bluuebunny/crossref_metadata_2025_split huggingface dataset:
Atomic Emission Method for Total Boron in Blood during Neutron-Capture Therapy
Authors: Laakso Juha , Kulvik Martti , Ruokonen Inkeri , Vähätalo Jyrki , Zilliacus Riitta , Färkkilä Markus , Kallio Merja 
Published: January 2020

Abstract
Background: Boron neutron-capture therapy (BNCT) is a drug-targeted binary radiotherapy for cancer. The 10B capture of thermal neutrons induces secondary radiation within cells during irradiation. The most widely used boron carrier is 4-dihydroxyborylphenylalanine (BPA). The duration and timing of the irradiation is adjusted by monitoring 10B concentrations in whole blood.
Methods: We developed a new method for boron determination that uses inductively coupled plasma atomic emission spectrometry (ICP-AES) and protein removal with trichloroacetic acid before analysis. This method was compared with the established but tedious inductively coupled plasma mass spectrometry (ICP-MS), which uses wet ashing as sample pretreatment. Erythrocyte boron concentrations were determined indirectly on the basis of plasma and whole blood boron concentrations and the hematocrit. The hematocrit was determined indirectly by measuring calcium concentrations in plasma and whole blood.
Results: Within- and between-day CVs were &lt;5%. The recoveries for boron in whole blood were 95.6–96.2%. A strong correlation was found between results of the ICP-AES and ICP-MS (r = 0.994). Marked differences in plasma and erythrocyte boron concentrations were observed during and after infusion of BPA fructose complex.
Conclusions: The present method is feasible, accurate, and one of the fastest for boron determination during BNCT. Our results indicate that it is preferable to determine boron in plasma and in whole blood. Indirect erythrocyte-boron determination thus becomes possible and avoids the impact of preanalytical confounding factors, such as the influence of the hematocrit of the patient. Such an approach enables a more reliable estimation of the irradiation dose.
Use of Up-Converting Phosphor Reporters in Lateral-Flow Assays to Detect Specific Nucleic Acid Sequences: A Rapid, Sensitive DNA Test to Identify Human Papillomavirus Type 16 Infection
Authors: Corstjens Paul , Zuiderwijk Michel , Brink Antoinette , Li Shang , Feindt Hans , Niedbala R Sam , Tanke Hans 
Published: January 2020

Abstract
Background: A lateral-flow (LF) device using the new reporter up-converting phosphor technology (UPTTM) was applied to DNA (hybridization) assays for the detection of specific nucleic acid sequences, thereby aiming to perform the test outside well-equipped laboratories. The methodology reported here is sensitive and provides a rapid alternative for more elaborate gel electrophoresis and Southern blotting. In a preliminary study, it was applied to screen for the presence of human papillomavirus type 16 (HPV16) in a defined series of cervical carcinomas.
Methods: A LF assay was used to capture haptenized DNA molecules and hybrids, which were immunolabeled (before LF) with 400-nm UPT particles. These particles emit visible light after excitation with infrared in a process called up-conversion. Because up-conversion occurs in only the phosphor lattice, autofluorescence of other assay components is virtually nonexistent.
Results: The use of the UPT reporter in LF-DNA tests, as compared with colloidal gold, improved the detection limit at least 100-fold. UPT LF-DNA tests were successfully applied to detect (in a blind test) the presence of HPV16 in DNA extracts obtained from cervical carcinomas. Test results matched 100% with previous characterization of these carcinomas.
Conclusions: The use of UPT in LF assays to detect specific nucleic acids provides low attamole-range sensitivity. Hybridization and consecutive detection of PCR-amplified HPV16 sequences were successful in a background of 10 μg of fish-sperm DNA. The sensitivity of UPT detection in these complex mixtures indicates that detection of viral infections without PCR or other amplification technique is achievable.
Rapid Quantification of Human ABCA1 mRNA in Various Cell Types and Tissues by Real-Time Reverse Transcription-PCR
Authors: Kielar Danuta , Dietmaier Wolfgang , Langmann Thomas , Aslanidis Charalampos , Probst Mario , Naruszewicz Marek , Schmitz Gerd 
Published: January 2020

