The impact of biomarkers in multivariate algorithms for bladder cancer diagnosis in patients with hematuria

BACKGROUND: We appraised 23 biomarkers previously associated with urothelial cancer in a case-control study. Our aim was to determine whether single biomarkers and/or multivariate algorithms significantly improved on the predictive power of an algorithm based on demographics for prediction of urothelial cancer in patients presenting with hematuria. METHODS: Twenty-two biomarkers in urine and carcinoembryonic antigen (CEA) in serum were evaluated using enzyme-linked immunosorbent assays (ELISAs) and biochip array technology in 2 patient cohorts: 80 patients with urothelial cancer, and 77 controls with confounding pathologies. We used Forward Wald binary logistic regression analyses to create algorithms based on demographic variables designated prior predicted probability (PPP) and multivariate algorithms, which included PPP as a single variable. Areas under the curve (AUC) were determined after receiver-operator characteristic (ROC) analysis for single biomarkers and algorithms. RESULTS: After univariate analysis, 9 biomarkers were differentially expressed (t test; P

Natural Toxicants: Tetrodotoxin

Tetrodotoxin (tetrodotoxin) is a potent neurotoxin, which shuts down electrical signaling in nerves by blocking the voltage-gated sodium channel proteins in nerve cell membranes. It was originally discovered in puffer fish but is found in a range of animal species and thought to be produced by bacteria. The toxin can be lethal to humans being 10 000 times more potent than cyanide. Human fatalities have been attributed to the ingestion of this toxin through consumption of puffer fish, a delicacy in Japan and other regions, and other marine species. The effects of tetrodotoxin poisoning onset quickly and include shortness of breath, numbness, tingling, light-headedness, paralysis, and irregular heartbeat. Treatment usually consists of respiratory assistance as no antidote has been developed. The accepted method of analysis for tetrodotoxin is the mouse bioassay, although recently more ethical assays have been developed including high performance liquid chromatography, biosensor and enzyme-linked immunosorbant assay.

Localization of polymorphic N-acetyltransferase (NAT2) in tissues of inbred mice

Like humans, mice exhibit polymorphism in the N-acetylation of aromatic amines, many of which are toxic and/or carcinogenic. Mice have three N-acetyltransferase (Nat) genes, Nat1, Nat2 and Nat3, and Nat2 is known to be polymorphic. There is a dramatic difference in the acetylation of NAT2 substrates by blood from fast (C57BL/6J) compared with slow acetylator (A/J) mice. However, the acetylation of these substrates by liver cytosols from the two strains is very similar. In order to determine whether the expression of the NAT2 protein corresponded with the activities measured, a polyclonal antipeptide antisera was raised against the C-terminal decapeptide of NAT2 and characterized using recombinant murine NAT2 antigen. Enzyme-linked immunosorbent assays (ELISAs) demonstrated that the anti-NAT2 antiserum bound in a concentration-dependent fashion to recombinant NAT2. Immunochemical analysis of mouse liver cytosols from C57BL/6J or A/J livers indicated that the level of NAT2 protein expressed in the two strains was similar. Immunohistochemical staining of C57BL/6J liver with anti-NAT2 antiserum showed that NAT2 was expressed in hepatocytes throughout the liver although the intensity of staining in the perivenous (centrilobular) region was higher than that in the periportal region. NAT2 was also detected in epithelial cells in the lung, kidney, bladder, small intestine and skin as well as in erythrocytes and lymphocytes in the spleen and hair follicles and sebaceous glands in the skin. Characterization of the distribution of NAT2 will be of value in elucidating the role of polymorphic N-acetylation in protecting the organism from environmental insults as well as in endogenous metabolism.

Development of a recombinant Fab-fragment based electrochemical immunosensor for deoxynivalenol detection in food samples

A reliable and cost-effective electrochemical method for the detection of deoxynivalenol (DON) in cereals and cereal-based food samples based on the use of a novel anti-DON Fab fragment is presented. The analytical system employed, Enzyme-Linked-Immunomagnetic-Electrochemical (ELIME) assay, is based on the use of immunomagnetic beads (IMBs) coupled with eight magnetized screen-printed electrodes (8-mScPEs) as electrochemical transducers.

