Increased quantitative proteome coverage with (13)C/(12)C-based, acid-cleavable isotope-coded affinity tag reagent and modified data acquisition scheme

Quantitative protein profiling using the isotope-coded affinity tag (ICAT) method and tandem mass spectrometry (MS) enables the pair-wise comparison of protein expression levels in biological samples. A new version of the ICAT reagent with an acid-cleavable bond, which allows removal of the biotin moiety prior to MS and which utilizes (13)C substitution for (12)C in the heavy-ICAT reagent rather than (2)H (for (1)H) as in the original reagent, was investigated. We developed and validated an MS data acquisition strategy using this new reagent that results in an increased number of protein identifications per experiment, without losing the accuracy of protein quantification. This was achieved by following a single survey (precursor) ion scan and serial collision induced dissociations (CIDs) of four different precursor ions observed in the prior survey scan. This strategy is common to many high-performance liquid chromatography-electrospray ionization (HPLC-ESI)-MS shotgun proteomic strategies, but heretofore not to ICAT experiments. This advance is possible because the new ICAT reagent uses (13)C as the "heavy" element rather than (2)H, thus, eliminating the slight delay in retention time of ICAT-labeled "light" peptides on a C18-based HPLC separation that occurs with (2)H and (1)H. Analyses using this new scheme of an ICAT-labeled trypsin-digested six protein mixture as well as a tryptic digest of a total yeast lysate, indicated that about two times more proteins were identified in a single analysis, and that there was no loss in accuracy of quantification.     

Evaluation of a Real-time PCR-based assay using the lightcycler system for detection of Toxoplasma gondii bradyzoite genes in blood specimens from patients with toxoplasmic retinochoroiditis

PCR based methods have advantages over traditional methods for the diagnosis of toxoplasmosis, especially when serology fails and clinical symptoms are not evident. However, current PCR-based assays are often labour-intensive and not readily quantifiable and have the potential for contamination due to a requirement for postamplification sample handling. Real-time PCR can address these limitations. We have developed and evaluated a highly sensitive Real-time PCR (Light-cycler, LC-PCR) to detect and quantify Toxoplasma gondii B1 and bradyzoite specific genes (SAG-4, MAG-1) in serum and peripheral blood mononuclear cells (PBMC) specimens, from five immunocompetent subjects with clinically suspected toxoplasmic retinochoroiditis (TRC) or without a suspected T. gondii infection. A standard curve for quantitation of parasitic load was generated using SYBR Green I fluorescent detection. The results were compared with those obtained with a nested PCR (n-PCR). In TRC patients, both PCR methods confirmed ophtalmoscopy and fluorangiographic findings. Among the TRC patients, the use of LC-PCR was more sensitive than n-PCR for detection and quantification of either B1 gene (P<0.001) or SAG-4/MAG-1 gene (P<0.05). LC-PCR has been shown particularly useful to accurately determine the parasite DNA load in follow-up specimens in whom the performance of either B1 or SAG-4 and MAG-1 in detecting T. gondii loads, varied with respect to specific antitoxoplasmic treatment.     

Effects of pH amendment on metal working fluid wastewater biological treatment using a defined bacterial consortium

The aim of this study was to determine whether pH amendment of a highly alkaline metal working fluid (MWF) wastewater would improve biological treatment in a bioreactor system following introduction of a bacterial inoculum (comprised of the following strains: Agrobacterium radiobacter, Comamonas testosteroni, Methylobacterium mesophilicum, Microbacterium esteraromaticum, and Microbacterium saperdae). The pH values tested were 6, 7, 8, and 9. Three replicate batch mode bioreactors inoculated with the bacterial inoculum (plus an abiotic control bioreactor) were operated for each of the four pH conditions. After 14 days, the final mean chemical oxygen demand (COD) reduction at pH 9 was 50 +/- 1.4%; at pH 8, 58 +/- 1.4%; pH 7, 65 +/- 1.0%; and pH 6, 75 +/- 2.7% of the initial COD (approximately 10,000 mg L(-1)), respectively. Interestingly, within 5 days, the pH in all inoculated bioreactors progressed toward pH 8. However, all abiotic control bioreactors remained at the pH at which they were amended. The fate of the inoculum, determined by denaturing gradient gel electrophoresis (DGGE) and by cluster analysis of the resulting DGGE profiles, revealed that the inocula survived throughout operation of all pH-amended bioreactors. Length-heterogeneity polymerase chain reaction (PCR) was used to track the population dynamics of individual strains. After 7 days of operation, M. esteraromaticum was the most abundant population in all bioreactors, regardless of pH. From our findings, it appears necessary to adjust the MWF wastewater from pH 9 to between 6 and 7, to achieve optimal biological treatment rates.     

