bluuebunny/crossref_metadata_2025_split huggingface dataset:
Efficient gene transfer into rhesus repopulating hematopoietic stem cells using a simian immunodeficiency virus–based lentiviral vector system
Authors: Hanawa Hideki , Hematti Peiman , Keyvanfar Keyvan , Metzger Mark E. , Krouse Allen , Donahue Robert E. , Kepes Steve , Gray John , Dunbar Cynthia E. , Persons Derek A. , Nienhuis Arthur W. 
Published: February 2004

Abstract
High-titer, HIV-1–based lentiviral vector particles were found to transduce cytokine-mobilized rhesus macaque CD34+ cells and clonogenic progenitors very poorly (&lt; 1%), reflecting the postentry restriction in rhesus cells to HIV infection. To overcome this barrier, we developed a simian immunodeficiency virus (SIV)–based vector system. A single exposure to a low concentration of amphotropic pseudotyped SIV vector particles encoding the green fluorescent protein (GFP) resulted in gene transfer into 68% ± 1% of rhesus bulk CD34+ cells and 75% ± 1% of clonogenic progenitors. Polymerase chain reaction (PCR) analysis of DNA from individual hematopoietic colonies confirmed these relative transduction efficiencies. To evaluate SIV vector–mediated stem cell gene transfer in vivo, 3 rhesus macaques underwent transplantation with transduced, autologous cytokine-mobilized peripheral blood CD34+ cells following myeloablative conditioning. Hematopoietic reconstitution was rapid, and an average of 18% ± 8% and 15% ± 7% GFP-positive granulocytes and monocytes, respectively, were observed 4 to 6 months after transplantation, consistent with the average vector copy number of 0.19 ± 0.05 in peripheral blood leukocytes as determined by real-time PCR. Vector insertion site analysis demonstrated polyclonal reconstitution with vector-containing cells. SIV vectors appear promising for evaluating gene therapy approaches in nonhuman primate models.
6-Amino-6-deoxychitosan. Preparation and Application as Plasmid Vector in COS-1 Cells
Authors: Satoh Taku , Nagasaki Takeshi , Sakairi Nobuo , Shinkai Seiji 
Published: February 2004

Abstract
6-Amino-6-deoxychitosan was prepared from 6-deoxy-6-halo-N-phthaloylchitosan via 6-azidation. The product has high stereoregularity because of the effective and regioselective reactions. To evaluate 6-amino-6-deoxychitosan as a gene transfer agent, transfection efficiencies of its complexes with plasmid DNA in COS-1 cells were examined.
Controlled Assembly of Dinuclear Metallorings into 1D Coordination Polymer and Mixed-metal Rare Earth Complexes with Red-to-Green Luminescence Properties
Authors: Cai Zheng-Hong , Tang Yu , Liu Wei-sheng , Tan Min-Yu 
Published: February 2004

Abstract
Novel rings connected by chains 1D coordination polymer of rare earth complex have been rationally synthesized by the reaction of Eu(NO3)3·6H2O and L, 1,4-bis{[(2′-benzylaminoformyl)phenoxyl]methyl}benzene. The functional terminal groups (N-benzylsalicylamide) can activate both Eu3+ and Tb3+ to give out their characteristic lights and the mixed-metal complexes of different ratio of these metals have also been prepared. According to the principle of paints, these complexes give out red-to-green luminescence under UV light, so the complexes with yellow and orange luminescence properties have been realized.
Spin-coating Preparation of High Quality Mesoporous Titania Nanofilms
Authors: Bao Ningzhong , Yanagisawa Kazumichi , Lu Xiaohua , Feng Xin 
Published: February 2004

Abstract
Highly ordered crack-free mesoporous titania nanofilms with crystalline anatase framework are obtained by spin-coating glass surface, using the very acidic solution containing ethanol, H2O, TiCl4, and P123 (Pluronic P123, an triblock copolymer H(OCH2CH2)20(OCH(CH3)CH2)70(OCH2CH2)20OH)).
Heck-type Benzylation of Olefins with Benzyl Trifluoroacetates
Authors: Narahashi Hirohisa , Yamamoto Akio , Shimizu Isao 
Published: February 2004

