bluuebunny/crossref_metadata_2025 huggingface dataset:
2. Jews, Gentiles, and Christians (200 bce–200 ce)
Authors: Cohen Charles L.
Published: January 2020

“Jews, Gentiles, and Christians (200 BCE–200 CE)” describes the period from 200 bce to 200 ce, when Christianity arose within the context of Second Temple Judaism. Jesus of Nazareth may be best understood as an itinerant preacher within Jewish apocalyptic tradition, and Christianity as initially a Jewish sect. But it soon became something else, attracting Gentiles while absorbing influences from the peoples it encountered. This intercultural dynamic is revealed in the writings that became Christians’ holy texts—the New Testament—and in their efforts to simultaneously recruit and differentiate themselves from Jews, who “desire signs,” and “Greeks,” who “desire wisdom” (1 Cor. 1:22).
4. From colony to competing states
Authors: Seth Michael J.
Published: January 2020

As the Second World War came to an end, most Koreans hoped that their nation would be an independent and prosperous state. ‘From colony to competing states’ shows that, instead, events took an unexpected turn. Korea became both free of Japanese colonial rule and simultaneously partitioned into two occupation zones by the United States and the Soviet Union. From these zones, two separate states were created: the Republic of Korea and the Democratic People’s Republic of Korea; two societies with different leaderships, political systems, and geopolitical orientations. When North Korea attempted to reunify the country in 1950, foreign powers again intervened resulting in the Korean War, a costly conflict that left the peninsula still divided.
Exploration of databases and methods supporting drug repurposing: a comprehensive survey
Authors: Tanoli Ziaurrehman , Seemab Umair , Scherer Andreas , Wennerberg Krister , Tang Jing , Vähä-Koskela Markus
Published: January 2020

Drug development involves a deep understanding of the mechanisms of action and possible side effects of each drug, and sometimes results in the identification of new and unexpected uses for drugs, termed as drug repurposing. Both in case of serendipitous observations and systematic mechanistic explorations, confirmation of new indications for a drug requires hypothesis building around relevant drug-related data, such as molecular targets involved, and patient and cellular responses. These datasets are available in public repositories, but apart from sifting through the sheer amount of data imposing computational bottleneck, a major challenge is the difficulty in selecting which databases to use from an increasingly large number of available databases. The database selection is made harder by the lack of an overview of the types of data offered in each database. In order to alleviate these problems and to guide the end user through the drug repurposing efforts, we provide here a survey of 102 of the most promising and drug-relevant databases reported to date. We summarize the target coverage and types of data available in each database and provide several examples of how multi-database exploration can facilitate drug repurposing.
Heterogeneous networks integration for disease–gene prioritization with node kernels
Authors: Tran Van Dinh , Sperduti Alessandro , Backofen Rolf , Costa Fabrizio
Published: January 2020

Motivation

The identification of disease–gene associations is a task of fundamental importance in human health research. A typical approach consists in first encoding large gene/protein relational datasets as networks due to the natural and intuitive property of graphs for representing objects’ relationships and then utilizing graph-based techniques to prioritize genes for successive low-throughput validation assays. Since different types of interactions between genes yield distinct gene networks, there is the need to integrate different heterogeneous sources to improve the reliability of prioritization systems.

Results

We propose an approach based on three phases: first, we merge all sources in a single network, then we partition the integrated network according to edge density introducing a notion of edge type to distinguish the parts and finally, we employ a novel node kernel suitable for graphs with typed edges. We show how the node kernel can generate a large number of discriminative features that can be efficiently processed by linear regularized machine learning classifiers. We report state-of-the-art results on 12 disease–gene associations and on a time-stamped benchmark containing 42 newly discovered associations.

Availability and implementation

Source code: https://github.com/dinhinfotech/DiGI.git.

Supplementary information

Supplementary data are available at Bioinformatics online.
LAMPA, LArge Multidomain Protein Annotator, and its application to RNA virus polyproteins
Authors: Gulyaeva Anastasia A , Sigorskih Andrey I , Ocheredko Elena S , Samborskiy Dmitry V , Gorbalenya Alexander E
Published: January 2020

Motivation

To facilitate accurate estimation of statistical significance of sequence similarity in profile–profile searches, queries should ideally correspond to protein domains. For multidomain proteins, using domains as queries depends on delineation of domain borders, which may be unknown. Thus, proteins are commonly used as queries that complicate establishing homology for similarities close to cutoff levels of statistical significance.

Results

In this article, we describe an iterative approach, called LAMPA, LArge Multidomain Protein Annotator, that resolves the above conundrum by gradual expansion of hit coverage of multidomain proteins through re-evaluating statistical significance of hit similarity using ever smaller queries defined at each iteration. LAMPA employs TMHMM and HHsearch for recognition of transmembrane regions and homology, respectively. We used Pfam database for annotating 2985 multidomain proteins (polyproteins) composed of >1000 amino acid residues, which dominate proteomes of RNA viruses. Under strict cutoffs, LAMPA outperformed HHsearch-mediated runs using intact polyproteins as queries by three measures: number of and coverage by identified homologous regions, and number of hit Pfam profiles. Compared to HHsearch, LAMPA identified 507 extra homologous regions in 14.4% of polyproteins. This Pfam-based annotation of RNA virus polyproteins by LAMPA was also superior to RefSeq expert annotation by two measures, region number and annotated length, for 69.3% of RNA virus polyprotein entries. We rationalized the obtained results based on dependencies of HHsearch hit statistical significance for local alignment similarity score from lengths and diversities of query-target pairs in computational experiments.

