HeLa cells grown on plates and suspended after trypsinization, we measured the efficiency of capture by retention and movement of the cell by the laser. Success for removing a captured cell by pipette was determined by PCR amplification of the 5S rRNA gene. After optimizing PCR amplification of a 2049-bp region of the p53 gene, we determined PCR fidelity by DNA sequencing.
Results: Addition of bovine serum albumin to suspended cells slowed reattachment from seconds to minutes and allowed efficient trapping. The success rate of removing a cell from the trap by pipette to a PCR tube was 91.5%. The 5S PCR assay also revealed that DNA and RNA that copurify with polymerases could give false-positive results. Sequence analysis of four clones derived from a single cell showed only three polymerase errors in 7200 bp of sequence read and revealed difficulties in reading the correct number in a run of 16 A:T. Comparison of the HeLa and wild-type human sequences revealed several previously unreported base differences and an (A:T)n length polymorphism in p53 introns.
Conclusions: These results represent the first use of optical trapping on adherent cells and demonstrate the high accuracy of DNA sequencing that can be achieved from a single cell.
PCR-based Calibration Curves for Studies of Quantitative Gene Expression in Human Monocytes: Development and Evaluation
Authors: Øvstebø Reidun , Haug Kari Bente Foss , Lande Knut , Kierulf Peter 
Published: February 2004

AbstractBackground: Quantitative reverse transcription-PCR (RT-PCR) used to detect small changes in specific mRNA concentrations is often associated with poor reproducibility. Thus, there is a need for stringent quality control in each step of the protocol.Methods: Real-time PCR-based calibration curves for a target gene, tissue factor (TF), and a reference gene, β-actin, generated from PCR amplicons were evaluated by running cDNA controls. In addition, the reverse transcription step was evaluated by running mRNA controls. Amplification efficiencies of calibrators and targets were determined. Variances within and between runs were estimated, and power statistics were applied to determine the concentration differences that could reliably be detected.Results: Within- and between-run variations (CVs) of cDNA controls (TF and β-actin), extrapolated from reproducible calibration curves (CVs of slopes, 4.3% and 2.7%, respectively) were 4–10% (within) and 15–38% (between) using both daily and “grand mean” calibration curves. CVs for the β-actin mRNA controls were 12% (within) and 19–28% (between). Estimates of each step’s contribution to the total variation were as follows: CVRT-PCR, 28%; CVPCR, 15%; CVRT, 23% (difference between CVRT-PCR and CVPCR). PCR efficiencies were as follows: β-actin calibrator/target, 1.96/1.95; TF calibrator/target, 1.95/1.93. Duplicate measurements could detect a twofold concentration difference (power, 0.8).Conclusions: Daily PCR calibration curves generated from PCR amplicons were reproducible, allowing the use of a grand mean calibration curve. The reverse transcription step contributes the most to the total variation. By determining a system’s total variance, power analysis may be used to disclose differences that can be reliably detected at a specified power.
Use of Capillary Zone Electrophoresis for Differentiating Excessive from Moderate Alcohol Consumption
Authors: Legros Franz J , Nuyens Vincent , Baudoux Michel , Zouaoui Boudjeltia Karim , Ruelle Jean-Luc , Colicis Jacques , Cantraine Francis , Henry Jean-Pol 
Published: February 2004