Abstract
A new synthetic method for 1-aryl-2-alkenes from 1-olefins by benzylation treating with benzyl trifluoroacetates using palladium-catalyst is developed on the basis of oxidative addition of benzyl carboxylates to Pd(0) complexes to give benzyl(carboxylato)palladium(II) complexes with cleavage of benzyl–oxygen bond.
A Tripodal Ligand Containing Three Imidazole Groups Inducing Spin Crossover in Both Fe(II) and Fe(III) Complexes; Structures and Spin Crossover Behaviors of the Complexes
Authors: Ohta Hiromi , Sunatsuki Yukinari , Kojima Masaaki , Iijima Seiichiro , Akashi Haruo , Matsumoto Naohide 
Published: February 2004

Abstract
A tripodal hexadentate ligand containing three imidazole groups gives spin-crossover iron complexes in both the +II and the +III oxidation states. The tetrafluoroborate salt of the Fe(II) complex shows a gradual spin transition in the temperature range 110–270 K. The corresponding Fe(III) complex shows a spin transition above 150 K, and at 300 K ca. 87% of the Fe(III) ion is in the high-spin state.
European Concerted Action on Anticoagulation. Quality Assessment of the CoaguChek Mini and TAS PT-NC Point-of-Care Whole-Blood Prothrombin Time Monitors
Authors: Poller Leon , Keown Michelle , Chauhan Nikhil , van den Besselaar Anton M H P , Tripodi Armando , Shiach Caroline , Jespersen Jorgen 
Published: February 2004

Abstract
Background: International Normalized Ratios (INRs) for prothrombin time obtained with the CoaguChek Mini and TAS (RapidPointCoag) PT-NC systems are markedly different and also differ from the “true” INR. There is therefore a need for local quality assessment (QA) of the two systems.
Methods: A set of 60 lyophilized artificially depleted and 60 lyophilized coumarin plasmas were tested at 10 centers on both point-of-care testing monitors. Subsets of three and five plasmas were selected as QA plasmas and compared with the remaining 55 to assess the relative ability of the systems to characterize performance at the individual centers. The incidence of aberrant results (outliers; &gt;15% deviation from the true INR) was also recorded. The expected incidence with the QA plasmas was calculated and compared.
Results: On both systems, INR with the common sets of 55 lyophilized plasmas varied considerably between centers. With the TAS PT-NC, subsets of five and three European Concerted Action on Anticoagulation (ECAA) artificially depleted plasmas gave good correlation with the 55 plasmas, but the coumarin plasmas performed less well. With the CoaguChek Mini, correlation was good with sets of five artificially depleted QA plasmas and reasonable with three but was less satisfactory with the coumarin plasmas. Outliers were detected with both types of plasmas on both test systems but with variable success.
Conclusions: With the TAS PT-NC, three ECAA artificially depleted lyophilized plasmas provided reliable QA, but five lyophilized coumarin plasmas were required. With the CoaguChek Mini, five artificially depleted plasmas gave reliable QA but coumarin plasmas gave poorer results. ECAA QA plasmas provide a local system for checking INRs obtained with monitors of both types.
De Novo Biosynthesis and Radiolabeling of Mammalian Digitalis-Like Factors
Authors: Qazzaz Hassan M A M , Cao (Tim) Zhimin , Bolanowski Duane D , Clark Barbara J , Valdes Roland 
Published: February 2004

