bluuebunny/crossref_metadata_2025 huggingface dataset:
Prevalence of Universal Medication Schedule prescribing and links to adherence
Authors: Wolf Michael S , Taitel Michael S , Jiang Jenny Z , Curtis Laura M , Wismer Guisselle A , Wallia Amisha , Parker Ruth M
Published: January 2020

Purpose

A Universal Medication Schedule (UMS) that uses explicit language to describe when to take medicine has been proposed as a patient-centered prescribing and dispensing standard. Despite widespread support, evidence of its actual use and efficacy is limited. We investigated the prevalence of UMS instructions and whether their use was associated with higher rates of medication adherence.

Methods

National pharmacy records were analyzed for a cohort of type 2 diabetic adults ≥18 years old (N = 676,739) new to ≥1 oral diabetes medications between January and June 2014. Prescription instructions (N = 796,909) dispensed with medications were classified as UMS or non-UMS. Instructions coded as UMS were further categorized as either providing precise UMS language (tier 1: “take 1 pill at morning, noon, evening, or bedtime”) or offering some explicit guidance (tier 2: “take 1 tablet by mouth before breakfast”; tier 3: “take 1 tablet twice daily with a meal”). Adherence over 12 months was measured by proportion of days covered.

Results

One-third of instructions (32.4%, n = 258,508) were classified as UMS (tier 1: 12.6%, n = 100,589; tier 2: 6.0%, n = 47,914; tier 3: 13.8%, n = 110,005). In multivariable analyses, UMS instructions (all tiers) exhibited better adherence compared to non-UMS instructions (relative risk [RR], 1.01; 95% confidence interval [CI], 1.00-1.02; P = 0.01). Patients older than 65 years who were less educated and taking medication more than once daily received greater benefit from tier 1 UMS instructions (RR, 1.14; 95% CI, 1.07-1.21; P < 0.001).

Conclusion

While infrequently used, the UMS could help older, less-educated patients adhere to more complex regimens with minimal investment.
Gravity in Midfacial Aging: A 3-Dimensional Study
Authors: Ramesh Sathyadeepak , Johnson Patricia , Sarcu David , Wulc Allan Edward
Published: January 2020

Background

Volume loss and volume descent are complementary aspects of facial aging, although the respective contribution of each is unclear.

Objectives

The aim of this study was to quantify in 3 dimensions the effects of gravity on the midface in both upright and supine positions in an older and a younger group of patients.

Methods

A prospective study was undertaken of 53 female patients who had not undergone treatment with dermal fillers or facial cosmetic surgery. Three-dimensional photography with a VECTRA H1 camera (Canfield Scientific, Fairfield, NJ) was taken in supine and sitting positions. Volume shifts and displacement of structures in 3 dimensions were measured and analyzed.

Results

Morphologically, upon shifting from sitting to supine position, the tail of the brow elevated, the tear trough filled, the buccal and malar fat shifted posteriosuperiorly, the earlobe decreased in height, the nasiolabial folds and marionette lines diminished, the modiolus shifted laterally, and the jowl diminished. Volumetric analysis revealed that the infraorbital region increased in volume by a mean [SD] of 0.59 [0.55] mL, the tear trough by 0.22 [0.19] mL, and the malar region by 1.2 [1.06] mL. With subjects in the supine position, all facial topographic landmarks displaced significantly from sitting position.

Conclusions

Facial aging in this cohort was predominantly due to tissue descent rather than volume loss. Reversal of the gravitational force restores the 3D position of the facial subunits and leads to volumization in desirable locations that approximates a more youthful appearance. The volume restored via redistribution of facial fat is comparable to that typically injected during direct volume transfer procedures.

Level of Evidence: 4
A Bayesian approach to accurate and robust signature detection on LINCS L1000 data
Authors: Qiu Yue , Lu Tianhuan , Lim Hansaim , Xie Lei
Published: January 2020

Motivation

LINCS L1000 dataset contains numerous cellular expression data induced by large sets of perturbagens. Although it provides invaluable resources for drug discovery as well as understanding of disease mechanisms, the existing peak deconvolution algorithms cannot recover the accurate expression level of genes in many cases, inducing severe noise in the dataset and limiting its applications in biomedical studies.

Results

Here, we present a novel Bayesian-based peak deconvolution algorithm that gives unbiased likelihood estimations for peak locations and characterize the peaks with probability based z-scores. Based on the above algorithm, we build a pipeline to process raw data from L1000 assay into signatures that represent the features of perturbagen. The performance of the proposed pipeline is evaluated using similarity between the signatures of bio-replicates and the drugs with shared targets, and the results show that signatures derived from our pipeline gives a substantially more reliable and informative representation for perturbagens than existing methods. Thus, the new pipeline may significantly boost the performance of L1000 data in the downstream applications such as drug repurposing, disease modeling and gene function prediction.

Availability and implementation

The code and the precomputed data for LINCS L1000 Phase II (GSE 70138) are available at https://github.com/njpipeorgan/L1000-bayesian.

Supplementary information

Supplementary data are available at Bioinformatics online.
Statistical inference of differential RNA-editing sites from RNA-sequencing data by hierarchical modeling
Authors: Tran Stephen S , Zhou Qing , Xiao Xinshu
Published: January 2020

Motivation

RNA-sequencing (RNA-seq) enables global identification of RNA-editing sites in biological systems and disease. A salient step in many studies is to identify editing sites that statistically associate with treatment (e.g. case versus control) or covary with biological factors, such as age. However, RNA-seq has technical features that incumbent tests (e.g. t-test and linear regression) do not consider, which can lead to false positives and false negatives.

