Evidence of complementarity between targeted and non-targeted analysis based on liquid and gas-phase chromatography coupled to mass spectrometry for screening halogenated persistent organic pollutants in environmental matrices.

This study explored the complementarity between targeted (TS) and non-targeted screening (NTS) based on liquid and gas-phase chromatography coupled to (high-resolution) mass spectrometry (LC-/GC-(HR)MS) for the comprehensive characterization of organohalogen fingerprints within a set of Lake Ontario lake trout samples. The concentrations of 86 legacy, emerging and novel halogenated compounds (HCs), were determined through 4 TS approaches involving no less than 6 hyphenated systems. In parallel, an innovative NTS strategy, involving both LC and GC-Q-Orbitrap, was implemented to specifically highlight halogenated signals. Non-targeted HRMS data were processed under the HaloSeeker software based on Cl and Br isotopic ratio and mass defect to extend the screening to unsuspected and unknown HCs. A total of 195 halogenated mass spectral features were characterized in the Lake Ontario lake trout, including well known HCs (PCBs, PBDEs, PBBs, DDT and their degradation products), emerging HCs (novel brominated flame retardants, short-, medium- and long-chain chlorinated paraffins) or suggested molecular formula (mainly polychlorinated ones). Among the 122 HCs highlighted by TS, only 21 were identified by NTS. These results fueled a discussion on the potential and limitations of both approaches, and the current position of NTS within environmental and health monitoring programs.

Delayed Slater determinant update algorithms for high efficiency quantum Monte Carlo.

Within ab initio Quantum Monte Carlo simulations, the leading numerical cost for large systems is the computation of the values of the Slater determinants in the trial wavefunction. Each Monte Carlo step requires finding the determinant of a dense matrix. This is most commonly iteratively evaluated using a rank-1 Sherman-Morrison updating scheme to avoid repeated explicit calculation of the inverse. The overall computational cost is, therefore, formally cubic in the number of electrons or matrix size. To improve the numerical efficiency of this procedure, we propose a novel multiple rank delayed update scheme. This strategy enables probability evaluation with an application of accepted moves to the matrices delayed until after a predetermined number of moves, K. The accepted events are then applied to the matrices en bloc with enhanced arithmetic intensity and computational efficiency via matrix-matrix operations instead of matrix-vector operations. This procedure does not change the underlying Monte Carlo sampling or its statistical efficiency. For calculations on large systems and algorithms such as diffusion Monte Carlo, where the acceptance ratio is high, order of magnitude improvements in the update time can be obtained on both multi-core central processing units and graphical processing units.

A model of technical variation of microarray signals.

We describe a mathematical model of signal from single-channel direct hybridization microarray platforms. The model establishes a linear relationship between microarray signals and their standard deviations from a minimum set of assumptions. We use the model to precisely define important microarray quality characteristics: resolved fold change and dynamic range. The definitions lead to closed form expressions relating these characteristics to physical parameters of the microarray experiment in the case when both specific and nonspecific binding of target to probe are governed by the Langmuir hybridization isotherm. The predictions of the model are in close agreement to data obtained from spike-in experiments. Given the generality of the model, the introduced definitions of dynamic range and resolved concentration fold-change can be used to conduct cross-platform comparisons and to guide improvement of the microarray platform.

Effects of chlorinated solvents on four species of North American amphibians.

Tetrachloroethylene (PCE), a dry cleaning and degreasing solvent, can enter groundwater through accidental leaks or spills, and concentrations as high as 75 mg/L have been reported in Canadian aquifers. Amphibians in wetlands receiving contaminated groundwater may be exposed to PCE and its degradation products, but little information is available on the impacts of these compounds on indigenous amphibian species. Acute (96-h static renewal) exposures to PCE and its major degradation products, trichloroethylene (TCE) and cisand trans-dichloroethylene, were conducted on embryos of four North American amphibian species: wood frogs (Rana sylvatica), green frogs (R. clamitans), American toads (Bufo americanus), and spotted salamanders (Ambystoma maculatum). Subsequently, chronic exposures to PCE and TCE were conducted with the larvae of American toads. Both PCE and TCE were teratogenic to amphibian embryos; median effective concentrations (EC50s) for developmental deformities produced by PCE and TCE exposure for wood frogs and green frogs were 12 and 40 mg/L, respectively. Embryonic survivorship, however, was not compromised at these concentrations. American toads were less sensitive; the EC50 for developmental abnormalities was not attained at the highest test concentrations, 45 and 85 mg/L PCE and TCE, respectively. These results are pertinent in assessing the impact of groundwater pollution on an aquifer-fed wetland.