AbstractBackground: The ABCA1 gene encodes for a member of subfamily A of the ATP-binding cassette transporters that plays an important role in cellular export of cholesterol and phospholipids; therefore, quantification of the ABCA1 mRNA is critical in many studies related to its expression and regulation by metabolic factors, nutritional status, and new antiatherogenic drug candidates. We developed a rapid, sensitive, specific, and reproducible real-time reverse transcription-PCR (RT-PCR) method for detection and quantification of ABCA1 transcripts in total RNA isolated from cultured human cells and tissues.Methods: To quantify ABCA1 mRNA, we generated a calibration curve from serial dilutions of in vitro-transcribed RNA corresponding to an amplified ABCA1 cDNA 205-bp fragment (homologous calibrator). Two pairs of fluorescent hybridization probes were used to detect the ABCA1 and porphobilinogen deaminase (PBGD) mRNAs; the latter served as an internal control. PCR was performed as real-time amplification of ABCA1 mRNA in 100 ng of total RNA isolated from various human tissues, and cultured cells were calculated from the calibration curve. In addition, normalized values of target (ABCA1/PBGD ratio) were calculated.Results: Using this method, we quantified ABCA1 transcripts in various human tissue samples as well as in monocytes, THP-1 cells, fibroblasts, and adipocytes. We demonstrated ABCA1 mRNA up-regulation during human adipocyte and monocyte differentiation. In addition, we examined the effect of cholesterol loading and deloading on ABCA1 expression in monocytes, THP-1 cells, and fibroblasts.Conclusions: Our RT-PCR assay allows the specific and highly reproducible detection and quantification of minute amounts of human ABCA1 mRNA. This new method is more accurate, more informative, and less laborious than the classic RT-PCR methods and Northern blot; it therefore could simplify all studies on ABCA1 mRNA expression.
Double-Gradient Denaturing Gradient Gel Electrophoresis Assay for Identification of L-Ferritin Iron-responsive Element Mutations Responsible for Hereditary Hyperferritinemia-Cataract Syndrome: Identification of the New Mutation C14G
Authors: Cremonesi Laura , Fumagalli Antonella , Soriani Nadia , Ferrari Maurizio , Levi Sonia , Belloli Silvana , Ruggeri Giuseppina , Arosio Paolo 
Published: January 2020

Abstract
Background: Hereditary hyperferritinemia-cataract syndrome is an autosomic dominant disorder caused by heterogeneous mutations on the iron-responsive element (IRE) of ferritin L-chain mRNA. The mutations described to date were identified by direct sequencing of DNA from probands with hyperferritinemia often associated to bilateral cataracts. A direct genetic approach on a large population is useful to recognize polymorphisms in the DNA region and the prevalence of mutations associated with minor increases in serum ferritin and subclinical cataracts. We developed a rapid DNA scanning technique to detect mutations in a single electrophoretic analysis.
Methods: The double-gradient denaturing gradient gel electrophoresis (DG-DGGE) method consisted of PCR amplification of the target genomic DNA with GC-clamped oligonucleotides. The sequence encoded the 5′ untranslated flanking region of ferritin L-chain mRNA, which includes an IRE stem-loop structure. The product was subjected to DG-DGGE (8.5–15% polyacrylamide and 50–95% denaturant) to separate the homo- and heteroduplexes.
Results: The method clearly identified all eight accessible mutations, including C-G transversions, which are the most difficult to detect. The method was applied to scan DNA samples from 50 healthy subjects and from 230 subjects with serum ferritin &gt;400 μg/L. The new mutation G14C was identified.
Conclusions: The DG-DGGE method detects all the mutations in the L-ferritin IRE sequence, is rapid and economical, and can be applied to scan large populations. The first population study indicated that the mutations are rare and may involve regions of the IRE structure not yet characterized.
High-Throughput Genotyping of Thiopurine S-Methyltransferase by Denaturing HPLC
Authors: Schaeffeler Elke , Lang Thomas , Zanger Ulrich M , Eichelbaum Michel , Schwab Matthias 
Published: January 2020

AbstractBackground: The thiopurine S-methyltransferase (TPMT) genetic polymorphism has a significant clinical impact on the toxicity of thiopurine drugs, which are used in the treatment of leukemia and as immunosuppressants. To date, 10 mutant alleles are known that are associated with intermediate or low TPMT activity. To facilitate rapid screening of clinically relevant TPMT mutations, we developed a strategy of high-throughput genotyping by applying denaturing HPLC (DHPLC).Methods: To test the specificity and efficiency of the DHPLC method, 98 DNA samples from a selected population of patients receiving thiopurine therapy or with previous thiopurine withdrawal were analyzed for the most frequent mutant TPMT alleles, *2 and *3A, which contain key mutations in exons 5, 7, and 10 to identify clearly different elution profiles. All fragments were examined by direct sequencing. Additionally, to test the sensitivity of DHPLC analysis, genotyping for the *2 and *3A alleles of all 98 DNA samples was performed by PCR-based methods (PCR-restriction fragment polymorphism analysis and allele-specific PCR).Results: The presence of mutations discriminating for alleles *2, *3A, *3C, and *3D, as well as various silent and intron mutations, were correctly predicted by DHPLC in 100% of the samples as confirmed by direct sequencing. Comparison with PCR-based methods for alleles *2 and *3 produced an agreement of 100% with no false-negative signals.Conclusions: DHPLC offers a highly sensitive, rapid, and efficient method for genotyping of the relevant TPMT mutations, discriminating at least for alleles *2 and *3, in clinical and laboratory practice. Additionally, DHPLC allows a simultaneous screening for novel genetic variability in the TPMT gene.
Preoperative Serum Prostate-specific Antigen (PSA) Below 10 μg/L Predicts Neither the Presence of Prostate Cancer Nor the Rate of Postoperative PSA Failure
Authors: Stamey Thomas A 
Published: January 2020

