bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Concentration of Circulating Corticotropin-releasing Hormone mRNA in Maternal Plasma Is Increased in Preeclampsia
Authors: Ng Enders K O , Leung Tse N , Tsui Nancy B Y , Lau Tze K , Panesar Nirmal S , Chiu Rossa W K , Lo Y M Dennis 
Published: February 2004

AbstractBackground: Increased fetal DNA in maternal plasma/serum has been reported in pregnancies complicated by preeclampsia. We hypothesize that fetal RNA may also be increased in maternal plasma in preeclampsia.Methods: We developed a real-time quantitative reverse transcription-PCR assay to measure the concentration of the mRNA of the corticotropin-releasing hormone (CRH) locus. Peripheral blood samples were obtained from healthy pregnant women both before and 2 h after delivery. Peripheral blood samples were also obtained from women suffering from preeclampsia and controls matched for gestational age. Plasma was harvested from these samples, and RNA was extracted. Plasma RNA was subjected to analysis by the reverse transcription-PCR assay.Results: CRH mRNA was detected in the plasma of 10 healthy pregnant women in the third trimester. CRH mRNA was found to be cleared very rapidly after cesarean section, with no detectable signal by 2 h postpartum. Plasma CRH mRNA concentrations were 1070 and 102 copies/mL, respectively, in 12 preeclamptic women and 10 healthy pregnant women matched for gestational age (Mann–Whitney test, P &lt;0.001).Conclusion: Plasma CRH mRNA represents a new molecular marker for preeclampsia. Maternal plasma RNA is gender- and polymorphism-independent and may allow noninvasive gene-expression profiling of an unborn fetus.
Purification of Human Kallikrein 6 from Biological Fluids and Identification of its Complex with α1-Antichymotrypsin
Authors: Hutchinson Shirley , Luo Liu-Ying , Yousef George M , Soosaipillai Antoninus , Diamandis Eleftherios P 
Published: February 2004

AbstractBackground: Human kallikrein 6 (hK6) is significantly increased in serum in many patients with ovarian cancer and may have a role in amyloid precursor processing and Alzheimer disease. The forms of hK6 in biological fluids are poorly characterized.Methods: hK6 protein was immunoaffinity-purified and positively identified by Western blotting, N-terminal sequencing, and mass spectrometry. hK6 in cerebrospinal fluid (CSF), milk, ascites, and serum was size-fractionated by chromatography and then measured by a highly sensitive and specific immunoassay. Hybrid assays were performed to detect the possible interactions between hK6 and proteinase inhibitors in CSF, milk, ascites fluid, and serum.Results: N-Terminal sequencing identified hK6 in the proform in both CSF and milk. hK6 exists in two forms in milk and ascites fluid: a free form with a molecular mass of ∼25 kDa and a higher molecular mass form. Hybrid sandwich assays (capture antibody for hK6 and detection antibody for inhibitors), utilizing a panel of known serine protease inhibitors, indicated that α1-antichymotrypsin forms a complex with hK6 in milk and ascites fluid. Only the free form of hK6 was detected in CSF and serum.Conclusions: hK6 exists mainly as a proenzyme in milk and CSF. A fraction of this enzyme is partially complexed with α1-antichymotrypsin in milk and ascites fluid of ovarian cancer patients.
Comprehensive Proteomic Profiling Identifies Serum Proteomic Signatures for Detection of Hepatocellular Carcinoma and Its Subtypes
Authors: Poon Terence C W , Yip Tai-Tung , Chan Anthony T C , Yip Christine , Yip Victor , Mok Tony S K , Lee Conrad C Y , Leung Thomas W T , Ho Stephen K W , Johnson Philip J 
Published: February 2004

