bluuebunny/crossref_metadata_2025 huggingface dataset:
Fusion Proteins for Combined Analysis of Autoantibodies to the 65-kDa Isoform of Glutamic Acid Decarboxylase and Islet Antigen-2 in Insulin-dependent Diabetes Mellitus
Authors: Rickert Mathias , Seissler Jochen , Dangel Werner , Lorenz Helga , Richter Wiltrud
Published: January 2020

Background: Prediction, risk assessment, and diagnosis of autoimmune diseases often rely on detection of autoantibodies directed to multiple target antigens, such as the 65-kDa isoform of glutamic acid decarboxylase (GAD65-abs) and the tyrosine phosphatase-like protein islet antigen-2 (IA2-abs), the two major subspecificities of islet cell antibodies (ICAs) associated with insulin-dependent diabetes mellitus. We hypothesized that a combination of autoantigens in a fusion protein unifying the important immunodominant epitopes could provide an efficient target for cost-effective, one-step screening of sera.

Methods: Chimeric proteins composed of GAD65 and IA2 residues were constructed, analyzed for their immune reactivity with monoclonal antibodies and sera, and used in a diagnostic assay with 35S-labeled protein as antigen.

Results: Length and order of GAD65 and IA2 sequences were critical for conservation of the conformational epitopes in the fusion protein. Among four chimera tested, only IA2(606–979)/GAD65(1–585) retained wild-type-like folding of GAD65 and IA2 domains and yielded a stable protein after baculovirus expression. Reactivity of GAD65 antibody- and IA2 antibody-positive sera from patients newly diagnosed with insulin-dependent diabetes mellitus, from ICA-positive prediabetics, and from ICA-positive first-degree relatives demonstrated conservation of the relevant autoreactive epitopes. The assay based on the in vitro translated fusion antigen had a sensitivity and specificity identical to those for detection of GAD65- and IA2-abs based on the two separate GAD65 and IA2 proteins.

Conclusions: Autoantigens such as GAD65 and IA2 can be combined successfully in a fusion protein of similar immune reactivity. This allows simultaneous detection of GAD65- and IA2-abs in a one-step screening assay and cost-effective identification of positive individuals at risk of diabetes or at onset of disease.
Management of Maternal Amanita phalloı̈des Poisoning during the First Trimester of Pregnancy: A Case Report and Review of the Literature
Authors: Boyer Jean-Christophe , Hernandez Francois , Estorc Jacques , De La Coussaye Jean-Emmanuel , Bali Jean-Pierre
Published: January 2020

Background: Amanita phalloı̈des poisoning produces acute liver failure and often death. Maternal poisonings are rare, and medical decisions of abortion or liver transplantation in this critical situation frequently are based on laboratory data. We report here the case of a 22-year-old-woman in the 11th week of pregnancy, who ingested mushrooms.

Case Report: The patient’s clinical symptoms (e.g., vomiting and diarrhea) and blood chemistry data (persistent increases of aspartate aminotransferase and alanine aminotransferase and severe decreases in prothrombin, factor V, factor II, factor VII, and factor X) indicated poisoning of medium severity. The management consisted of intravenous hydration, and administration of silymarine and N-acetylcysteine. No fetal damage was observed, and birth and development of the infant (now 2 years of age) proceeded without incident.

Conclusion: Abortion is not necessarily indicated in maternal poisoning by A. phalloı̈des, even in the first trimester of pregnancy.
Influence of Vitamin-optimized Plasma Homocysteine Cutoff Values on the Prevalence of Hyperhomocysteinemia in Healthy Adults
Authors: Fokkema M Rebecca , Weijer Jacomijn M , Dijck-Brouwer D A Janneke , van Doormaal Jasper J , Muskiet Frits A J
Published: January 2020

Background: Hyperhomocysteinemia is a cardiovascular disease (CVD) risk factor. We determined plasma homocysteine (Hcy) reference values at optimized vitamin status and investigated their influence on the prevalence of hyperhomocysteinemia in healthy adults. Results were compared with those obtained using European Concerted Action Project (ECAP) cutoff values.

Methods: Healthy adults (n = 101) received folic acid (5 mg/day) and vitamin B12 (1 mg/day) for 2 weeks and the same dosages of folic acid and vitamin B12 plus vitamin B6 (1 mg · kg−1 · day−1) during the following 2 weeks. Hcy concentrations, both fasting and 6-h post-methionine load, were determined at baseline and after 4 weeks.

Results: Baseline (4 weeks) fasting and 6-h postload Hcy reference values were 4.7–14.6 (4.1–9.3) and 18.8–49.7 (12.9–35.1) μmol/L, respectively. Mean fasting and 6-h postload Hcy decreased after 4 weeks of vitamin supplementation by 3.5 μmol/L (33.5%) and 8.5 μmol/L (26.3%), respectively. The percentages of subjects exhibiting significant decreases in fasting Hcy following vitamin supplementation were 88% (all subjects), 92% (non-vitamin users), and 72% (vitamin users). The prevalences of hyperhomocysteinemia with use of ECAP cutoff values were 29% for all groups, 29% for men, 27% for premenopausal women, and 53% for postmenopausal women. With vitamin-optimized cutoff values, prevalences were 58%, 58%, 76%, and 89%, respectively. Use of vitamin-optimized cutoff values increased the diagnostic value of fasting Hcy and decreased that of a 6-h postload Hcy compared with use of ECAP cutoff values.

