An enantio-selective chromatographic stationary phase for S-ibuprofen prepared by stoichiometric molecular imprinting

Molecularly Imprinted Polymers (MIPs) against S-ibuprofen were synthesised using a tailor made functional monomer, 2-acrylamido-4-methylpyridine, following extensive pre-polymerisation studies of template-monomer complexation. An apparent association constant of 340 +/- 22 M-1 was calculated that was subsequently corrected to account for dimerisation of ibuprofen (K-dim = 320 +/- 95 M-1) resulting in an intrinsic association constant of 715 +/- 16 M-1, consistent with previously reported values. Using the synthesised imprinted polymer as a stationary phase, complete resolution of a racemic mixture of ibuprofen was achieved in predominantly aqueous mobile phases. An imprinting factor of 10 was observed, and was found to be in agreement with the difference in the average number of binding sites between MIP and blank polymers, calculated by staircase frontal chromatography. The imprinted polymers exhibited enhanced selectivity for the templated drug over structurally related NSAIDs. When applied as sorbents in solid-phase extraction of ibuprofen from commercial tablets, urine and blood serum samples, recoveries up to 92.2% were achieved. © The Royal Society of Chemistry 2012

Preparation of a molecularly imprinted polymer for the solid-phase extraction of scopolamine with hyoscyamine as a dummy template molecule

Molecularly imprinted polymers (MIPs) selective for scopolamine were produced using hyoscyamine (a close structural analogue) as template molecule. The produced polymers were used as media for solid-phase extraction, exhibiting selective binding properties for the analyte from biological samples. Human and calf urine and serum were processed on the MIP under various extraction protocols. The best performance was observed after loading the analyte in aqueous environment facilitating retention on the MIP by non-selective hydrophobic interactions. The MIPs were subsequently washed using an optimised solvent system to enable selective desorption of the analyte. Other related and non-related compounds were accessed to evaluate molecular recognition properties. Recoveries of up to 79% were achieved for the analyte of interest from biological samples.

A pilot survey for Fusarium mycotoxin biomarkers in women from Golestan, northern Iran

Fusarium mycotoxins are frequent contaminants of cereals in many world regions, and are suggested risk factors for various acute and chronic human diseases. To date a lack of exposure tools has restricted epidemiological studies of the potential health effects. Recently established exposure biomarkers for deoxynivalenol (DON) and fumonisins are now available and here a pilot biomarker survey of 110 women (aged 39 to 72 years) from Golestan, northern Iran was conducted on samples collected at one time point during August-September 2007. Urinary DON and DON-glucuronide combined were detected frequently (79/110, 72%), mean 1.3 ng DON/ml urine, range not detected (nd)-6.5 ng/ml; mean creatinine adjusted levels were 1.5 ng DON/mg creatinine, range nd-7.1 ng/mg). Neither urinary de-epoxy DON (DOM-1) and DOM-1 glucuronide combined, nor urinary fumonisin B-1 were detected. This study is the first reported biomarker based exposure assessment of DON and fumonisins in this region. Overall DON exposure at this time point appears modest compared to other world regions where data are available.

Study of aflatoxin exposure in a Brazilian population using an aflatoxin - albumin biomarker

The evaluation of exposure to aflatoxins (AF) by measurement of the level of contamination in food is hampered due to the heterogeneous distribution of AF in food. Therefore, an alternative is to estimate the exposure using specific biological markers (biomarkers) based on an understanding of the metabolism of the compound. For AF, these include aflatoxin-N-7-guanine in the urine, or AFB(1)-albumin (AF-alb) in the blood. This study assessed the level of exposure to AF in Brazilian individuals using a biomarker approach, i.e. the AF-alb adducts. Blood samples were collected from urban residents (n=50; aged 18-52) in June 1999, at the Blood Center of Antonio Carlos de Camargo Hospital, Sao Paulo, Brazil. AF-alb adduct levels were determined, by ELISA following serum albumin extraction and digestion. AF-alb adducts were detected in 31/50 (62%) samples [range 0-57.3 pg AFB(1)-lys adducts/mg of blood albumin (pg/mg)]. The mean level of positives was 14.9 pg/mg and males had the two highest levels measured (57.1 and 57.3 pg/mg). There was no correlation with age or profession. This is the first study of Brazilian, or indeed South American, individuals that has determined exposure to AF at the individual level using a biomarker approach. These levels are similar to those observed in the Philippines. These data warrant further investigation of both the sources and consequences of exposure to this potent toxin in Brazil.