Abstract
Background: Detection of hepatocellular carcinoma (HCC) in patients with chronic liver disease (CLD) is difficult. We investigated the use of comprehensive proteomic profiling of sera to differentiate HCC from CLD.
Methods: Proteomes in sera from 20 CLD patients with α-fetoprotein (AFP) &lt;500 μg/L (control group) and 38 HCC patients (disease group) were profiled by anion-exchange fractionation (first dimension), two types (IMAC3 copper and WCX2) of ProteinChip® Arrays (second dimension), and time-of-flight mass spectrometry (third dimension). Bioinformatic tests were used to identify tumor-specific proteomic features and to estimate the values of the tumor-specific proteomic features in the diagnosis of HCC. Cross-validation was performed, and we also validated the models with pooled sera from the control and disease groups, serum from a CLD patient with AFP &gt;500 μg/L, and postoperative sera from two HCC patients.
Results: Among 2384 common serum proteomic features, 250 were significantly different between the HCC and CLD cases. Two-way hierarchical clustering differentiated HCC and CLD cases. Most HCC cases with advanced disease were clustered together and formed two subgroups that contained significantly more cases with lymph node invasion or distant metastasis. For differentiation of HCC and CLD by an artificial network (ANN), the area under the ROC curve was 0.91 (95% confidence interval, 0.82–1.01; P &lt;0.0005) for all cases and 0.954 (95% confidence interval, 0.881–1.027; P &lt;0.0005) for cases with nondiagnostic serum AFP (&lt;500 μg/L). At a specificity of 90%, the sensitivity was 92%. Both cluster analysis and ANN correctly classified the pooled serum samples, the CLD serum sample with increased AFP, and the HCC patient in complete remission.
Conclusion: Tumor-specific proteomic signatures may be useful for detection and classification of hepatocellular cancers.
Scanning for Mutations of the Ryanodine Receptor (RYR1) Gene by Denaturing HPLC: Detection of Three Novel Malignant Hyperthermia Alleles
Authors: Tammaro Angela , Bracco Adele , Cozzolino Santolo , Esposito Maria , Di Martino Antonietta , Savoia Gennaro , Zeuli Laura , Piluso Giulio , Aurino Stefania , Nigro Vincenzo 
Published: February 2004

AbstractBackground: Malignant hyperthermia (MH) is a fatal autosomal dominant pharmacogenetic disorder characterized by skeletal muscle hypertonicity that causes a sudden increase in body temperature after exposure to common anesthetic agents. The disease is genetically heterogeneous, with mutations in the gene encoding the skeletal muscle ryanodine receptor (RYR1) at 19q13.1 accounting for up to 80% of the cases. To date, at least 42 RYR1 mutations have been described that cause MH and/or central core disease. Because the RYR1 gene is huge, containing 106 exons, molecular tests have focused on the regions that are more frequently mutated. Thus the causative defect has been identified in only a fraction of families as linked to chromosome 19q, whereas in others it remains undetected.Methods: We used denaturing HPLC (DHPLC) to analyze the RYR1 gene. We set up conditions to scan the 27 exons to identify both known and unknown mutations in critical regions of the protein. For each exon, we analyzed members from 52 families with positive in vitro contracture test results, but without preliminary selection by linkage analysis.Results: We identified seven different mutations in 11 MH families. Among them, three were novel MH alleles: Arg44Cys, Arg533Cys, and Val2117Leu.Conclusion: Because of its sensitivity and speed, DHPLC could be the method of choice for the detection of unknown mutations in the RYR1 gene.
Rapid Screening of Multiple β-Globin Gene Mutations by Real-Time PCR on the LightCycler: Application to Carrier Screening and Prenatal Diagnosis of Thalassemia Syndromes
Authors: Vrettou Christina , Traeger-Synodinos Joanne , Tzetis Maria , Malamis George , Kanavakis Emmanuel 
Published: February 2004

Abstract
Background: Hemoglobinopathies are priority genetic diseases for prevention programs. Rapid genotype characterization is fundamental in the diagnostic laboratory, especially when offering prenatal diagnosis for carrier couples.
Methods: As a model, we designed a protocol based on the LightCycler™ technology to screen for a spectrum of β-globin gene mutations in the Greek population. Design was facilitated by dual fluorochrome detection and close proximity of many mutations. Three probe sets were capable of screening 95% of β-globin gene mutations in the Greek population, including IVSII-745C→G, HbS, Cd5-CT, Cd6-A, Cd8-AA, IVSI-1G→A, IVSI-5G→A, IVSI-6T→C, IVSI-110G→A, and Cd39 C→T.
Results: The protocol, standardized by analysis of 100 β-thalassemia heterozygotes with known mutations, was 100% reliable in distinguishing wild-type from mutant alleles. Subsequent screening of 100 Greek β-thalassemia heterozygotes with unknown mutations found 96 of 100 samples heterozygous for 1 of the 10 mutations, although melting curves were indistinguishable for mutations ΗbS/Cd6 and IVSI-5/IVSI-1, indicating a need of alternative methods for definitive diagnosis. One sample demonstrating a unique melting curve was characterized by sequencing as Cd8/9+G. Three samples carried mutations outside the gene region covered by the probes. The protocol was 100% accurate in 25 prenatal diagnosis samples, with 14 different genotype combinations diagnosed. The protocol was also flexible, detecting five β-globin gene mutations from other population groups (IVSI-1G→T, IVSI-5G→C, IVSI-116T→G, Cd37 TGG→TGA, and Cd41/42 −TCTT).
Conclusions: The described LightCycler system protocol can rapidly screen for many β-globin gene mutations. It is appropriate for use in many populations for directing definitive mutation diagnosis and is suited for rapid prenatal diagnosis with low cost per assay.
Rapid Detection of β-Globin Gene Mutations and Polymorphisms by Temporal Temperature Gradient Gel Electrophoresis
Authors: Shaji Ramachandran V , Edison Eunice Sindhuvi , Poonkuzhali Balasubramanian , Srivastava Alok , Chandy Mammen 
Published: February 2004