Conclusions: Use of vitamin-optimized cutoff values gives rise to high hyperhomocysteinemia pretest probabilities in the general population and, therefore, precludes any meaningful role for Hcy testing. Future demonstration of a beneficial effect of decreasing Hcy on CVD risk would justify use of vitamin-optimized cutoff values for assessment of CVD risk.
Cardiac Troponin T and Creatine Kinase MB Are Not Increased in Exterior Oblique Muscle of Patients with Renal Failure
Authors: Fredericks Salim , Murray Joanne F , Bewick Michael , Chang René , Collinson Paul O , Carter Nicholas D , Holt David W
Published: January 2020

Background: Serum cardiac troponin T (cTnT) concentrations may be increased in patients with renal dysfunction without evidence of cardiac damage, as assessed by conventional methods. It has been suggested that these positive measurements result from the expression in skeletal muscle of fetal isoforms of cTnT, which are detected by the cTnT immunoassay.

Methods: Skeletal muscle (exterior oblique) biopsies were taken from healthy living kidney donors (n = 5) and transplant recipients (n = 19). The amounts of cTnT and creatine kinase (CK) isoenzymes in skeletal muscle of healthy controls were compared with those in patients with renal failure (Wilcoxon–Mann–Whitney test). cTnT was measured quantitatively by a second-generation assay, with a limit of detection of 1 μg/g of protein, and qualitatively by immunohistochemistry and immunoblotting. CK-MB was measured by quantitative electrophoresis.

Results: Minute quantities of cTnT were detected in 2 of the 5 (40%) control samples and 9 of the 19 (47%) renal failure samples, respectively, at mean concentrations of <5 μg/g of protein for both subject groups. This was <1/6000th that found in heart muscle. There was no significant difference in cTnT or CK-MB content in skeletal muscle between healthy controls and patients with renal failure. Increased serum cTnT did not predict detectable cTnT in skeletal muscle. cTnT was not detected qualitatively by immunoblotting or immunohistochemistry in any skeletal muscle samples.

Conclusions: Uremia does not affect the content of cTnT or CK-MB in exterior oblique muscle, suggesting that cTnT detected in serum from patients with renal failure does not originate from skeletal muscle.
Electrospray Tandem Mass Spectrometry for Analysis of Acylcarnitines in Dried Postmortem Blood Specimens Collected at Autopsy from Infants with Unexplained Cause of Death
Authors: Chace Donald H , DiPerna James C , Mitchell Brenda L , Sgroi Bethany , Hofman Lindsay F , Naylor Edwin W
Published: January 2020

Background: Deaths from inherited metabolic disorders may remain undiagnosed after postmortem examination and may be classified as sudden infant death syndrome. Tandem mass spectrometry (MS/MS) may reveal disorders of fatty acid oxidation in deaths of previously unknown cause.

Methods: We obtained filter-paper blood from 7058 infants from United States and Canadian Medical Examiners. Acylcarnitine and amino acid profiles were obtained by MS/MS. Specialized interpretation was used to evaluate profiles for disorders of fatty acid, organic acid, and amino acid metabolism. The analyses of postmortem blood specimens were compared with the analyses of bile specimens, newborn blood specimens, and specimens obtained from older infants at risk for metabolic disorders.

Results: Results on 66 specimens suggested diagnoses of metabolic disorders. The most frequently detected disorders were medium-chain and very-long-chain acyl-CoA dehydrogenase deficiencies (23 and 9 cases, respectively), glutaric acidemia type I and II deficiencies (3 and 8 cases, respectively), carnitine palmitoyl transferase type II/translocase deficiencies (6 cases), severe carnitine deficiency (4 cases), isovaleric acidemia/2-methylbutyryl-CoA dehydrogenase deficiencies (4 cases), and long-chain hydroxyacyl-CoA dehydrogenase/trifunctional protein deficiencies (4 cases).

Conclusions: Postmortem metabolic screening can explain deaths in infants and children and provide estimates of the number of infant deaths attributable to inborn errors of metabolism. MS/MS is cost-effective for analysis of postmortem specimens and should be considered for routine use by Medical Examiners and pathologists in unexpected/unknown infant and child death.
Accumulation of Free 3-Hydroxy Fatty Acids in the Culture Media of Fibroblasts from Patients Deficient in Long-Chain l-3-Hydroxyacyl-CoA Dehydrogenase: A Useful Diagnostic Aid
Authors: Jones Patricia M , Moffitt Monica , Joseph Delanie , Harthcock Pamela A , Boriack Richard L , Ibdah Jamal A , Strauss Arnold W , Bennett Michael J
Published: January 2020

Background: The diagnosis of long-chain l-3-hydroxy-acyl-coenzyme A dehydrogenase (LCHAD) deficiency frequently requires the study of cultured fibroblasts. We developed such a test that does not require disruption and loss of the cells.

Methods: We measured free 3-hydroxy fatty acids (3-OHFAs) in media of skin fibroblasts cultures from 11 patients with a genetic deficiency of LCHAD and the associated disorder of mitochondrial trifunctional protein (MTFP). Fibroblasts were cultured for 24 h with 100 μmol/L nonisotopic palmitate added. 3-OHFAs were measured by selected-ion monitoring, stable-isotope dilution gas chromatography-mass spectrometry with [13C]-labeled internal standards.

Results: 3-OH-hexadecanoic and 3-OH-tetradecanoic FAs were increased 14- and 11-fold, respectively, in all patients with LCHAD or MTFP deficiency when compared with control fibroblast cell lines after overnight incubation with palmitate. 3-OH-dodecanoic FA demonstrated a modest, fivefold increase in LCHAD-deficient cells. The concentrations of all 3-OHFAs were similar whether or not the medium samples were hydrolyzed to release conjugated species such as acylcarnitines, suggesting that 3-OHFAs accumulate in the media as free FAs.

