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Dilution Series (dilution + series)
Selected AbstractsHepatitis B virus markers in anti-HBc only positive individuals,JOURNAL OF MEDICAL VIROLOGY, Issue 3 2001Bernard Weber Abstract Isolated reactivity to hepatitis B virus (HBV) core antigen (anti-HBc) is observed relatively frequently in immunocompromised individuals, intravenous drug abusers (IVDA), and in the presence of HCV infection. The reason for the lack of HBsAg is not clear. The aim of the present study was to investigate which factors (genetic variability of S gene, low-level HBsAg, and immune complexes may be responsible for the failure of HBsAg detection with commercial HBsAg screening assays. Dilution series of two recombinant HBsAg escape mutants and dilutions of serum samples from chronic HBV carriers with multiple insertions in the a determinant and different HBsAg subtypes were tested with a highly sensitive assay that detects wild-type HBsAg (Elecsys HBsAg, Roche Diagnostics, Penzberg, Germany) and two assays that detect HBV wild-type and escape mutants (Murex HBsAg Version 3, Murex and Enzygnost HBsAg 5.0, Dade Behring, Marburg, Germany). Elecsys HBsAg showed in comparison to Murex HBsAg Version 3 and Enzygnost HBsAg 5.0 a reduced sensitivity for escape mutant detection. On the other hand, the best performance for HBsAg subtype detection was obtained with Elecsys HBsAg. In the second part of the study, a selected panel of isolated anti-HBc reactive (n,=,104) serum samples (AxSYM Core) was submitted to testing by Elecsys HBsAg, Murex HBsAg Version 3, Enzygnost HBsAg 5.0, and HBsAg detection after immune complex dissociation (ICD) and anti-HBs determination with two different assays (AxSYM Ausab and Elecsys Anti-HBs). To assess the specificity of anti-HBc test results, all the samples were tested by a second anti-HBc assay (Elecsys Anti-HBc). Quantitative HBV DNA detection was undertaken with a commercially available HBV PCR assay (Amplicor HBV Monitor). HCV infection was present in 65.4% of anti-HBc only reactive individuals. Five AxSYM Core positive samples were negative by Elecsys Anti-HBc. Overall, 15 (14.4%) AxSYM Ausab negative samples gave positive results with Elecsys Anti-HBs (median value: 21 IU/ml). No low-level HBsAg carrier was detected among the isolated anti-HBc reactive individuals with Elecsys HBsAg. There was no evidence for the presence of immune complexes. Only one sample was repeatedly reactive by the Murex HBsAg, suggesting that the a mutant form of HBsAg was responsible for the isolated anti-HBc reactivity, however neutralisation assay was not interpretable and HBV DNA PCR was negative. Fifteen (14.4%) anti-HBc only positive individuals were HBV DNA carriers with concentrations ranging from 800 to more than >4,000,000 copies of viral DNA/ml. In conclusion, the most probable explanations for isolated anti-HBc reactivity in our study group are a possible interference of HBsAg synthesis by HCV infection (65.4%) and divergence of results of anti-HBs assays (14.4%). There is no evidence for the presence of low-level HBsAg carriers and immune complexes. HBsAg mutants cannot be excluded definitively by the test strategy used in the present evaluation. J. Med. Virol. 64:312,319, 2001. © 2001 Wiley-Liss, Inc. [source] DNA sequence analysis of interlocus recombination between the human T-cell receptor gamma variable (GV) and beta diversity-joining (BD/BJ) sequences on chromosome 7 (inversion 7)ENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 2 2002Scott W. Ballinger Abstract V(D)J recombinase-mediated recombination between the T-cell receptor (TCR) gamma variable (GV) genes at chromosome 7p15 and the TCR beta joining (BJ) genes at 7q35 leads to the formation of a hybrid TCR gene. These TCR gamma/beta interlocus rearrangements occur at classic V(D)J recombination signal sequences (RSS) and, because the loci are in an inverted orientation, result in inversion events that are detectable in the chromosome structure as inv(7)(p15;q35). Similar rearrangements involving oncogenes and either TCR or immunoglobulin genes mediated by the V(D)J recombinase are found in lymphoid malignancies. Oligonucleotide primers that allow polymerase chain reaction (PCR) amplification across the inv(7) genomic recombination junction sequence have been described. Southern blot analysis has been primarily used to confirm the GV/BJ hybrid nature of the product, with limited information on the DNA sequence of these recombinations. We have modified this PCR method using total genomic DNA from the mononuclear cells in