bluuebunny/crossref_metadata_2025_split huggingface dataset:
Silicon-based Biosensors for Rapid Detection of Protein or Nucleic Acid Targets
Authors: Jenison Robert , La Helen , Haeberli Ayla , Ostroff Rachel , Polisky Barry 
Published: January 2020

AbstractBackground: We developed a silicon-based biosensor that generates visual, qualitative results or quantitative results for the detection of protein or nucleic acid targets in a multiplex format.Methods: Capture probes were immobilized either passively or covalently on the optically coated surface of the biosensor. Intermolecular interactions of the immobilized capture probe with specific target molecules were transduced into a molecular thin film. Thin films were generated by enzyme-catalyzed deposition in the vicinity of the surface-bound target. The increased thickness on the surface changed the apparent color of the biosensor by altering the interference pattern of reflected light.Results: Cytokine detection was achieved in a 40-min multiplex assay. Detection limits were 4 ng/L for interleukin (IL)-6, 31 ng/L for IL1-β, and 437 ng/L for interferon-γ. In multianalyte experiments, cytokines were specifically detected with signal-to-noise ratios ranging from 15 to 80. With a modified optical surface, specificity was also demonstrated in a nucleic acid array with unambiguous discrimination of single-base changes in a 15-min assay. For homozygous wild-type and homozygous mutant samples, signal-to-noise ratios of ∼100 were observed. Heterozygous samples yielded approximately equivalent signals for wild-type and mutant capture probes.Conclusions: The thin-film biosensor allows rapid, sensitive, and specific detection of protein or nucleic acid targets in an array format with results read visually or quantified with a charge-coupled device camera. This biosensor is suited for multianalyte detection in clinical diagnostic assays.
New Thiocholine Ester Substrates for the Assay of Human Serum Cholinesterase
Authors: Yamada Magohei , Marui Yoji , Hayashi Chozo , Miki Yasuyoshi , Takemura Shoji 
Published: January 2020

Abstract
Background: Several thiocholine alkanoyl esters were newly synthesized and explored as substrates for the assay of human serum cholinesterase after being subjected to the Ellman reaction (Arch Biochem Biophys 1958;74:443–50 and Arch Biochem Biophys 1959;82:70–7).
Methods: We synthesized thiocholine ester iodides by the method of Renshow et al. (J Am Chem Soc 1938;60:1765–70). We examined solubility in H2O, substrate specificity serum for cholinesterase, (spontaneous) self-hydrolysis, storage stability, and reaction conditions for measurement of the activity of the enzyme.
Results: Isobutyryl and cyclohexane-carboxyl esters showed the best efficiency for the specific and stable assay of human serum cholinesterase. Aqueous solubility of each was &gt;10 mmol/L, and the reactivity with acetylcholinesterase was negligible. For isobutyryl and cyclohexane-carboxyl esters, respectively, spontaneous hydrolysis in the aqueous phase was ∼1/25 and ∼1/175 slower than the enzymatic hydrolysis, and assays with these substrates were linear to 1800 and 3000 U/L, respectively. The Km values of these acylthiocholines with human cholinesterase were almost equivalent (6.9 × 10−3 mmol/L). The substrates were stable in aqueous solution and in the solid state as the iodides for at least 5 years at 5 °C.
Conclusions: The isobutyrate and cyclohexane-carboxylate of thiocholine are suitable for the specific assay of human serum cholinesterase.
Concentration Ratios of Morphine to Codeine in Blood of Impaired Drivers as Evidence of Heroin Use and not Medication with Codeine
Authors: Ceder Gunnel , Jones Alan Wayne 
Published: January 2020