Conclusions: Measurement of 3-OHFA excretion from LCHAD- or MTFP-deficient cell lines can be used as a diagnostic tool. Free FAs are the predominant form of these abnormal metabolic intermediates in culture media.
Interference of Transferrin Isoform Types with Carbohydrate-deficient Transferrin Quantification in the Identification of Alcohol Abuse
Authors: Helander Anders , Eriksson Gunne , Stibler Helena , Jeppsson Jan-Olof
Published: January 2020

Background: Isoforms of transferrin interfere with measurement of carbohydrate-deficient transferrin (CDT) as a marker of heavy alcohol consumption. We evaluated the rate of inaccurate CDT results by immunoassays.

Methods: We studied 2360 consecutive sera (1614 individuals) submitted for CDT assay without clinical information as well as samples from 1 patient with a congenital disorder of glycosylation (CDG Ia) and from 6 healthy carriers of CDG Ia. The CDTect, %CDT-TIA, and new %CDT immunoassays were compared with HPLC (%CDT-HPLC). Transferrin isoform pattern were evaluated by isoelectric focusing (IEF).

Results: Transferrin BC and CD heterozygotes were found at frequencies of ∼0.7% and ∼0.2%, respectively. Another transferrin C subtype, where di- and trisialotransferrin partly coeluted (tentatively identified as C2C3), was observed in ∼0.6%. Compared with the %CDT-HPLC method, the immunoassays often produced low results for transferrin BC and high results for transferrin CD and “C2C3”. A very high trisialotransferrin value (frequency ∼1%) often produced high CDT immunoassay results. In four of six healthy carriers of CDG Ia, a- and disialotransferrin were highly increased and the HPLC and IEF isoform patterns were indistinguishable from those in alcohol abuse.

Conclusions: Rare transferrin isoform types and abnormal amounts of trisialotransferrin (total frequency ∼2–3%) may cause incorrect determination of CDT with immunoassays. The observed variants were readily identified by HPLC and IEF, which can be recommended for verification of CDT immunoassay results in doubtful cases. In healthy carriers of CDG Ia, CDT is high by all assays.
Direct and Indirect Free Thyroxine Assay Methods: Theory and Practice
Authors: Midgley John E M
Published: January 2020

Background: For the diagnosis of thyroid disease, measurement of “free hormone” is generally accepted as an appropriate measure. However, valid assays measuring the free fraction of thyroxine (FT4) ideally must perform without bias, despite large variations in the concentrations and affinities of serum T4-binding proteins in the population. Several approaches have been taken to overcome such bias, and these have created considerable controversy in the field over the past decade.

Approach: This review, from both a historical and an analytical standpoint, charts the progress made over more than 30 years in improvements to the performance of assays in common use for the measurement of FT4 in serum or plasma. It reexamines the theory behind early approaches to such assays [for example, the free thyroxine index (FTI) method], that preceded more accurate, two-step immunoassays or one-step analog techniques. It evaluates the continuous refinements to the latter assays that by now have largely supplanted the FTI approach and where the deficiencies that so exercised clinical chemists in the past have been virtually eliminated in the leading assays.

Content: The basic Mass Action theory underpinning all such methods is discussed by assessing how far each particular approach obeys the criteria the theory imposes. In this, it is not the intention of the review to dissect individual commercial or academic assays, but rather to give guidance where appropriate as to how any assay said to measure FT4 can be conveniently evaluated by those intending to use it. Examples are given where inappropriate tests may wrongly imply assay invalidity by misinterpreting how FT4 assays work.

Summary: Detailed knowledge of the underlying theory is essential when devising tests for direct FT4 assays, to ensure that such tests do not overstep the practical limits of assay validity.
Genotyping Single-Nucleotide Polymorphisms by the Invader Assay with Dual-Color Fluorescence Polarization Detection
Authors: Hsu Tony M , Law Scott M , Duan Shenghui , Neri Bruce P , Kwok Pui-Yan
Published: January 2020

Background: The PCR-Invader® assay is a robust, homogeneous assay that has been shown to be highly sensitive and specific in genotyping single-nucleotide polymorphism (SNP) markers. In this study, we introduce two changes to improve the assay: (a) we streamline the PCR-Invader method by assaying both alleles for each SNP in one reaction; and (b) we reduce the cost of the method by adopting fluorescence polarization (FP) as the detection method.

Methods: PCR product was incubated with Invader oligonucleotide and two primary probes at 93 °C for 5 min. Signal probes corresponding to the cleaved flaps of the primary probes [labeled with fluorescein and 6-carboxytetramethylrhodamine (TAMRA) dye] and Cleavase® VIII enzyme (a flap endonuclease) were then added to the mixture. This reaction mixture was incubated at 63 °C for 5 min. FP measurements were made with a fluorescence plate reader.

Results: Eighty-eight individuals were genotyped across a panel of 10 SNPs, using PCR product as template, for a total of 880 genotypes. An average “no call” rate of 3.2% was observed after first round of experiments. PCR products were remade in those samples that failed to produce any genotype in the first round, and all gave clear-cut genotypes. When the genotypes determined by the PCR-Invader assay and template-directed dye-terminator incorporation assay with FP were compared, they were in 100% concordance for all SNP markers and experiments.