New approaches for validation of lethal phenotypes and genetic reversion in Helicobacter pylori.

BACKGROUND: Because of limited genetic tools for use in Helicobacter pylori, tests routinely applied in other bacteria for demonstrating a gene's role in viability and other phenotypes have not been applied to this organism. In a mutational study of putative response regulator genes, we aimed to develop such tools for H. pylori. MATERIALS AND METHODS: We attempted to mutate five response regulator genes by allelic exchange insertional mutagenesis. For genes that yielded no viable mutants, a second copy of the gene was inserted into the chromosome via a suicide vector, and it was seen if providing the second copy would permit the gene's disruption. For genes that yielded mutants with selectable phenotypes, a strategy was developed for reversion whereby an intact copy of the gene is introduced to the organism by transformation with PCR products. Following this procedure, revertants were selected by phenotypic tests then tested for genetic reversion. RESULTS: After failure to attain transformants upon attempted mutation of genes HP0166 and HP1365, we inserted a second copy of each gene within the H. pylori chromosome. In each case the second copy relieved the block of transformation. Mutation of genes HP0703 and HP1021 gave non-motile and small-colony phenotypes, respectively. Following transformation with PCR products containing intact copies of the genes, both phenotype and genotype had reverted following phenotypic selections. CONCLUSIONS: The methods used in this study provide new approaches for confirming suspected genotype/phenotype associations and should be widely applicable in the study of H. pylori.

Effects of sonic toothbrush use on permanent dental luting cements.

The aim of this project was to assess the effects of sonic toothbrushes on commonly used permanent luting cements. While results showed differences between the tensile bond strengths of the three cements, the differences were similar between the two groups: sonic and nonsonic toohbrush exposure. These findings suggest that the sonic toothbrush had no significant effect on the tensile bond strengths of any of the three tested cements.

Assessment of rates of deformity in wild frog populations using in situ cages: a case study of Leopard Frogs (Rana pipiens) in Ontario, Canada.

High rates of deformity in wild amphibian populations from north-eastern North America have been increasingly reported since 1995. In the St Lawrence River basin (Canada) elevated frequencies of limb and eye deformities in mudpuppies (Necturus maculosus) and leopard frogs (Rana pipiens) were recorded in the early 1990s. A caging study was conducted during 1998 to verify the rates recorded in leopard frogs and pursue the potential causes of deformities seen in juveniles and adults. Week-old leopard frog tadpoles were collected from a reference wetland and maintained through to metamorphosis in cages in previously identified high risk wetlands. Deformity frequencies were measured and compared with frequencies measured in wild populations of leopard frogs inhabiting the same wetlands. The results of caging studies and sampling of wild populations were also compared with corresponding data collected from a reference wetland. No deformities were observed in caged or wild reference animals. Very low deformity frequencies (up to 2.2%) were observed in frogs caged in high risk wetlands, but greater frequencies (3.4-10%) were observed in wild young-of-the-year frogs captured at the same sites. The types of deformities were similar among groups; they included fused, missing or extra digits and disproportionate hindlimb length or eye pupil size. In addition, mortality rates were elevated in two cages in high risk wetlands. In general, the caging procedure was effective in establishing the potential for production of deformities in the waters of a given wetland, but tended to underestimate the rates calculated for samples of wild populations. The ramifications of the first-year findings for similar assessments of amphibian deformity rates and establishment of cause-effect linkages are discussed.

A whole-genome microarray reveals genetic diversity among Helicobacter pylori strains.

Helicobacter pylori colonizes the stomach of half of the world's population, causing a wide spectrum of disease ranging from asymptomatic gastritis to ulcers to gastric cancer. Although the basis for these diverse clinical outcomes is not understood, more severe disease is associated with strains harboring a pathogenicity island. To characterize the genetic diversity of more and less virulent strains, we examined the genomic content of 15 H. pylori clinical isolates by using a whole genome H. pylori DNA microarray. We found that a full 22% of H. pylori genes are dispensable in one or more strains, thus defining a minimal functional core of 1281 H. pylori genes. While the core genes encode most metabolic and cellular processes, the strain-specific genes include genes unique to H. pylori, restriction modification genes, transposases, and genes encoding cell surface proteins, which may aid the bacteria under specific circumstances during their long-term infection of genetically diverse hosts. We observed distinct patterns of the strain-specific gene distribution along the chromosome, which may result from different mechanisms of gene acquisition and loss. Among the strain-specific genes, we have found a class of candidate virulence genes identified by their coinheritance with the pathogenicity island.