Availability and implementation

LAMPA 1.0.0 R package is placed at github (https://github.com/Gorbalenya-Lab/LAMPA).

Supplementary information

Supplementary data are available at Bioinformatics online.
Lowering cutoffs for initial and confirmation testing for cocaine and marijuana: large-scale study of effects on the rates of drug-positive results
Authors: Wingert William E
Published: January 2020

A large-scale study was conducted to determine whether lowering the initial testing and confirmation testing cutoffs in urine would significantly affect the positive rates for cocaine (COC) and marijuana (THC). Customary cutoffs for COC are 300 μg/L and 150 μg/L for initial testing (screening) and gas chromatography–mass spectrometry (GC-MS; confirmation), respectively; for THC, the usual respective cutoffs are 50 μg/L and 10 μg/L. By applying a screening cutoff of 100 μg/L for COC and lowering the GC-MS cutoff to 50 μg/L, the COC-positive rate increased from 1.2% to 2.1%. For THC, lowering the screening cutoff to 20 μg/L while leaving the GC-MS cutoff at 10 μg/L increased the THC-positive rate from 2.8% to 4.1%. These increases appear noteworthy.
A case of sulfhemoglobinemia and emergency measurement of sulfhemoglobin with an OSM3 CO-oximeter
Authors: Wu Caicai , Kenny Margaret A
Published: January 2020

We describe a case of sulfhemoglobinemia associated with toxic paint ingestion. Blood gases, oxygen content, and fractional hemoglobin derivatives were assayed with Radiometer 520 and OSM3 instruments. Although the CO-oximeters indicated the presence of sulfhemoglobin (SulfHb), the results were not quantitative. An OSM3 service software program was activated to obtain the actual concentrations of the hemoglobin fractions. Subsequently, we evaluated the performance of the OSM3 service program for the analysis of SulfHb by performing precision studies and comparing OSM3 results with those of an AVL 912 CO-oximeter. Retrospectively, we determined that the patient’s specimens contained 6% SulfHb. There was an obvious deviation between standard OSM3 oxyhemoglobin fraction measurements and those obtained by using its service program—the effect of a high SulfHb content.
Counterpoint The inseparable triad: analytical sensitivity, measurement uncertainty, and quantitative resolution
Authors: Pardue Harry L
Published: January 2020

The formal definition of sensitivity associates the term with the change in the response of a system for a small change of the stimulus causing the response, i.e., the ratio of the response of a system to the stimulus causing it. One interpretation of sensitivity associates the rate of change of the response for a small change of the stimulus as the slope of a calibration plot of response vs stimulus. An alternative interpretation associates sensitivity with the smallest value of the stimulus that can be resolved with a given degree of confidence, i.e., the detection limit. Applications of the first usage to analytical chemistry date at least to the beginning of this century; applications of the second interpretation are of more recent origin. The accompanying paper argues in favor of the second interpretation on the basis that, among other things, the “slope” interpretation conflicts with the formal definition of sensitivity and is meaningless as a descriptor of the performance of a measuring system. In this paper I offer arguments to support my belief that the slope definition of sensitivity is consistent with both formal definitions and accepted usage in analytical chemistry and, more importantly, that it is an invaluable descriptor of one of the most important characteristics of any analytical method. I include information to support my belief that proper use of the slope definition yields much more information than is available in the “detection limit” interpretation.
Molecular diagnostics of infectious diseases
Authors: Tang Yi-Wei , Procop Gary W , Persing David H
Published: January 2020

Over the past several years, the development and application of molecular diagnostic techniques has initiated a revolution in the diagnosis and monitoring of infectious diseases. Microbial phenotypic characteristics, such as protein, bacteriophage, and chromatographic profiles, as well as biotyping and susceptibility testing, are used in most routine laboratories for identification and differentiation. Nucleic acid techniques, such as plasmid profiling, various methods for generating restriction fragment length polymorphisms, and the polymerase chain reaction (PCR), are making increasing inroads into clinical laboratories. PCR-based systems to detect the etiologic agents of disease directly from clinical samples, without the need for culture, have been useful in rapid detection of unculturable or fastidious microorganisms. Additionally, sequence analysis of amplified microbial DNA allows for identification and better characterization of the pathogen. Subspecies variation, identified by various techniques, has been shown to be important in the prognosis of certain diseases. Other important advances include the determination of viral load and the direct detection of genes or gene mutations responsible for drug resistance. Increased use of automation and user-friendly software makes these technologies more widely available. In all, the detection of infectious agents at the nucleic acid level represents a true synthesis of clinical chemistry and clinical microbiology techniques.
Evaluation of a near-patient test for C-reactive protein used in daily routine in primary healthcare by use of difference plots
Authors: Dahler-Eriksen Bjarne Steen , Lassen Jens Flensted , Petersen Per Hyltoft , Lund Erik Dalsgaard , Lauritzen Torsten , Brandslund Ivan
Published: January 2020