Conclusions: The improvements introduced in this study make PCR-Invader assay simpler and more cost-effective, and therefore more suitable for high-throughput genotyping.
Integrated Patient Data for Optimal Patient Management: The Value of Laboratory Data in Quality Improvement
Authors: Emons Matthew F
Published: January 2020

Managed care organizations are shifting from traditional utilization management programs to focus on initiatives that improve the health of an insured population. This strategy requires sophisticated data integration to identify at-risk individuals and track outcomes. Laboratory data are becoming increasingly valuable tools for managed care organizations and healthcare providers. The HEDIS® Effectiveness of Care measures have incorporated laboratory data into several key performance indicators. By building a comprehensive repository of laboratory data that includes both procedure codes and laboratory values, managed care organizations can realize substantial savings by avoiding the costly medical record reviews required when administrative data are incomplete. In addition to tracking clinical outcomes, laboratory data provide the ability to risk-stratify a population to target high-risk individuals for case management and disease management interventions. Healthcare organizations face several challenges in the integration of laboratory data into medical databases and practice management software. Confidentiality is a key consideration in view of recent healthcare regulations. Providers of laboratory services should work collaboratively with organizations setting standards for healthcare informatics to facilitate the pooling of data for quality improvement and outcomes research. Health Level Seven, Inc. (HL7), Logical Observation Identifier Names and Codes (LOINC®), and Systematized Nomenclature of Medicine (SNOMED) will likely play a key role in this process.
Genotyping Energy-Transfer-Cassette-labeled Short-Tandem-Repeat Amplicons with Capillary Array Electrophoresis Microchannel Plates
Authors: Medintz Igor L , Berti Lorenzo , Emrich Charles A , Tom Jennifer , Scherer James R , Mathies Richard A
Published: January 2020

Background: Genetic analysis of microsatellite DNA is a powerful tool used in linkage analysis, gene mapping, and clinical diagnosis. To address the expanding needs of studies of short tandem repeats (STRs), we demonstrated high-performance STR analysis on a high-throughput microchannel plate-based platform.

Methods: Energy-transfer-cassette-labeled STR amplicons were separated and typed on a microfabricated 96-channel radial capillary array electrophoresis (CAE) microchannel plate system. Four-color detection was accomplished with a laser-excited confocal fluorescence rotary scanner.

Results: Multiplex STR analysis with single base-pair resolution was demonstrated on denaturing polyacrylamide gel media. The high-throughput multiplex capabilities of this genetic analysis platform were demonstrated by the simultaneous separation of STR amplicons representing 122 samples in ninety-six 5.5-cm-long channels in <8 min. Sizing values obtained for these amplicons on the CAE microchannel plate were comparable to those measured on a conventional commercial CAE instrument and exhibit <1% sizing variance.

Conclusions: Energy-transfer-cassette labeling and microfabricated CAE microchannel plates allow high-performance multiplex STR analyses.
Haptoglobin Polymorphism and Iron Homeostasis
Authors: Beutler Ernest , Gelbart Terri , Lee Pauline
Published: January 2020

Background: There is a marked difference in the degree of expression of the homozygous C282Y HFE genotype that is associated with hereditary hemochromatosis. It has been reported that individuals with the haptoglobin 2-2 type manifest increased iron concentrations, including serum iron, transferrin saturation, and ferritin.

Methods: We studied 232 patients, 115 homozygous for the c.845G→A (C282Y) mutation and 117 matched controls with the wild-type HFE genotype, for haptoglobin phenotypes. Haptoglobin types were determined by electrophoresis of the denatured protein. The HFE genotype was determined by allele-specific oligonucleotide hybridization. Ferritin and transferrin saturation were measured by standard methods.

Results:There was no relationship between haptoglobin type and ferritin concentration or transferrin saturation.

Conclusions: The effect of haptoglobin type on iron homeostasis cannot account for the marked phenotypic variation that is seen in patients homozygous for the HFE C282Y mutation.
Digoxin Assays: Frequent, Substantial, and Potentially Dangerous Interference by Spironolactone, Canrenone, and Other Steroids
Authors: Steimer Werner , Müller Christine , Eber Barbara
Published: January 2020

Background: A case of digoxin toxicity resulted from falsely low values with the MEIA II assay for digoxin (AxSYM®; Abbott). The low results were caused by negative interference from canrenone and spironolactone, the latter of which has recently been advocated for the treatment of severe heart failure. Analytical interference from spironolactone has been reported, but little information is available for this effect with newer digoxin assays.

Methods: We examined nine assays (AxSYM, IMx®, TDx®, Emit®, Dimension®, aca®, TinaQuant®, Elecsys®, and Vitros®) for interference by spironolactone, canrenone, and three metabolites. Additionally, all routine digoxin measurements (AxSYM) over a period of 16.5 months (n = 3089) were monitored for interference.

Results: Suppression of the expected values by canrenone (3125 μg/L) was observed for the AxSYM (42% of expected value), IMx (51%), and Dimension (78%) assays. A positive bias was observed for the aca (0.7 μg/L), the TDx (0.62 μg/L), and the Elecsys (>0.58 μg/L). Twenty-five of 669 routinely monitored patients had falsely low results. Nineteen of these had potentially toxic concentrations of digoxin (Emit; >2.0 μg/L), although the AxSYM assay indicated therapeutic or less severe toxic concentrations (Δmax = 7.1 μg/L). Except for two unresolved cases, this was attributable to spironolactone, canrenone, hydrocortisone, or prednisolone. Standard doses of spironolactone (up to 50 mg/day) in patients with heart failure displayed inhibition <11%.