The locus of enterocyte effacement (LEE)-encoded regulator controls expression of both LEE- and non-LEE-encoded virulence factors in enteropathogenic and enterohemorrhagic Escherichia coli.

Regulation of virulence gene expression in enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) is incompletely understood. In EPEC, the plasmid-encoded regulator Per is required for maximal expression of proteins encoded on the locus of enterocyte effacement (LEE), and a LEE-encoded regulator (Ler) is part of the Per-mediated regulatory cascade upregulating the LEE2, LEE3, and LEE4 promoters. We now report that Ler is essential for the expression of multiple LEE-located genes in both EPEC and EHEC, including those encoding the type III secretion pathway, the secreted Esp proteins, Tir, and intimin. Ler is therefore central to the process of attaching and effacing (AE) lesion formation. Ler also regulates the expression of LEE-located genes not required for AE-lesion formation, including rorf2, orf10, rorf10, orf19, and espF, indicating that Ler regulates additional virulence properties. In addition, Ler regulates the expression of proteins encoded outside the LEE that are not essential for AE lesion formation, including TagA in EHEC and EspC in EPEC. delta ler mutants of both EPEC and EHEC show altered adherence to epithelial cells and express novel fimbriae. Ler is therefore a global regulator of virulence gene expression in EPEC and EHEC.

Effects of intermittent UVA exposure on cultured lens epithelial cells.

PURPOSE: This paper addresses the question of whether a single non-lethal dose of UVA radiation or the same dose divided three daily one-third doses have like or unlike effects on the growth and the catalase activity of cultured rabbit epithelial (RLE) and immortalized human epithelial (HLE) cells. METHODS: Non-confluent cultured RLE and HLE cells were used to study the effects of UVA radiation on their growth. The effects of three doses of 3 J/cm(2) given one day apart on cell growth (i.e. live cell number) over a three day period were compared with those of a single 9 J/cm(2 ) exposure. Estimation of live cell numbers was done 24 hrs after each exposure by counting trypan blue exclusive cells using a hemocytometer. Confluent cultures of RLE cells were used to study the effects of UVA radiation on their catalase activity. The effects of three doses of 1.25 J/cm(2) each given one day apart on catalase activity (breakdown of H( 2) O( 2) measured spectro-photometrically at 240 nm) were compared with that of a single 3.75 J/cm(2) exposure. RESULTS: A single 9 J/cm(2) dose of UVA reduced the cell growth to only 10% of controls after 24 hrs. By three days after a single 9 J/cm( 2) exposure, the number of live cells was only 70% of controls. Three intermittent exposures of 3 J/cm(2) each for three consecutive days reduced the cell number to 76% of controls. Similar results were obtained for HLE cells. Little if any recovery of cell numbers occurred after three intermittent exposures. A single dose of 3.7 J/cm(2) reduced RLE catalase activity to 20% of controls by one day. By three days after exposure, catalase activity returned to 90% of controls. After three intermittent exposures to 1.25 J/cm(2) each, over three days, RLE catalase activity was reduced to 75% of the controls. There appeared to be no recovery within two additional days. CONCLUSIONS: While single below lethal doses of UVA allow no recovery of RLE or HLE cell growth, partial recovery does occur after several daily intermittent exposures. Recovery of the catalase activity by RLE cells does occur after single sub lethal exposures, but intermittent exposures allow no recovery. The recovery of lens epithelial cell growth and catalase activity from UVA damage varies with the exposure regimen.

Sequence anomalies in the Cag7 gene of the Helicobacter pylori pathogenicity island.

The severity of Helicobacter pylori-related disease is correlated with a pathogenicity island (the Cag region of about 26 genes) whose presence is associated with the up-regulation of an IL-8 cytokine inflammatory response in gastric epithelial cells. Statistical analysis of the Cag gene sequences calculated from the complete genome of strain 26695 revealed several unusual features. The Cag7 sequence (1,927 aa) has two repeat regions. Repeat region I runs 317 aa in a form of AAA proximal to the protein N terminal; repeat region II extends 907 aa in the middle of the protein sequence consisting of 74 contiguous segments composed from selections among six consensus sequences and includes 58 regularly distributed cysteine residues with consecutive cysteines mostly 12, 18, or 24 aa apart. This "regular" cysteine arrangement may provide a scaffolding of linker elements stabilized by disulfide bridges. When Cag7 homologues from different strains are compared, differences were found almost exclusively in the repeat regions, resulting from deletion and/or insertion of repeating units. These observations suggest that the anomalous repetitive structure of the sequence plays an important role in the conformation of Cag7 gene product and potentially in the function of the pathogenicity island. Other facets of the Cag7 sequence show significant charge clusters, high multiplet count, and extremes of amino acid usage.