Abstract
Background: Both the illicit drug heroin and the prescription drug codeine are metabolized to morphine, which tends to complicate interpretation of opiate-positive samples. We report here the concentrations of morphine and codeine, the morphine/codeine ratios, and 6-acetylmorphine (6-AM) in blood specimens from individuals arrested for driving under the influence of drugs (DUID) in Sweden. The results were compared with positive findings of 6-AM in urine as evidence of heroin intake.
Methods: In 339 DUID suspects, both blood and urine specimens were available for toxicologic analysis. In another 882 cases, only blood was available. All specimens were initially analyzed by immunoassay, and the positive results were verified by isotope-dilution gas chromatography–mass spectrometry. In routine casework, the limits of quantification (LOQs) for unconjugated opiates were 5 ng/g for blood and 20 μg/L for urine.
Results: The median concentration of morphine in blood was 30 ng/g with 2.5 and 97.5 percentiles of 5 and 230 ng/g, respectively (n = 979). This compares with a median codeine concentration of 20 ng/g and 2.5 and 97.5 percentiles of 5 and 592 ng/g, respectively (n = 784). The specific metabolite of heroin, 6-AM, was identified in only 16 of 675 blood specimens (2.3%). This compares with positive findings of 6-AM in 212 of 339 urine samples (62%) from the same population of DUID suspects. When 6-AM was identified in urine, the morphine/codeine ratio in blood was always greater than unity (median, 6.0; range, 1–66). In 18 instances, 6-AM was present in urine, although morphine and codeine were below the LOQ in blood. The morphine/codeine ratio in blood was greater than unity in 85% of DUID cases when urine was not available (n = 506), and the median morphine and codeine concentrations were 70 ng/g and 10 ng/g, respectively. When morphine/codeine ratios in blood were less than unity (n = 76), the median morphine and codeine concentrations were 10 ng/g and 180 ng/g, respectively.
Conclusions: Only 2.3% of opiate-positive DUID suspects were verified as heroin users on the basis of positive findings of 6-AM in blood. A much higher proportion (62%) were verified heroin users from 6-AM identified in urine. When urine was not available for analysis, finding a morphine/codeine concentration ratio in blood above unity suggests heroin use and not medication with codeine. This biomarker indicated that 85% of opiate-positive DUID blood samples were from heroin users.
Identification of α1-Antitrypsin Variants in Plasma with the Use of Proteomic Technology
Authors: Mills Kevin , Mills Philippa B , Clayton Peter T , Johnson Andrew W , Whitehouse David B , Winchester Bryan G 
Published: January 2020

Abstract
Background: Proteomic technology permits the investigation of genetic metabolic diseases at the level of protein expression. Changes in the expression, polypeptide structure, and posttranslational modification of individual proteins can be detected in complex mixtures of proteins.
Methods: We used high-resolution two-dimensional polyacrylamide gel electrophoresis to separate isoforms of plasma proteins and detect abnormalities of mass and/or charge. We confirmed the identity of the separated proteins by in-gel digestion with proteases and N-glycanases and then analyzed the released peptides and glycans by matrix-assisted laser-desorption ionization–time-of-flight mass spectrometry.
Results: Complete characterization of the polypeptide sequences and glycosylation of α1-antitrypsin isoforms was achieved in plasma from controls and from patients with three different known α1-antitrypsin deficiencies and congenital disorder of glycosylation type Ia.
Conclusions: This study shows that proteomic techniques are a powerful and sensitive means of detecting changes in the amino acid sequence and abnormal posttranslational modifications of specific proteins in a complex biologic matrix.
Hurler-like Phenotype
Authors: Chamoles Néstor A , Blanco Mariana B , Gaggioli Daniela , Casentini Carina 
Published: January 2020

Abstract
Background: Clinical differentiation among mucopolysaccharidosis, oligosaccharidosis, and mucolipidosis II and III is difficult. We describe methods for the assay of 8 lysosomal enzymes in dried blood spots on filter paper that allow screening for 12 lysosomal storage diseases that present with a Hurler-like phenotype.
Methods: To test tubes containing 3-mm blood spots, we added elution liquid and fluorescent or radioactive substrate solution. After incubation at 37 °C, the reaction was terminated by the addition of a stop buffer. The amount of hydrolyzed product was compared with a calibrator to allow the quantification of enzyme activity. Sample stability was studied during storage for 21 days and during shipment of samples. We measured enzyme activities in 85 healthy controls (35 newborn, 50 adult), 57 patients suffering from 11 lysosomal storage diseases, and 46 obligate carriers.
Results: Intra- and interassay CVs were &lt;9% and &lt;15%, respectively. Mean activity losses during transportation or storage for up to 21 days at 4 °C were ≤27%. Enzyme activities in all patients were outside the ranges of values seen for carriers and controls.
Conclusions: The described methodology distinguishes between patients and controls with samples that are sufficiently stable to be mailed to the testing laboratory.
Early Blood Chemistry in Britain and France
Authors: Coley Noel G 
Published: January 2020