Conclusions: The frequency and magnitude of the false-negative results particularly compromise the use of both microparticle enzyme immunoassays. Not only may toxic concentrations remain unidentified, but intoxication could occur should dosage be increased because of falsely low results. With 11 million digoxin tests/year ordered in the US, conceivably many patients could be adversely affected.
Assessment of the Diagnostic Accuracy of the TDx-FLM II to Predict Fetal Lung Maturity
Authors: Fantz Corinne R , Powell Cynthia , Karon Brad , Parvin Curtis A , Hankins Kelly , Dayal Molina , Sadovsky Yoel , Johari Vandita , Apple Fred S , Gronowski Ann M
Published: January 2020

Background: Because respiratory distress syndrome (RDS) affects 1% of live births, accurate and rapid assessment of markers of fetal lung maturity is critical to clinicians in deciding whether to deliver a preterm infant. Our objective was to determine the optimal diagnostic cutoff value for the TDx-FLM II assay (Abbott Laboratories) for predicting clinically significant RDS.

Methods: Amniotic fluid TDx-FLM II data were collected retrospectively over 4 years. Women were included in the study if they had delivered within 72 h of TDx-FLM II testing and both the mother and infant charts could be reviewed. Women who had been treated with steroids and delivered unaffected infants were excluded from the analysis. The diagnosis of RDS was defined as infants who either were treated with surfactant and/or were placed on a ventilator and/or required continuous positive airway pressure for >1 day.

Results: A total of 185 women met all entry criteria (15 RDS, 170 non-RDS). A cutoff value for a mature result of ≥45 mg/g gave a sensitivity of 100% (95% confidence interval, 82–100%) and a specificity of 90% (95% confidence interval, 78–89%).

Conclusions: The TDx-FLM II appears to predict clinically significant RDS when a cutoff of ≥45 mg/g is used for mature results. Further studies will be required to confirm these findings.
Quantitative Analysis of a MDR1 Transcript for Prediction of Drug Resistance in Acute Leukemia
Authors: Fujimaki Shin-ichi , Funato Tadao , Harigae Hideo , Fujiwara Junko , Kameoka Junichi , Meguro Kuniaki , Kaku Mitsuo , Sasaki Takeshi
Published: January 2020

Background: Assessing the drug resistance of leukemic cells is important for treatment of leukemia. We developed a quantitative reverse transcription (RT)-PCR method for multidrug resistance 1 (MDR1) and multidrug resistance-related protein 1 (MRP1) transcripts to evaluate drug resistance, and applied it to clinical samples.

Methods: The cutoffs for copy numbers of MDR1 and MRP1 transcripts were defined based on copy numbers in healthy bone marrow mononuclear cells. To confirm that the cutoffs reflected biological resistance, we established vincristine (VCR)-resistant K562 sublines that showed various degrees of drug resistance and examined the correlation between the copy numbers of these transcripts and the biological resistance of these clones. In addition, we compared the sensitivity and specificity of quantitative RT-PCR to a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometric (FCM) analysis.

Results: The defined cutoff for copy numbers of MDR1 transcripts corresponded with the degree of biological resistance of VCR-resistant K562 sublines. Clinical study revealed that the concentrations of MDR1 mRNA in all relapsed patients with acute myelogenous leukemia (AML) were above the cutoff. Moreover, both AML and acute lymphoblastic leukemia patients with high MDR1 mRNA expression at diagnosis tended to show a low remission rate and short remission periods. No association was observed between the amounts of MRP1 transcripts and clinical outcomes. The specificity and sensitivity of quantitative RT-PCR for MDR1 were superior to the MTT assay and FCM analysis.

Conclusion: These results suggest the efficacy of this quantitative analysis of MDR1 transcripts for the prediction of clinical drug resistance in acute leukemia.
Detection of Human Kallikrein 4 in Healthy and Cancerous Prostatic Tissues by Immunofluorometry and Immunohistochemistry
Authors: Obiezu Christina V , Soosaipillai Antoninus , Jung Klaus , Stephan Carsten , Scorilas Andreas , Howarth David H C , Diamandis Eleftherios P
Published: January 2020

Background: Human kallikrein 4 (gene, KLK4; protein, hK4), a recently discovered member of the kallikrein gene family, shares many characteristics with prostate-specific antigen, the best available marker for prostate cancer. Because the protein has not been detected in any human tissue, we attempted to develop immunologic methods for hK4 analysis and use them to detect hK4 in healthy and cancerous tissue extracts and biological fluids.

Methods: We extracted total RNA from 20 pairs of matched (healthy–cancer) prostate tissue samples. KLK4 cDNA was amplified by reverse transcription-PCR (RT-PCR) and cloned in a pPICZαA expression vector. We then transformed the construct product into Pichia pastoris yeast strains and induced secreted recombinant protein production by addition of methanol. We purified the recombinant protein by nickel ion-affinity chromatography and used it as an immunogen in rabbits and mice to generate polyclonal anti-hK4 antibodies. These antibodies were used to develop a sandwich-type immunoassay suitable for hK4 quantification in biological fluids and tissue extracts.

Results: The immunoassay had a detection limit of 0.1 μg/L. We detected hK4 in 10 of 21 matched (healthy–cancer) prostate tissues, and hK4 was frequently higher in healthy tissues. In one matched-sample pair, the hK4 content was relatively high in both the healthy [4.62 μg/g of total protein (TP)] and the cancerous (1.22 μg/g of TP) prostate tissue. Among tissue extracts, we found the highest concentrations of hK4 in healthy (0.0–4.62 μg/g of TP) and cancerous (0.0–1.72 μg/g of TP) prostatic extracts and in placental extracts (0.0–0.05 μg/g of TP). We also detected traces of hK4 protein immunoreactivity in amniotic fluid (<0.1–0.6 μg/L), human breast milk (<0.1–0.75 μg/L), and seminal plasma (0.2–0.9 μg/L). Immunohistochemical studies showed cytoplasmic staining for hK4 protein in both malignant and benign epithelial cells of the prostate. However, we did not detect hK4 in cerebrospinal fluid, healthy and cancerous ovarian tissue extracts, and many other human tissue extracts.