Identification and quantitation of species in complex DNA mixtures by real-time polymerase chain reaction

Six TaqMan real-time polymerase chain reaction (PCR) systems using minor groove binding (MGB) probes have been developed for the detection quantitation of bovine, porcine, lamb, chicken, turkey, and ostrich DNA in complex samples. Species-specific amplification was achieved by combining only two fluorogenic probes and 10 oligonucleotide primers targeting mitochondrial sequences, decreasing the cost of the assay significantly. The limits of detection ranged from 0.03 to 0.80 pg of template DNA. Analysis of experimental mixtures containing two to four different species showed the suitability of the assay for detection of more than 1% of pork, chicken, or turkey and of more than 5% of cattle or lamb. The quantitation accuracy in samples containing 10-100% of beef or pork DNA was close to 90%. The system is complemented with one additional TaqMan MGB detector based on consensus sequence segments of the nuclear 18S ribosomal RNA gene. A method to evaluate the presence of unknown eukaryotic DNA in a mixture, where data derived from the species-specific detection are compared with the experimental values obtained from the general 18S detector, is presented. This method allows the validation of the quantitative measurements, providing an internal control of the total content of PCR-amplifiable DNA in the sample. The system was tested on DNA mixtures containing different shares of up to four different species and on DNA extracted from processed commercial food samples.     

Yeast population dynamics of industrial fuel-ethanol fermentation process assessed by PCR-fingerprinting

Yeast population used in industrial production of fuel-ethanol may vary according to the plant process condition and to the environmental stresses imposed to yeast cells. Therefore, yeast strains isolated from a particular industrial process may be adapted to such conditions and should be used as starter strain instead of less adapted commercial strains. This work reports the use of PCR-fingerprinting method based on microsatellite primer (GTG)₅ to characterize the yeast population dynamics along the fermentation period in six distilleries. The results show that indigenous fermenting strains present in the crude substrate can be more adapted to the industrial process than commercial strains. We also identified new strains that dominate the yeast population and were more present either in molasses or sugar cane fermenting distilleries. Those strains were proposed to be used as starters in those industrial processes. This is the first report on the use of molecular markers to discriminate Saccharomyces cerevisiae strains from fuel-ethanol producing process.     

Study of Tissue-Specific CpG Methylation of DNA in Extended Genomic Loci

Modern approaches for studies on genome functioning include investigation of its epigenetic regulation. Methylation of cytosines in CpG dinucleotides is an inherited epigenetic modification that is responsible for both functional activity of certain genomic loci and total chromosomal stability. This review describes the main approaches for studies on DNA methylation. Under consideration are site-specific approaches based on bisulfite sequencing and methyl-sensitive PCR, whole-genome approaches aimed at searching for new methylation hot spots, and also mapping of unmethylated CpG sites in extended genomic loci.     

Fasciola hepatica: humoral and cytokine responses to a member of the saposin-like protein family following delivery as a DNA vaccine in mice

The humoral and cellular responses to DNA vaccination of BALB/c mice with a novel antigen from the Fasciola hepatica saposin-like protein family (FhSAP-2) have been investigated. Two constructs were produced containing the FhSAP-2 DNA sequence, one intended for extracellular secretion of FhSAP-2 protein, and one expressing FhSAP-2 in the cytoplasm of a transfected cell. The constructs were tested in HEK 293T cells, with the secretory construct producing less detectable FhSAP-2 relative to cytoplasmic construct when observed by fluorescence. The size of expressed protein was confirmed by Western blot of cell lysate, but FhSAP-2 was undetectable in cell supernatants. Both, secretory and cytoplasmic constructs as well as FhSAP-2 recombinant protein were tested in mice. The antibody response elicited in mice vaccinated with the rFhSAP-2 induced high levels of IgG(1), IgG(2), and IgE as well as high levels of IL-10 and IFNgamma indicating a mixed Th1/Th2 response. Vaccination of mice intramuscularly with the cytoplasmic FhSAP-2 construct resulted in a dominant IgG(2a) isotype antibody as well as a dominant IFNgamma cytokine, with significant IgE, IgG(1), and IL-10 responses also present, suggesting a mixed Th1/Th2 profile. Isotype and cytokine profiles elicited by the FhSAP-2 secretory construct were similar to those obtained with the cytoplasmic construct but at levels that were significantly lower. The results demonstrate that FhSAP-2 can be delivered as a DNA vaccine construct and induces a stronger Th1 response than the recombinant protein alone. This could result in an improvement in the immunoprophylactic potential of this candidate vaccine against F. hepatica.     

Entamoeba histolytica: the serine-rich gene polymorphism-based genetic variability of clinical isolates from Georgia

It is generally accepted that a majority of individuals infected by Entamoeba histolytica do not develop symptomatic disease. However, the parasite and the host factors contributing to the development of the disease, remain undetermined. It is also unclear why certain individuals develop extra-intestinal amebiasis without exhibiting apparent intestinal symptoms. An outbreak of amebic liver abscess in Tbilisi, Georgia in 1998-1999 suggested that the causative E. histolytica strain had an unusual propensity for extra-intestinal spread. To correlate the genetic differences with pathogenic potential of the parasite, we have examined the SREHP gene polymorphisms among Georgian E. histolytica isolates. Comparison of polymorphic patterns revealed the presence of several different genotypes of E. histolytica, thus preventing an association of a single genotype with hepatic disease, but supporting the previous finding of extensive genetic diversity among E. histolytica isolates from the same geographic origin.