Low vitamin D status adversely affects bone health parameters in adolescents

Background: The effects of subclinical vitamin D deficiency on bone mineral density (BMD) and bone turnover in adolescents, especially in boys, are unclear.

Objective: We aimed to investigate the relations of different stages of vitamin D status and BMD and bone turnover in a representative sample of adolescent boys and girls.

Design: BMD was measured by dual-energy X-ray absorptiometry at the nondominant forearm and dominant heel in a random sample of 12- (n = 260) and 15-y-old (n = 239) boys and 12- (n = 266) and 15-y-old (n = 250) girls. Serum 25-hydroxyvitamin D, parathyroid hormone, osteocalcin, and type I collagen cross-linked C-telopeptide were assessed by using enzyme-linked immunoassays. Relations between vitamin D status and bone health indexes were assessed by using regression modeling.

Results: Using multivariate regression to adjust for potential physical, lifestyle, and dietary confounding factors, we observed that 12-and 15-y-old girls with high vitamin D status (>= 74.1 nmol/L) had significantly greater forearm (but not heel) BMD (beta = 0.018; SE = 0.008; P < 0.05 for each age group) and lower serum parathyroid hormone concentrations and bone turnover markers than did those with low vitamin D status. These associations were evident in subjects sampled throughout the year and in winter only. There was no significant relation between vitamin D status and BMD in boys.

Conclusions: Maintaining serum 25-hydroxyvitamin D concentrations above approximate to 50 nmol/L throughout the year may be a cost-effective means of improving bone health. Increased emphasis on exploring strategies for improving vitamin D status in adolescents is needed.

Fetal and maternal leptin in pre-gestational diabetic pregnancy

Objective: To compare maternal and fetal leptin among women without diabetes, women with type 1 diabetes, and women with type 2 diabetes. 

Methods: In a prospective study at the National Maternity Hospital, Dublin, 40 women with type 1 diabetes, 10 with type 2 diabetes, and 30 without diabetes were enrolled between July 2006 and July 2008. Maternal (36-week) and cord blood leptin was measured by enzyme-linked immunoassay. 

Results: No difference was found in maternal leptin among the groups: without diabetes (mean, range): 325 pg/mL, 36-1492 pg/mL; type 1 diabetes: 343.2 pg/mL, 55.5-1108.2 pg/mL; type 2 diabetes: 2022 pg/mL, 35.1-1553.3 pg/mL (P>0.05). Leptin levels were higher among fetuses of women with type 1 (223 pg/mL, 25.7-810 pg/mL) and type 2 (447.2 pg/mL, 1363-679 pg/mL) diabetes than among women without diabetes (803 pg/mL, 273-623.1 pg/mL; P<0.05). The single significant predictor of fetal leptin for the whole cohort was maternal body mass index (BMI; r=039, P=0.01). Only third-trimester glycosylated hemoglobin (HbA1c) was significantly related to fetal leptin after controlling for maternal BMI among women with diabetes (r=028, P=0.04).

 Conclusion: Fetuses of women with diabetes might have some degree of leptin resistance. This might be important in appetite regulation in extrauterine life. (C) 2012 International Federation of Gynecology and Obstetrics. 

A PLATE ASSAY FOR THE DETECTION OF ORGANOPHOSPHONATE MINERALIZATION BY ENVIRONMENTAL BACTERIA, AND ITS MODIFICATION AS AN ACTIVITY STAIN FOR IDENTIFICATION OF THE CARBON-PHOSPHORUS BOND-CLEAVAGE ENZYME PHOSPHONOACETATE HYDROLASE

A replica plate screening technique, based on the acid molybdate assay for detection of phosphate has been developed to permit the detection of microorganisms capable of mineralizing organophosphonates. The method was further adapted as the basis of an activity stain for the detection of the carbon - phosphorus bond cleavage enzyme phosphonoacetate hydrolase in PAGE gels.