We have assessed the technical performance and robustness of NycoCard® CRP Whole Blood, a near-patient test for C-reactive protein (CRP), when used in realistic daily routine situations in general practice clinics (GPC). Thirteen GPCs participated, five of them with technician staff. From 898 patients, split-sample measurements for CRP were made. Results from GPCs were compared with results from a turbidimetric laboratory method, traceable to international reference preparations (IFCC CRM 470). Results were evaluated in difference plots where the expected distribution, due to an estimated analytical variation, was compared with measured differences. Of all difference points, 91.5% (n = 819) were within a 95% prediction interval based on the imprecision of both methods. Mean bias (95% confidence interval) was −0.3 mg/L (−0.9 to 0.3). No differences in analytic quality were found between GPCs with technician staffs and GPCs without, and between test results obtained within the first and second week, compared with the rest of the study period. We find the test as good when used in GPCs as could be expected from laboratory testing, and consequently robust, which is a necessity for use in routine situations in general practice. General application of difference plots in test evaluations are discussed in detail.
Differentiation between naproxen, naproxen–protein conjugates, and naproxen–lysine in plasma via micellar electrokinetic capillary chromatography—a new approach in the bioanalysis of drug targeting preparations
Authors: Albrecht Christiane , Reichen Jürg , Visser Jan , Meijer Dirk K F , Thormann Wolfgang
Published: January 2020

Pharmacotherapy through the targeting of drugs is a promising new approach that requires adequate analytical methods capable of differentiating between the free drug, the drug carrier, and metabolites. Using micellar electrokinetic capillary chromatography (MECC), we report the separation of naproxen (NAP) from NAP covalently coupled to human serum albumin or to mannosylated serum albumin and the metabolite naproxen–lysine. An assay for selective analysis of the different forms of NAP by direct plasma injection was developed with salicylate as internal standard and solute detection by laser-induced fluorescence. Compared with previously applied techniques, including HPLC and total plasma fluorescence, MECC offers the advantage that free and covalently bound NAP can be differentiated in one run and can be accurately monitored in microliter quantities of plasma. Summation of all NAP equivalents determined by MECC revealed data that compare well with those produced by total plasma fluorescence and HPLC.
Quantification of c-erbB-2 gene expression in breast cancer by competitive RT-PCR
Authors: Révillion Françoise , Hornez Louis , Peyrat Jean-Philippe
Published: January 2020

We adapted competitive reverse transcription-polymerase chain reaction (RT-PCR) for quantitative evaluation of c-erbB-2 expression in breast cancer. A fixed amount of cDNA target was coamplified with dilutions of a nonhomologous DNA sequence used as an internal standard (IS). The IS and the target shared the same primer sequences but yielded PCR products of different sizes (348 and 340 bp, respectively). A fluorescent sense primer was used so that the PCR products separated on denaturing polyacrylamide gels could be quantified with an automated DNA sequencer. In human breast cancer cell lines, c-erbB-2 expression was found to range from 0.151 to 652 zmol/μg total RNA (i.e., 91 to 391 200 molecules/μg total RNA; 1 zmol = 10−3 amol), with the two highest values corresponding to the c-erbB-2 overexpressing cell lines MDA-MB-453 and SK-BR-3. In a series of 39 breast cancer biopsies, the concentrations ranged from 0.117 zmol/μg to 1.15 amol/μg total RNA (i.e., 70 to 690 000 molecules/μg total RNA). The c-erbB-2 oncoprotein (p185) was determined in 30 samples by an enzyme immunoassay. A close correlation was found between c-erbB-2 gene and oncoprotein expression (P = 0.0067). Thus, this competitive RT-PCR method appears to be a reliable way to evaluate the expression of c-erbB-2 in small tumor samples.
Fiber-optic immunosensor for measurement of myoglobin
Authors: Hanbury C Michael , Miller W Greg , Harris Robert B
Published: January 2020

A self-contained fiber-optic immunosensor was developed to measure the 16 500-Da protein myoglobin. The sensing element was constructed by entrapment of Cascade Blue-labeled antibody within polyacrylamide gel at the distal face of an optical fiber 300 μm in core diameter. The polyacrylamide gel composition was optimized to allow diffusion of myoglobin but to exclude hemoglobin and higher-molecular-mass proteins from the sensing area. The analytical signal was derived from fluorescence energy transfer between Cascade Blue and the heme group of myoglobin. Fluorescence quenching occurred when myoglobin bound to labeled antibody. The total amount of fluorescence quench was dependent on the antibody labeling conditions and the amount of antibody incorporated in the sensor gel matrix. Myoglobin concentrations >5 nmol/L (83 μg/L) were measurable with response times of 15 to 130 min limited by diffusion into the sensing element. This report demonstrates the technical feasibility for a self-contained immunosensor to measure a protein analyte.
Thyroxine, thyrotropin, and age in a euthyroid hospital patient population
Authors: Davey Richard
Published: January 2020

The diagnosis of thyroid disease now often can be achieved reliably by measuring thyrotropin (TSH) alone. Thyroxine (T4), triiodothyronine, and other analytes are only needed if TSH and the accompanying clinical condition are discordant. We describe here work that confirms the age independence of TSH in both inpatient and outpatient euthyroid hospital populations between ages 20 and at least 80 years, and demonstrates that although free T4 does vary with age, the range of variation remains within the T4reference interval. On this basis, TSH-based thyroid diagnostic algorithms can be used reliably in adults without reference to age-related reference intervals.
Effects of uremic toxins and fatty acids on serum protein binding of furosemide: possible mechanism of the binding defect in uremia
Authors: Takamura Norito , Maruyama Toru , Otagiri Masaki
Published: January 2020