peripheral blood samples to increase specificity and to allow direct sequencing of the translocation junction that results from the recombination between the GV1 and BJ1 families of TCR genes in 25 examples from 11 individuals (three adults, one child, six newborns, and one ataxia telangiectasia (AT) patient). We focused on samples from newborns based on previous studies indicating that the predominant hypoxanthine-guanine phosphoribosyl transferase (HPRT) mutations in newborns are V(D)J recombinase-mediated deletion events and that the frequency of these mutations decreases with increasing age. Although the dilution series-based PCR assay utilized does not yield sharply defined quantitative endpoints, results of this study strongly suggest that inv(7) recombinations in newborns occur at equal or lower frequencies than those seen in adults. Consistent with the PCR primer pairs, all sequenced products contain a GV1 and a BJ1 segment and most also contain a BD1 segment. GV1s2 and 1s4 were the most frequently found GV1 genes (8 and 9 examples, respectively) and BJ1s5 and 1s6 were the most frequently found BJ1 genes (9 and 10 examples, respectively). These results demonstrate the effectiveness of this methodology for assessing GV/BJ interlocus rearrangements mediated by V(D)J recombinase. Environ. Mol. Mutagen. 40:85,92, 2002. © 2002 Wiley-Liss, Inc. [source] Dominant sugar utilizers in sediment of Lake Constance depend on syntrophic cooperation with methanogenic partner organismsENVIRONMENTAL MICROBIOLOGY, Issue 6 2008Nicolai Müller Summary Six strains of novel bacteria were isolated from profundal sediment of Lake Constance, a deep freshwater lake in Germany, by direct dilution of the sediment in mineral agar medium containing a background lawn of the hydrogen-scavenging Methanospirillum hungatei as a syntrophic partner. The numbers of colony-forming units obtained after incubation for more than 2 months were in the same range as those of total bacterial counts determined by DAPI staining (up to 108 cells per millilitre) suggesting that these organisms were dominant members of the community. Identical dilution series in the absence of methanogenic partners yielded numbers that were lower by two to three orders of magnitude. The dominant bacteria were isolated in defined co-culture with M. hungatei, and were further characterized. Growth was slow, with doubling times of 22,28 h at 28°C. Cells were small, 0.5 × 5 ,m in size, Gram-positive, and formed terminal oval spores. At 20°C, glucose was fermented by the co-culture strain BoGlc83 nearly stoichiometrically to 2 mol of acetate and 1 mol of methane plus CO2. At higher temperatures, also lactate and traces of succinate were formed. Anaerobic growth depended strictly on the presence of a hydrogen-scavenging partner organism and was inhibited by bromoethane sulfonate, which together indicate the need for a syntrophic partnership for this process. Strain BoGlc83 grew also aerobically in the absence of a partner organism. All enzymes involved in ATP formation via glycolysis and acetyl CoA were found, most of them at activities equivalent to the physiological substrate turnover rate. This new type of sugar-fermenting bacterium appears be the predominant sugar utilizer in this environment. The results show that syntrophic relationships can play an important role also for the utilization of substrates which otherwise can be degraded in pure culture. [source] Three lines of evidence in a sediment toxicity evaluation for hexachlorobutadieneENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 9 2000Phyllis C. Fuchsman Abstract Three approaches were used in a site-specific sediment toxicity evaluation for hexachlorobutadiene (HCBD), a chemical not previously tested for toxicity in sediment. The results of a sediment dilution study, spiked sediment toxicity tests, and a probabilistic model based on equilibrium partitioning theory were used to estimate ecological effects thresholds for HCBD in sediments of a Gulf Coast estuary. Twenty-nine sediment samples, including 11 undiluted samples and six dilution series, were tested for toxicity under estuarine conditions (10%0 salinity) using Hyalella azteca and Leptocheirus plumulosus. Site sediment was used as diluent, and all samples were assayed for a range of organic and inorganic chemicals. A logistic relationship was observed between HCBD concentrations and organism response, and nonlinear regression explained approximately 90% of the observed variation in amphipod survival as a function of HCBD. Spiked sediment toxicity test results generally agreed