An assay for angiotensin-converting enzyme using capillary zone electrophoresis

A sensitive and rapid method was developed for angiotensin-converting enzyme (ACE) activity determination by capillary zone electrophoresis. Hippuryl-View the MathML source-histidyl-View the MathML source-leucine, a synthetic tripeptide, was used as the ACE-specific substrate. Capillary zone electrophoresis was employed to separate the products of the enzymatic reaction and the ACE activity was determined by quantification of hippuric acid, a result of the enzymatic reaction on the tripeptide. The capillary electrophoresis was performed in a 27 cm × 75 μm i.d. fused-silica capillary using 200 mM boric acid–borate buffer (pH 9.0) as a run buffer with an applied voltage of 8.1 kV at a capillary temperature of 23°C. The electrophoresis was monitored at 228 nm. Each electrophoretic run requires only a nanoliter of the enzymatic reactant solution, at only 6 min, rendering a powerful tool for the ACE assay.

Is there an association between angiotensin-converting enzyme gene variants and chronic nonproductive cough?

Background: It is unclear why some patients develop a chronic nonproductive cough. Angiotensin-converting enzyme (ACE) inactivates tussive peptides in the airways such as bradykinin and tachykinins. An insertion/deletion polymorphism in the ACE gene accounts for variation in ACE levels, and patients with the II genotype have lowest serum ACE levels compared with ID and DD genotypes. We hypothesized that the II genotype would be associated with increased risk of developing a chronic cough.

Materials and methods: We recruited 47 patients (33 women), referred for evaluation of cough (median cough duration, 24 months; range, 2 to 240 months). Cough patients were evaluated using a comprehensive diagnostic protocol, and cough reflex sensitivity was measured using a capsaicin inhalation challenge. ACE genotyping was performed on DNA samples from patients using the polymerase chain reaction followed by agarose gel electrophoresis. ACE genotypes in patients with chronic cough were compared with those in 199 healthy control subjects. Serum ACE levels were determined using a colorimetric assay.

Results: Genotype frequencies for the ACE gene were similar between patients and control subjects. There was no correlation between capsaicin sensitivity and ACE genotypes or serum ACE levels.

Conclusion: Susceptibility to develop chronic cough is not associated with ACE genotype.

A nonradioactive method for the assay of polyphosphate kinase activity and its application in the study of polyphosphate metabolism in Burkholderia cepacia

Studies of polyphosphate (polyP) metabolism in microorganisms have been hampered by the lack of a convenient method for the assay in cell extracts of the activity of polyphosphate kinase (PPK), the enzyme principally responsible for microbial polyP biosynthesis. We report the development of such an assay, based on the well-established metachromatic reaction, with toluidine blue, of the polyP formed during the PPK-catalyzed reaction. The method was successfully used in the characterization of PPK activity in crude extracts of an environmental Burkholderia cepacia isolate. The development of a protocol for the physical recovery of polyP from solution is also reported. (C) 2002 Elsevier Science (USA). All rights reserved.

Detection of monensin residues in poultry liver using an enzyme immunoassay

Monensin, a carboxylic acid ionophore, is commonly fed to poultry to control coccidiosis. A method for the detection and quantification of monensin residues in liver has been developed, Samples (3 g) were extracted with acetonitrile-water and applied to a competitive enzyme immunoassay using a polyclonal antiserum raised against a monensin-transferrin conjugate, The limit of detection (mean + 3s) calculated from the analysis of 12 known negative samples was 2.91 ng g(-1). Intra- and inter-assay RSD were determined as 8.5 and 10.6%, respectively, using a liver sample fortified with 20 ng g(-1) monensin, A pharmacokinetic study in which 70 six week old broilers were fed monensin at a rate of 120 mg kg(-1) in their feed for 14 d resulted in mean monensin liver residues of 102 ng g(-1). However these had fallen below the limit of detection of the assay within the 3 d withdrawal period recommended by the manufacturer.