Conclusions: hK4 protein is present in some prostatic tissue extracts but at relatively low concentrations, although KLK4 mRNA is readily detectable by RT-PCR. We propose that the protein either is not synthesized efficiently or is degraded very quickly.
Commutability Assessment of Potential Reference Materials Using a Multicenter Split-Patient-Sample Between-Field-Methods (Twin-Study) Design: Study within the Framework of the Dutch Project “Calibration 2000”
Authors: Baadenhuijsen Henk , Steigstra Herman , Cobbaert Christa , Kuypers Aldy , Weykamp Cas , Jansen Rob
Published: January 2020

Background: The Dutch project “Calibration 2000” aims at harmonization of laboratory results via calibration by development of commutable, matrix-based, secondary reference materials. An alternative approach to the NCCLS EP14 protocol for studying commutability of reference materials is presented, the “twin-study design”, which in essence is a multicenter, split-patient-sample, between-field-methods protocol.

Methods: The study consisted of the simultaneous analysis of fresh patient sera and potential reference materials (PRMs) for HDL-cholesterol (HDL-C) by 86 laboratories forming 43 laboratory couples. Six subgroups of method combinations were formed. The patient sera were selected and interchanged by each laboratory couple. The PRMs consisted of three types: C37, prepared according to the NCCLS C37 protocol; Fro, frozen selectively pooled human serum; and Lyo, which was the same serum pool as Fro but lyophilized in the presence of sucrose. All PRMs were provided in three HDL-C concentrations. The regression line residuals for the PRMs were normalized by expressing them as multiples of the state-of-the-art within laboratory SD (SDSA). In addition, the extra contribution of each PRM to the total measurement uncertainty, CVNetto, was calculated.

Results: Averaged over the three PRM concentrations, 1.6% of the C37 residuals were outside the 3 SDSA limit. For the Fro and Lyo PRMs, these values were 2.4% and 11.1%. CVNetto values for C37, Fro, and Lyo were 2.9%, 4.3%, and 5.3%, respectively.

Conclusions: The present twin-study design, as a practical alternative to the NCCLS EP14 protocol, is a viable way of studying commutability characteristics of PRMs. The study suggests that the C37 PRMs are the best candidates for a future reference material.
Artificial intelligence in medical imaging: A radiomic guide to precision phenotyping of cardiovascular disease
Authors: Oikonomou Evangelos K , Siddique Musib , Antoniades Charalambos
Published: January 2020

Rapid technological advances in non-invasive imaging, coupled with the availability of large data sets and the expansion of computational models and power, have revolutionized the role of imaging in medicine. Non-invasive imaging is the pillar of modern cardiovascular diagnostics, with modalities such as cardiac computed tomography (CT) now recognized as first-line options for cardiovascular risk stratification and the assessment of stable or even unstable patients. To date, cardiovascular imaging has lagged behind other fields, such as oncology, in the clinical translational of artificial intelligence (AI)-based approaches. We hereby review the current status of AI in non-invasive cardiovascular imaging, using cardiac CT as a running example of how novel machine learning (ML)-based radiomic approaches can improve clinical care. The integration of ML, deep learning, and radiomic methods has revealed direct links between tissue imaging phenotyping and tissue biology, with important clinical implications. More specifically, we discuss the current evidence, strengths, limitations, and future directions for AI in cardiac imaging and CT, as well as lessons that can be learned from other areas. Finally, we propose a scientific framework in order to ensure the clinical and scientific validity of future studies in this novel, yet highly promising field. Still in its infancy, AI-based cardiovascular imaging has a lot to offer to both the patients and their doctors as it catalyzes the transition towards a more precise phenotyping of cardiovascular disease.
Coronary microvascular dysfunction in hypertrophy and heart failure
Authors: Camici Paolo G , Tschöpe Carsten , Di Carli Marcelo F , Rimoldi Ornella , Van Linthout Sophie
Published: January 2020

Left ventricular (LV) hypertrophy (LVH) is a growth in left myocardial mass mainly caused by increased cardiomyocyte size. LVH can be a physiological adaptation to physical exercise or a pathological condition either primary, i.e. genetic, or secondary to LV overload. Patients with both primary and secondary LVH have evidence of coronary microvascular dysfunction (CMD). The latter is mainly due to capillary rarefaction and adverse remodelling of intramural coronary arterioles due to medial wall thickening with an increased wall/lumen ratio. An important feature of this phenomenon is the diffuse nature of this remodelling, which generally affects the coronary microvessels in the whole of the left ventricle. Patients with LVH secondary to arterial hypertension can develop both heart failure with preserved ejection fraction (HFpEF) and heart failure with reduced ejection fraction (HFrEF). These patients can develop HFrEF via a ‘direct pathway’ with an interval myocardial infarction and also in its absence. On the other hand, patients can develop HFpEF that can then progress to HFrEF with or without interval myocardial infarction. A similar evolution towards LV dysfunction and both HFpEF and HFrEF can occur in patients with hypertrophic cardiomyopathy, the most common genetic cardiomyopathy with a phenotype characterized by massive LVH. In this review article, we will discuss both the experimental and clinical studies explaining the mechanisms responsible for CMD in LVH as well as the evidence linking CMD with HFpEF and HFrEF.
Prognostic power of left atrial strain in patients with acute heart failure
Authors: Park Jae-Hyeong , Hwang In-Chang , Park Jin Joo , Park Jun-Bean , Cho Goo-Yeong
Published: January 2020

Aims

Left atrial (LA) dysfunction can be associated with left ventricular (LV) disorders; however, its clinical significance has not been well-studied in patients with acute heart failure (AHF). We evaluated prognostic power of peak atrial longitudinal strain (PALS) of the left atrium according to heart failure (HF) phenotypes and atrial fibrillation (AF).