AbstractBackground: Digoxin-like immunoreactive factors (DLIFs) are endogenous mammalian cardenolides with structural features similar to those of the plant-derived digitalis compounds. DLIFs and their structurally related forms (Dh-DLIFs) may serve as effectors of ion-transport activity mediated by their interaction with Na,K-ATPase and thus play a role as a new hormonal axis. Although some evidence implicates the adrenal gland as a tissue source for the DLIFs, little is known about the biosynthetic pathway producing these compounds. We now demonstrate de novo biosynthesis of DLIF by incorporation of radioactive carbon (14C) into the structures of both DLIF and Dh-DLIF.Methods: We used a combination of reversed-phase HPLC techniques to separate the radioactive DLIF components after incorporation of 14C into their structure by use of either [1,2-14C]acetic acid or [4-14C]cholesterol as precursors and a Y-1 mouse adrenocortical tumor cell line. We also stimulated and suppressed production of steroidogenesis by use of cAMP analogs and Mevastatin, respectively, to demonstrate the dependence of DLIF production on the cholesterol-dependent biosynthetic pathway. A combination of chromatographic mobility, immunoassays specific for digoxin and dihydrodigoxin, and deglycosylation using 5-sulfosalicylic acid were used to identify the DLIF and Dh-DLIF components.Results: With cholesterol as precursor, the cells produced DLIF (7.5 mCi/mmol) with a labeling efficiency of 10%, whereas with acetate the cells produced DLIF (72.2 mCi/mmol) with a labeling efficiency of 0.08% of the total DLIF produced. The radiolabeled DLIF and Dh-DLIF molecules had identical chromatographic mobilities and stoichiometric removal of sugars as the previously characterized DLIFs isolated from different mammalian species and tissues. With radioactive cholesterol as precursor, the 14C was incorporated into the DLIF-genin portion of the compounds and not the sugars. Interestingly, treatment of Y-1 cells with 8-bromoadenosine 3′:5′-cAMP to stimulate steroidogenesis did not increase production of DLIF or Dh-DLIF but did increase production of progesterone. Mevastatin (5 μmol), an inhibitor of the enzyme hydroxymethylglutaryl-CoA reductase and thus of cholesterol biosynthesis, gave an 85% decrease in the production of 14C-DLIF and progesterone, but only a modest 15% decrease in 14C-Dh-DLIF production.Conclusions: These data demonstrate that the adrenal cell has the cellular machinery necessary for de novo biosynthesis of DLIF and Dh-DLIF starting from a simple carbon pool and also support the concept that cholesterol is a major precursor of the DLIF compounds. This cell culture model provides a source of radiolabeled DLIF compounds for future experimental work.
Interlaboratory Comparison of Fetal Male DNA Detection from Common Maternal Plasma Samples by Real-Time PCR
Authors: Johnson Kirby L , Dukes Kimberly A , Vidaver John , LeShane Erik S , Ramirez Idania , Weber William D , Bischoff Farideh Z , Hahn Sinuhe , Sharma Arun , Dang Dianne X , Hire Lisa M , Bianchi Diana W , Simpson Joe Leigh , Holzgreve Wolfgang , Elias Sherman , Klinger Katherine W 
Published: February 2004

Abstract
Background: Analysis of fetal DNA from maternal plasma by PCR offers great potential for noninvasive prenatal genetic diagnosis. To further evaluate this potential, we developed and validated a standard protocol to determine whether fetal DNA sequences could be reproducibly amplified and measured across multiple laboratories in a common set of specimens.
Methods: Each of five participating centers in a National Institute of Child Health and Human Development consortium collected 20 mL of peripheral blood from 20 pregnant women between 10 and 20 weeks of gestation. The plasma fraction was separated according to a common protocol, divided, and frozen in five aliquots. One aliquot was shipped to each participating laboratory, where DNA was extracted according to a standard protocol. All plasma samples (n = 100) were then analyzed blindly for the presence and quantity of total DNA (GAPDH) and male fetal DNA (SRY) by real-time PCR. Genomic DNA was isolated from female and male cells at one center, quantified, and shipped to the others to serve as calibrators for GAPDH and SRY, respectively.
Results: The amplification of known quantities of DNA was consistent among all centers. The mean quantity of male DNA amplified from maternal plasma when the fetus was male ranged from 51 to 228 genome equivalents (GE)/mL. Qualitative concordance was found overall among centers. The sensitivity of the assay for detection of male DNA when the fetus was male varied from 31% to 97% among centers. Specificity was more consistent (93–100%) with only four false-positive results obtained across the entire study.
Conclusions: All centers were able to consistently amplify frozen and shipped DNA. The PCR procedure used here is reliable and reproducible. Centers that extracted and amplified more DNA per milliliter of maternal plasma had superior sensitivities of Y chromosome sequence detection. The specificity of the assay was more consistent among centers. A robust and thoroughly optimized protocol for the extraction of DNA from maternal plasma is needed to make testing of fetal DNA in maternal plasma a clinically relevant analytical tool.
Rapid Detection of K-ras Mutations in Bile by Peptide Nucleic Acid-mediated PCR Clamping and Melting Curve Analysis: Comparison with Restriction Fragment Length Polymorphism Analysis
Authors: Chen Chiung-Yu , Shiesh Shu-Chu , Wu Sheu-Jen 
Published: February 2004