To elucidate the mechanism of impaired serum binding of furosemide observed in patients with renal dysfunction, we examined in vitro the serum protein binding of furosemide in the absence and presence of uremic toxins that are endogenously retained solutes in uremic serum and act as inhibitors of drug binding. Analysis of the binding data of furosemide at its therapeutic concentration (6.6 mg/L) indicated that, among the four uremic toxins studied, 3-carboxy-4-methyl-5-propyl-2-furanpropionate (CMPF) showed the greatest inhibitory potency for the binding of furosemide to serum; moreover, the inhibition was competitive. CMPF thus most likely represents the primary determinant for the serum binding defect of furosemide in uremia. However, CMPF and oleate appear to exert a synergistic effect on the inhibition of furosemide serum binding—perhaps through a cascade effect on furosemide-binding inhibition in the oleate–CMPF–furosemide system, in which the binding of oleate to its low-affinity sites indirectly displaces furosemide from albumin and thus increases the transiently liberated CMPF molecules. Similar cascade effects on furosemide binding in the presence of CMPF were also originated by other long-chain (C18) fatty acids, linoleate and stearate, although to a lesser extent. Because CMPF is not effectively removed by ordinary hemodialysis treatment, the combined direct and cascade effects of CMPF and fatty acids appear to contribute to the increase in the free fraction of furosemide during hemodialysis.
Protein tyrosine phosphatase-like protein IA2-antibodies plus glutamic acid decarboxylase 65 antibodies (GADA) indicates autoimmunity as frequently as islet cell antibodies assay in children with recently diagnosed diabetes mellitus
Authors: Borg Henrik , Fernlund Per , Sundkvist Göran
Published: January 2020

Islet cell antibodies (ICA), the classical autoimmunity marker for insulin-dependent diabetes mellitus (IDDM), are detected in ∼85% of children with recently diagnosed diabetes. Because the ICA assay is semiquantitative and difficult to standardize, alternative assays are needed. When glutamic acid decarboxylase 65 (GAD 65) was discovered as a major islet antigen, the measurement of antibodies to GAD 65 (GADA) was considered a good alternative to ICA. Recently, however, we showed that 1 in 3 ICA-positive diabetic patients do not have GADA. Now, antibodies against the protein tyrosine phosphatase-like protein IA2 (IA2-ab) have been detected in IDDM. To find out whether measurements of IA2-ab combined with those of GADA could detect autoimmunity to the same extent as ICA, we have measured all three kinds of antibodies (using radioligand binding assays for IA2-ab and GADA) in 100 recently diagnosed diabetic and 100 control children: ICA were found in 87, IA2-ab in 69, and GADA in 66 of the 100 diabetic patients, whereas in the 100 control children ICA were found in 2, IA2-ab in 1, and GADA in 3. Among the 87 ICA-positive patients, 45 (52%) had both IA2-ab and GADA, 21 (24%) had only IA2-ab, and 16 (18%) had only GADA, whereas 5 (6%) lacked both IA2-ab and GADA. Among the 13 ICA-negative patients, 1 (8%) had both IA2-ab and GADA, 2 (15%) had only IA2-ab, and 4 (31%) had only GADA. Thus, 6 of the 100 patients had neither ICA, IA2-ab, nor GADA. Combining the IA2-ab and GADA assays gave positive results for autoimmunity in 89 of the 100 patients, compared with 87 by the ICA assay. The combination of the IA2-ab and GADA assays appears to be an effective alternative to the ICA assay.
Kinetics and proposed mechanism of the reaction of an immunoinhibition, particle-enhanced immunoassay
Authors: Thompson John C , Craig Alan R , Davey Carol L , Newman David J , Lonsdale Michele L , Bucher William J , Nagle Patrick D , Price Christopher P
Published: January 2020

We report kinetic studies on the reaction of a latex agglutination immunoassay used to quantify phenytoin in serum. In this assay, polystyrene particles with a covalently attached analog of phenytoin react with an antiphenytoin monoclonal antibody to form light-scattering aggregates, with the rate of this reaction being decreased by addition of phenytoin from sample. In the absence of free (sample) phenytoin, this reaction did not exhibit a maximum rate of agglutination in the presence of excess antibody, i.e., an equivalence point. Furthermore, agglutination was inhibitable by free phenytoin even when the latter was added after agglutination of particles with antibody had begun. Most significantly, the immunoagglutination proceeded in an identical fashion with monovalent F(ab) fragment. These data are consistent with low-affinity immunospecific particle–antibody complexation, which then induces colloidal aggregation, without requiring immunospecific bridging by antibody molecules. The described mechanism is not generalizable to all latex agglutination immunoassays, although disturbance of colloidal stability may be a component in most assays.
The relation between chemically measured total iron-binding capacity concentrations and immunologically measured transferrin concentrations in human serum
Authors: Gambino Raymond , Desvarieux Edouard , Orth Michael , Matan Holly , Ackattupathil Tomy , Lijoi Elenore , Wimmer Cathy , Bower John , Gunter Elaine
Published: January 2020

We sought to determine if serum total iron-binding capacity (TIBC) is equivalent to serum transferrin (TRF) so that a low-cost colorimetric chemical assay for unsaturated iron-binding capacity (UIBC) could be substituted for a high-cost immunologic assay for TRF. Our study design included independent and blinded measurements of UIBC, serum iron, and TRF concentrations in human serum samples. Data from five independent correlation studies carried out at three different Quest Diagnostics laboratories were combined into one data set containing 570 paired results for TIBC and TRF. r2 was 0.941 when three outliers were eliminated from the 570-sample data set. Scatter about the regression line was fully accounted for by the CVs for the TIBC and TRF assays. When each test is measured precisely and without bias, the ratio of TIBC (μmol/L) to TRF (g/L) in SI units is close to the theoretically expected value of 25.0.
Screening children exposed to lead: an assessment of the capillary blood lead fingerstick test
Authors: Parsons Patrick J , Reilly Andrew A , Esernio-Jenssen Debra
Published: January 2020

