Immunological detection of Bacteroides fragilis in clinical-samples

A monospecific polyclonal antiserum, prepared against Bacteroides fragilis common polysaccharide antigen purified by polyacrylamide gel immunoblot detected B. fragilis, B. thetaiotaomicron, B. ovatus and Prevotella melaninogenica in pus samples from various anatomical sites by immunofluorescence microscopy of the pus. With standard clinical laboratory culture methods, 36% of 147 samples were positive for one or more of the above bacteria. Of these, B. fragilis accounted for 33%. By immunofluorescent labelling of pus with the common antigen antiserum the detection of these bacteria in the samples increased to 50%. All nine of the blood cultures in which B. fragilis was detected by culture contained bacteria positive for the common antigen. Immunofluorescent labelling of pus samples with a selection of monoclonal antibodies specific for surface polysaccharides which are known to be antigenically variable in culture in vitro and in an animal model of infection showed that these polysaccharides are also variable in natural infection. The results indicate that the common polysaccharide antigen, in contrast to the variable surface polysaccharides, is a suitable target for the immunodetection of B, fragilis in clinical samples from a range of anatomical sites.

Parallel Peptidome and Transcriptome Analyses of Amphibian Skin Secretions Using Archived Frozen Acid-Solvated Samples

Amphibian skin secretions are unique sources of bioactive peptides and their donor species are currently rapidly disappearing from the biosphere. Here, we report that both peptides and polyadenylated mRNAs from skin granular glands remain amenable to study in samples of stimulated skin secretions following their storage in 0.1 % aqueous trifluoroacetic acid at -20 °C for many years. Frozen acidified solutions of toad (Bombina variegata) skin secretions, stored for 12 years, were thawed and samples removed for direct reverse phase HPLC fractionation. Additional samples were removed, snap frozen and lyophilised for construction of cDNA libraries following polyadenylated mRNA capture using magnetic oligo-dT beads and reverse transcription. Using the bombesin and bradykinin peptides found in bombinid toad skin as models, individual variant peptides of each type were located in reverse phase HPLC fractions and their corresponding biosynthetic precursor-encoding mRNA transcripts were cloned from the cDNA library using a RACE PCR strategy. This study illustrates unequivocally that both amphibian skin secretion peptides and their biosynthetic precursor-encoding polyadenylated mRNAs are stable in frozen acid-solvated skin secretion samples for considerable periods of time-a finding that may have fundamental implications in the study of archived materials but also in the wider field of molecular biology.

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.

Quantifying contributions of domestic wastewater treatment systems to nutrient fluxes in stream headwaters

Effective implementation of the Water Framework Directive requires a reappraisal of conventional approaches to water quality monitoring. Quantifying the impact of domestic wastewater treatment systems (DWWTS) in Irish catchments is further complicated by high levels of natural heterogeneity. This paper presents a numerical model that couples attenuation to flow along different hydrological pathways contributing to river discharge; this permits estimation of the impact of DWWTS to overall nutrient fluxes under a range of geological conditions. Preliminary results suggest high levels of attenuation experienced
before DWWTS effluent reaches bedrock play a significant role in reducing its ecological impact on aquatic receptors. Conversely, low levels of attenuation in systems discharging directly to surface water may affect water quality more significantly, particularly during prolonged dry periods in areas underlain by low productivity aquifers (>60% of Ireland), where dilution capacity is limited.

A field study assessing the impact of on-site wastewater treatment systems on surface water quality in a Co. Monaghan catchment

Poorly functioning on-site wastewater treatment systems (OSWTS) can be among the many sources of pollution to groundwater and surface water, which are of critical concern owing to potential human and ecological health risks. An investigation into the effects of on-site wastewater treatment systems (OSWTS) on surface water quality has been undertaken at several sites within a catchment in Co. Monaghan. The study sites were located in areas of 'low’ permeability, suggesting that run-off usually dominates over infiltration. Poor treatment performance of OSWTS within the catchment were found to be the result of several factors, including location in areas with unsuitable soil and site characteristics, incorrect installation, poor maintenance and inappropriate operation by the home owner.

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.