In Vitro UDP-Sugar:Undecaprenyl-Phosphate Sugar-1-Phosphate Transferase Assay and Product Detection by Thin Layer Chromatography

In vitro assays are invaluable for the biochemical characterization of UDP-sugar:undecaprenyl-phosphate sugar-1-phosphate transferases. These assays typically involve the use of a radiolabeled substrate and subsequent extraction of the product, which resides in a lipid environment. Here, we describe the preparation of bacterial membranes containing these enzymes, a standard in vitro transferase assay with solvents containing chloroform and methanol, and two methods to measure product formation: scintillation counting and thin layer chromatography.

High population prevalence of cardiac troponin I measured by a high-sensitivity assay and cardiovascular risk estimation: the MORGAM Biomarker Project Scottish Cohort

Aims
Our aim was to test the prediction and clinical applicability of high-sensitivity assayed troponin I for incident cardiovascular events in a general middle-aged European population.

Methods and results
High-sensitivity assayed troponin I was measured in the Scottish Heart Health Extended Cohort (n = 15 340) with 2171 cardiovascular events (including acute coronary heart disease and probable ischaemic strokes), 714 coronary deaths (25% of all deaths), 1980 myocardial infarctions, and 797 strokes of all kinds during an average of 20 years follow-up. Detection rate above the limit of detection (LoD) was 74.8% in the overall population and 82.6% in men and 67.0% in women. Troponin I assayed by the high-sensitivity method was associated with future cardiovascular risk after full adjustment such as that individuals in the fourth category had 2.5 times the risk compared with those without detectable troponin I (P < 0.0001). These associations remained significant even for those individuals in whom levels of contemporary-sensitivity troponin I measures were not detectable. Addition of troponin I levels to clinical variables led to significant increases in risk prediction with significant improvement of the c-statistic (P < 0.0001) and net reclassification (P < 0.0001). A threshold of 4.7 pg/mL in women and 7.0 pg/mL in men is suggested to detect individuals at high risk for future cardiovascular events.

Conclusion
Troponin I, measured with a high-sensitivity assay, is an independent predictor of cardiovascular events and might support selection of at risk individuals.

Development and single laboratory validation of an optical biosensor assay for tetrodotoxin detection as a tool to combat emerging risks in European seafood

Tetrodotoxin (TTX) is a potent neurotoxin emerging in European waters due to increasing ocean temperatures. Its detection in seafood is currently performed as a consequence of using the Association of Analytical Communities (AOAC) mouse bioassay (MBA) for paralytic shellfish poisoning (PSP) toxins, but TTX is not monitored routinely in Europe. Due to ethical and performance-related issues associated with this bioassay, the European Commission has recently published directives extending procedures that may be used for official PSP control. An AOAC-accredited high-performance liquid chromatography (HPLC) method has now been accepted by the European Union as a first action screening method for PSP toxins to replace the MBA. However, this AOAC HPLC method is not capable of detecting TTX, so this potent toxin would be undetected; thereby, a separate method of analysis is required. Surface plasmon resonance (SPR) optical biosensor technology has been proven as a potential alternative screening method to detect PSP toxins in seafood. The addition of a similar SPR inhibition assay for TTX would complement the PSP assay in removing the MBA. The present report describes the development and single laboratory validation in accordance with AOAC and IUPAC guidelines of an SPR method to be used as a rapid screening tool to detect TTX in the sea snail Charonia lampas lampas, a species which has been implicated in 2008 in the first case of human TTX poisoning in Europe. As no current regulatory limits are set for TTX in Europe, single laboratory validation was undertaken using those for PSP toxins at 800 µg/kg. The decision limit (CCa) was 100 µg/kg, with the detection capability (CCß) found to be =200 µg/kg. Repeatability and reproducibility were assessed at 200, 400, and 800 µg/kg and showed relative standard deviations of 8.3, 3.8, and 5.4 % and 7.8, 8.3, and 3.7 % for both parameters at each level, respectively. At these three respective levels, the recovery of the assay was 112, 98, and 99 %.

Novel hydrolysis-probe based qPCR assay to detect saxitoxin transcripts of dinoflagellates in environmental samples

Paralytic Shellfish Poisoning (PSP) is a serious human illness caused by ingestion of seafood enriched with paralytic shellfish toxins (PSTs). PSTs are neurotoxic compounds produced by marine dinoflagellates, specifically by Alexandrium spp., Gymnodinium catenatum and Pyrodinium bahamense. Every year, massive monitoring of PSTs and their producers is undertaken worldwide to avoid PSP incidences. Here we developed a sensitive, hydrolysis probe-based quantitative PCR (qPCR) assay to detect a gene essential for PST synthesis across different dinoflagellate species and genera and tested it on cDNA generated from environmental samples spiked with Alexandrium minutum or Alexandrium fundyense cells. The assay was then applied to two environmental sample series from Norway and Spain and the results were complemented with cell counts, LSU-based microarray data and toxin measurements (enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) biosensor method). The overall agreement between the results of the qPCR assay and the complementary data was good. The assay reliably detected sxtA transcripts from Alexandrium spp. and G. catenatum, even though Alexandrium spp. cell concentrations were mostly so low that they could not be quantified microscopically. Agreement between the novel assay and toxin measurements or cell counts was generally good; the few inconsistencies observed were most likely due to disparate residence times of sxtA transcripts and PSTs in seawater, or, in the case of cell counts, to dissimilar sxtA4 transcript numbers per cell in different dinoflagellate strains or species. © 2013 Elsevier B.V.