Abstract
Recent information on the relationship of serum prostate-specific antigen (PSA) to prostate cancer and new reports on death rates in men warrant a reassessment of how we diagnose and treat prostate cancer. We now know for the first time that the annual death rate from prostate cancer in men ≥65 years of age is only 226 per 100 000 men. At least 40 000 of 100 000 men over age 65 (40%) have invasive prostate cancer as judged by examination of prostates in 3- to 4-mm step-sections. Thus, only 1 of every 177 men 65 years of age or older (226 in 40 000) with invasive prostate cancer dies annually from his cancer. Serum PSA between 2 and 10 μg/L is used almost universally as an indication to biopsy the prostate. When 10–20 biopsies are commonly taken, it is not surprising that ∼40% of men are biopsy-positive for prostate cancer. Despite this reliance on serum PSA as an indication for biopsy, data at Stanford show no clinically useful relationship between preoperative serum PSA (in the range 2–10 mg/L) and the volume of Gleason grade 4/5 cancer or the volume of Gleason grades 3, 2, and 1 cancer, nor can we show any useful relationship of such preoperative PSA concentrations (2–10 μg/L) to biochemical PSA failure rates after radical prostatectomy. We urgently need a better serum marker for prostate cancer. Because PSA biochemical failure rates after radical prostatectomy are directly proportional to the amount of Gleason grade 4/5 cancer in the prostate, a serum marker of Gleason grade 4/5 carcinoma could be ideal.
Improved Diagnostic Classification of Alcohol Abusers by Combining Carbohydrate-deficient Transferrin and γ-Glutamyltransferase
Authors: Sillanaukee Pekka , Olsson Ulf 
Published: January 2020

Abstract
Background: Biochemical markers can provide objective evidence of high alcohol consumption. However, currently available markers have limitations in their diagnostic performance.
Methods: The diagnostic values of the most frequently used markers [carbohydrate-deficient transferrin (CDT), γ-glutamyltransferase (GGT), aspartate aminotransferase, alanine aminotransferase, and mean corpuscular volume] were studied in an analysis of six different clinical studies (n = 1412) on alcohol abusers and social drinkers. The purpose of the analyses was to determine whether a combination of markers would improve the diagnosis of subjects.
Results: Discrimination between alcohol abusers and social drinkers, as measured by the areas under nonparametric ROC plots, was significantly better (P &lt;0.001) for the new combined marker [γ-CDT = 0.8 · ln(GGT) + 1.3 · ln(CDT)] than for any of the separate markers or combination of CDT or GGT with other markers. The cutoff values for γ-CDT (6.5) can be taken to be the same among males and females.
Conclusions: The combined variable γ-CDT is a powerful tool to discriminate alcohol abusers from social drinkers and is recommended for clinical use.
Fatty Acid Ethyl Esters in Liver and Adipose Tissues as Postmortem Markers for Ethanol Intake
Authors: Salem Raneem O , Refaai Majed A , Cluette-Brown Joanne E , Russo Joshua W , Laposata Michael 
Published: January 2020