Abstract
Background: Inherited hemoglobin disorders represent the most common Mendelian disease worldwide. Prevention programs based on molecular diagnosis of heterozygous carriers and/or patients require the use of reliable mutation scanning methods in at-risk populations.
Methods: We developed a rapid and highly specific mutation-screening test based on temporal temperature gradient gel electrophoresis (TTGE). We analyzed 889 β-thalassemia genes from homozygous β-thalassemia patients and unrelated individuals with heterozygous β-thalassemia. Previously reported common mutations were screened by reverse dot blots using allele-specific probes. The rare mutations were analyzed by TTGE.
Results: We found common mutations in 753 β-thalassemia genes. TTGE analysis in the rest of the genes showed the presence of mutations in different regions of the β-globin gene in 134 of them, and these mutations were characterized by DNA sequencing. In the two genes in which mutations were not identified, large deletions spanning β-globin gene were suspected.
Conclusions: Compared with other approaches for comprehensive mutation screening, the reported method is rapid, highly sensitive, cost-effective, and suitable for high-throughput screening of a large number of samples.
External Quality Assurance Program for PCR Amplification of Genomic DNA: An Italian Experience
Authors: Raggi Claudia Casini , Pinzani Pamela , Paradiso Angelo , Pazzagli Mario , Orlando Claudio 
Published: February 2004

AbstractBackground: External quality assurance (EQA) programs for diagnostic tests based on nucleic acid amplification have not been widely implemented in clinical laboratories and remain limited to few tests. Development of specific EQA programs based on application-based proficiency testing for any diagnostic molecular target is challenging. Development of EQA trials based on methodologic proficiency testing and directed to the evaluation of analytical aspects common to the majority of PCR-based tests may be valuable.Methods: We developed an EQA program for evaluation of DNA extraction and amplification and analysis of products after PCR. Participants received a package containing primers and reference materials to evaluate three specific controls for, respectively, DNA extraction (quality and quantity), PCR performance (specificity and efficiency), and interpretation of results after electrophoresis. Each participant was asked to return to the organizers a form with their numerical results and an aliquot of all amplified samples for joint evaluation.Results: Results varied in all phases of the experimental procedure: preamplification, amplification, and post-PCR interpretation. To give a general estimation on the quality of performances for each laboratory, we designed a score scheme in which the results of any specific action were evaluated on the basis of the distribution around the median consensus values. The maximum possible score was 84. On the basis of total score obtained by each laboratory, we created a qualitative ranking list that provided the final interpretation of results as excellent (&gt;63 points; n = 4 laboratories), good (53–63 points; n = 13), sufficient (42–52 points; n = 15), poor (31–41 points; n = 3), and not acceptable (&lt;31 points; n = 4).Conclusions: This survey demonstrates the importance of EQA trials based on methodologic proficiency testing directed to evaluation of analytical aspects common to the majority of PCR-based tests.
Indicators of Erythrocyte Damage after Microwave Warming of Packed Red Blood Cells
Authors: Hirsch Jan , Menzebach Axel , Welters Ingeborg Dorothea , Dietrich Gerald Volker , Katz Norbert , Hempelmann Gunter 
Published: February 2004