with the results of the dilution study, demonstrating the causality of the observed concentration,response relationship. Effects thresholds were estimated as HCBD concentrations corresponding to 80% amphipod survival. The most conservative effects thresholds from the spiked sediment and dilution studies were 0.63 mg/kg normalized to 1% total organic carbon (mg/kg1%OC) for H. azteca and 1.4 mg/kg1%OC for L. plumulosus. Aquatic LC50s for 10 species and a measured acute,chronic ratio from the published literature were used to predict a distribution of sediment effects thresholds for HCBD, with 10th and 90th percentile values of 2.6 and 45 mg/kg1%OC, respectively. The predicted and observed sediment effects thresholds thus agreed relatively well, although the H. azteca and L. plumulosus test results from this study seem to be somewhat more conservative than the majority of published aquatic toxicity test results. [source] Statistical tests and power analysis for three in-vivo bioassays to determine the quality of marine sedimentsENVIRONMETRICS, Issue 3 2002Nelly van der Hoeven Abstract Statistical tests are recommended for three marine sediment in-vivo bioassays. In two bioassays (Corophium volutator and Echinocardium cordatum), the mortality in the sediment is compared with that in a control. An unconditional 2,×,2 test is recommended. For one bioassay (Rotoxkit MTM with Brachionus plicatilis), mortality in a dilution series of pore water is compared with the mortality in a control. The Williams test for trends is recommended. For each of these tests the power to assess an effect has been calculated. The number of replicates recommended in the standardized test protocol only allows large effects to be observed in almost all (95 per cent) of the experiments. Given the control mortality rates estimated from a large set of controls, a power of 95 per cent will only be reached if the mortality rate in the tested sediment is over 30 per cent for C. volutator and almost 60 per cent for E. cordatum. To reach this power for bioassays with B. plicatilis, where five concentrations are compared with a control, the mortality rate in the lowest effect concentration should be about 35 per cent. As an alternative to no effect testing, it is suggested that whether the effect of a treatment remains below some chosen minimal relevant effect (MRE) should be tested. Given an MRE at a fixed mortality rate of 25 per cent and ,,=,0.05, at least 55 individuals are necessary to be reasonably sure (95 per cent) that a mortality of 10 per cent will not be declared toxic incorrectly. The tests for mortality are based on the assumption that the survival probabilities of individuals within a test vessel are independent. We have described a method to test this assumption and applied it to the data on C. volutator. Copyright © 2002 John Wiley & Sons, Ltd. [source] Direct examination of soil for sporangia of Synchytrium endobioticum using chloroform, calcium chloride and zinc sulphate as extraction reagentsEPPO BULLETIN, Issue 1 2005G. C. M. Van Leeuwen Fields infested with Synchytrium endobioticum can be descheduled when the soil is found free from sporangia of S. endobioticum. For direct examination, EPPO Standard PM 3/59 describes a soil extraction technique based on the use of a sieve shaker with six sieves. We compared recovery of sporangia between this (modified) method and an extraction method employed by the Dutch Plant Protection Service (PPS method). Recovery was determined using an inoculum dilution series: 125, 25, 5, 1, 0.2 or 0.04 sporangia per g soil. Extraction reagents used were chloroform and calcium chloride in the method described by EPPO, calcium chloride and zinc sulphate in the PPS method. At 125 sporangia per g soil, the mean density determined for the modified EPPO method was 228 sporangia per g soil when chloroform was used. Using calcium chloride, recovery percentage was higher for the modified EPPO method than for the PPS method (286, 136%, n.s. P < 0.05). The advantage of the modified EPPO method was the larger soil volume to be processed; its disadvantages were use of complex equipment and noxious reagents (chloroform). Both extraction methods showed high variation in recovery between samples, making accurate estimation of sporangial densities in soil awkward. [source] Temperature dependence of Fe(III) and sulfate reduction rates and its effect on growth and composition of bacterial enrichments from an acidic pit lake neutralization experimentGEOBIOLOGY, Issue 4 2005J. MEIER ABSTRACT Microbial Fe(III) and sulfate reduction are important