Results

In this study, we demonstrate the limitations of currently used tests and introduce the method, RNA-editing tests (REDITs), a suite of tests that employ beta-binomial models to identify differential RNA editing. The tests in REDITs have higher sensitivity than other tests, while also maintaining the type I error (false positive) rate at the nominal level. Applied to the GTEx dataset, we unveil RNA-editing changes associated with age and gender, and differential recoding profiles between brain regions.

Availability and implementation

REDITs are implemented as functions in R and freely available for download at https://github.com/gxiaolab/REDITs. The repository also provides a code example for leveraging parallelization using multiple cores.
Working with Restrictions: A Scoping Review of Social Work and Human Service Practice with People Seeking Asylum in the Global North
Authors: Field Rebecca Soraya , Chung Donna , Fleay Caroline
Published: January 2020

With a record number of people reported to be forcibly displaced worldwide and an increasingly anti-asylum policy environment in Global North host nations, it is critical to examine social work and human services with people seeking asylum and people with refugee status. This scoping review sought to identify and review the existing qualitative research on such practice from the perspectives of social work and human service practitioners, people seeking asylum and people with refugee status and clients in Global North host nations. The review presents the key findings and trends emerging from twenty publications and identifies areas for future research to further develop knowledge of this field of practice as there is a dearth of research on this topic. Through thematic analysis of very different publications, it was found that Global North host nations have varying forms of restrictive policy and dominant anti-asylum discourses. This and other factors result in many potential areas for improvement of social work and human services. The findings include recommendations for more inclusive and compassionate policy, person-centred and strength-based practice approaches, and further qualitative research with people seeking asylum.
Relational Autonomy and Service Choices in Social Worker–Client Conversations in an Outpatient Clinic for People Using Drugs
Authors: Juhila Kirsi , Ranta Johanna , Raitakari Suvi , Banks Sarah
Published: January 2020

This article focuses on how clients’ self-determination is accomplished in social worker–client conversations when discussing choices of clients’ future services in a low-threshold outpatient clinic in Finland targeted at people who use drugs. Self-determination is approached from the point of view of relational autonomy, meaning that choices are never made completely independently but within certain societal and interactional contexts. The article applies interactional analysis to data from ten social worker–client conversations, which include forty-eight instances of ‘choice talk’. The results demonstrate how social workers work hard to promote clients’ self-determination, and how this is carried out with different emphases within the frame of relational autonomy. Social workers do not perform ethically questionable manipulation practices. Quite the reverse, their contributions in the conversations can be interpreted as endeavours to increase clients’ self-confidence and autonomy competencies. However, a concern from an ethical point of view is that real service options are rather scarce for the clinic’s clients. This considerably reduces the clients’ capacity for self-determination. Furthermore, it also reduces the autonomy of social workers, who have limited opportunities to organise the services their clients desire and that the social workers themselves consider are the best options.
Temporal Gating of Synaptic Competition in the Amygdala by Cannabinoid Receptor Activation
Authors: Madeira Natália , Drumond Ana , Fonseca Rosalina
Published: January 2020

The acquisition of fear memories involves plasticity of the thalamic and cortical pathways to the lateral amygdala (LA). In turn, the maintenance of synaptic plasticity requires the interplay between input-specific synaptic tags and the allocation of plasticity-related proteins. Based on this interplay, weakly activated synapses can express long-lasting forms of synaptic plasticity by cooperating with strongly activated synapses. Increasing the number of activated synapses can shift cooperation to competition. Synaptic cooperation and competition can determine whether two events, separated in time, are associated or whether a particular event is selected for storage. The rules that determine whether synapses cooperate or compete are unknown. We found that synaptic cooperation and competition, in the LA, are determined by the temporal sequence of cortical and thalamic stimulation and that the strength of the synaptic tag is modulated by the endocannabinoid signaling. This modulation is particularly effective in thalamic synapses, supporting a critical role of endocannabinoids in restricting thalamic plasticity. Also, we found that the availability of synaptic proteins is activity-dependent, shifting competition to cooperation. Our data present the first evidence that presynaptic modulation of synaptic activation, by the cannabinoid signaling, functions as a temporal gating mechanism limiting synaptic cooperation and competition.
Lipopolysaccharide-Induced Inflammatory Cytokine Expression in Taste Organoids
Authors: Feng Shan , Achoute Leyitha , Margolskee Robert F , Jiang Peihua , Wang Hong
Published: January 2020

Inflammatory cytokines are signaling molecules that regulate numerous physiological processes, from tissue homeostasis to metabolism and food intake. Expression of certain cytokines can be markedly induced in subsets of taste bud cells under acute and chronic inflammation. This may contribute to altered taste perception and preference associated with many diseases. Although the pathways of cytokine induction are well studied in immune cells, they remain poorly characterized in taste cells, in part due to the difficulties of performing biochemical analyses with a limited number of taste cells. The recently developed taste organoid model provides an opportunity to carry out these mechanistic studies in vitro. However, it was unknown whether taste organoids respond to inflammatory stimuli as do in vivo native taste buds. Here we analyze lipopolysaccharide (LPS)-induced expression and secretion of two inflammatory cytokines, tumor necrosis factor (TNF), and interleukin-6 (IL-6). We show that, similarly to native mouse taste epithelia, organoids derived from mouse circumvallate stem cells express several toll-like receptors (TLRs), including TLR4—the primary receptor for LPS. Organoids and native taste epithelia express all five genes in the nuclear factor-κb (Nfkb) family that encode the transcription factor NF-κB, a critical regulator of inflammatory responses. LPS stimulates fast induction of TNF and IL-6 with similar induction kinetics in organoids and native taste epithelia. These results show that taste epithelial cells possess necessary components for inflammatory cytokine induction and secretion and suggest that the organoid model can be a useful tool to dissect the underlying mechanisms.
Liver and Adipose Tissue Fatty Acid Ethyl Esters Obtained at Autopsy Are Postmortem Markers for Premortem Ethanol Intake
Authors: Refaai Majed A , Nguyen Phan N , Steffensen Thora S , Evans Richard J , Cluette-Brown Joanne E , Laposata Michael
Published: January 2020

