Comparison of PCR and clinical laboratory tests for diagnosing <Emphasis Type="Italic">H. pylori</Emphasis> infection in pediatric patients

Background

Histology and/or culture are generally considered the gold standard for the detection of H. pylori infection. Especially in children, these tests may result in a false negative outcome because of patchy distribution of the organism in the stomach mucosa. We have developed a PCR assay utilizing nested primer pairs directed against a subunit of the H. pylori urease gene (ureA). As part of a prospective evaluation of diagnostic tests to aid in detecting H. pylori infection in children, the aim of this study was to compare our PCR and Western blot assays with results obtained from histologic examination of biopsy specimens, rapid urease tests, and an FDA approved serologic assay and published PCR results to determine if we could validate the assays for diagnostic use on our patient population.

Results

Gastric biopsy specimens obtained from 101 pediatric patients were evaluated for the presence of H. pylori using histologic techniques, rapid urease (CLOtest) test and the PCR assay. Serum samples from each patient were assayed using both ELISA and Western Blot for antibodies to H. pylori. A total of 32 patients tested were positive by at least one of the methods evaluated. Thirteen patients had positive histology, 13 had a positive CLOtest, and 17 patients had positive H. pylori PCR. Out of the 13 CLO positive patients, 12 were positive by histologic analysis and all 13 were positive by PCR. Results of serologic tests on the same population did not correlate well with other assays. Twenty-eight patients showed serologic evidence of H. pylori infection, of which 9 were both CLO and histology positive and 12 were positive by PCR. Of the seropositive patients, 26 were ELISA positive, 13 were positive by Western blot, and 11 by both serologic methods.

Conclusions

The results obtained suggest that our nested PCR assay has the specificity and sensitivity necessary for clinical application when compared to standard histologic examination and rapid urease test. In addition, we found the current commercially available approved ELISA method appears unable to accurately detect H. pylori in this population. The Western blot assay yielded better concordance with CLOtest and histology, but not as good as the nested PCR assay.

     

Multiple male traits interact: attractive bower decorations facilitate attractive behavioural displays in satin bowerbirds

Sexually selected male courtship displays often involve multiple behavioural and physical traits, but little is known about the function of different traits in mate choice. Here, we examine female courtship behaviours to learn how male traits interact to influence female mating decisions. In satin bowerbirds (Ptilonorhynchus violaceus), successful males give highly aggressive, intense behavioural displays without startling females. Males do this by modulating their displays in response to female crouching, which signals the display intensity that females will tolerate without being startled. Females typically visit multiple males for multiple courtships before choosing a mate, and females show differing tolerance for intense displays during their first courtship with each male. We test three hypotheses that may explain this: (i) familiarity with the courting male; (ii) the order of the courtship in mate-searching; and (iii) the attractiveness of the courting male. We found that females are more tolerant of intense displays during first courtships with attractive males; this increased female tolerance may allow attractive males to give higher intensity courtship displays that further enhance their attractiveness. We then examined why this is so, finding evidence that females are less likely to be startled by males with better physical displays (bower decorations), and this reduced startling then contributes to male courtship success. This role of physical displays in facilitating behavioural displays suggests a novel mechanism by which multiple physical and behavioural traits may influence female choice.     

An obligatory intermediate in the folding pathway of cytochrome c552 from Hydrogenobacter thermophilus

The folding mechanism of many proteins involves the population of partially organized structures en route to the native state. Identification and characterization of these intermediates is particularly difficult, as they are often only transiently populated and may play different mechanistic roles, being either on-pathway productive species or off-pathway kinetic traps. Following different spectroscopic probes, and employing state-of-the-art kinetic analysis, we present evidence that the folding mechanism of the thermostable cytochrome c552 from Hydrogenobacter thermophilus does involve the presence of an elusive, yet compact, on-pathway intermediate. Characterization of the folding mechanism of this cytochrome c is particularly interesting for the purpose of comparative folding studies, because H. thermophilus cytochrome c552 shares high sequence identity and structural homology with its homologue from the mesophilic bacterium Pseudomonas aeruginosa cytochrome c551, which refolds through a broad energy barrier without the accumulation of intermediates. Analysis of the folding kinetics and correlation with the three-dimensional structure add new evidence for the validity of a consensus folding mechanism in the cytochrome c family.     

