A critical appraisal of tools available for monitoring epigenetic changes in clinical samples from patients with myeloid malignancies

Research over the past decade has confirmed that epigenetic alterations act in concert with genetic lesions to deregulate gene expression in acute myeloid leukemia and myelodysplastic syndromes. In addition, we now have the capability to pharmaceutically target epigenetic modifications, and there is an urgent need forearly validation of the efficacy of the drugs. Also, an improved understanding of the functionality of epigenetic modifications may further pave the road towards an individualized therapy. Here, we provide the pros and cons of the currently most feasible methods used for characterizing the methylome in clinical samples, and give a brief introduction to novel approaches to sequencing that may revolutionize our abilities to characterize the genomes and epigenomes in acute myeloid leukemia and myelodysplastic syndrome patients.

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.

Development of a Broad-Specificity Monoclonal Antibody-Based Immunoaffinity Chromatography Cleanup for Organophosphorus Pesticide Determination in Environmental Samples

An immunoaffinity chromatographic (IAC) method for the selective extraction and concentration of 13 organophosphorus pesticides (OPs, including coumaphos, parathion, phoxim, quinalphos, dichlofenthion, triazophos, azinphos-ethyl, phosalone, isochlorthion, parathion-methyl, cyanophos, disulfoton, and phorate) prior to analysis by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was developed. The IAC column was prepared by covalently immobilizing a monoclonal antibody with broad specificity for OPs on CNBr-activated Sephrose 4B. The column capacity ranged from 884 to 2641 ng/mL of gel. The optimum elution solvent was 0.01 M phosphate-buffered saline containing 80% methanol. The breakthrough volume of the IAC column was found to be 400 mL. Recoveries of OPs from spiked environmental samples by IAC cleanup and HPLC-MS/MS analysis ranged from 60.2 to 107.1%, with a relative standard deviation below 11.1%. The limit of quantitation for 13 OPs ranged from 0.01 to 0.13 ng/mL (ng/g). The application of IAC cleanup coupled to HPLC-MS/MS in real environmental samples demonstrated the potential of this method for the determination of OP residues in environmental samples at trace levels.

Mass spectrometric analysis of muscle samples to detect potential antibiotic growth promoter misuse in broiler chickens

Mass spectrometric methods were developed and validated for the analysis in chicken muscle of a range of antibiotic growth promoters: spiramycin, tylosin, virginiamycin and bacitracin, and separately for two marker metabolites of carbadox (quinoxaline-2-carboxylic acid and 1,4-bisdesoxycarbadox), and a marker metabolite of olaquindox (3-methyl-quinoxaline-2-carboxylic acid). The use of these compounds as antibiotic growth promoters has been banned by the European Commission. This study aimed to develop methods to detect their residues in muscle samples as a means of checking for the use of these drugs during the rearing of broiler chickens. When fed growth-promoting doses for 6 days, spiramycin (31.4 mu g kg(-1)), tylosin (1.0 mu g kg(-1)), QCA (6.5 mu g kg(-1)), DCBX (71.2 mu g kg(-1)) and MQCA (0.2 mu g kg(-1)) could be detected in the muscle 0 days after the withdrawal of fortified feed. Only spiramycin could consistently be detected beyond a withdrawal period of 1 day. All analytes showed stability commercial cooking process, therefore raw or cooked muscle could be used for monitoring purposes.

The prediction of intake potential and organic matter digestibility of grass silages by near infrared spectroscopy analysis of undried samples

A study was undertaken to examine a range of sample preparation and near infrared reflectance spectroscopy (NIPS) methodologies, using undried samples, for predicting organic matter digestibility (OMD g kg(-1)) and ad libitum intake (g kg(-1) W-0.75) of grass silages. A total of eight sample preparation/NIRS scanning methods were examined involving three extents of silage comminution, two liquid extracts and scanning via either external probe (1100-2200 nm) or internal cell (1100-2500 nm). The spectral data (log 1/R) for each of the eight methods were examined by three regression techniques each with a range of data transformations. The 136 silages used in the study were obtained from farms across Northern Ireland, over a two year period, and had in vivo OMD (sheep) and ad libitum intake (cattle) determined under uniform conditions. In the comparisons of the eight sample preparation/scanning methods, and the differing mathematical treatments of the spectral data, the sample population was divided into calibration (n = 91) and validation (n = 45) sets. The standard error of performance (SEP) on the validation set was used in comparisons of prediction accuracy. Across all 8 sample preparation/scanning methods, the modified partial least squares (MPLS) technique, generally minimized SEP's for both OMD and intake. The accuracy of prediction also increased with degree of comminution of the forage and with scanning by internal cell rather than external probe. The system providing the lowest SEP used the MPLS regression technique on spectra from the finely milled material scanned through the internal cell. This resulted in SEP and R-2 (variance accounted for in validation set) values of 24 (g/kg OM) and 0.88 (OMD) and 5.37 (g/kg W-0.75) and 0.77 (intake) respectively. These data indicate that with appropriate techniques NIRS scanning of undried samples of grass silage can produce predictions of intake and digestibility with accuracies similar to those achieved previously using NIRS with dried samples. (C) 1998 Elsevier Science B.V.

Molecularly imprinted polymers for the extraction of imiquimod from biological samples using a template analogue strategy

Molecularly Imprinted Polymers (MIPs) against imiquimod, a highly potent immune response modifier used in the treatment of skin cancer, were synthesised using a template analogue strategy and were compared with imprints of the drug itself. An investigation of the complexation between the functional monomer and the template analogue revealed an association constant of 1,376 ± 122 M-1, significantly higher than previously reported values for similar systems. The binding characteristics of the synthesised imprinted polymers were evaluated and extremely strong binding for imiquimod was observed while imprinting factors as high as 17 were calculated. When applied as sorbents in solid-phase extraction of imiquimod from aqueous, urine and blood serum samples, clean extracts and recoveries up to 95% were achieved, and it is concluded that while imiquimod imprints exhibited higher capacity for the drug, template analogue imprints are more selective. The results obtained suggest potential applications of imiquimod imprints as sorbents in rapid extraction and monitoring of undesirable systemic release of the drug.

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.