Background: Fatty acid ethyl esters (FAEEs) are nonoxidative ethanol metabolites that have been implicated as mediators of alcohol-induced organ damage. FAEEs are detectable in the blood after ethanol ingestion, and on that basis have been proposed as markers of ethanol intake. Because blood is not always available at autopsy, in this study we quantified FAEEs in human liver and adipose tissue as potential postmortem markers of premortem ethanol intake.

Methods: Twenty-four sets of samples were collected at the Massachusetts State Medical Examiner’s Office, and 7 sets of samples were obtained from the Pathology Department of Massachusetts General Hospital. Samples of liver and adipose tissue were collected at autopsy, and FAEEs were isolated and quantified from these organs as mass per gram of wet weight. Postmortem analysis of blood involved assessment for ethanol and other drugs.

Results: The study shows a substantial difference in FAEE concentrations in liver and adipose tissue of patients with detectable blood ethanol at the time of autopsy vs those with no detectable blood ethanol, who were either chronic alcoholics or social drinkers. In addition, a specific FAEE, ethyl arachidonate, was found at concentrations >200 pmol/g almost exclusively in the liver and adipose tissue of individuals with detectable blood ethanol at the time of death, providing an additional FAEE-related marker for prior ethanol intake.

Conclusions: The mass of FAEEs in liver and adipose tissue and the presence of ethyl arachidonate can serve as postmortem markers of premortem ethanol intake when no blood sample can be obtained.
Determination of Coumarin-type Anticoagulants in Human Plasma by HPLC-Electrospray Ionization Tandem Mass Spectrometry with an Ion Trap Detector
Authors: Kollroser Manfred , Schober Caroline
Published: January 2020

Background: Coumarin-type anticoagulants are used for the long-term treatment and prevention of thromboembolic disorders. The identification of these drugs is crucial in patients with an increased prothrombin time of unknown origin. The aim of this study was to develop a sensitive and specific method for the simultaneous determination of phenprocoumon, acenocoumarol, and warfarin in human plasma by HPLC-electrospray ionization tandem mass spectrometry.

Methods: After addition of the internal standard, p-chlorowarfarin, plasma samples were extracted using Oasis® MCX solid-phase extraction cartridges. The compounds were separated on a Symmetry C18 column (Waters) with a mobile phase of acetonitrile–1 g/L formic acid (75:25 by volume) at a flow rate of 0.5 mL/min.

Results: Extraction and separation of the three drugs and the internal standard were accomplished in 9 min. The overall extraction efficiency was >89% for all three compounds. The limits of detection were 1 μg/L for phenprocoumon and warfarin and 10 μg/L for acenocoumarol. Regression analysis of the calibration data revealed good correlation (r2 ≥0.995) for all compounds. Within-run accuracies for quality-control samples were ± 1% to 7% of the target concentration, with CVs <9%.

Conclusions: The proposed method enables the unambiguous identification and quantification of phenprocoumon, warfarin, and acenocoumarol in both clinical and forensic specimens. This method combines a new, rapid solid-phase extraction procedure with an extremely fast chromatographic analysis, which is especially advantageous for clinical laboratories.
Polymorphisms of TLR4: Rapid Genotyping and Reduced Response to Lipopolysaccharide of TLR4 Mutant Alleles
Authors: Schmitt Christopher , Humeny Andreas , Becker Cord-Michael , Brune Kay , Pahl Andreas
Published: January 2020

Background: Pathogen recognition receptors such as Toll-like receptors (TLRs), which recognize pathogen-associated molecular patterns, lead to the activation of innate immunity. Genetic variations in these receptors may lead to an altered host immune response to pathogens.

Methods: We developed homogeneous fluorescence-based PCR assays as well as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) genotyping assays to detect TLR4 polymorphisms. These assays were compared with restriction fragment length polymorphism (RFLP) analysis. Peripheral blood monocytes from donors with differing genotypes were prepared and exposed to bacterial products in vitro. The abundance of mRNAs of the proinflammatory cytokines interleukin (IL)-1β, IL-6, and tumor necrosis factor-α from these monocytes were monitored by real-time reverse transcription-PCR.

Results: By our homogeneous PCR method, the allele frequencies were 5.6% for the TLR4 Asp299Gly and 6.0% for the TLR4 Thr399Ile polymorphism in 116 healthy German Caucasians. Nine incorrect genotype calls were detected in the RFLP analysis and two in the TaqMan genotype analysis. MALDI-TOF-MS allowed clear detection of all TLR4 alleles. Monocytes from donors homozygous for the TLR4 mutant alleles Asp299Gly and Thr399Ile were lipopolysaccharide hyporesponsive and exhibited median effective concentrations (EC50s) approximately fourfold higher than those of monocytes carrying wild-type or heterozygous alleles. In contrast, a TLR2 agonist elicited similar responses in monocytes irrespective of the TLR4 genotype.