We describe results of a 3-year study in which 499 paired venous and capillary blood specimens, collected by fingerstick on the same day, were analyzed for lead (BPb) and erythrocyte protoporphyrin (EP). False-positive rates (FPRs) and the proportion of false positives were calculated at four BPb thresholds. At the 100 μg/L threshold, the FPR for all data was 13%, but the proportion of false positives was only 5%. The log ratios of capillary-to-venous BPb data indicate that, with the exception of eight outliers, two subpopulations exist that follow a log-normal distribution. These two subpopulations, the “core” (n = 303) and “shifted” (n = 188) groups, on average generated a positive bias at 100 μg/L BPb of 8.6% and 30.3%, respectively. The log ratios of capillary-to-venous EP data followed a normal distribution, indicating that capillary EP is not statistically different from venous EP.
Uniform solid-phase extraction procedure for toxicological drug screening in serum and urine by HPLC with photodiode-array detection
Authors: Lai Chi-Kong , Lee Ting , Au Kam-Ming , Chan Albert Yan-Wo
Published: January 2020

In this HPLC–diode-array detection method for toxicological drug screening, a mixed-mode solid-phase extraction procedure is optimized for isolation of a broad range of drugs from serum and urine. Basic, neutral, and weakly acidic drugs are uniformly recovered. The extract from the solid-phase cartridge is readily injected to a reversed-phase HPLC column for separation by gradient elution. Unknown drugs and metabolites in urine and serum samples from acute drug poisoning cases are rapidly identified by matching their retention times and ultraviolet spectra with hundreds of reference compounds in the library. Urine metabolites of common toxicants from various medications and drugs of abuse are recorded, with their changes of retention times and ultraviolet spectra as related to their metabolic transformations. Glucuronide conjugates of common benzodiazepines, tricyclic antidepressants, and beta-blockers are examined directly without chemical or enzymatic hydrolysis. The system is reliable for diverse clinical investigations of drug overdoses, drug-induced psychoses, and substance abuse.
Increased serum concentration of carbohydrate-deficient transferrin in patients with combined pancreas and kidney transplantation
Authors: Arndt Torsten , Hackler Rolf , Müller Thomas , Kleine Tilman O , Gressner Axel M
Published: January 2020

Serum concentration of carbohydrate-deficient transferrin (cCDT) is used for laboratory diagnosis and follow-up of chronic alcohol abuse. In analyzing by CDTect-RIA (Pharmacia) sera from outpatients with combined pancreas and kidney transplantation and no excessive alcohol consumption, we found above-normal values for cCDT and CDT/transferrin ratios (CDT/Tf) in more than half of the samples. Isoelectric focusing of these samples showed distinct bands of carbohydrate-deficient isotransferrins, supporting the abnormal findings from the CDTect assay. In contrast, diabetics and outpatients who had received only kidney transplants showed normal values for cCDT, CDT/Tf, and isotransferrin patterns. Increased serum Tf, sialidase-producing microorganisms, and immunosuppressive medication were eliminated as causes of these abnormal cCDT and CDT/Tf results. Successful pancreas transplantation leads to hyperinsulinemia and normoglycemia, in contrast to hypoinsulinemia and hyperglycemia in the patients who receive kidney transplants alone. These factors may have pathogenic importance for CDT increase, yielding results falsely interpreted as positive with respect to alcohol abuse in patients with combined pancreas and kidney transplantation.
Comparison of methods for measurement of apolipoprotein B and cholesterol in low-density lipoproteins
Authors: Vrga Ljubica , Contacos Christine , Li Stephen C H , Sullivan David R
Published: January 2020

We describe a new method for the direct measurement of LDL-apolipoprotein (apo) B by using a commercial kit that isolates LDL by immunoseparation. We evaluated immunoseparation of LDL for apo B and cholesterol measurement in 46 dyslipidemic patients with LDL-cholesterol (LDL-C) between 1.5 and 8.2 mmol/L, 11 of whom had plasma triglyceride (TG) concentrations >4.0 mmol/L. There was a reasonable correlation (r = 0.94, n = 40) between LDL-apo B obtained after immunoseparation and d >1.006 kg/L apo B obtained after ultracentrifugation. LDL-C by the immunoseparation method also correlated well (r = 0.98, n = 46) with the d >1.006 kg/L cholesterol after ultracentrifugation. These results show that immunoseparation can be used to determine LDL-apo B, even in hypertriglyceridemic samples. This method may provide a quick and simple alternative for the identification of hyperapobetalipoproteinemia, even when TG concentrations are high.
Improved reverse transcriptase–polymerase chain reaction protocol with exogenous internal competitive control for prostate-specific antigen mRNA in blood and bone marrow
Authors: Corey Eva , Arfman Edward W , Liu Alvin Y , Vessella Robert L
Published: January 2020