Abstract
Background: The poorly sialylated transferrin isoforms in serum were analyzed by capillary zone electrophoresis (CZE) to differentiate moderate from heavy alcohol consumption.
Methods: We enrolled 614 volunteers, classified after interviews, self-reported drinking habits, and AUDIT scores as alcohol abusers (consuming &gt;50 g/day ethanol for the previous 3 months or longer; n = 413) or moderate drinkers (&lt;30 g/day ethanol; n = 201). Serum transferrin isoforms were separated at 28 kV and monitored at 214 nm on a P/ACE 5500 CZE with use of fused-silica capillaries and the related CEofix CDT reagent set. Immunosubtraction by anti-human transferrin and electrophoretic migration times identified the isoforms. Previous markers of alcohol abuse and an assay combining anion-exchange minicolumn chromatography with immunoturbidimetry (%CDT) were included in the study. Sensitivities and specificities were compared by ROC analysis.
Results: The asialylated isoform was missing in 95% of moderate drinkers but present in 92% of alcohol misusers. Disialotransferrin had a specificity and sensitivity of 0.75 at a cutoff of 0.7% of total transferrin, whereas the sum (asialo- + disialotransferrin) at a threshold of 1.2% had a sensitivity of 0.73 and a specificity of 0.92. Trisialotransferrin values did not distinguish between the two populations. Sensitivities and specificities of %CDT averaged 0.77 and 0.74, respectively, at a 2.6% cutoff; 0.67 and 0.83 at 2.8%; and 0.63 and 0.90 at 3%. CDT data were more sensitive and specific for males. Conventional biomarkers appeared less discriminating.
Conclusions: Asialotransferrin detected by CZE in sera of alcohol abusers offers the highest discrimination between excessive and moderate drinking.
Validation of the FibroTest Biochemical Markers Score in Assessing Liver Fibrosis in Hepatitis C Patients
Authors: Rossi Enrico , Adams Leon , Prins Alexander , Bulsara Max , de Boer Bastiaan , Garas George , MacQuillan Gerry , Speers David , Jeffrey Gary 
Published: February 2004

Abstract
Background: Determining the stage of fibrosis by liver biopsy is important in managing patients with hepatitis C virus infection. We investigated the predictive value of the proprietary FibroTest score to accurately identify significant fibrosis in Australian hepatitis C patients.
Methods: Serum obtained from 125 confirmed hepatitis C patients before antiviral therapy was analyzed for haptoglobin, α2-macroglobulin, apolipoprotein A1, bilirubin, and γ-glutamyltransferase activity, and the FibroTest score was computed. Liver fibrosis pathology was staged according to a defined system on a scale of F0 to F4. We used predictive values and a ROC curve to assess the accuracy of FibroTest scores.
Results: The prevalence of significant fibrosis defined by liver biopsy was 0.38. The most useful single test for predicting significant fibrosis was serum α2-macroglobulin (cutoff value, 2.52 g/L; sensitivity, 75%; specificity, 67%). The negative predictive value of a FibroTest score &lt;0.1 was 85%, and the positive predictive value of a score &gt;0.6 was 78%. Although 33 of the 125 patients had FibroTest scores &lt;0.1 and were therefore deemed unlikely to have fibrosis, 6 (18%) had significant fibrosis. Conversely, of the 24 patients with scores &gt;0.6 who were likely to have significant fibrosis, 5 (21%) had mild fibrosis. Of the 125 patients in the cohort, 57 (46%) could have avoided liver biopsy, but discrepant results were recorded in 11 of those 57 (19%).
Conclusion: The FibroTest score could not accurately predict the presence or absence of significant liver fibrosis.
Certification of Standard Reference Material 970, Ascorbic Acid in Serum, and Analysis of Associated Interlaboratory Bias in the Measurement Process
Authors: Margolis Sam A , Vangel Mark , Duewer David L 
Published: February 2004

Abstract
Background: The accurate and reproducible measurement of ascorbic acid is essential in delineating the role of ascorbic acid as a diagnostic tool for human disease and for the comparison of data acquired by different laboratories. A stabilized pair of standards of ascorbic acid in human serum, which is compatible with most analytical methods, have been prepared.
Methods: The certification was based on the gravimetric addition of ascorbic acid to metaphosphoric acid-stabilized, ascorbic acid-depleted serum and NIST liquid chromatography–electrochemical measurements. The NIST results were analyzed statistically for homogeneity, and the expanded uncertainty of each SRM was calculated using all of the NIST data. An interlaboratory comparison exercise was also performed.
Results: These materials, Standard Reference Material (SRM) 970 Ascorbic Acid in Serum, Level I and Level II, are homogeneous and are certified to contain (10.07 ± 0.21) and (30.57 ± 0.28) mmol ascorbic acid/L of solution (expanded uncertainty), respectively. In the interlaboratory comparison (n = 17), the relative SDs for the two materials were 22% and 19%.
Conclusions: Two lots of serum, each containing different amounts of ascorbic acid stabilized in metaphosphoric acid, have been prepared and characterized. Many laboratories provide inaccurate results.
Toxicity of Free p-Cresol: A Prospective and Cross-Sectional Analysis
Authors: De Smet Rita , Van Kaer Jacqueline , Van Vlem Bruno , De Cubber Antoine , Brunet Philippe , Lameire Norbert , Vanholder Raymond 
Published: February 2004