Conclusions: Homogeneous fluorescence-based PCR assays provide a specific and sensitive method for high-throughput genotyping of TLR4 mutations. The newly developed PCR and MALDI-TOF-MS assays may be useful to evaluate the presence of TLR4 polymorphisms in patients to predict susceptibility to bacterial infection.
European Concerted Action on Anticoagulation. Evaluation of a Method for International Sensitivity Index Calibration of Two Point-of-Care Prothrombin Time (PT) Monitoring Systems (CoaguChek Mini and TAS PT-NC) with Fresh Plasmas Based on Whole-Blood Equivalent PT
Authors: Poller Leon , Keown Michelle , Chauhan Nikhil , van den Besselaar Anton MHP , Tripodi Armando , Shiach Caroline , Jespersen Jorgen
Published: January 2020

Background: The International Sensitivity Index (ISI) calibration of whole-blood prothrombin time (PT) monitors for point-of-care testing (POCT) described by Tripodi et al. (Thromb Haemost 1993;70:921–4) has been shown to be dependable but is too complex and demanding. The use of plasma would simplify calibration of whole-blood POCT PT monitors, but important differences may exist between the ISI for whole blood and plasma calibrations.

Methods: In a 10-center calibration study of two POCT whole-blood monitoring systems (CoaguChek Mini and TAS PT-NC), we characterized the relationship between the log PT for whole blood and fresh plasma with use of single lots of test strips/cards. This relationship (linear) was used to correct the difference between the whole-blood and plasma ISI. The reliability of the correction with different lots of test strips/cards was assessed at three centers. The linear relationship was used to correct the difference in the whole-blood and plasma ISI with four other lots of TAS PT-NC cards and with two additional lots of CoaguChek Mini test strips.

Results: The correction decreased the ISI difference from 13.3% to 0.9% for the TAS PT-NC and from 5.7% to 0.6% for the CoaguChek Mini. In assessments at three centers, which included different lots of test strips/cards, the mean ISI difference was markedly decreased with the TAS PT-NC but not with the CoaguChek Mini, for which the mean ISI difference increased slightly.

Conclusions: The proposed correction resolves the discrepancy between whole-blood and fresh plasma ISI calibrations with TAS PT-NC test cards. The CoaguChek Mini systems could be calibrated without this correction.
Development and Evaluation of an ELISA for Human Trefoil Factor 3
Authors: Vestergaard Else Marie , Poulsen Steen Seier , Grønbæk Henning , Larsen Rune , Nielsen Anette Mertz , Ejskjær Kirsten , Clausen Jes Thorn , Thim Lars , Nexø Ebba
Published: January 2020

Background: The three trefoil factors (TFF1, TFF2, and TFF3) are small peptides believed to cross-link mucous glycoproteins and to play a role in the maintenance and repair of the gastrointestinal mucosa. To define the physiologic and potential diagnostic values of TFF3, assays able to measure TFF3 are warranted.

Methods: An ELISA was developed that uses two antibodies from rabbits immunized with recombinant human TFF3 and a calibrator (3–100 pmol/L) prepared from recombinant human TFF3.

Results: The ELISA had a detection limit of 3.0 pmol/L. The imprecision (CV) was 5–9% for mean concentrations of 13–65 pmol/L, corresponding to serum concentrations of 65–330 pmol/L. There was no cross-reaction toward human TFF1 and TFF2 (40 nmol/L). Neither food intake nor the menstrual cycle influenced the values of TFF3 significantly. The central 95% reference interval for TFF3 in serum from healthy blood donors (n = 300) was 91–250 pmol/L and showed no variation with age and limited variation with sex. TFF3 was increased in serum from patients (n = 12) with inflammation and/or ulceration of the upper gastrointestinal tract (P <0.05), whereas in serial measurements of serum from three patients with severe exacerbation of chronic inflammatory bowel disease restricted to the colon, normal concentrations and only minor variations during treatment and tapering were observed.

Conclusions: The ELISA measures TFF3 in human serum and represents a specific and precise method for measurement of TFF3, which will be of value for further studies of TFF3 in health and disease.
Therapeutic Drug Monitoring of Anticonvulsant Drugs by Micellar HPLC with Direct Injection of Serum Samples
Authors: Martinavarro-Domínguez Adrián , Capella-Peiró Maria-Elisa , Gil-Agustí Mayte , Marcos-Tomás José V , Esteve-Romero Josep
Published: January 2020

Background: We developed a micellar liquid chromatographic (MLC) procedure for the determination of three extensively monitored antiepileptics in serum samples: carbamazepine, phenobarbital, and phenytoin.

Methods: We determined the composition of the mobile phase after modeling the elution behavior of the antiepileptics in hybrid micellar mobile phases of sodium dodecyl sulfate (SDS) with different organic modifiers (propanol, butanol, or pentanol) in an experimental design that used five mobile phases, a C18 column, and ultraviolet detection. In the micellar chromatographic system, the serum samples can be injected directly.

Results: The optimum mobile phase was 70 mL/L butanol in 0.05 mol/L SDS, pH 7, in which the three antiepileptics were resolved in <10 min. Intra- and interday precision was evaluated at four different drug concentrations within the therapeutic range (n =10); CVs were <2.1%. The method was applied to the analysis of 120 serum samples, and results were similar to those obtained by the TDx® method.