Comparison of the mouse spleen cell assay and a radioimmunoassay for the measurement of serum erythropoietin

The mouse spleen cell assay (MSCA) has been compared with a radioimmunoassay for the measurement of serum erythropoietin (Ep). In 20 normal subjects the serum values ranged from 15 to 73 mU/ml for the MSCA compared with 5-30 mU/ml for the RIA. For normal sera there was no correlation between the results of the two assays. In 37 patients with anaemias of differing aetiologies and at various stages of treatment values ranged from 10 to 3645 mU/ml for the MSCA and 13-10,000 mU/ml for the RIA. Although patient values from the two assays were highly correlated (r = 0.98, P less than 0.001), the MSCA results were generally lower. These discrepancies can be largely accounted for by two factors. Firstly the MSCA is sensitive to non-specific matrix effects. Secondly, heat inactivation of serum, a prerequisite for the MSCA, but not for the RIA, destroys a variable and unpredictable proportion of the Ep in the test sera leading to an underestimation of Ep in the MSCA. We conclude that the RIA is more reliable than the MSCA which, in its present form, cannot be recommended for the accurate measurement of serum erythropoietin.

Identification and Validation of an Anthracycline/Cyclophosphamide–Based Chemotherapy Response Assay in Breast Cancer

Background: There is no method routinely used to predict response to anthracycline and cyclophosphamide–based chemotherapy in the clinic; therefore patients often receive treatment for breast cancer with no benefit. Loss of the Fanconi anemia/BRCA (FA/BRCA) DNA damage response (DDR) pathway occurs in approximately 25% of breast cancer patients through several mechanisms and results in sensitization to DNA-damaging agents. The aim of this study was to develop an assay to detect DDR-deficient tumors associated with loss of the FA/BRCA pathway, for the purpose of treatment selection.

Methods: DNA microarray data from 21 FA patients and 11 control subjects were analyzed to identify genetic processes associated with a deficiency in DDR. Unsupervised hierarchical clustering was then performed using 60 BRCA1/2 mutant and 47 sporadic tumor samples, and a molecular subgroup was identified that was defined by the molecular processes represented within FA patients. A 44-gene microarray-based assay (the DDR deficiency assay) was developed to prospectively identify this subgroup from formalin-fixed, paraffin-embedded samples. All statistical tests were two-sided.

Results: In a publicly available independent cohort of 203 patients, the assay predicted complete pathologic response vs residual disease after neoadjuvant DNA-damaging chemotherapy (5-fluorouracil, anthracycline, and cyclophosphamide) with an odds ratio of 3.96 (95% confidence interval [Cl] =1.67 to 9.41; P = .002). In a new independent cohort of 191 breast cancer patients treated with adjuvant 5-fluorouracil, epirubicin, and cyclophosphamide, a positive assay result predicted 5-year relapse-free survival with a hazard ratio of 0.37 (95% Cl = 0.15 to 0.88; P = .03) compared with the assay negative population.

Conclusions: A formalin-fixed, paraffin-embedded tissue-based assay has been developed and independently validated as a predictor of response and prognosis after anthracycline/cyclophosphamide–based chemotherapy in the neoadjuvant and adjuvant settings. These findings warrant further validation in a prospective clinical study.

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.

Use of the γ-H2AX Assay to Investigate DNA Repair Dynamics Following Multiple Radiation Exposures

Radiation therapy is one of the most common and effective strategies used to treat cancer. The irradiation is usually performed with a fractionated scheme, where the dose required to kill tumour cells is given in several sessions, spaced by specific time intervals, to allow healthy tissue recovery. In this work, we examined the DNA repair dynamics of cells exposed to radiation delivered in fractions, by assessing the response of histone-2AX (H2AX) phosphorylation (γ-H2AX), a marker of DNA double strand breaks. γ-H2AX foci induction and disappearance were monitored following split dose irradiation experiments in which time interval between exposure and dose were varied. Experimental data have been coupled to an analytical theoretical model, in order to quantify key parameters involved in the foci induction process. Induction of γ-H2AX foci was found to be affected by the initial radiation exposure with a smaller number of foci induced by subsequent exposures. This was compared to chromatin relaxation and cell survival. The time needed for full recovery of γ-H2AX foci induction was quantified (12 hours) and the 1:1 relationship between radiation induced DNA double strand breaks and foci numbers was critically assessed in the multiple irradiation scenarios.