Abstract
Background: Localized overheating of packed red blood cells (PRBCs) after microwave warming with consequent damage to erythrocytes has been reported. We therefore compared possible cellular markers of erythrocyte damage, as measured by flow cytometry, with laboratory indicators of hemolysis to evaluate the effects of microwave warming on PRBCs.
Methods: PRBC samples were warmed to room temperature or to 37, 42, 47, 52, or 57 °C in a water bath. Flow cytometry was performed after fluorescein labeling using antibodies to spectrin, Ca2+-ATPase, and Na+-K+-ATPase. The forward-to-sideward scatter (FSC/SSC) ratio and antibody binding were evaluated. Plasma free hemoglobin (FHb) and α-hydroxybutyrate dehydrogenase (HBDH) were measured immediately after heating and after 48 h. In addition, all measurements were made before and after the heating of PRBCs to 35 °C by a microwave blood warmer.
Results: Analysis of 15 000 erythrocytes showed a decrease in the FSC/SSC ratio and antibody binding above 47 °C [at 37 °C, median (SD) of 94.2 (7.4) with 0.07 (0.05)% fluorescein-positive; at 52 °C, median (SD) of 177.0 (19.0) with 18.5 (6.4)% positively gated; P &lt;0.001]. FHb [room temperature, 0.3 (0.2) g/L] was increased 2-fold at 37 and 42 °C, 4-fold at 47 °C, and 25-fold at 52 °C. HBDH increased in parallel. Hemolysis markers showed an additional twofold increase 48 h after heating to 42 and 47 °C. Microwave heating to 35 °C did not produce significant changes of any marker.
Conclusions: All markers of cellular damage were altered after heating to &gt;47 °C, and a substantial part of hemolysis was delayed. The methodology can be used for future testing of other blood warming devices.
High-Resolution Genotyping by Amplicon Melting Analysis Using LCGreen
Authors: Wittwer Carl T , Reed Gudrun H , Gundry Cameron N , Vandersteen Joshua G , Pryor Robert J 
Published: February 2004

AbstractBackground: High-resolution amplicon melting analysis was recently introduced as a closed-tube method for genotyping and mutation scanning (Gundry et al. Clin Chem 2003;49:396–406). The technique required a fluorescently labeled primer and was limited to the detection of mutations residing in the melting domain of the labeled primer. Our aim was to develop a closed-tube system for genotyping and mutation scanning that did not require labeled oligonucleotides.Methods: We studied polymorphisms in the hydroxytryptamine receptor 2A (HTR2A) gene (T102C), β-globin (hemoglobins S and C) gene, and cystic fibrosis (F508del, F508C, I507del) gene. PCR was performed in the presence of the double-stranded DNA dye LCGreen, and high-resolution amplicon melting curves were obtained. After fluorescence normalization, temperature adjustment, and/or difference analysis, sequence alterations were distinguished by curve shape and/or position. Heterozygous DNA was identified by the low-temperature melting of heteroduplexes not observed with other dyes commonly used in real-time PCR.Results: The six common β-globin genotypes (AA, AS, AC, SS, CC, and SC) were all distinguished in a 110-bp amplicon. The HTR2A single-nucleotide polymorphism was genotyped in a 544-bp fragment that split into two melting domains. Because melting curve acquisition required only 1–2 min, amplification and analysis were achieved in 10–20 min with rapid cycling conditions.Conclusions: High-resolution melting analysis of PCR products amplified in the presence of LCGreen can identify both heterozygous and homozygous sequence variants. The technique requires only the usual unlabeled primers and a generic double-stranded DNA dye added before PCR for amplicon genotyping, and is a promising method for mutation screening.
Diagnostic Accuracy of Fecal Calprotectin Assay in Distinguishing Organic Causes of Chronic Diarrhea from Irritable Bowel Syndrome: A Prospective Study in Adults and Children
Authors: Carroccio Antonio , Iacono Giuseppe , Cottone Mario , Di Prima Lidia , Cartabellotta Fabio , Cavataio Francesca , Scalici Calogero , Montalto Giuseppe , Di Fede Gaetana , Rini GiovamBattista , Notarbartolo Alberto , Averna Maurizio R 
Published: February 2004