The possibility of improving diagnosis of micrometastases from prostate cancer by further enhancing the detection of prostate-specific antigen-producing cells in circulation is being evaluated. We have developed a reverse transcriptase-PCR protocol with the desirable characteristics of low limit of detection, high specificity, reproducibility of response, and ease of performance. Among the procedural alterations that have contributed to these improvements are longer PCR primers, a two-step amplification cycle, and hot-start PCR. We have lowered the limit of detection to one LNCaP prostate-cancer cell in 108 peripheral blood mononuclear cells, and samples of blood and bone marrow from healthy donors have yielded no false positives. Because PCR procedures frequently exhibit tube-to-tube variability, we have incorporated a set of internal and external controls into the protocol—a significant advance in assuring assay reliability.
Improved troponin T ELISA specific for cardiac troponin T isoform: assay development and analytical and clinical validation
Authors: Müller-Bardorff Margit , Hallermayer Klaus , Schröder Angelika , Ebert Christoph , Borgya Anneliese , Gerhardt Willie , Remppis Andrew , Zehelein Jörg , Katus Hugo A
Published: January 2020

The first generation of troponin T ELISA (TnT 1) can yield false-positive results in patients with severe skeletal muscle injury. Therefore, a cardiac-specific second-generation troponin T ELISA (TnT 2) was developed, in which the cross-reactive antibody 1B10 has been replaced by a high-affinity cardiac-specific antibody M11.7. No cross-reactivity of TnT 2 was observed with purified skeletal muscle troponin T (1000 μg/L) or in test samples from 43 marathon runners and 24 patients with rhabdomyolysis and highly increased creatine kinase. TnT 2 was increased >0.2 μg/L in 5 of 40 patients with renal failure and in 4 of 20 muscular dystrophy patients. The detection limit is 0.012 μg/L. Day-to-day imprecision (CV) within the range 0.19–14.89 μg/L was <5.8%. In 4955 patients without myocardial damage, 99.6% had TnT <0.10 μg/L. Assay comparison (TnT 1 vs TnT 2) over the whole concentration range (i.e., in 323 samples from AMI-suspected patients) showed a slope, intercept, and standard error of estimate (Sey) of 1.18, 0.01 μg/L, and 0.81 μg/L, respectively.
Novel bispecific immunoprobe for rapid and sensitive detection of prostate-specific antigen
Authors: Kreutz Fernando T , Suresh Mavanur R
Published: January 2020

Prostate-specific antigen (PSA) is one of the most useful tumor markers for the screening and follow-up of prostate cancer. Bispecific monoclonal antibodies (bsMAbs) are unique immunoprobes that incorporate two different binding sites in the same antibody molecule. This antibody designing can bring important advantages in the development of new immunoassays. We have developed a new hybrid hybridoma that secretes bsMAb anti-PSA × anti-horseradish peroxidase. This bsMAb has shown rapid kinetics and an excellent detection limit in a sandwich single-step assay with a total incubation time of 15 min and a 5-min substrate development. This assay in a manual format has a detection limit of 0.028 μg/L. Comparison with the Hybritech Tandem-E® PSA assay yielded a regression equation with slope = 0.433 [95% confidence interval (CI) = 0.415–0.451], intercept = 0.88 (CI = 0.45–1.31), and Sy|x = 1.83 μg/L (r = 0.98). This new immunoprobe can be used to develop a new generation of assays for clinical laboratories and can be adapted to screening devices for physicians’ offices and even home diagnostics.
Telomeres and telomerase: biological and clinical importance
Authors: Dahse Regine , Fiedler Wolfgang , Ernst Günther
Published: January 2020

We review the present knowledge of telomeres and telomerase with special attention to their role in cell proliferation, cellular senescence, and human aging. We summarize the functional aspects of telomerase in cancer, as well as its role as a useful diagnostic and prognostic tumor marker, and discuss possible approaches to telomerase inhibition as a target for cancer therapy.
Convenient chromatographic prepurification step before measurement of urinary cortisol by radioimmunoassay
Authors: Morineau Gilles , Gosling James , Patricot Marie-Claude , Soliman Hany , Boudou Philippe , Halnak Akram Al , Le Brun Gaëlle , Brérault Jean-Louis , Julien René , Villette Jean-Marie , Fiet Jean
Published: January 2020

We applied various prepurification protocols (extraction with different solvents, liquid/solid separation on bonded silica media, Celite, and Sephadex LH20 chromatography) with a range of commercially available RIA kits to measure cortisol in urine samples. We then compared the results with the concentrations measured by a HPLC method validated with reference to isotope dilution gas chromatography–mass spectrometry. We conclude that chromatography on a commercial, prepacked diol minicolumn (Waters™ Sep-Pak Vac RC) in combination with dichloromethane extraction is a convenient and very effective purification step before RIA of urinary cortisol in patients not receiving corticoid medication. We tested numerous steroids for interference and found that free polar cortisol derivatives (hydroxylated or hydrogenated) could only partially account for the overestimations routinely encountered when free urinary cortisol concentrations are measured by direct RIA.
Screening blood spots for inborn errors of metabolism by electrospray tandem mass spectrometry with a microplate batch process and a computer algorithm for automated flagging of abnormal profiles
Authors: Rashed Mohamed S , Bucknall Martin P , Little Douglas , Awad Amin , Jacob Minnie , Alamoudi Mohamed , Alwattar Mona , Ozand Pinar T
Published: January 2020