AbstractBackground: Current methods for detection of K-ras gene mutations are time-consuming. We aimed to develop a one-step PCR technique using fluorescent hybridization probes and competing peptide nucleic acid oligomers to detect K-ras mutations in bile and to compare the efficacy with restriction fragment length polymorphism (RFLP) analysis.Methods: Bile samples were obtained from 116 patients with biliary obstruction, including gallstones (n = 64), benign biliary strictures (n = 6), pancreatic cancer (n = 20), and cholangiocarcinoma (n = 26). The DNA was extracted and subjected to K-ras mutation analysis by real-time PCR and RFLP analysis. Mutations were confirmed by direct sequencing. The sensitivity and specificity were calculated according to the clinical results.Results: The analysis time for real-time PCR was &lt;1 h, whereas RFLP analysis took more than 2 days. With the sensor probe designed for the GAT (G12D) mutant in codon 12 of the K-ras gene, the real-time PCR method also detected the GTT (G12V) mutant. In contrast, a specific sensor probe for the TGT (G12C) mutant detected GAT (G12D), AGT (G12S), and GTT (G12V) mutants in addition to the TGT mutant. The real-time PCR assay allowed the detection of mutation in a 3000-fold excess of wild-type bile DNA. In bile, K-ras codon 12 mutations were detected in 16 of 46 malignant cases by real-time PCR with the TGT probe and 15 by RFLP analysis. All benign cases were wild type.Conclusion: Real-time PCR with a cysteine-specific (TGT) sensor probe can rapidly detect K-ras gene mutations in bile and diagnose malignant biliary obstruction with high specificity.
Measurement of Plasma Free Metanephrine and Normetanephrine by Liquid Chromatography–Tandem Mass Spectrometry for Diagnosis of Pheochromocytoma
Authors: Lagerstedt Susan A , O’Kane Dennis J , Singh Ravinder J 
Published: February 2004

AbstractBackground: Quantification of plasma free metanephrines is usually accomplished by HPLC with electrochemical detection, but sample preparation is labor-intensive and time-consuming, run times are long, and interfering substances sometimes obscure the relevant peaks. The aim of this study was to develop a sensitive and specific LC-MS/MS method for plasma free metanephrines.Methods: After solid-phase extraction, chromatographic separation of normetanephrine (NMN) and metanephrine (MN) was accomplished by use of a cyano analytical column. NMN, MN, d3-NMN, and d3-MN positive ions were detected in the multiple-reaction monitoring mode using the specific transitions m/z 166→134, 180→148, 169→137, and 183→151, respectively, with an atmospheric pressure chemical ionization source.Results: Multiple calibration curves exhibited consistent linearity and reproducibility. Interassay imprecision values (CV; n = 20) for NMN at 0.64, 1.9, and 2.7 nmol/L were 6.6%, 7.8%, and 13%, respectively. Interassay CV for MN at 0.60, 1.2, and 2.1 nmol/L (n = 20) were 9.2%, 6.8%, and 9.8%, respectively. The mean recoveries of NMN and MN relative to the internal standard were 100% and 96%, respectively. The assays were linear between 0.20 and 10.0 nmol/L. Deming regression of HPLC and LC-MS/MS results yielded slopes of 0.93 (95% confidence interval, 0.89–0.98) and 0.89 (0.85–0.93) and y-intercepts of −0.16 and 0.03 nmol/L for NMN (n = 132) and MN (n = 92), respectively.Conclusions: This novel LC-MS/MS approach provides a precise, rapid, and specific alternative method to HPLC for the quantification of the low nanomolar concentrations of free metanephrines in plasma.
Quality Assessment of Interpretative Commenting in Clinical Chemistry
Authors: Lim Ee Mun , Sikaris Ken A , Gill Janice , Calleja John , Hickman Peter E , Beilby John , Vasikaran Samuel D 
Published: February 2004

Abstract
Background: Clinical interpretation of laboratory results is an integral part of clinical chemistry. However, the performance goals for assessing interpretative commenting in this discipline have not been as well established as for the quality of analytical requirements.
Methods: We present a review of the 10 case reports circulated in the 2002 Patient Report Comments Program by the Royal College of Pathologists of Australasia (RCPA) and the Australasian Association of Clinical Biochemists Chemical Pathology Group of RCPA-Quality Assurance Programs Pty Ltd. Participants were expected to add an interpretative comment to a set of results accompanied by brief clinical details. Comments received were broken down into components that were translated into key phrases. An expert panel evaluated the appropriateness of the key phrases and proposed a suggested composite comment. A case summary/rationale was also returned to participants.
Results: There was considerable diversity in the range of interpretative comments received for each case report. Although the majority of comments received were felt to be acceptable by the expert panel, some comments were felt to be inappropriate, misleading, or in a few instances, dangerous.
Conclusion: The golden rule in medicine is “do no harm”. Although there is no objective evidence that interpretive comments help to improve patient outcomes, if comments are added to reports it is important that they reflect accepted practice and current guidelines. It is of concern that a large proportion of comments returned were considered to be inappropriate and/or misleading. The Patient Report Comments Program has highlighted the need to consider limiting commenting to persons with clear expertise.
Potential Clinical Utility of a New IRMA for Parathyroid Hormone in Postmenopausal Patients with Primary Hyperparathyroidism
Authors: Carnevale Vincenzo , Dionisi Simona , Nofroni Italo , Romagnoli Elisabetta , Paglia Federica , De Geronimo Simona , Pepe Jessica , Clemente Giovanni , Tonnarini Gianfranco , Minisola Salvatore 
Published: February 2004