AbstractBackground: Fatty acid ethyl esters (FAEEs) are nonoxidative metabolites of ethanol. FAEEs are found in liver, pancreas, and adipose tissues up to 24 h after consumption of ethanol, and on that basis, they are potentially useful markers for ethanol intake. In this study with rats, we investigated the efficacy of using FAEEs in liver and in adipose tissue as postmortem markers for premortem ethanol ingestion.Methods: An animal study was conducted in which test rats received injections of ethanol and control rats received injections of normal saline. The rats were killed 2 h after the injections. The bodies of the animals were stored at 4 °C up to 12 h, and samples of liver and adipose tissues were collected at different time intervals and processed for FAEE quantification. In another set of experiments, the rats received injections and were killed as described above, but bodies of animals from both groups were stored at 4, 25, or 37 °C for up to 72 h, and liver samples were collected and processed for FAEE quantification.Results: FAEEs were detected up to 12 h after death in liver and adipose tissue samples from the bodies of ethanol-treated animals stored at 4 °C; negligible amounts were detected in the bodies of animals that received normal saline. Adipose tissues contained higher amounts of FAEEs than liver, as well as more species: eight FAEE species in adipose tissue and five in liver tissue. Higher concentrations of FAEEs were detected in livers of treated animals stored at 25 °C for up to 48 h than in livers of controls stored under the same conditions.Conclusions: For at least 12 h after death, FAEEs in liver and adipose tissues are useful postmortem markers of premortem ethanol ingestion.
Semiautomated DNA Mutation Analysis Using a Robotic Workstation and Molecular Beacons
Authors: Smit Maarten L , Giesendorf Belinda A J , Vet Jacqueline A M , Trijbels Frans J M , Blom Henk J 
Published: January 2020

Abstract
Background: Our increasing knowledge of the genetic basis of inheritable diseases requires the development of automated reliable methods for high-throughput analyses.
Methods: We investigated the combination of semiautomated DNA extraction from blood using a robotic workstation, followed by automated mutation detection using highly specific fluorescent DNA probes, so-called molecular beacons, which can discriminate between alleles with as little as one single-base mutation. We designed two molecular beacons, one recognizing the wild-type allele and the other the mutant allele, to determine genotypes in a single reaction. To evaluate this procedure, we examined the C677T mutation in the methylenetetrahydrofolate reductase (MTHFR) gene, which is associated with an increased risk for cardiovascular disease and neural tube defects. DNA was isolated from 10 μL of fresh EDTA-blood samples by use of a robotic workstation. The DNA samples were analyzed using molecular beacons as well as conventional methods.
Results: Both methods were compared, and no differences were found between outcomes of genotyping.
Conclusions: The described assay enables robust and automated extraction of DNA and analysis of up to 96 samples (10 μL of blood per sample) within 5 h. This is superior to conventional methods and makes it suitable for high-throughput analyses.
Pharmacokinetic Basis for the Efficient and Safe Use of Low-Dose Mycophenolate Mofetil in Combination with Tacrolimus in Kidney Transplantation
Authors: Mourad Michel , Malaise Jacques , Chaib Eddour Djamila , De Meyer Martine , König Josiane , Schepers Raf , Squifflet Jean Paul , Wallemacq Pierre 
Published: January 2020

AbstractBackground: Mycophenolate mofetil (MMF) is an effective posttransplantation immunosuppressive agent used in combination with cyclosporin A (CsA) or tacrolimus (Tc). An increase in plasma mycophenolic acid (MPA) has been shown in patients receiving Tc-MMF combination therapy compared with CsA-MMF combination therapy at the same dose of MMF. The aim of this prospective study was to assess the pharmacokinetic/pharmacodynamic (PK/PD) relationship for MPA in kidney transplant patients receiving low-dose MMF (500 mg twice a day) in combination with Tc.Methods: Adult kidney transplant recipients (n = 51) were included. MPA-PK profiles (blood sampling at 0, 0.5, 1, 2, 4, 6, and 12 h after MMF oral dose) were obtained within the first 2 weeks after transplantation, 3 months after grafting, and at every adverse clinical event [side effect or acute rejection (AR)]. All patients received Tc, MMF (500 mg twice a day), and steroids.Results: Thirty patients (59%) had uneventful outcomes, and 21 patients had 33 episodes of MPA-related side effects; only 3 patients had AR. A total of 78 MPA-PK profiles were obtained. The following PK parameters were increased in the side-effects group compared with the non-side effects group: mean MPA cmin, 2.63 ± 1.58 vs 1.75 ± 0.82 mg/L (P = 0.016); mean c30, 10.47 ± 6.27 vs 7.66 ± 8.95 mg/L (P = 0.009); mean c60, 9.67 ± 5.42 vs 5.83 ± 2.6 mg/L (P = 0.0002); mean area under the MPA time-concentration curve from 0 to 12 h [MPA-AUC(0–12)], 48.38 ± 18.5 vs 36.04 ± 10.82 mg · h/L (P = 0.0006); mean dose-normalized MPA-AUC, 0.16 ± 0.05 vs 0.12 ± 0.04 (mg · h/L)/(mg/m2) (P = 0.0015). For the three AR patients, MPA concentrations obtained at the time of AR revealed MPA cmin values of 1.86, 1.76, and 3.83 mg/L, respectively, and MPA-AUC(0–12) values of 37.7, 24.9, and 104.9 mg · h/L. The threshold of toxicity was 3 mg/L (sensitivity, 38.7%; specificity, 91.5%) for cmin, 8.09 mg/L for maximum MPA concentration during the first hour (sensitivity, 77.8%; specificity, 67.4%), and 37.6 mg · h/L for MPA-AUC(0–12) (sensitivity, 83.3%; specificity, 59.6%).Conclusions: These results demonstrate the relationship between plasma MPA concentrations and toxicity. High cmin, c30, and c60 values as well as AUC(0–12) are associated with increased risk for side effects. These values may have an importance in a routine monitoring program.
Vitamin B12 Treatment Normalizes Metabolic Markers But Has Limited Clinical Effect: A Randomized Placebo-controlled Study
Authors: Hvas Anne-Mette , Ellegaard Jørgen , Nexø Ebba 
Published: January 2020