Abstract
I review here key research in the early years of the field of blood chemistry. The review includes successes and limitations of animal chemistry in the critical period of the eighteenth and nineteenth centuries. Eighteenth century medical theories emphasized the primacy of body solids. Body fluids were governed by the tenets of humoral pathology. After Boerhaave sparked interest in the chemistry of the body fluids, a new humoralism developed. With the rise of animal chemistry in the eighteenth century, two complementary ideas came into play. The concept of vital force was introduced in 1774, and the chemical composition of animal matters, including the blood, began to be investigated. In the early nineteenth century, the development of new methods of analysis encouraged such chemical studies. Prominent chemists led the field, and physicians also became involved. Physiologists were often opposed to the chemical tradition, but François Magendie recognized the importance of chemistry in physiology. Liebig linked the formation and functions of the blood to general metabolism and so extended the scope of animal chemistry from 1842. About the same time, microscopic studies led to discoveries of the globular structure of the blood, and Magendie’s famous pupil, Claude Bernard, began the animal chemistry studies that led him to new discoveries in hematology. This review addresses discoveries, controversies, and errors that relate to the foundations of clinical chemistry and hematology and describes contributions of instrumental investigators.
K-ras Mutation Detection by Hybridization to a Polypyrrole DNA Chip
Authors: Lopez-Crapez Evelyne , Livache Thierry , Marchand Joseph , Grenier Jean 
Published: January 2020

Abstract
Background: Detection of mutations in cancer-related genes is of major importance for both basic knowledge and clinical practice. Several strategies have been developed to diagnose these alterations. We describe a method based on polypyrrole DNA chip technology to detect K-ras gene mutations in tumors.
Methods: An oligodeoxynucleotide array was constructed on a silicon device by copolymerization of 5′-pyrrole-labeled oligodeoxynucleotides and pyrrole. The samples to be analyzed were then amplified by PCR, and the single-stranded biotin-labeled amplified DNA was specifically hybridized to the addressed probes. Perfectly matched duplexes were detected by fluorescence microscopy using R-phycoerythrin as the detection label. The developed methodology was applied to genotype assignment of K-ras in human samples. The genotypes of 75 DNA genomic samples from colorectal cancer patients were analyzed side by side using direct DNA sequencing and a polypyrrole DNA chip.
Results: The chip method unequivocally defined all of the genotypes. Mutations present at &lt;10% of the wild-type DNA concentration could be distinguished.
Conclusions: This probe array assay is a rapid and reliable procedure that may be used to detect mutations.
Quality Specifications for Glucose Meters: Assessment by Simulation Modeling of Errors in Insulin Dose
Authors: Boyd James C , Bruns David E 
Published: January 2020

Abstract
Background: Proposed quality specifications for glucose meters allow results to be in error by 5–10% or more of the “true” concentration. Because meters are used as aids in the adjustment of insulin doses, we aimed to characterize the quantitative effect of meter error on the ability to identify the insulin dose appropriate for the true glucose concentration.
Methods: Using Monte Carlo simulation, we generated random “true” glucose values within defined intervals. These values were converted to “measured” glucose values using mathematical models of glucose meters having defined imprecision (CV) and bias. For each combination of bias and imprecision, 10 000–20 000 true and measured glucose concentrations were matched with the corresponding insulin doses specified by selected insulin-dosing regimens. Discrepancies in prescribed doses were counted and their frequencies plotted in relation to bias and imprecision.
Results: For meters with a total analytical error of 5%, dosage errors occurred in ∼8–23% of insulin doses. At 10% total error, 16–45% of doses were in error. Large errors of insulin dose (two-step or greater) occurred &gt;5% of the time when the CV and/or bias exceeded 10–15%. Total dosage error rates were affected only slightly by choices of sliding scale among insulin dosage rules or by the range of blood glucose. To provide the intended insulin dosage 95% of the time required that both the bias and the CV of the glucose meter be &lt;1% or &lt;2%, depending on mean glucose concentrations and the rules for insulin dosing.
Conclusions: Glucose meters that meet current quality specifications allow a large fraction of administered insulin doses to differ from the intended doses. The effects of such dosage errors on blood glucose and on patient outcomes require study.
Determination of Carbohydrate-deficient Transferrin Using Capillary Zone Electrophoresis
Authors: Wuyts Birgitte , Delanghe Joris R , Kasvosve Ishmael , Wauters Annick , Neels Hugo , Janssens Jacques 
Published: January 2020