Comparing periodontal disease in identical twins: a case report.

Previous investigators have shown that numerous environmental and genetic variables may contribute to the pathogenesis of periodontal disease. This case report presents clinical and laboratory findings of a set of Caucasian female identical twins. One patient presented clinically with mild gingivitis and no clinical or radiographic signs of periodontitis. The other exhibited gingivitis with localized, moderate-to-severe periodontitis. Neither patient reported a history of systemic conditions that might influence their periodontal health, and neither presented other known risk factors, such as tobacco use. The only apparent variable was related to their oral hygiene. The periodontally involved patient exhibited higher plaque scores than her twin in all clinical visits. Subgingival plaque cultures revealed the presence of Porphyromonas gingivalis and Bacteroides forsythus only in the diseased twin. Both patients had low colony counts of Prevotella intermedia and Eikenella corrodens, but only the healthy twin harbored small quantities of Fusobacterium nucleatum. This case report offers an opportunity to assess etiology of periodontitis in two genetically identical patients whose only obvious difference was their oral hygiene.

Intraocular transplantation of E1A-immortalized retinal precursor cells.

The purpose of this study was to examine the effect of the ocular environment on the survival, tumorigenicity, and phenotypic marker expression of immortalized retinal precursor cells transplanted into immunocompetent adult and neonatal Sprague-Dawley rats. EIA-NR.3, a rat immortalized retinal precursor cell culture, was used as an inexhaustible source of experimental graft material. These cells were prelabeled with the fluorescent marker dil (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) and transplanted intravitreally (50,000 cells per microL) into 11 adult and 31 neonatal Sprague-Dawley rat eyes. At 1 mo posttransplant, animals were sacrificed and retinal tissue sections examined histologically for the presence of grafted cells, signs of tumor formation, and retinal phenotypic marker expression. No obvious signs of tumor formation or rejection were seen in a total of 42 eyes in the immunocompetent hosts. Our results indicate that EIA-NR.3 cells survive at least 1 month in vivo, and can migrate from the vitreous into neuroretinal cell layers. Subpopulations of surviving grafted cells were seen to express photoreceptor markers rhodopsin and recoverin comparably between in vitro and in vivo conditions. However, the number of cells immunoreactive for vimentin and E1A decreased significantly under in vivo conditions. This report represents the first experimental intravitreal transplantation of E1A-immortalized retinal precursor cells into adult and neonatal rats. The intraocular location and environment appears to affect phenotypic expression of surviving grafted cells, especially with respect to vimentin and E1A expression. The fact that E1A-NR.3 cells survived intraocularly at least 1 mo without tumor formation suggests that the cells may continue to be useful for further in vivo studies of experimental retinal transplantation, and effects of histological location on retinal cell phenotype and histogenesis in immunocompetent hosts.

Ice incubation step allows adherence of archival histological specimens on gelatin-coated slides for TUNEL staining.

Gelatin-coated slides provide poor tissue adherence during histological procedures which require 37 degreesC incubations, such as terminal deoxynucleotidyl transferase nick-end labelling (TUNEL) analysis. We encountered this difficulty during attempts to analyze archival ocular tissue sections which had been previously sectioned and mounted on gelatin-coated slides. The solution to this problem turned out to be relatively straightforward: Immediately after the 37 degreesC terminal deoxynucleotide transferase step, we incubated the slides on ice for 30 min before continuing with the remainder of the protocol. This ice-incubation step re-solidified the melted gelatin, which allowed for continued adherence of the tissue specimen for further manipulations. This modified TUNEL staining protocol has been used successfully to analyze 14 archival specimens thus far, which would have been nearly impossible to accomplish otherwise. We believe that this ice re-solidification step for gelatin-coated slides has broad applications for procedures which require 37 degreesC incubations, including TUNEL staining, as well as other in situ hybridization and immunohistochemistry protocols.