AbstractBackground: Fecal calprotectin (FC) has been proposed as a marker of inflammatory bowel disease (IBD), but few studies have evaluated its usefulness in patients with chronic diarrhea of various causes. We evaluated the diagnostic accuracy of a FC assay in identifying “organic” causes of chronic diarrhea in consecutive adults and children.Methods: We consecutively enrolled 70 adult patients (30 males, 40 females; median age, 35 years) and 50 children (20 males, 30 females; median age, 3.5 years) with chronic diarrhea of unknown origin. All patients underwent a complete work-up to identify the causes of chronic diarrhea. FC was measured by ELISA.Results: In adult patients, FC showed 64% sensitivity and 80% specificity with 70% positive and 74% negative predictive values for organic causes. False-positive results (8 of 40 cases) were associated with the use of aspirin (3 cases) or nonsteroidal antiinflammatory drugs (1 case) and with the presence of concomitant liver cirrhosis (3 cases). False-negative results mainly included patients suffering from celiac disease (5 cases). Patients with IBD (9 cases) were identified with 100% sensitivity and 95% specificity. In pediatric patients, sensitivity was 70%, specificity was 93%, and positive and negative predictive values were 96% and 56%. False-negative results (11 of 35 cases) were associated mainly with celiac disease (6 cases) or intestinal giardiasis (2 cases).Conclusions: FC assay is an accurate marker of IBD in both children and adult patients. In adults, false negatives occur (e.g., in celiac disease) and false-positive results are seen in cirrhosis or users of nonsteroidal antiinflammatory drugs. Diagnostic accuracy is higher in children.
Convenient and Effective Method for Removing Fibrinogen from Serum Specimens before Protein Electrophoresis
Authors: Qiu Ling L , Levinson Stanley S , Keeling Kristen L , Elin Ronald J 
Published: February 2004

AbstractBackground: Fibrinogen in serum specimens can be misinterpreted on protein electrophoresis as a monoclonal protein. We evaluated selective precipitation of fibrinogen with ethanol.Methods: Pooled human plasma was mixed with absolute ethanol or saline (final concentrations of 40, 80, 100, 120, and 160 mL/L) and incubated at 4 °C overnight or placed in an ice bath for 15 min. After centrifugation, the supernatants and resuspended pellets were used for protein electrophoresis and quantitative measurements of protein and fibrinogen.Results: The fibrinogen band was effectively eliminated from the electrophoretic pattern in the plasma samples treated with ethanol at 100 mL/L and incubated in an ice bath for 15 min without a significant change in immunoglobulin concentrations. The 100 mL/L ethanol did not noticeably change the electrophoretic pattern of monoclonal immunoglobulins. This approach allowed analysis of a sample collected from an arteriovenous shunt kept open with heparin.Conclusions: Ethanol, 100 mL/L, can selectively precipitate fibrinogen without significantly interfering with the immunoglobulins. The precipitation process can be completed in 15 min at 0–4 °C and can avoid the need to obtain another blood sample.
Strategy for Analysis of Cardiac Troponins in Biological Samples with a Combination of Affinity Chromatography and Mass Spectrometry
Authors: Labugger Ralf , Simpson Jeremy A , Quick Michelle , Brown Heather A , Collier Christine E , Neverova Irina , Van Eyk Jennifer E 
Published: February 2004

AbstractBackground: Cardiac troponins are modified during ischemic injury and are found as a heterogeneous mixture in blood of patients with cardiovascular diseases. We present a strategy to isolate cardiac troponins from human biological material, by use of affinity chromatography, and to provide samples ready for direct analysis by mass spectrometry.Methods: Cardiac troponins were isolated from human left ventricular tissue by affinity chromatography. Isolated troponins were either eluted and analyzed by Western blot or enzymatically digested while bound to affinity beads. The resulting peptide mixture was subjected to mass spectrometry for protein identification and characterization. The same method was used to analyze serum from patients with acute myocardial infarction (AMI).Results: Affinity chromatography with antibodies specific for one cardiac troponin subunit facilitated the isolation of the entire cardiac troponin complex from myocardial tissue. The three different proteases used for enzymatic digestion increased the total protein amino acid sequence coverage by mass spectrometry for the three cardiac troponin subunits. Combined amino acid sequence coverages for cardiac troponin I, T, and C (cTnI, cTnT, cTnC) were 54%, 48%, and 40%, respectively. To simulate matrix effects on the affinity chromatography–mass spectrometry approach, we diluted tissue homogenate in cardiac troponin-free serum. Sequence coverages in this case were 44%, 41%, and 19%, respectively. Finally, affinity chromatography–mass spectrometry analysis of AMI serum revealed the presence of cardiac troponins in a wide variety of its free and/or complexed subunits, including the binary cTnI-cTnC and cTnI-cTnC-cTnT complexes.Conclusions: Affinity chromatography–mass spectrometry allows the extraction and analysis of cardiac troponins from biological samples in their natural forms. We were, for the first time, able to directly confirm the presence of cardiac troponin complexes in human serum after AMI. This approach could assist in more personalized risk stratification as well as the search for reference materials for cardiac troponin diagnostics.