Determining mycotoxins and mycotoxigenic fungi in food and feed:Developing biomarkers of human exposure to mycotoxins

Exposure assessment is a critical part of epidemiological studies into the effect of mycotoxins on human health. Whilst exposure assessment can be made by estimating the quantity of ingested toxins from food analysis and questionnaire data, the use of biological markers (biomarkers) of exposure can provide a more accurate measure of individual level of exposure in reflecting the internal dose. Biomarkers of exposure can include the excreted toxin or its metabolites, as well as the products of interaction between the toxin and macromolecules such as protein and DNA. Samples in which biomarkers may be analysed include urine, blood, other body fluids and tissues, with urine and blood being the most accessible for human studies. Here we describe the development of biomarkers of exposure for the assessment of three important mycotoxins; aflatoxin, fumonisin and deoxynivalenol. A number of different biomarkers and methods have been developed that can be applied to human population studies, and these approaches are reviewed in the context of their application to molecular epidemiology research.

Isolation and detection of Campylobacter jejuni from chicken fecal samples by immunomagnetic separation–PCR

Campylobacter jejuni (C. jejuni) is one of the leading causes of bacterial food-borne disease worldwide. The presence of Campylobacter in chicken feces poses a high risk for contamination of chicken meat and for Campylobacter infections in human. Detection of this bacterium in chicken fecal specimens before slaughter is therefore vital to prevent disease transmission. By combining two techniques – immunomagnetic separation (IMS) and polymerase chain reaction (PCR), this study developed a reliable and specific method for rapid detection of C. jejuni in chicken fecal samples. The specificity of the assay was assured by two selection steps: 1) Dynabeads®M-270 Amine microbeads (2.8 µm in diameter) coated with C. jejuni monoclonal antibodies were used as the primary selection to isolate bacteria from fecal samples. 2) A PCR assay amplifying the Hippuricase gene was performed as the specific selection to accurately confirm the presence of C. jejuni. Without pre-enrichment, this method was able to detect approximately 10 CFU of C. jejuni in 1 µl of spiked feces within 3 h.

Dual-colour HER2/chromosome 17 chromogenic in situ hybridisation assay enables accurate assessment of HER2 genomic status in gastric cancer and has potential utility in HER2 testing of biopsy samples

Determination of HER2 protein expression by immunohistochemistry (IHC) and genomic status by fluorescent in situ hybridisation (FISH) are important in identifying a subset of high HER2-expressing gastric cancers that might respond to trastuzumab. Although FISH is considered the standard for determination of HER2 genomic status, brightfield ISH is being increasingly recognised as a viable alternative. Also, the impact of HER2 protein expression/genomic heterogeneity on the accuracy of HER2 testing has not been well studied in the context of gastric biopsy samples.

3-Deoxyfructose concentrations are increased in human plasma and urine in diabetes

3-Deoxyglucosone (3-DG) is a reactive dicarbonyl sugar thought to be a key intermediate in the nonenzymatic polymerization and browning of proteins by glucose. 3-DG may be formed in vivo from fructose, fructose 3-phosphate, or Amadori adducts to protein, such as N epsilon-fructoselysine (FL), all of which are known to be elevated in body fluids or tissues in diabetes. Modification of proteins by 3-DG formed in vivo is thought to be limited by enzymatic reduction of 3-DG to less reactive species, such as 3-deoxyfructose (3-DF). In this study, we have measured 3-DF, as a metabolic fingerprint of 3-DG, in plasma and urine from a group of diabetic patients and control subjects. Plasma and urinary 3-DF concentrations were significantly increased in the diabetic compared with the control population (0.853 +/- 0.189 vs. 0.494 +/- 0.072 microM, P <0.001, and 69.9 +/- 44.2 vs. 38.7 +/- 16.1 nmol/mg creatinine, P <0.001, respectively). Plasma and urinary 3-DF concentrations correlated strongly with one another, with HbA1c (P <0.005 in all cases), and with urinary FL (P <0.02 and P = 0.005, respectively). The overall increase in 3-DF concentrations in plasma and urine in diabetes and their correlation with other indexes of glycemic control suggest that increased amounts of 3-DG are formed in the body during hyperglycemia in diabetes and then metabolized to 3-DF. These observations are consistent with a role for increased formation of the dicarbonyl sugar 3-DG in the accelerated browning of tissue proteins in diabetes.