Metabolic profiling of amino acids and acylcarnitines from blood spots by automated electrospray tandem mass spectrometry (ESI-MS/MS) is a powerful diagnostic tool for inborn errors of metabolism. New approaches to sample preparation and data interpretation have helped establish the methodology as a robust, high-throughput neonatal screening method. We introduce an efficient 96-well-microplate batch process for blood-spot sample preparation, with which we can obtain high-quality profiles from 500-1000 samples per day per instrument. A computer-assisted metabolic profiling algorithm automatically flags abnormal profiles. We selected diagnostic parameters for the algorithm by comparing profiles from patients with known metabolic disorders and those from normal newborns. Reference range and cutoff values for the diagnostic parameters were established by measuring either metabolite concentrations or peak ratios of certain metabolite pairs. Rigorous testing of the algorithm demonstrates its outstanding clinical sensitivity in flagging abnormal profiles and its high cumulative specificity.
Serum concentrations of free ubiquitin and multiubiquitin chains
Authors: Takada Koji , Nasu Hidekazu , Hibi Nozomu , Tsukada Yutaka , Shibasaki Toshiaki , Fujise Kiyotaka , Fujimuro Masahiro , Sawada Hitoshi , Yokosawa Hideyoshi , Ohkawa Kiyoshi
Published: January 2020

Ubiquitin, which can conjugate with cellular proteins, is classified into two forms: free ubiquitin and multiubiquitin chains. The latter is active as a signal for degradation of the targeted proteins. We found both forms in human serum and, using two immunoassays, quantitated them in sera from healthy subjects and patients with some diseases. Because of putative leakage of erythrocyte ubiquitin, hemolytic serum and serum obtained after long incubation (>1–2 h) of blood at room temperature were excluded. Serum concentrations of multiubiquitin chains and free ubiquitin were substantially higher in rheumatoid arthritis and hemodialysis patients, respectively, than healthy subjects. Additionally, in acute viral hepatitis, serum multiubiquitin chain concentrations were increased in the acute phase, decreased in the recovery phase, and correlated with alanine and aspartate aminotransferase activities (r = 0.676 and 0.610, P <0.0001 and <0.001, respectively). Therefore, serum ubiquitin may have prognostic value.
Analytical performance of the Tandem®-R free PSA immunoassay measuring free prostate-specific antigen
Authors: Woodrum David L , French Chester M , Hill Timothy M , Roman Steven J , Slatore Harold L , Shaffer Jennifer L , York Lisa G , Eure Kathy L , Loveland Kathleen G , Gasior Gail H , Southwick Paula C , Shamel L Blair
Published: January 2020

The analytical performance of the Tandem®-R free PSA assay available from Hybritech Inc. was evaluated. Comparison of recoveries of purified free (unbound) prostate-specific antigen (PSA) diluted in female serum in the Tandem-R free PSA assay and the Tandem-R (total) PSA assay demonstrated a link in calibration between the assays and an accurate determination of percent free PSA. The cross-reactivity of the assay to purified PSA–α1-antichymotrypsin was determined to be <1%. The minimum-detectable concentration was <0.05 μg/L. The within-run and between-day CVs were ≤5% for samples with >0.3 μg/L free PSA. Dilution and recovery showed no significant deviations from linearity across the assay range. The assay was insensitive to interference from blood components. The Tandem-R free PSA kit was shown to be an accurate, precise, and reliable assay for the measurement of free PSA.
Issues in detecting abuse of xenobiotic anabolic steroids and testosterone by analysis of athletes’ urine
Authors: Catlin Don H , Hatton Caroline K , Starcevic Sanja H
Published: January 2020

Over the last decade the number of laboratories accredited by the International Olympic Committee (IOC) has grown to 25. Nearly half of the ∼90 000 samples tested annually are collected on short notice—the most effective means to deter the use of anabolic androgenic steroids (AAS). The major urinary metabolites of AAS have been characterized and are identified by their chromatographic retention times and full or partial mass spectra. The process of determining if an athlete has used testosterone (T) begins with finding a T to epitestosterone (E) ratio >6 and continues with a review of the T/E–time profile. For the user who discontinues taking T, the T/E reverts to baseline (typically ∼ 1.0). For the extremely rare athlete with a naturally increased T/E ratio, the T/E remains chronically increased. Short-acting formulations of T transiently increase T/E, and E administration lowers it. Among ancillary tests to help discriminate between naturally increased T/E values and those reflecting T use, the most promising is determination of the carbon isotope ratio.
Diagnosis of lysosomal storage disorders: evaluation of lysosome-associated membrane protein LAMP-1 as a diagnostic marker
Authors: Meikle Peter J , Brooks Doug A , Ravenscroft Elaine M , Yan Miao , Williams Ruth E , Jaunzems Alvis E , Chataway Timothy K , Karageorgos Litsa E , Davey Richard C , Boulter Christine D , Carlsson Sven R , Hopwood John J
Published: January 2020

Early diagnosis of lysosomal storage disorders (LSDs), before the onset of irreversible pathologies, will be a key factor in the development of effective therapies for many of these disorders. Newborn screening offers a potential mechanism for the early detection of these disorders. From studies of both normal and LSD-affected human skin fibroblasts we identified the lysosome-associated membrane protein LAMP-1 as a potential diagnostic marker. We have developed a sensitive method for the quantification of this protein with a time-resolved fluorescence immunoassay. A soluble form of LAMP-1 was observed in plasma samples, and determination of 152 unaffected individuals gave a median value of 303 μg/L with the 5th and 95th percentile at 175 and 448 μg/L respectively. Plasma samples from 320 LSD-affected individuals representing 25 different disorders were assayed. We observed that 17 of the 25 disorder groups tested had >88% of individuals above the 95th percentile of the control population, with 12 groups having 100% above the 95th percentile. Overall, 72% of patients had LAMP-1 concentrations above the 95th percentile of the unpartitioned control population. We suggest that LAMP-1 may be a useful marker in newborn screening for LSDs.
Effect of anti-thyroid peroxidase (TPO) antibodies on TPO activity measured by chemiluminescence assay
Authors: Kaczur V , Vereb Gy , Molnár I , Krajczár G , Kiss E , Farid N R , Balázs Cs
Published: January 2020