Methods and results

From an AHF registry with 4312 patients, we analysed PALS in 3818 patients. Patients were categorized into PALS tertiles. We also divided the patients according to HF phenotypes [HF with reduced ejection fraction (HFrEF), HF with mid-range ejection fraction (HFmrEF), or HF with preserved ejection fraction (HFpEF)] and presence of AF. The primary outcomes were all-cause mortality and HF hospitalization. PALS was weakly but significantly correlated with LA volume index (r = −0.310, P < 0.001), E/e′ (r = −0.245, P < 0.001), and LV ejection fraction (r = 0.371, P < 0.001). A total of 2016 patients (52.8%) experienced adverse clinical events during median follow-up duration of 30.6 months (interquartile ranges 11.6–54.4 months). In the multivariate analysis, PALS was a significant predictor of events [hazard ratio (HR) 0.984, 95% confidence interval (CI) 0.971–0.996; P = 0.012]. Patients with the lowest tertile (HR 1.576, 95% CI 1.219–2.038; P < 0.001) had a higher number of events than those with the highest tertile in the multivariate analysis. In the subgroup analysis, however, PALS was not a prognosticator (HR 0.987, 95% CI 0.974–1.000; P = 0.056) in AF patients. The prognostic power of PALS was not different between HFrEF (HR 0.977, 95% CI 0.969–0.974; P < 0.001), HFmrEF (HR 0.984, 95% CI 0.972–0.996; P = 0.008), and HFpEF (HR 0.980, 95% CI 0.973–0.987; P < 0.001, P for interaction = 0.433).

Conclusion

PALS was a significant prognostic marker in AHF patients. The prognostic power was similar regardless of HF phenotypes, but PALS was not associated with clinical events in AF patients.
Preclosing of the femoral artery allows total percutaneous venoarterial extracorporeal membrane oxygenation and prevents groin wound infection after lung transplantation
Authors: Pellenc Quentin , Girault Antoine , Roussel Arnaud , Aguir Sonia , Cerceau Pierre , Longrois Dan , Mal Hervé , Mordant Pierre , Castier Yves
Published: January 2020

OBJECTIVES

In lung transplantation (LT), femoral venoarterial extracorporeal membrane oxygenation (VA-ECMO) usually requires an open approach that may be associated with severe groin wound infection. In endovascular aortic procedures, preclosing of the femoral artery (PFA) with vascular closure devices allows the percutaneous insertion and withdrawal of large-bore cannulae. We sought to evaluate whether this innovative technique could be applied in the specific setting of LT to achieve total percutaneous VA-ECMO and decrease groin wound infection.

METHODS

We conducted a retrospective study of a prospective database including patients who underwent LT in our centre from January 2011 to December 2017. Patients who underwent peripheral VA-ECMO using the PFA technique after January 2014 (PFA group, n = 106) were compared to those who underwent peripheral VA-ECMO using open cannulation and/or decannulation before January 2014 (non-PFA group, n = 48). The primary end point was the rate of technical success defined as total percutaneous VA-ECMO. Secondary end points included groin wound infections and delayed vascular complications.

RESULTS

The PFA technique was technically successful in 98 patients (92.5%). As compared with the non-PFA group, the PFA group was characterized by a similar rate of vascular complications (16.6% vs 11.3%, P = 0.360) and a decreased rate of groin wound infection (18.9% vs 0%, P < 0.001). In multivariate analysis, risk factors associated with vascular complications following PFA included female sex, peripheral arterial disease and ECMO duration.

CONCLUSIONS

In LT patients, PFA is associated with a high rate of total percutaneous VA-ECMO, thus preventing the occurrence of groin wound infection.
Early protein expression profile in bronchoalveolar lavage fluid and clinical outcomes in primary graft dysfunction after lung transplantation
Authors: Frick Anna E , Verleden Stijn E , Ordies Sofie , Sacreas Annelore , Vos Robin , Verleden Geert M , Vanaudenaerde Bart M , Claes Sandra , Schols Dominique , Van Raemdonck Dirk E , Neyrinck Arne P
Published: January 2020

OBJECTIVES

Primary graft dysfunction (PGD) remains a major post-transplant complication and is associated with increased morbidity and mortality. Mechanisms evoking PGD are not completely clear, but inflammation plays a central role. We investigated the association between PGD and inflammatory proteins present in immediate postoperative bronchoalveolar lavage.

METHODS

All double-lung recipients transplanted at our institution from 2002 to 2018 were included in our study. We retrospectively selected 80 consecutive lung transplant recipients with different PGD grades (n = 20 for each PGD grades 0–1 to 2–3). In bronchoalveolar lavage performed within the first 24 h after donor aortic cross-clamping following lung transplantation, concentrations of 30 cytokines, chemokines and growth factors were assessed by enzyme-linked immunosorbent assay (ELISA) and correlated with donor and recipient demographics and outcomes. For analysis, 2 groups were defined: ‘mild’ PGD (grade 0–1) and ‘severe’ PGD (grades 2–3).

RESULTS

Significant differences between mild and severe PGD were found in 8 biomarkers [interleukin (IL)-6, IL-10, IL-13, eotaxin, granulocyte colony-stimulating factor, interferon γ, macrophage inflammatory protein 1α, surfactant protein D (SP-D); P < 0.05]. Increased IL-10 and IL-13, but none of the other proteins, were associated with short-term outcome (longer time to extubation; P = 0.005 and P < 0.0001; increased intensive care unit stay; P = 0.012 and P < 0.0001; and hospital stay; P = 0.041 and P = 0.002). There were no significant differences in donor and recipient characteristics between the groups.