AbstractBackground: Current methods for carbohydrate-deficient transferrin (CDT) often suffer from low precision, complexity, or risk of false positives attributable to genetic variants. In this study, a new capillary zone electrophoresis (CZE) method for CDT was developed.Methods: CZE was performed on a P/ACE 5000 using fused-silica capillaries [50 μm (i.d.) × 47 cm] and the CEOFIX CDT buffer system with addition of 50 μL of anti-C3c and 10 μL of anti-hemoglobin. Native sera were loaded by high-pressure injection for 3 s, separated at 28 kV over 12 min, and monitored at 214 nm.Results: CDT was completely resolved by differences in migration times (di-trisialotransferrin, 9.86 ± 0.05 min; monosialotransferrin, 9.72 ± 0.05 min; asialotransferrin, 9.52 ± 0.04 min), with a CV of 0.15%. The number of theoretical plates was 312 000 ± 21 000 for the mono- and 199 000 ± 6500 for the di-trisialylated transferrin. Genetic CB and CD variants showed prominent peaks with migration times of 10.12 ± 0.06 and 9.89 ± 0.03 min, respectively, and the carbohydrate-deficient glycoprotein syndrome could be detected, excluding false-positive results. CZE results (as a percentage; y) correlated with the Axis %CDT TIATM (x) values by Deming regression analysis: y = 1.92x − 7.29; r = 0.89. CDT values in 130 healthy nonalcoholics were determined. The 2.5th and 97.5th percentiles were 1.84% and 6.79%.Conclusions: CZE without sample pretreatment can determine CDT with good precision, allows detection of variants, and correlates with ion-exchange chromatography.
HPLC Analysis of Reduced and Oxidized Coenzyme Q10 in Human Plasma
Authors: Tang Peter H , Miles Michael V , DeGrauw Antonius , Hershey Andrew , Pesce Amadeo 
Published: January 2020

AbstractBackground: The percentage of reduced coenzyme Q10 (CoQ10H2) in total coenzyme Q10 (TQ10) is decreased in plasma of patients with prematurity, hyperlipidemia, and liver disease. CoQ10H2 is, however, easily oxidized and difficult to measure, and therefore reliable quantification of plasma CoQ10H2 is of clinical importance.Methods: Venous blood was collected into evacuated tubes containing heparin, which were immediately placed on ice and promptly centrifuged at 4 °C. The plasma was harvested and stored in screw-top polypropylene tubes at −80 °C until analysis. After extraction with 1-propanol and centrifugation, the supernatant was injected directly into an HPLC system with coulometric detection.Results: The in-line reduction procedure permitted transformation of CoQ10 into CoQ10H2 and avoided artifactual oxidation of CoQ10H2. The electrochemical reduction yielded 99% CoQ10H2. Only 100 μL of plasma was required to simultaneously measure CoQ10H2 and CoQ10 over an analytical range of 10 μg/L to 4 mg/L. Intra- and interassay CVs for CoQ10 in human plasma were 1.2–4.9% across this range. Analytical recoveries were 95.8–101.0%. The percentage of CoQ10H2 in TQ10 was ∼96% in apparently healthy individuals. The method allowed analysis of up to 40 samples within an 8-h period.Conclusions: This optimized method for CoQ10H2 analysis provides rapid and precise results with the potential for high throughput. This method is specific and sufficiently sensitive for use in both clinical and research laboratories.
Year-Long Validation Study and Reference Values for Urinary Amino Acids Using a Reversed-Phase HPLC Method
Authors: Venta Rafael 
Published: January 2020

Abstract
Background: Reversed-phase HPLC (RP-HPLC) has become an alternative to ion-exchange chromatography for amino acid analysis in biological fluids. However, validation studies for its urine application are limited, and the corresponding reference values have not been reported extensively. We studied the long-term performance of a commercial HPLC method for urine amino acid analysis and established specific age-related reference values for urine amino acid excretion.
Methods: Method performance was continuously assessed by recovery and precision studies with urine samples and controls, respectively. Healthy individuals were prospectively analyzed throughout a 5-year period. Excretion of individual amino acids, expressed as mmol/mol of creatinine, was included in six age-related groups for random urine samples (0–1 month, 1–12 months, 1–3 years, 3–8 years, 8–16 years, and &gt;16 years) and in two groups for 24-h urine collections (8–16 years and &gt;16 years).
Results: Over a 1-year period, CVs for retention times were &lt;0.5% and 3.3% for within- and between-run imprecision, respectively. For amino acid concentrations, within-run CVs were 2.9–17% and between-run CVs were 7.1–46% for the same period. Amino acid recoveries were 78–122%. Reference intervals for 35 amino acids were calculated and compared with the concentrations observed in patients diagnosed with specific pathologies. A few statistically significant differences were found between the reference intervals derived using random and 24-h urine collections.
Conclusions: Long-term reliability of the RP-HPLC method for urine amino acid analysis has been demonstrated. Representative age-related reference intervals for the RP-HPLC method in both random urine and 24-h urine collections have been established, and their feasibility for diagnosis of aminoaciduria has been shown. These intervals could serve as a guide for laboratories changing to HPLC methods.
ζ-, ε-, and γ-Globin mRNA in Blood Samples and CD71+ Cell Fractions from Fetuses and from Pregnant and Nonpregnant Women, with Special Attention to Identification of Fetal Erythroblasts
Authors: Høgh Anne Mette , Hviid Thomas Vauvert F , Christensen Britta , Sørensen Steen , Larsen Rasmus D , Smidt-Jensen Steen , Bang Jens , Philip John 
Published: January 2020