Abstract
Background: A new commercially available (so-called second-generation) IRMA for parathyroid hormone (PTH) separately detects intact PTH and its N-truncated fragments; however, no studies have compared the first- and second-generation IRMAs for PTH in patients with primary hyperparathyroidism (PHPT) to assess their respective diagnostic accuracies.
Methods: We concomitantly investigated 39 postmenopausal patients with PHPT and a control group of 70 healthy postmenopausal women matched for age, renal function, and vitamin D status. In all individuals, PTH was measured with a classic IRMA (PTH-S; DiaSorin Inc.), which uses antibodies directed against epitopes 1–34 and 39–84, and a new method (Scantibodies Laboratory. Inc.), which uses antibodies against epitopes 1–4 and 39–84 (PTH-W) and epitopes 7–34 and 39–84 (PTH-T). We also assayed serum PTH in 10 PHPT patients every 24 h for 5 days after successful surgery.
Results: The different assays gave serum PTH values that were &gt;2 SD higher than values for the control population in 59% (PTH-S), 77% (PTH-W), and 82% (PTH-T) of patients with PHPT. However, ROC curve analysis showed no significant differences among the three PTH assays, demonstrating overlapping diagnostic sensitivities. In PHPT patients, the correlation among the assays was highly significant (r = 0.91–0.92; P &lt;0.001). The ratio PTH-W:PTH-T × 100 showed a gaussian distribution in both PHPT patients and controls, whose mean (SD) values [63.4 (13.3)% vs 64.5 (9.5)%, respectively] did not differ significantly. After parathyroidectomy, the mean percentages of variation in PTH detected with all of the assays were quite similar.
Conclusions: The distribution of the PTH-W:PTH-T ratio in patients and controls suggests that PHPT does not markedly influence the rate at which biologically inactive fragments are generated by central or peripheral cleavage of PTH. The similar postoperative curves seem to contradict the hypothesized effect of acute hypocalcemia in modulating the central secretion of hormonal fragments. Our results indicate that the three investigated assays have similar diagnostic sensitivities in PHPT.
Pyrosequencing Technology as a Method for the Diagnosis of Multiple Endocrine Neoplasia Type 2
Authors: Kruckeberg Kent E , Thibodeau Stephen N 
Published: February 2004

Abstract
Background: Multiple endocrine neoplasia type 2 (MEN2) is a cancer syndrome with well-characterized causative mutations. Missense mutations in ∼15 codons of the RET gene have been linked to disease phenotypes in the vast majority of cases. These missense mutations range from very simple single nucleotide base changes to more numerous changes at a given codon; they therefore are often tested for by more than one DNA-based diagnostic method. We developed and evaluated a Pyrosequencing™ technology-based approach for MEN2 mutation testing that allows both simple and complex mutations to be analyzed on one platform.
Methods: Archived DNA from peripheral blood of patients referred to the Mayo Clinic Molecular Genetics laboratory for MEN2 testing was selected. One to all of codons 609, 611, 618, 620, 630, 634, 768, 804, and 918 were analyzed by Pyrosequencing technology to match the original analysis of each patient. Template PCRs were set up using an automated liquid handler; the subsequent post-PCR preparation step was performed manually, and the sequencing was performed by a PSQ 96 instrument. Samples were tested in batch sizes expected to occur routinely.
Results: We analyzed samples from 217 patients who previously tested negative for MEN2 and 230 patients who previously tested positive, for a total of 1449 sequencing reactions. One discrepant result was found (100% concordant for negatives and 99.6% concordant for positives). A total of 37 unique mutations or alterations of unknown significance were analyzed.
Conclusion: Pyrosequencing technology offers an accurate, nonisotopic, simple, and rapid method for the analysis of DNA from patients suspected of having MEN2.