CONCLUSIONS

Expression profiles of key inflammatory mediators in bronchoalveolar lavage fluid differed significantly between lung transplant recipients with severe versus mild PGD and correlated with clinical outcome variables. Further research should focus on the early mechanisms leading to PGD.
Exploring Community-Dwelling Older Adults’ Considerations About Values and Preferences for Future End-of-Life Care: A Study from Sweden
Authors: Eneslätt Malin , Helgesson Gert , Tishelman Carol
Published: January 2020

Background and Objectives

There is a substantial body of research on advance care planning (ACP), often originating from English-speaking countries and focused on health care settings. However, studies of content of ACP conversations in community settings remain scarce. We therefore explore community-dwelling, older adults’ reasoning about end-of-life (EoL) values and preferences in ACP conversations.

Research Design and Methods

In this participatory action research project, planned and conducted in collaboration with national community-based organizations, we interviewed 65 older adults without known EoL care needs, about their values and preferences for future EoL care. Conversations were stimulated by sorting and ranking statements in a Swedish version of GoWish cards, called the DöBra cards, and verbatim transcripts were analyzed inductively.

Results

While participants shared some common preferences about EoL care, there was great variation among individuals in how they reasoned. Although EoL preferences and prioritizations could be identical, different individuals explained these choices very differently. We exemplify this variation using data from four participants who discussed their respective EoL preferences by focusing on either physical, social, existential, or practical implications.

Discussion and Implications

A previously undocumented benefit of the GoWish/DöBra cards is how the flexibility of the card statements support substantial discussion of an individual’s EoL preferences and underlying values. Such in-depth descriptions of participants’ reasoning and considerations are important for understanding the very individual nature of prioritizing EoL preferences. We suggest future users of the DöBra/GoWish cards consider the underlying reasoning of individuals’ prioritizations to strengthen person-centeredness in EoL conversations and care provision.
A highly contiguous genome assembly of the bat hawkmoth Hyles vespertilio (Lepidoptera: Sphingidae)
Authors: Pippel Martin , Jebb David , Patzold Franziska , Winkler Sylke , Vogel Heiko , Myers Gene , Hiller Michael , Hundsdoerfer Anna K
Published: January 2020

Background

Adapted to different ecological niches, moth species belonging to the Hyles genus exhibit a spectacular diversity of larval color patterns. These species diverged ~7.5 million years ago, making this rather young genus an interesting system to study a wide range of questions including the process of speciation, ecological adaptation, and adaptive radiation.

Results

Here we present a high-quality genome assembly of the bat hawkmoth Hyles vespertilio, the first reference genome of a member of the Hyles genus. We generated 51× Pacific Biosciences long reads with an average read length of 8.9 kb. Pacific Biosciences reads longer than 4 kb were assembled into contigs, resulting in a 651.4-Mb assembly consisting of 530 contigs with an N50 value of 7.5 Mb. The circular mitochondrial contig has a length of 15,303 bp. The H. vespertilio genome is very repeat-rich and exhibits a higher repeat content (50.3%) than other Bombycoidea species such as Bombyx mori (45.7%) and Manduca sexta (27.5%). We developed a comprehensive gene annotation workflow to obtain consensus gene models from different evidence including gene projections, protein homology, transcriptome data, and ab initio predictions. The resulting gene annotation is highly complete with 94.5% of BUSCO genes being completely present, which is higher than the BUSCO completeness of the B. mori (92.2%) and M. sexta (90%) annotations.

Conclusions

Our gene annotation strategy has general applicability to other genomes, and the H. vespertilio genome provides a valuable molecular resource to study a range of questions in this genus, including phylogeny, incomplete lineage sorting, speciation, and hybridization. A genome browser displaying the genome, alignments, and annotations is available at https://genome-public.pks.mpg.de/cgi-bin/hgTracks?db=HLhylVes1.
Compartment and hub definitions tune metabolic networks for metabolomic interpretations
Authors: Waller T Cameron , Berg Jordan A , Lex Alexander , Chapman Brian E , Rutter Jared
Published: January 2020

Background

Metabolic networks represent all chemical reactions that occur between molecular metabolites in an organism's cells. They offer biological context in which to integrate, analyze, and interpret omic measurements, but their large scale and extensive connectivity present unique challenges. While it is practical to simplify these networks by placing constraints on compartments and hubs, it is unclear how these simplifications alter the structure of metabolic networks and the interpretation of metabolomic experiments.

Results

We curated and adapted the latest systemic model of human metabolism and developed customizable tools to define metabolic networks with and without compartmentalization in subcellular organelles and with or without inclusion of prolific metabolite hubs. Compartmentalization made networks larger, less dense, and more modular, whereas hubs made networks larger, more dense, and less modular. When present, these hubs also dominated shortest paths in the network, yet their exclusion exposed the subtler prominence of other metabolites that are typically more relevant to metabolomic experiments. We applied the non-compartmental network without metabolite hubs in a retrospective, exploratory analysis of metabolomic measurements from 5 studies on human tissues. Network clusters identified individual reactions that might experience differential regulation between experimental conditions, several of which were not apparent in the original publications.

Conclusions

Exclusion of specific metabolite hubs exposes modularity in both compartmental and non-compartmental metabolic networks, improving detection of relevant clusters in omic measurements. Better computational detection of metabolic network clusters in large data sets has potential to identify differential regulation of individual genes, transcripts, and proteins.