Insertion site of the locus of enterocyte effacement in enteropathogenic and enterohemorrhagic Escherichia coli differs in relation to the clonal phylogeny of the strains.

The locus of enterocyte effacement pathogenicity island confers the attaching and effacing histopathology on epithelial cells infected with enteropathogenic and enterohemorrhagic Escherichia coli. We investigated the site of insertion of the locus of enterocyte effacement in E. coli strains in relation to their evolution based on conservation of housekeeping proteins in these strains. The results indicate that the insertion site of the locus of enterocyte effacement varies according to the evolutionary lineage, suggesting that it has inserted at multiple times and sites during the evolution of these pathogens.

Effects of toothbrush design and brushing proficiency on plaque removal.

A single-blind, short-term cross-over clinical trial compared the plaque removal performance of three commercially available manual toothbrushes. A sample of 25 dental hygiene students, 19 to 42 years old, served as participants. On 3 separate occasions, participants were instructed to refrain from toothbrushing or flossing for 24 hours before clinical trials. A prebrushing plaque index using disclosing solution was performed on each participant. One of the 3 test brushes was then randomly dispensed to each participant, and they were allowed to brush for 90 seconds without the aid of a mirror. A postbrushing plaque index was then performed on each participant. This procedure was repeated 2 more times at 2-week intervals so that each participant was tested with all 3 toothbrushes. Previous studies of this kind using random participant samples have suggested that brush design does indeed affect the efficacy of plaque removal. This study used participants who were well versed on efficient toothbrushing technique to determine if improved brushing skills would overshadow advantages in toothbrush design. No significant differences in performance were detected between the test brushes for any of the three scored areas.

Son preference and the sex ratio at birth in China: a provincial level analysis.

In this paper we use data from the 1 Per Cent Sample of the 1990 Census of China to calculate sex ratios for the Chinese provinces that are specific not only to parity, but also to the sex composition of previously-born children. We analyze the degree of relationship among and between the SRB's and then consider the phenomenon of son preference. We find the variation in sex ratios at birth among the Chinese provinces is related in an important way to variation among the provinces in the degree of son preference. Our analysis shows abnormally high SRB's in most of the provinces of China, especially at parities 2 and higher when the prior births were daughters. Other societies, namely Taiwan and South Korea, with rapid fertility decline and strong son preference also manifest abnormally high SRB's at parities beyond the first.

A cloned pathogenicity island from enteropathogenic Escherichia coli confers the attaching and effacing phenotype on E. coli K-12.

Attaching and effacing (AE) bacteria are a diverse group of gastrointestinal pathogens, comprising members of four genera, that cause the intestinal epithelial microvilli to be replaced with raised clusters of filamentous actin that conform to the surface of attached bacteria. We have cloned a 35.4 kb 'pathogenicity island' from the prototype AE bacterium, enteropathogenic Escherichia coli, containing all previously described AE genes. Transfer of this pathogenicity island to avirulent E. coli converts the recipients into strains that secrete virulence proteins, induce host signal-transduction pathways, and cause AE lesions on cultured epithelial cells. These results demonstrate that this pathogenicity island contains all pathogen-specific genes necessary for inducing AE lesions, and that the defining feature of this class of pathogens can be acquired by an avirulent bacterium in a single genetic step.

Cloning of the RDEC-1 locus of enterocyte effacement (LEE) and functional analysis of the phenotype on HEp-2 cells.

EPEC Escherichia coli are an important cause of epidemic diarrhea in infants. The disease is characterized by attaching and effacing (A/E) lesions where the bacteria attach intimately to the enterocyte surface resulting in localized destruction of microvilli. A 35-kb chromosomal locus termed LEE (locus of enterocyte effacement) in an EPEC strain (E2348/69) has recently been found and is thought to contain all the necessary genes for A/E lesions. RDEC-1 is a strain of E. coli that causes diarrhea in rabbits by a similar mechanism and serves as a model for human EPEC disease. We report 1) the cloning of the RDEC-1 LEE, 2) show that the RDEC-1 LEE is similar in size to the LEE in E2348/69, 3) the RDEC-1 LEE possesses all four regions of the E2348/69 LEE, 4) there are restriction site polymorphisms between the RDEC-1 LEE and that of E2348/69, and 5) the RDEC-1 LEE clone is functionally similar to E2348/69 in its fluorescent actin staining test and suggests that the LEE may be sufficient for the production of A/E lesions by these strains.