Abstract
Background: Information about the appearance of γ-, ε-, and ζ-globin mRNAs in fetal erythroblasts during gestation and about the presence and amounts of these mRNAs in pregnant and nonpregnant women is important from the perspective of using these molecules as a marker of fetal erythroblasts. A specific marker is necessary for isolation and identification of fetal nucleated red blood cells from maternal blood samples for use in antenatal diagnosis of fetal genetic or chromosomal abnormalities.
Methods: We used a very sensitive reverse transcription-PCR (RT-PCR) method, coamplification analysis of γ- and ε-globin cDNA, and quantitative analysis of γ-globin mRNA based on competitive RT-PCR to investigate these aspects.
Results: All adult whole-blood samples were negative for ε- and ζ-globin mRNA. Analyses of CD71+ cell fractions showed that specimens from 19 of 20 nonpregnant and 10 of 14 pregnant women (at 9–13 weeks of gestation) were positive for γ-globin mRNA (Fisher’s exact test, P = 0.13), and those from 3 of 20 nonpregnant and 5 of 14 pregnant women were positive for ζ-globin mRNA (Fisher’s exact test, P = 0.23). No ε-globin mRNA was detected in CD71+ cell fractions from 1-mL blood samples from adults. CD71+ cell fractions from eight fetal blood samples (at 17–20 weeks of gestation) were positive for all three globin mRNAs. We found no statistically significant difference between the amounts of γ-globin mRNA in pregnant and nonpregnant women.
Conclusions: This study indicates that ε-globin mRNA might function as a marker for fetal CD71+ cells early in pregnancy. Although γ-globin mRNA can be detected in CD71+ cell fractions from most adults, these transcripts also may be of use because of a marked difference between adult and fetal values.
Polymorphic Short Tandem Repeats for Diagnosis of the Charcot-Marie-Tooth 1A Duplication
Authors: Latour Philippe , Boutrand Laetitia , Levy Nicolas , Bernard Rafaëlle , Boyer Amandine , Claustrat Francine , Chazot Guy , Boucherat Michel , Vandenberghe Antoon 
Published: January 2020

Abstract
Background: A 1.5-Mb microduplication containing the gene for peripheral myelin protein 22 (PMP22) on chromosome 17p11.2-12 is responsible for 75% of cases of the demyelinating form of Charcot-Marie-Tooth disease (CMT1A). Methods for molecular diagnosis of CMT1A use Southern blot and/or amplification by PCR of polymorphic poly(AC) repeats (microsatellites) located within the duplicated region, or the detection of junction fragments specific for the duplication. Difficulties with both strategies have led us to develop a new diagnostic strategy with highly polymorphic short tandem repeats (STRs) located inside the CMT1A duplicated region.
Methods: We tested 10 STRs located within the duplication for polymorphic behavior. Three STRs were selected and used to test a set of 130 unrelated CMT1A patients and were compared with nonduplicated controls. The study was then extended to a larger population of patients. Alleles of interest were sequenced. A manual protocol using polyacrylamide electrophoresis and silver staining and an automated capillary electrophoresis protocol to separate fluorescently labeled alleles were validated.
Results: We identified three new STRs covering 0.55 Mb in the center of the CMT1A duplication. One marker, 4A, is located inside the PMP22 gene. The two others, 9A and 9B, more telomerically positioned, have the highest observed heterozygosity reported to date for CMT1A markers: 0.80 for 9A, and 0.79 for 9B. Tetra- and pentanucleotide repeats offered clear amplification, accurate sizing, and easy quantification of intensities.
Conclusions: Combined use of the three STRs allows robust diagnosis with almost complete informativeness. In our routine diagnosis for CMT1A, they have replaced the use of other polymorphic markers, either in a manual adaptation or combined with fluorescence labeling and allele sizing on a DNA sequencer.