A chemiluminescence method was developed to measure thyroid peroxidase (TPO) activity and the inhibitory effect of anti-TPO antibodies in purified porcine TPO. The TPO preparation was characterized kinetically and controlled by Western-blotting technique. The chemiluminescence method proved to be reproducible and much more sensitive than the widely used guaiacol method, being able to detect TPO concentrations of 2.21 × 10−5 g/L vs 6.63 × 10−2g/L with the latter. Otherwise, the determinations with the two methods correlated well (r = 0.76). Investigating the effect of IgGs from 23 hypothyroid patients on measured TPO activity, we detected inhibition in 19 cases with the chemiluminescence technique (15 with the guaiacol method). Anti-TPO antibodies showed competitive inhibition of TPO activity with respect to the substrate guaiacol. In both systems, the inhibition is present in the IgG F(ab′)2 fragment. We conclude that the high sensitivity of chemiluminescence detection allows routine determination of the inhibition of TPO activity by anti-TPO antibodies.
Fatty acid ethyl esters: short-term and long-term serum markers of ethanol intake
Authors: Laposata Michael
Published: January 2020

This review includes a description of short-term and long-term markers of ethanol intake and their clinical utility. The major portion of this report is a summary of studies on fatty acid ethyl ester, a new marker for monitoring both acute and chronic ethanol intake. With the markers described in the review, algorithms to assess recent ethanol intake, chronic ethanol intake, and end organ damage are included to provide a practical approach to the evaluation of the patient.
Novel analytical approach to monitoring advanced glycosylation end products in human serum with on-line spectrophotometric and spectrofluorometric detection in a flow system
Authors: Wróbel Kazimierz , Wróbel Katarzyna , Garay-Sevilla Ma Eugenia , Nava Laura Eugenia , Malacara Juan Manuel
Published: January 2020

We proposed a simple analytical procedure for measurement of serum advanced glycosylation end products (AGEs) based on simultaneous detection of low-molecular-mass peptides and AGEs with a flow system and two detectors connected on-line: spectrophotometric for peptides (λ = 280 nm) and spectrofluorometric for AGEs (λex = 247 nm, λem = 440 nm). Sample pretreatment was carried out in microcentrifuge tubes: Serum (20 μL) was deproteinized with trichloroacetic acid (480 μL, 0.15 mol/L) and lipids were extracted with chloroform (100 μL). Twenty microliters of the filtered aqueous layer was injected to the flow system and the relation between fluorescence and absorption signals was measured. A peptide-derived AGE calibrator was used for calibration. Within-day and between-day CVs were 6.7% and 9.1%, respectively, at an AGE concentration corresponding approximately to that in healthy individuals. Mean results (±SD) in 10 healthy individuals were 10.1% ± 1.0%, in 21 patients with diabetes without complications 18.0% ± 6.2%, in 25 patients with complications 24.1% ± 15.4%, and in 12 diabetic patients in end-stage renal disease 92% ± 30%. Comparison with an ELISA procedure (x, in arbitrary units/L) yields a regression equation y = 0.713x + 1.24 (Sy‖x = 6777, r = 0.8477, n = 41).
Point-of-care fibrinolytic tests: the other side of blood coagulation
Authors: Oberhardt Bruce J , Mize Patrick D , Pritchard Cynthia G
Published: January 2020

Point-of-care (POC) coagulation tests with paramagnetic iron oxide particles have provided alternatives to testing previously done only in the laboratory. With this technology, POC fibrinolytic tests have followed quietly the trail blazed by POC clotting tests and have found specific applications. These include rapid verification of in vivo thrombolytic drug action by in vitro testing with subsequent quantitative drug monitoring of the systemic lytic state, and also the determination of in vitro thrombolytic drug response before the drug is actually administered, to individualize therapy by selection of the most appropriate drug. Other applications include POC coagulation factor assays associated with fibrinolysis, and most recently the POC screening of patients with fibrinolytic defects. In this latter application, plasma from cardiac catheterization (n = 19) and venous thrombosis (n = 47) patient groups were tested. Controls consisted of two independent donor pools (n = 10, n = 21) as negatives and two plasma samples with known genetic defects in the fibrinogen molecule (Aα554 Arg → Cys) as positives.
Coagulation monitor based on serum migration through absorbent materials
Authors: Wilson Paul E
Published: January 2020

I describe a method to measure blood coagulation properties, based on the hypothesis that the distance the serum component of a clotted plasma sample moves through a suitable absorbent material should be proportional to the blood’s ability to clot. A simple apparatus was constructed to test this principle, in which an absorbent strip contacts clotted plasma samples. At various times, the liquid migration distance into the strip was measured and correlated with clotting times determined by well-accepted procedures. Use of this device to test lyophilized normal and barium-absorbed plasmas, factor VII-deficient plasma, frozen normal plasmas, plasmas from patients undergoing oral anticoagulation, and saline-diluted plasmas (as for Quick Percent assays) showed that clotting times correlated with migration distances for all types of samples (r2 = 0.93–0.99). The device distinguishes between samples from normal and coumadin-treated subjects. The concept can be integrated into an inexpensive, point-of-care coagulation monitor.