AbstractBackground: Uremic syndrome is the consequence of the retention of solutes usually cleared by the healthy kidneys. p-Cresol can be considered a prototypic protein-bound uremic toxin. It is conceivable, analogous with drugs, that the non-protein-bound fraction of p-cresol exerts toxicity. This aspect had never been evaluated, nor have the factors influencing the free fraction of p-cresol.Methods: In a transsectional study we evaluated the relationship between prehemodialysis free p-cresol and the ratio of free to total p-cresol (F:T) to clinical and biological factors in 44 chronic renal failure patients. The evolution of free p-cresol was assessed prospectively in 12 patients showing a change in serum albumin of at least 5 g/L over time. Hospitalization days attributable to infection and the free p-cresol concentrations were noted over a 1-year period. The impact of free p-cresol in vitro on leukocyte functional capacity was evaluated by chemiluminescence.Results: We observed a correlation between total and free p-cresol (r = 0.84; P &lt;0.001). In the multivariate analyses, free p-cresol and F:T showed a negative correlation with albumin. A shift from normal serum albumin to hypoalbumininemia in 12 patients led to an increase in free p-cresol from 5.9 ± 3.2 to 8.2 ± 4.5 μmol/L (P &lt;0.05; 0.64 ± 0.35 to 0.89 ± 0.49 mg/L). Free p-cresol (P &lt;0.05) was higher in the patients hospitalized for infectious disease. In vitro, free p-cresol was higher in a 25 g/L than in a 50 g/L albumin solution (P &lt;0.05). Leukocyte chemiluminescence production was more inhibited in the low albumin (high free p-cresol) solution (28% ± 6% vs 21% ± 8%; P &lt;0.05).Conclusions: Hypoalbuminemia and total p-cresol increase the free fraction of p-cresol. Patients hospitalized for infections have higher free p-cresol. In vitro, high free p-cresol has a negative impact on leukocyte chemiluminescence production. These data demonstrate the toxicity of free p-cresol.
Low-Positive Anti-Hepatitis C Virus Enzyme Immunoassay Results: An Important Predictor of Low Likelihood of Hepatitis C Infection
Authors: Dufour D Robert , Talastas Mageli , Fernandez Maria D A , Harris Barbara , Strader Doris B , Seeff Leonard B 
Published: February 2004

Abstract
Background: Tests for hepatitis C antibodies (anti-HCV enzyme immunoassays) are usually described as positive or negative. Several studies, mainly in blood donors, have found that specimens with low signal/cutoff (S/C) ratios are commonly negative when tested with a recombinant immunoblot assay (RIBA) or for HCV RNA.
Methods: We retrospectively reviewed 17 418 consecutive anti-HCV results from a screening program for high-risk veterans; 2986 (17.1%) samples were anti-HCV-positive, and 490 (16.4%) had S/C ratios ≤3.7 (low positive). Additional tests were performed in 1814 anti-HCV-positive individuals.
Results: RIBA was performed in 263 patients with low-positive anti-HCV; results were negative in 86%, indeterminate in 12%, and positive in 2%. Only 16 of 140 individuals (11%) with low-positive anti-HCV values were HCV RNA-positive, whereas HCV RNA was positive in 90% of 1435 individuals with high-positive anti-HCV values (P &lt;0.0001). Compared with those with high-positive anti-HCV, individuals with low-positive anti-HCV values were older (P &lt;0.0001) and were less likely to have risk factors for HCV (P &lt;0.0001 for most), multiple increased alanine aminotransferase (ALT) activity values (30% vs 81%; P &lt;0.0001), or positive anti-hepatitis B core antigen (19% vs 59%; P &lt;0.0002). Among 634 individuals with high anti-HCV titers and multiple increased ALT activity values, 95% were HCV RNA-positive.
Conclusions: The S/C ratio is important even in high-risk individuals; laboratories should report the S/C ratio along with anti-HCV EIA results and perform supplemental RIBA testing in those with low-positive values to avoid reporting false-positive results.
Cerebrospinal Fluid Lactate and Pyruvate Concentrations and Their Ratio in Children: Age-related Reference Intervals
Authors: Benoist Jean-François , Alberti Corinne , Leclercq Sandrine , Rigal Odile , Jean-Louis Rosalie , Ogier de Baulny Hélene , Porquet Dominique , Biou Daniel 
Published: February 2004