Conclusions: The MLC method allows chromatographic determination of three antiepileptics, using an interpretative strategy of optimization, without pretreatment of the serum samples and with direct injection in a hybrid micellar mobile phase of SDS–butanol. The method provides complete resolution and quantification of mixtures of two and three antiepileptics.
Antibodies Directed against the E Region of Pro-Insulin-like Growth Factor-II Used to Evaluate Non-Islet Cell Tumor-induced Hypoglycemia
Authors: van Doorn Jaap , Hoogerbrugge Cok M , Koster Johanna G , Bloemen Ruud J , Hoekman Klaas , Mudde Aart H , van Buul-Offers Sylvia C
Published: January 2020

Background: Detection of incompletely processed precursor forms of insulin-like growth factor-II (“big” IGF-II) in plasma is essential for both the diagnosis and follow-up of non-islet cell tumor-induced hypoglycemia (NICTH) and may be relevant to other diseases as well. RIA using an antibody raised against a synthetic peptide consisting of the first 21 amino acids of the E domain [E(68–88)] of human pro-IGF-II cannot distinguish between E-peptide-containing big IGF-II and cleaved E domain or fragments. We therefore developed and validated an ELISA that specifically detects big IGF-II in plasma.

Methods: The ELISA used a solid-phase antibody to E(68–88) and a liquid-phase monoclonal hIGF-II antibody. Pro-IGF-II purified from normal human plasma was used as a calibrator. Acid Sep-Pak C18 extracts of plasma from NICTH patients were analyzed, and the results were compared with those obtained for plasma samples from healthy individuals. In addition, blood specimens derived from dialyzed patients with chronic renal failure, which contained relatively high concentrations of cleaved E domain or fragments, were studied. The results were validated by acid Sephadex G-50 gel filtration.

Results: Results from this ELISA indicated that the concentration of big IGF-II in NICTH plasma was higher (mean ± SD, 22.6 ± 9.4 nmol/L) than in normal plasma (3.8 nmol/L). Conversely, the concentrations in pooled CRF plasma (2.0 ± 0.8 nmol/L) were low. Antibodies directed against either E(68–88) or E(13–134) of pro-IGF-II could be used to detect these peptides in tumor tissue by immunohistochemistry.

Conclusions: The possibility of quantifying pro-IGF-II by ELISA in plasma represents a potentially useful tool for the diagnosis and follow-up of NICTH and should facilitate further in vitro and in vivo studies on its regulation and function in humans.
Holo-Transcobalamin Is an Early Marker of Changes in Cobalamin Homeostasis. A Randomized Placebo-controlled Study
Authors: Nexo Ebba , Hvas Anne-Mette , Bleie Øyvind , Refsum Helga , Fedosov Sergey N , Vollset Stein Emil , Schneede Jorn , Nordrehaug Jan Erik , Ueland Per Magne , Nygard Ottar Kjell
Published: January 2020

Background: We examined the effect of oral vitamin B12 treatment on fluctuations in plasma total cobalamin and its binding proteins transcobalamin (TC) and haptocorrin (HC).

Methods: Patients (n = 88; age range, 38–80 years) undergoing coronary angiography (part of the homocysteine-lowering Western Norway B-Vitamin Intervention Trial) were allocated to daily oral treatment with (a) vitamin B12 (0.4 mg), folic acid (0.8 mg), and vitamin B6 (40 mg); (b) vitamin B12 and folic acid; (c) vitamin B6; or (d) placebo. EDTA blood was obtained before treatment and 3, 14, 28, and 84 days thereafter.

Results: The intraindividual variation for patients not treated with B12 was ∼10% for plasma total cobalamin, total TC, apo-TC, and apo-HC, and <20% for holo-TC and TC saturation. In B12-treated patients, the maximum change in concentrations was observed already after 3 days for total TC (−16%), holo-TC (+54%), and TC saturation (+82%). At this time holo-HC (+20%) and plasma total cobalamin (+28%) showed an initial burst, but had increased further at 84 days. All changes were highly significant compared with the control group (P <0.0001).

Conclusions: Oral vitamin B12 treatment produces maximal effects on total TC, holo-TC, and TC saturation within 3 days, whereas maximal increases in holo-HC and plasma total cobalamin occur later. The results support the view that holo-TC is an early marker of changes in cobalamin homeostasis.
Guanidinoacetate and Creatine plus Creatinine Assessment in Physiologic Fluids: An Effective Diagnostic Tool for the Biochemical Diagnosis of Arginine:Glycine Amidinotransferase and Guanidinoacetate Methyltransferase Deficiencies
Authors: Carducci Claudia , Birarelli Maurizio , Leuzzi Vincenzo , Carducci Carla , Battini Roberta , Cioni Giovanni , Antonozzi Italo
Published: January 2020

Background: Disorders of creatine metabolism arise from genetic alterations of arginine:glycine amidinotransferase (AGAT), guanidinoacetate methyltransferase (GAMT), and the creatine transporter. We developed a strategy for the detection of AGAT and GAMT defects by measurement of guanidinoacetate (GAA) and creatine plus creatinine (Cr+Crn) in biological fluids.

Methods: Three patients with AGAT deficiency from the same pedigree and their eight relatives, as well as a patient affected by a GAMT defect and his parents were analyzed by a new HPLC procedure in comparison with 90 controls. The method, which uses precolumn derivatization with benzoin, separation with a reversed-phase column, and fluorescence detection, has shown good precision and sensitivity and requires minimal sample handling.