Validation and application of a reporter gene assay for the determination of estrogenic endocrine disruptor activity in milk

Endocrine disruptors (EDs) are compounds known to interfere with the endocrine system by disturbing the action or pathways of natural hormones which may lead to infertility or cancer.Our diet is considered to be one of the main exposure routes to EDs. Since milk and dairy products are major components of our diet they should be monitored for ED contamination. Most assays developed to date utilise targeted, chromatography based methods which lack information on the biological activity and mixture effects of the monitored compounds.A biological reporter gene assay (RGA) was developed to assess the total estrogen hormonal load in milk. It has been validated according to EU decision 2002/657/EC. Analytes were extracted by liquid-liquid extraction with acetonitrile followed by clean up on a HLB column which yielded good recovery and small matrix effects. The method has been shown to be estrogen specific, repeatable and reproducible, with covariance values below 20%. In conclusion, this method enables the detection of low levels of estrogen hormonal activity in milk with a detection capability of 36pgg EEQ and has been successfully applied in testing a range of milk samples. © 2014 Elsevier Ltd.

Biofilm Eradication Kinetics of the Ultrashort Lipopeptide C12-OOWW-NH2 Utilizing a Modified MBEC Assay™

In this study, we report the antimicrobial planktonic and biofilm kill kinetics of ultrashort cationic lipopeptides previously demonstrated by our group to have a minimum biofilm eradication concentration (MBEC) in the microgram per mL (μg/mL) range against clinically relevant biofilm-forming micro-organisms. We compare the rate of kill for the most potent of these lipopeptides, dodecanoic (lauric) acid-conjugated C₁₂-Orn-Orn-Trp-Trp-NH₂ against the tetrapeptide amide H-Orn-Orn-Trp-Trp-NH₂ motif and the amphibian peptide Maximin-4 via a modification of the MBEC Assay™ for Physiology & Genetics (P&G). Improved antimicrobial activity is achieved upon N-terminal lipidation of the tetrapeptide amide. Increased antimicrobial potency was demonstrated against both planktonic and biofilm forms of Gram-positive micro-organisms. We hypothesize rapid kill to be achieved by targeting of microbial membranes. Complete kill against established 24-h Gram-positive biofilms occurred within 4 h of exposure to C₁₂-OOWW-NH₂ at MBEC values [methicillin-resistant Staphylococcus epidermidis (ATCC 35984): 15.63 μg/mL] close to the values for the planktonic minimum inhibitory concentration (MIC) [methicillin-resistant Staphylococcus epidermidis (ATCC 35984): 1.95 μg/mL]. Such rapid kill, especially against sessile biofilm forms, is indicative of a reduction in the likelihood of resistant strains developing with the potential for quicker resolution of pathogenic infection. Ultrashort antimicrobial lipopeptides have high potential as antimicrobial therapy.

A rapid PCR-based assay for identification of cryptic Myotis spp. (M. mystacinus, M. brandtii and M. alcathoe)

The development of a quick PCR-based method to distinguish European cryptic Myotis spp., Myotis mystacinus, Myotis brandtii and Myotis alcathoe is described. Primers were designed around species-specific single nucleotide polymorphisms (SNP's) in the ND1 mitochondrial gene, and a pair of control primers was designed in the 12S mitochondrial gene. A multiplex of seven primer combinations produces clear species-specific bands using gel electrophoresis. Robustness of the method was tested on 33 M. mystacinus, 16 M. brandtii and 15 M. alcathoe samples from across the European range of these species. The method worked well on faecal samples collected from maternity roosts of M. mystacinus. The test is intended to aid collection of data on these species through a rapid and easy identification method with the ability to use DNA obtained from a range of sources including faecal matter.

Identification of Pseudomonas aeruginosa by a duplex real-time polymerase chain reaction assay targeting the ecfX and the gyrB genes

Phenotypic identification of Gram-negative bacteria from respiratory specimens of patients with cystic fibrosis carries a high risk of misidentification. Molecular identification techniques that use single-gene targets are also susceptible to error, including cross-reaction issues with other Gram-negative organisms. In this study, we have designed a Pseudomonas aeruginosa duplex real-time polymerase chain reaction (PCR) (PAduplex) assay targeting the ecfX and the gyrB genes. The PAduplex was evaluated against a panel of 91 clinical and environmental isolates that were presumptively identified as P. aeruginosa. The results were compared with those obtained using a commercial biochemical identification kit and several other P. aeruginosa PCR assays. The results showed that the PAduplex assay is highly suitable for routine identification of P. aeruginosa isolates from clinical or environmental samples. The 2-target format provides simultaneous confirmation of P. aeruginosa identity where both the ecfX and gyrB PCR reactions are positive and may also reduce the potential for false negatives caused by sequence variation in primer or probe targets.