Inflammatory and immunological biomarkers are not related to survival in adults with Cystic Fibrosis

Background
Chronic Pseudomonas aeruginosa pulmonary infection is associated with a decline in lung function and reduced survival in people with Cystic Fibrosis (CF). Damaging inflammatory and immunological mediators released in the lungs can be used as markers of chronic infection, inflammation and lung tissue damage.

Methods
Clinical samples were collected from CF patients and healthy controls. Serum IgG and IgA anti-Pseudomonas antibodies, sputum IL-8 and TNFα, plasma IL-6 and urine TNFr1 were measured by ELISA. Sputum neutrophil elastase (NE), cathepsin S and cathepsin B were measured by spectrophotometric and fluorogenic assays. The relationship between IgG and IgA, inflammatory mediators and long-term survival was determined.

Results
IgG and IL-6 positively correlated with mortality. However, multivariate analysis demonstrated that after adjusting for FEV1, IgG was not independently related to mortality. A relationship was observed between IgG and IL-6, TNFα, TNFr1 and between IgA and IL8, cathepsin S and cathepsin B.

Conclusions
These data indicate that biomarkers of inflammation are not independent predictors of survival in people with CF.

Empirical assessment of non-invasive population genetics in bats:Comparison of DNA quality from faecal and tissue samples

Non-invasive population genetics has become a valuable tool in ecology and conservation biology, allowing genetic studies of wild populations without the need to catch, handle or even observe the study subjects directly. We address some of the concerns regarding the limitations of using non-invasive samples by comparing the quality of population genetic information gained through DNA extracted from faecal samples and biopsy samples of two elusive bat species, Myotis mystacinus and Myotis nattereri. We demonstrate that DNA extracted from faeces and tissue samples gives comparable results for frequency based population genetic analyses, despite the occurrence of genotyping errors when using faecal DNA. We conclude that non-invasive genetic sampling for population genetic analysis in bats is viable, and although more labour-intensive and expensive, it is an alternative to tissue sampling, which is particularly pertinent when specimens are rare, endangered or difficult to capture. © 2012 Museum and Institute of Zoology PAS.

Multiplex biotoxin surface plasmon resonance method for marine biotoxins in algal and seawater samples

A multiplex surface plasmon resonance (SPR) biosensor method for the detection of paralytic shellfish poisoning (PSP) toxins, okadaic acid (and analogues) and domoic acid was developed. This method was compared to enzyme-linked immunosorbent assay (ELISA) methods. Seawater samples (n?=?256) from around Europe were collected by the consortia of an EU project MIcroarrays for the Detection of Toxic Algae (MIDTAL) and evaluated using each method. A simple sample preparation procedure was developed which involved lysing and releasing the toxins from the algal cells with glass beads followed by centrifugation and filtering the extract before testing for marine biotoxins by both multi-SPR and ELISA. Method detection limits based on IC20 values for PSP, okadaic acid and domoic acid toxins were 0.82, 0.36 and 1.66 ng/ml, respectively, for the prototype multiplex SPR biosensor. Evaluation by SPR for seawater samples has shown that 47, 59 and 61 % of total seawater samples tested positive (result greater than the IC20) for PSP, okadaic acid (and analogues) and domoic acid toxins, respectively. Toxic samples were received mainly from Spain and Ireland. This work has demonstrated the potential of multiplex analysis for marine biotoxins in algal and seawater samples with results available for 24 samples within a 7 h period for three groups of key marine biotoxins. Multiplex immunological methods could therefore be used as early warning monitoring tools for a variety of marine biotoxins in seawater samples.

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.