electron transport processes in acidic pit lakes and stimulation by the addition of organic substrates is a strategy to remove acidity, iron and sulfate. This principle was applied in a pilot-scale enclosure in pit lake 111 (Brandenburg, Germany). Because seasonal and spatial variation of temperature may affect the performance of in situ experiments considerably, the influence of temperature on Fe(III) and sulfate reduction was investigated in surface sediments from the enclosure in the range of 4,28 °C. Potential Fe(III) reduction and sulfate reduction rates increased exponentially with temperature, and the effect was quantified in terms of the apparent activation energy Ea measuring 42,46 kJ mol,1 and 52 kJ mol,1, respectively. Relatively high respiration rates at 4 °C and relatively low Q10 values (,2) indicated that microbial communities were well adapted to low temperatures. In order to evaluate the effect of temperature on growth and enrichment of iron and sulfate-reducing bacterial populations, MPN (Most Probable Number) dilution series were performed in media selecting for the different bacterial groups. While the temperature response of specific growth rates of acidophilic iron reducers showed mesophilic characteristics, the relatively high specific growth rates of sulfate reducers at the lowest incubation temperature indicated the presence of moderate psychrophilic bacteria. In contrast, the low cell numbers and low specific growth rates of neutrophilic iron reducers obtained in dilution cultures suggest that these populations play a less significant role in Fe and S cycling in these sediments. SSCP (Single-Strand Conformation Polymorphism) or DGGE (Denaturing Gradient Gel Electrophoresis) fingerprinting based on 16S rRNA genes of Bacteria indicated different bacterial populations in the MPN dilution series exhibiting different temperature ranges for growth. [source] Social Stress Alters Expression of Large Conductance Calcium-Activated Potassium Channel Subunits in Mouse Adrenal Medulla and Pituitary GlandsJOURNAL OF NEUROENDOCRINOLOGY, Issue 3 2009O. Chatterjee Large conductance calcium-activated potassium (BK) channels are very prominently expressed in adrenal chromaffin and many anterior pituitary cells, where they shape intrinsic excitability complexly. Stress- and sex-steroids regulate alternative splicing of Slo-,, the pore-forming subunit of BK channels, and chronic behavioural stress has been shown to alter Slo splicing in tree shrew adrenals. In the present study, we focus on mice, measuring the effects of chronic behavioural stress on total mRNA expression of the Slo-, gene, two key BK channel , subunit genes (,2 and ,4), and the ,STREX' splice variant of Slo-,. As a chronic stressor, males of the relatively aggressive SJL strain were housed with a different unfamiliar SJL male every 24 h for 19 days. This ,social-instability' paradigm stressed all individuals, as demonstrated by reduced weight gain and elevated corticosterone levels. Five quantitative reverse transcriptase-polymerase chain assays were performed in parallel, including ,-actin, each calibrated against a dilution series of its corresponding cDNA template. Stress-related changes in BK expression were larger in mice tested at 6 weeks than 9 weeks. In younger animals, Slo-, mRNA levels were elevated 44% and 116% in the adrenal medulla and pituitary, respectively, compared to individually-housed controls. ,2 and ,4 mRNAs were elevated 162% and 194% in the pituitary, but slightly reduced in the adrenals of stressed animals. In the pituitary, dominance scores of stressed animals correlated negatively with , and , subunit expression, with more subordinate individuals exhibiting levels that were three- to four-fold higher than controls or dominant individuals. STREX variant representation was lower in the subordinate subset. Thus, the combination of subunits responding to stress differs markedly between adrenal and pituitary glands. These data suggest that early stress will differentially affect neuroendocrine cell excitability, and call for detailed analysis of functional consequences. [source] Detection of Puccinia striiformis in Latently Infected Wheat Leaves by Nested Polymerase Chain ReactionJOURNAL OF PHYTOPATHOLOGY, Issue 7-8 2009Xiaojie Wang Abstract Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most devastating wheat diseases worldwide, especially in temperate regions with cool moist weather conditions. The identification of the pathogen in infected plants based on morphological or physiological criteria before sporulation is labour-intensive