Results: In the three AGAT patients, plasma GAA was 0.01–0.04 μmol/L [mean (SD) for neurologically normal controls was 1.16 (0.59) μmol/L], Cr+Crn was 15–29 μmol/L [reference limit in our laboratory, 79 (38) μmol/L]. Urinary GAA was 2.4–5.8 μmol/L [reference, 311 (191) μmol/L], and Cr+Crn was 2.1–3.3 mmol/L [reference, 9.9 (4.1) mmol/L]. We found a smaller decrease in GAA and Cr+Crn in some carriers of an AGAT defect. In the patient with GAMT deficiency, plasma and urine GAA was increased (18.6 and 1783 μmol/L, respectively), and Cr+Crn was decreased in plasma (10.7 μmol/L) and urine (2.1 mmol/L). GAA was increased in the parents’ plasmas and in the mother’s urine.

Conclusion: The assessment of GAA is a new tool for the detection of both GAMT and AGAT deficiencies.
Comparison of Logistic Regression and Neural Net Modeling for Prediction of Prostate Cancer Pathologic Stage
Authors: Veltri Robert W , Chaudhari Manisha , Miller M Craig , Poole Edward C , O’Dowd Gerard J , Partin Alan W
Published: January 2020

Background: Prostate cancer (PCa) pathologic staging remains a challenge for the physician using individual pretreatment variables. We have previously reported that UroScoreTM, a logistic regression (LR)-derived algorithm, can correctly predict organ-confined (OC) disease state with >90% accuracy. This study compares statistical and neural network (NN) approaches to predict PCa stage.

Methods: A subset (756 of 817) of radical prostatectomy patients was assessed: 434 with OC disease, 173 with capsular penetration (NOC-CP), and 149 with metastases (NOC-AD) in the training sample. Additionally, an OC + NOC-CP (n = 607) vs NOC-AD (n = 149) two-outcome model was prepared. Validation sets included 120 or 397 cases not used for modeling. Input variables included clinical and several quantitative biopsy pathology variables. The classification accuracies achieved with a NN with an error back-propagation architecture were compared with those of LR statistical modeling.

Results: We demonstrated >95% detection of OC PCa in three-outcome models, using both computational approaches. For training patient samples that were equally distributed for the three-outcome models, NNs gave a significantly higher overall classification accuracy than the LR approach (40% vs 96%, respectively). In the two-outcome models using either unequal or equal case distribution, the NNs had only a marginal advantage in classification accuracy over LR.

Conclusions: The strength of a mathematics-based disease-outcome model depends on the quality of the input variables, quantity of cases, case sample input distribution, and computational methods of data processing of inputs and outputs. We identified specific advantages for NNs, especially in the prediction of multiple-outcome models, related to the ability to pre- and postprocess inputs and outputs.
Differential Reactivity of Two Homogeneous LDL-Cholesterol Methods to LDL and VLDL Subfractions, as Demonstrated by Ultracentrifugation and HPLC
Authors: Usui Shinichi , Kakuuchi Hajime , Okamoto Motoi , Mizukami Yuki , Okazaki Mitsuyo
Published: January 2020

Background: The analytical and clinical performance of homogeneous LDL-cholesterol assays has been reported, but their reactions with subfractions of LDL and VLDL have not been described in detail.

Methods: We evaluated reaction selectivity of two homogeneous LDL-cholesterol assays, LDLk (Kyowa Medex) and LDLd (Daiichi Pure Chemical), with ultracentrifugally isolated VLDL and LDL subfractions to identify the lipoprotein particles from which the cholesterol recognized by these assays originates.

Results: The LDLd (y) and LDLk (x) methods correlated highly for whole serum samples: y = 0.986x − 39.5 mg/L (r = 0.966; n = 34). In isolated VLDL, the LDLk and the LDLd methods recovered 17.3% and 23.8% of cholesterol, respectively; but correlation analysis revealed differential reactivity to small and large VLDL particles. For the isolated LDL subfraction of density 1.019–1.040 kg/L, the LDLd method had significantly higher reactivity (95.6–98.7%) than the LDLk (88.4–92.0%). Both methods, however, demonstrated poor recovery (∼50%) for the 1.050–1.063 kg/L fraction, indicating incomplete reactivity with small, dense LDL. Reactivity with lipoprotein(a) was better (71.2–90.8%) for both methods than with small LDL. For intermediate-density lipoprotein (IDL), there was no significant difference in recovery between the two methods (71.7% for LDLk and 68.9% for LDLd), but the LDLk method appeared to be more sensitive to IDL particle size.