Detection of ivermectin residues in bovine liver using an enzyme immunoassay

Ivermectin, a member of the avermectin group, is frequently used to control parasites in many food producing animal species. A method for the detection and quantification of ivermectin residues in bovine liver has been developed. Liver samples (4 g) were extracted with acetonitrile and applied to a competitive enzyme immunoassay using a polyclonal antiserum raised in rabbits against an ivermectin-transferrin conjugate, The limit of detection of the assay (mean +/- 3s) calculated from the analysis of 24 known negative samples was 1.6 ng g(-1), Intra- and inter-assay RSDs were determined as 8.8 and 14.6%, respectively, using a negative bovine liver sample fortified with 100 ng g(-1) of ivermectin. Four Friesian steers were treated with a pour-on application of ivermectin at a dose rate of 0.5 mg kg(-1) body mass then withdrawn and killed at 7, 14, 21 and 28 d, Livers mere removed and ivermectin residue concentrations determined using the proposed immunoassay procedure, Seven days post-treatment the ivermectin liver concentration was determined as 52.7 ng g(-1), decreasing to 4.1 ng(-1) at 28 d, All immunoassay results were confirmed using high-performance liquid chromatography (HPLC), The immunoassay and HPLC results for invermectin ranged from 1 to 58 ng g(-1) and were in close correlation (r = 0.99).

DETERMINATION OF THE CONCENTRATIONS OF THE STEROIDS ESTRADIOL, PROGESTERONE AND TESTOSTERONE IN BOVINE SERA - COMPARISON OF COMMERCIAL DISSOCIATION ENHANCED LANTHANIDE FLUORESCENCE IMMUNOASSAY KITS WITH CONVENTIONAL RADIO AND ENZYME IMMUNOASSAYS

The performance of three conventional enzyme and radioimmunoassays routinely used to detect residues of anabolic steroids in cattle sera were compared with dissociation enhanced lanthanide fluorescence immunoassay (DELFIA) kits designed for the hospital market. Slight modifications to the kit reagents were required for the analysis of bovine sera. Owing to the large sample volumes used in conventional assays, detection limits were generally better than those obtained with DELFIA kits, however, assay reproducibility was enhanced using the DELFIA technology. Comparison of sera obtained from cattle implanted with anabolic steroids revealed a good correlation between alternate methods (r(2) from 0.91 to 0.97). The DELFIA kits offer a faster method for measuring estradiol, progesterone and testosterone with adequate sensitivity and in a safer environment than that encountered using radioimmunoassays.

Engineering N-linked protein glycosylation with diverse O antigen lipopolysaccharide structures in Escherichia coli

Campylobacter jejuni has a general N-linked protein glycosylation system that can be functionally transferred to Escherichia coli. In this study, we engineered E. coli cells in a way that two different pathways, protein N-glycosylation and lipopolysaccharide (LPS) biosynthesis, converge at the step in which PglB, the key enzyme of the C. jejuni N-glycosylation system, transfers O polysaccharide from a lipid carrier (undecaprenyl pyrophosphate) to an acceptor protein. PglB was the only protein of the bacterial N-glycosylation machinery both necessary and sufficient for the transfer. The relaxed specificity of the PglB oligosaccharyltransferase toward the glycan structure was exploited to create novel N-glycan structures containing two distinct E. coli or Pseudomonas aeruginosa O antigens. PglB-mediated transfer of polysaccharides might be valuable for in vivo production of O polysaccharides-protein conjugates for use as antibacterial vaccines.

Development of a solid-phase extraction coupling chemiluminescent enzyme immunoassay for determination of organophosphorus pesticides in environmental water samples

Solid-phase extraction (SPE) and direct competitive chemiluminescence enzyme immunoassay (dcCL-EIA) were combined for the detection of organophosphorus pesticides (OPs) in environmental water samples. dcCL-EIA based on horseradish peroxidase labeled with a broad-specificity monoclonal antibody against OPs was developed, and the effects of several physicochemical parameters on dcCL-EIA performance were studied. SPE was used for the pretreatment of water samples to remove interfering substances and to concentrate the OP analytes. The coupling of SPE and dcCL-EIA can detect seven OPs (parathion, coumaphos, phoxim, quinalphos, triazophos, dichlofenthion, and azinphos-ethyl) with the limit of quantitation below 0.1 ng/mL. The recoveries of OPs from spiked water samples ranged from 62.5% to 131.7% by SPE-dcCL-EIA and 69.5% to 112.3% by SPE-HPLC-MS/MS. The screening of OP residues in real-world environmental water samples by the developed SPE-dcCL-EIA and their confirmatory analysis using SPE-HPLC-MS/MS demonstrated that the assay is ideally suited as a monitoring method for OP residues prior to chromatographic analysis.