and time-consuming. To accelerate and simplify the process of detection, a nested Polymerase Chain Reaction (PCR) assay was developed for specific and sensitive detection of Pst. Specific primers Psta-Psts were designed according to a genome-specific sequence of Pst. In nested PCR, with a 10-fold dilution series of template DNA, the detection limit was 2 pg DNA in the first PCR with the primers Psta-Psts. The second round PCR was then performed using amplified products from the first PCR as the template and Nesta-Nests as the primers. An amplification signal was detectable even when only 2 fg of P. striiformis f. sp. tritici DNA was used as the template in nested PCR. With nested PCR, the sensitivity of detection was enhanced 1000 fold. Using extracts from stripe rust-infected wheat leaves, the fungus could be determined in the leaves before symptom appearance. The assay provides a rapid and sensitive method for detection of P. striiformis f. sp. tritici in latently infected leaves of overwintering wheat plants. [source] Resolution of trisomic mosaicism in prenatal diagnosis: estimated performance of a 50K SNP microarrayPRENATAL DIAGNOSIS, Issue 13 2007Jillian Cross Abstract Objective To evaluate the ability of a DNA single nucleotide polymorphism (SNP) microarray to detect chromosome mosaicism for trisomy in prenatal samples in order to compare this with conventional cytogenetics. Method We created a dilution series of mock mosaic samples, by mixing measured amounts of fibroblast cells containing trisomy 8 from a male with aliquots of cells with a normal female karyotype. DNAs were extracted from these mosaic mixtures, then analysed on the Affymetrix 50K Xba SNP chip. Duplicate aliquots of each mosaic sample were probed using interphase FISH, with centromeric probes for chromosomes X, Y and 8, to estimate independently the proportion of male trisomy 8 in each sample. Data from the arrays were analysed using publicly available analysis tools. Statistical calculations were then performed using a Student's t -test to determine if there was a significant difference between the copy numbers of each chromosome. Results These experiments using the Affymetrix 50K Xba SNP microarray showed mosaicism to be obvious at 20% and with additional statistical calculations, the lower limit for detection is about 10%. Conclusion The SNP microarray platform tested can detect mosaicism for trisomy in prenatal samples at levels comparable with conventional cytogenetic techniques in routine use. Copyright © 2007 John Wiley & Sons, Ltd. [source] Methyldibromoglutaronitrile in rinse-off products causes allergic contact dermatitis: an experimental studyBRITISH JOURNAL OF DERMATOLOGY, Issue 1 2004C.D. Jensen Summary Background The frequency of sensitivity to the cosmetic preservative methyldibromoglutaronitrile (MDBGN) has increased significantly in Europe. Most cases of allergic contact dermatitis from MDBGN are caused by leave-on cosmetic products. The risk of developing allergic contact dermatitis from rinse-off products has been less studied. Objectives To investigate the allergic response elicited in presensitized individuals from exposure to a rinse-off product preserved with the maximum permitted level of MDBGN. Methods Nineteen contact allergic individuals and nine controls participated in a double-blind, randomized repeated open application test (ROAT) using two coded liquid soaps with and without MDBGN. Areas of 50 cm2 on the lower arms were washed with the soaps twice a day for up to 28 days; two of the subjects continued for 34 days. The subjects were also patch tested with a dilution series of MDBGN to determine their patch test threshold values. Results Seven presensitized individuals (37%) developed allergic contact dermatitis from the soap containing MDBGN. The mean dose of MDBGN per application was 2·2 µg cm,2 and the reactions appeared between days 6 and 34. All nine controls had negative ROATs. The difference in reactivity between test subjects and controls was significant (one-sided Fisher's exact test, P = 0·04). Patch test threshold values ranged from <,0·001% to 0·2% MDBGN in ethanol/water. Conclusions This study shows that the exposure to a rinse-off product containing the maximum permitted level of MDBGN can easily elicit an allergic response in presensitized individuals. Along with reported cases of induction and elicitation caused by MDBGN in rinse-off products the study indicates that the permitted level of MDBGN in rinse-off products is too high. We recommend that this level should be re-evaluated. [source] | |||||||||||||