Abstract
Background: Lactate (L) and pyruvate (P) concentrations in cerebrospinal fluid (CSF) and the L/P ratio have diagnostic value in numerous primary and acquired disorders affecting the central nervous system, but age-related reference values are not available for children.
Methods: We analyzed CSF and blood lactate and pyruvate concentrations and their ratio in a 4-year retrospective survey of a children’s hospital laboratory database. Reference intervals (10th–90th percentiles) were established from data on 197 hospitalized children. A recent regression modeling method was used to normalize and smooth values against age. The model equation of best fit was calculated for each variable.
Results: Slight age-related variations were shown by the model, with an increase in lactate, a decrease in pyruvate, and a resulting increase in the L/P ratio with increasing age. However, the SD did not vary with age. We defined the upper limit of the reference intervals as the 90th percentiles, which from birth to 186 months of age varied continuously from 1.78 to 1.88 mmol/L (6%), 148 to 139 μmol/L (6%), and 16.9 to 19.2 (14%) for lactate, pyruvate, and the L/P ratio, respectively. At a threshold of 2 (in Z-score units), the sensitivity for a subgroup of inborn errors of metabolism (respiratory chain disorders) was 73%, 42%, and 31% for lactate, pyruvate, and the L/P ratio, respectively.
Conclusions: In children, CSF lactate and pyruvate concentrations and their ratio appear to vary slightly with age. Average 90th percentile values of 1.8 mmol/L, 147 μmol/L, and 17, respectively, could be used in infants up to 24 months of age. In older children, age-adjusted reference intervals should be used, especially when values are close to the 90th percentile.
Comparison of Two CYP2D6 Genotyping Methods and Assessment of Genotype-Phenotype Relationships
Authors: Chou Wen-Hwei , Yan Feng-Xiang , Robbins-Weilert Doris K , Ryder Thomas B , Liu Wei Wei , Perbost Clotilde , Fairchild Maureen , de Leon Jose , Koch Walter H , Wedlund Peter J 
Published: February 2004

Abstract
Background: There have been no published reports comparing the CYP450 GeneChip® microarray assay with more standard methods of genetic testing.
Methods: We collected 20-mL blood samples from 236 volunteers for DNA isolation and testing before each individual ingested 60 mg of dextromethorphan, and collected their urine. CYP2D6 alleles *3 to *7, *9, *17, and *41, and multiple CYP2D6 gene copies were tested by allele-specific PCR (AS-PCR), whereas alleles *2 to *4 and *6 to *11 were tested by the Affymetrix CYP450 GeneChip assay. Five of the CYP2D6 alleles (*3, *4, *6, *7, and *9) were tested by both AS-PCR and the CYP450 GeneChip assay in an independent and blinded fashion in 232 of the 236 healthy volunteers. The combined CYP2D6 genotype from both methods was used to divide the population into four subgroups, poor metabolizers (PMs), intermediate metabolizers (IMs), extensive metabolizers (EMs), and ultrarapid metabolizers (UMs), based on their relative function and ability to express the CYP2D6 gene. The urinary elimination of dextromethorphan was assessed in each of these CYP2D6 subgroups.
Results: The CYP2D6*3, *4, *6, *7, and *9 alleles showed a high degree of concordance between the CYP450 GeneChip and AS-PCR methods (&gt;99% concordance). The mean (SD) of the log[dextromethorphan metabolic ratio (MR)] in the four CYP2D6 subgroups was PM = 0.49 (0.38); IM = −1.24 (0.53); EM = −2.35 (0.61); and UM = −2.43 (0.38).
Conclusions: Oligonucleotide microarray technology is an efficient and reliable way to test for CYP2D6 gene variation based on five alleles compared by separate methods. The methodology is influenced by the quality and amount of DNA present. The log(dextromethorphan MR) is a highly variable index that appears to reflect the crude nature of the dextromethorphan MR as an indicator of CYP2D6 in vivo enzyme activity.