Conclusions: The two homogeneous assays for LDL-cholesterol demonstrate only partial reactivity to small, dense LDL and nonspecific reactions to VLDL particles. Modification will be required in the homogeneous methods to obtain LDL-cholesterol values equivalent to those obtained by ultracentrifugation.
Lack of Association between Increased Carotid Intima-Media Thickening and Decreased HDL-Cholesterol in a Family with a Novel ABCA1 Variant, G2265T
Authors: Hong Seung Ho , Riley Ward , Rhyne Jeffrey , Friel Gina , Miller Michael
Published: January 2020

Low HDL-cholesterol (HDL-C) concentrations are inversely correlated with cardiovascular disease, and previous studies have demonstrated that variants in the ATP-binding cassette transporter, ABCA1, are responsible for a proportion of HDL-C deficiency states. We identified a novel variant in ABCA1 in a kindred with decreased HDL-C. This variant was not identified in >200 chromosomes of healthy individuals. The proband, a heterozygote for G2265T, developed premature coronary artery disease. In addition to low HDL-C, six biological family members heterozygous for the ABCA1 variant exhibited low HDL-C concentrations compared with unaffected family members (0.83 ± 0.32 vs 1.33 ± 0.36 mmol/L; P = 0.009). Despite the decreased HDL-C, carotid artery B-mode ultrasound studies failed to reveal increased intima-media thickening in affected individuals compared with age- and sex-matched controls. Although these data extend previous observations that a single defective ABCA1 allele may lead to decreased HDL-C, associated evidence of early atherosclerosis was not confirmed.
Epitope Mapping of Antibodies against Prostate-specific Antigen with Use of Peptide Libraries
Authors: Leinonen Jari , Wu Ping , Stenman Ulf-Håkan
Published: January 2020

Background: Prostate-specific antigen (PSA) is the most important marker for prostate cancer, but PSA concentrations determined by various assays can differ significantly because of differences in specificity of the antibodies used. To identify epitopes recognized by various monoclonal antibodies (MAbs) to PSA, we have isolated peptides that react with the paratopes of these.

Methods: Six anti-PSA MAbs representing three major epitope groups were screened with five cyclic phage display peptide libraries. After selection, the peptide sequences were determined by sequencing of the relevant part of viral DNA. Binding of the phage peptides to the MAbs was monitored by immunoassay.

Results: For each MAb, several paratope-binding peptides with distinct sequence motifs were identified, but only ∼10% showed similarity with the PSA sequence. Some of these correctly predicted the location of the epitopes. By sequential panning of the library with two closely related MAbs, we identified peptides reacting equally with both MAbs. When analyzed against a large panel of PSA MAbs, the peptides generally showed restricted specificity toward the MAb used for selection, but some peptides bound to several related MAbs.

Conclusions: Most of the cyclic peptides selected with PSA MAbs are specific for the MAb used for selection and do not resemble any sequence on the antigen. Peptides reactive with two MAbs recognizing the same epitope can be obtained by sequential panning. This method can be used to predict the location of some epitopes, but additional methods are needed to confirm the result.
Methods for Measurement of LDL-Cholesterol: A Critical Assessment of Direct Measurement by Homogeneous Assays versus Calculation
Authors: Nauck Matthias , Warnick G Russell , Rifai Nader
Published: January 2020

Background: Because LDL-cholesterol (LDL-C) is a modifiable risk for coronary heart disease, its routine measurement is recommended in the evaluation and management of hypercholesterolemia. We critically examine here the new homogeneous assays for direct determination of LDL-C.

Approach: This review relies on published studies and data of the authors using research and routine methods for LDL-C determination. We review experience with methods from their earlier use in lipid research laboratories through the transition to routine clinical testing and the recent development of homogeneous assays. We focus on comparative evaluations and characterizations and the performance of the assays.

Content: Homogeneous assays seem to be able to meet current National Cholesterol Education Program (NCEP) requirements for LDL-C testing for precision (CV <4%) and accuracy (bias <4%), when samples collected from nonfasting individuals are used. In addition, all five currently available assays have been certified by the Cholesterol Reference Methods Laboratory Network. The homogeneous methods also appear to better classify individuals into NCEP cutpoints than the Friedewald calculation. However, the limited evaluations to date raise questions about their reliability and specificity, especially in samples with atypical lipoproteins.

Conclusions: Available evidence supports recommending the homogeneous assays for LDL-C to supplement the Friedewald calculation in those cases where the calculation is known to be unreliable, e.g., triglycerides >4000 mg/L. Before the homogeneous assays can be confidently recommended to replace the calculation in routine practice, more evaluation is needed.
Inaccuracy of Lipid Measurements with the Portable Cholestech L·D·X Analyzer in Patients with Hypercholesterolemia
Authors: Stein James H , Carlsson Cynthia M , Papcke-Benson Kristi , Einerson Jean A , McBride Patrick E , Wiebe Donald A
Published: January 2020

Background: Although total cholesterol concentrations measured by portable lipid analyzers have acceptable bias and precision in young and middle-aged adults, clinically relevant differences in HDL-cholesterol (HDL-C) and triglyceride values have been described. Furthermore, the accuracy of portable lipid analyzers in older hyperlipidemic individuals, who have a high incidence of coronary heart disease, has not been validated. This study determined the biases and variability in portable lipid measurements in older patients with hypercholesterolemia and related them to National Cholesterol Education Program Adult Treatment Panel III guidelines.

Methods: Participants were ≥70 years of age with fasting serum LDL-cholesterol (LDL-C) concentrations >1.40 g/L. Fasting fingerstick samples were analyzed on a Cholestech L·D·X desktop analyzer. Antecubital venous samples were analyzed in a proficiency-certified clinical laboratory.

Results: Portable measurements systematically overestimated triglycerides (0.296 g/L; P <0.001) and HDL-C (0.015 g/L; P = 0.026). LDL-C concentrations were underestimated (0.043 g/L; P = 0.046). Total and non-HDL cholesterol calculations based on the portable lipid device provided unbiased estimates, but wide variability was present. Significant variability in lipid determinations limited their clinical usefulness in individual patients, especially because 2 SD of the mean bias between the laboratory and the portable determinations of LDL-C and non-HDL cholesterol exceeded the 0.30 g/L cutoff that defines treatment targets in the current lipid guidelines.