AbstractBackground: The clinical significance of increased plasma methylmalonic acid (P-MMA) is unclear. We assessed the efficacy of vitamin B12 treatment in reducing P-MMA and plasma total homocysteine compared with the clinical benefits of treatment.Methods: We studied 140 individuals with mildly to modestly increased P-MMA (0.40–2.00 μmol/L), not previously treated with vitamin B12, in a randomized, placebo-controlled study. A detailed medical history was obtained, and laboratory tests as well as an objective neurologic disability score were performed at baseline and 3 months after the start of intervention.Results: P-MMA (P &lt;0.001) or plasma total homocysteine (P &lt;0.001) decreased in the treatment group vs the placebo group, but no significant difference was found in the change of blood hemoglobin (P = 0.18) and mean cell volume (P = 0.71). Changes in symptom scores did not differ between the groups for symptoms of anemia (P = 0.63), neurologic symptoms (P = 0.21), gastroenterologic symptoms (P = 0.32), or the Neurological Disability Score (P = 0.85).Conclusions: Treatment with vitamin B12 reduces P-MMA and plasma total homocysteine, but individuals with a mild to modest increase in P-MMA may have only limited clinical benefit from vitamin B12 treatment, at least in the short term.
Clinical Evaluation of the Elecsys β-CrossLaps Serum Assay, a New Assay for Degradation Products of Type I Collagen C-Telopeptides
Authors: Okabe Reiko , Nakatsuka Kiyoshi , Inaba Masaaki , Miki Takami , Naka Hiroshi , Masaki Hideki , Moriguchi Atushi , Nishizawa Yoshiki 
Published: January 2020

AbstractBackground: The Elecsys β-CrossLaps serum assay measures type I collagen degradation fragments (β-CTx) that contain the β-isomerized octapeptide EKAHD-β-GGR. We investigated the analytical performance of the assay and changes in β-CrossLaps in patients with metabolic bone diseases.Methods: The electrochemiluminescent sandwich immunoassay uses two monoclonal antibodies directed against different regions of the linear EKAHD-β-GGR.Results: β-CrossLaps (β-CTx) immunoreactivity was stable in serum and plasma stored at 4 °C for 24 h or at room temperature for 4 h, and it did not decrease appreciably in samples stored at −30 °C for 12 weeks. Nine cycles of repeated freezing-thawing did not affect serum β-CTx. The intra- and interassay imprecision (CVs) for four samples was ≤2.6% (n = 10) and ≤4.1% (n = 10), respectively. The mean day-to-day biological variation (CV) was 20% in 10 postmenopausal women (n = 10 days). Serum β-CTx and osteocalcin were correlated in patients with hyperparathyroidism (r = 0.796; P &lt;0.0001; n = 28), chronic renal failure on hemodialysis (r = 0.784; P = 0.0003; n = 16), hypoparathyroidism (r = 0.950; P = 0.0001; n = 11), and pseudohypoparathyroidism (r = 0.987; P = 0.130; n = 4). Serum β-CTx decreased by 47.4% ± 8.8% (mean ± SD) and 60.7% ± 6.5% at 3 and 6 months, respectively, after initiation of estrogen replacement therapy in 34 women. These decreases were greater than the decreases in urinary excretion of deoxypyridinoline (31.8% ± 3.9% and 38.1% ± 4.4%, respectively) or pyridinoline cross-linked C-terminal telopeptide of type I collagen (15.9% ± 3.9% and 16.9% ± 4.6%, respectively).Conclusions: The Elecsys β-CrossLaps serum assay provides a potentially useful tool for assessing bone resorption state, including its response to estrogen replacement therapy.