Conclusions: Lipid values obtained from portable lipid analyzers may be useful for screening, but they should not be used to make clinical decisions regarding the diagnosis and management of dyslipidemia in individual patients.
Diagnostic Biochip Array for Fast and Sensitive Detection of K-ras Mutations in Stool
Authors: Prix Lothar , Uciechowski Peter , Böckmann Beatrix , Giesing Michael , Schuetz Andreas J
Published: January 2020

Background: Tumor cells that shed into stool are attractive targets for molecular screening and early detection of colon or pancreatic malignancies. We developed a diagnostic test to screen for 10 of the most common mutations of codons 12 and 13 of the K-ras gene by hybridization to a new biochip array.

Methods: DNA was isolated from 26 stool samples by column-based extraction from 9 cell lines. Peptide nucleic acid (PNA)-mediated PCR clamping was used for mutant-specific amplification. We used a biochip, consisting of a small plastic support with covalently immobilized 13mer oligonucleotides. The read out of the biochip was done by confocal time-resolved laser scanning. Hybridization, scanning, and data evaluation could be performed in <2 h.

Results: Approximately 80 ng of DNA was obtained from 200-mg stool samples. No inhibition of the PCR by remaining impurities from stool was observed. Mutation detection was possible in 1000-fold excess of wild-type sequence. Discrimination ratios between the mutations were >19 as demonstrated by hybridization with tumor cell line DNA. Stool samples (n = 26) were analyzed in parallel with PNA-PCR, restriction assay for K-ras codon 12 mutations, sequencing, and hybridization to the biochip. Nine mutations were found by hybridization, all confirmed by sequencing. PNA-PCR alone leads to an overestimation of mutations because suppression of the wild type is not effective enough with high concentrations of wild-type DNA. The restriction assay found only four mutations.

Conclusions: The K-ras biochip is well suited for fast mutation detection from stool in colorectal cancer screening.
Guidelines and Recommendations for Laboratory Analysis in the Diagnosis and Management of Diabetes Mellitus
Authors: Sacks David B , Bruns David E , Goldstein David E , Maclaren Noel K , McDonald Jay M , Parrott Marian
Published: January 2020

Background: Multiple laboratory tests are used in the diagnosis and management of patients with diabetes mellitus. The quality of the scientific evidence supporting the use of these assays varies substantially.

Approach: An expert committee drafted evidence-based recommendations for the use of laboratory analysis in patients with diabetes. An external panel of experts reviewed a draft of the guidelines, which were modified in response to the reviewers’ suggestions. A revised draft was posted on the Internet and was presented at the AACC Annual Meeting in July, 2000. The recommendations were modified again in response to oral and written comments. The guidelines were reviewed by the Professional Practice Committee of the American Diabetes Association.

Content: Measurement of plasma glucose remains the sole diagnostic criterion for diabetes. Monitoring of glycemic control is performed by the patients, who measure their own plasma or blood glucose with meters, and by laboratory analysis of glycated hemoglobin. The potential roles of noninvasive glucose monitoring, genetic testing, autoantibodies, microalbumin, proinsulin, C-peptide, and other analytes are addressed.

Summary: The guidelines provide specific recommendations based on published data or derived from expert consensus. Several analytes are of minimal clinical value at the present time, and measurement of them is not recommended.
Determination of Anti-Acetylcholine Receptor Antibodies in Myasthenic Patients by Use of Time-resolved Fluorescence
Authors: Říčný Jan , Šimková Libuše , Vincent Angela
Published: January 2020

Background: Autoantibodies against nicotinic acetylcholine receptor (nAChR) in myasthenia gravis (MG) patients are usually detected by radioimmunoprecipitation assays using extracted acetylcholine receptors labeled irreversibly with 125I-α-bungarotoxin (α-BuTx). To provide a nonradioactive immunoassay, we established an assay using nAChRs labeled with Eu3+-α-cobratoxin (α-CTx).

Methods: We derivatized α-CTx with a diethylenetriaminepentaacetate moiety and formed a complex with Eu3+. The complex was purified by HPLC, and the fractions were tested for binding to Torpedo and human nAChRs. The most active fractions were used to label nAChRs for the immunoprecipitation assay, and the bound Eu3+ was quantified by time-resolved fluorescence.

Results: Eu3+-labeled α-CTx competed with 125I-α-BuTx for binding to Torpedo nAChRs and saturated the binding sites of human nAChRs, with a Kd of 7.2 × 10−9 mol/L. Results of the immunoassay performed with Eu3+-labeled α-CTx were similar to those obtained with 125I-α-BuTx, with a slightly higher limit of detection [0.3 nmol/L (n = 6) vs ∼0.1 nmol/L for isotopic assay]. None of 34 negative sera tested (16 healthy controls, 10 patients with nonmyasthenia-related disease, 8 patients seronegative for MG) gave a value >0.3 nmol/L. Of the 35 positive myasthenic sera (with antibody values, previously determined by isotopic assay, of 0.4–1290 nmol/L) compared in the two assays, 32 tested positive with the Eu3+ assay. Linear regression analysis yielded the equation: y = 1.035x − 0.013 nmol/L; Sy|x = 0.172 nmol/L; r2 = 0.977.

Conclusions: The new time-resolved fluorescence method for quantification of antibodies to nAChRs in MG patients provides a performance similar to that of the widely used isotopic assay and could be used in laboratories with restricted use of isotopes.