Modulation of triple-helical stability and subsequent melanoma cellular responses by single-site substitution of fluoroproline derivatives

Collagen is a multifunctional protein, serving as a structural scaffold and a modulator of cellular responses. Prior work has identified distinct regions from several collagen types that promote cell adhesion, spreading, migration, and signal transduction. One of these regions, alpha1(IV)1263-1277 from type IV collagen, mediates these responses via melanoma cell CD44-chondrotin sulfate proteoglycan receptors. In the study presented here, we have used a triple-helical model of alpha1(IV)1263-1277 to evaluate (a) conformational stability and (b) cellular responses based on single-site incorporation of trans-4-fluoro-L-proline (trans-Flp) or cis-4-fluoro-L-proline (cis-Flp) for trans-4-hydroxy-L-proline (trans-Hyp). The structural effects of cis-Flp and trans-Flp substitution were studied by circular dichroism and NMR spectroscopies. The peptide containing a single trans-Flp instead of trans-Hyp was slightly more thermally stable than the parent peptide (T(m) = 37 vs 34 degrees C), while the peptide containing cis-Flp was considerably less stable than the parent peptide (T(m) = 30 degrees C). Melanoma cell adhesion and spreading were examined under conditions where the trans-Hyp-, trans-Flp-, and cis-Flp-containing ligands were approximately 15, <10, and approximately 65% denatured, respectively. Adhesion to each of the three ligands was remarkably sensitive to the respective ligand conformation, with EC(50) values of approximately 2.5, approximately 0.35, and >5.0 microM for the trans-Hyp-, trans-Flp-, and cis-Flp-containing ligands, respectively. Melanoma cell spreading was quantitated over a ligand concentration range of 0.01-50 microM and, in a fashion similar to adhesion, was more extensive on the trans-Flp ligand than on the trans-Hyp ligand. Very low levels of spreading were observed with the cis-Flp-containing ligand at all concentrations tested. Melanoma cell adhesion to and spreading on the three ligands suggested the dramatic biological consequence of even subtle changes in relative triple-helical content. Such subtle changes may model those occurring in the basement membrane during the tumor cell invasion process, and thus provide mechanistic insight into this stage of metastasis.     

Ultrastructural study of the yeast-mycelium transition in Histoplasma capsulatum. II. Changes at 34 degrees C

The ultrastructural changes which occur during the mycelium to yeast transition in Histoplasma capsulatum induced by a temperature shift from 25 degrees C to 34 degrees C are described and compared to those observed after a temperature shift from 25 degrees C to 37 degrees C. 24 hours after the temperature shift to 34 degrees C only 8% of the cells are lysed. However, many mitochondria have lost their characteristic elongated form and have become rounded. Vesicular cristae which are no longer oriented parallel to the long axis of the mitochondria are also observed. In contrast a temperature shift from 25 degrees C to 37 degrees C induces lysis of 70% of the cells; mitochondria are rarely observed in the remaining cells. These ultrastructural changes can be correlated with the uncoupling of oxidative phosphorylation and the production of heat shock proteins.     

Charge effect on the photoinactivation of Gram-negative and Gram-positive bacteria by cationic meso-substituted porphyrins

Background  

In recent times photodynamic antimicrobial therapy has been used to efficiently destroy Gram (+) and Gram (-) bacteria using cationic porphyrins as photosensitizers. There is an increasing interest in this approach, namely in the search of photosensitizers with adequate structural features for an efficient photoinactivation process. In this study we propose to compare the efficiency of seven cationic porphyrins differing in meso-substituent groups, charge number and charge distribution, on the photodynamic inactivation of a Gram (+) bacterium (Enterococcus faecalis) and of a Gram (-) bacterium (Escherichia coli). The present study complements our previous work on the search for photosensitizers that might be considered good candidates for the photoinactivation of a large spectrum of environmental microorganisms.     

Female preferences drive the evolution of mimetic accuracy in male sexual displays

Males in many bird species mimic the vocalizations of other species during sexual displays, but the evolutionary and functional significance of interspecific vocal mimicry is unclear. Here we use spectrographic cross-correlation to compare mimetic calls produced by male satin bowerbirds (Ptilonorhynchus violaceus) in courtship with calls from several model species. We show that the accuracy of vocal mimicry and the number of model species mimicked are both independently related to male mating success. Multivariate analyses revealed that these mimetic traits were better predictors of male mating success than other male display traits previously shown to be important for male mating success. We suggest that preference-driven mimetic accuracy may be a widespread occurrence, and that mimetic accuracy may provide females with important information about male quality. Our findings support an alternative hypothesis to help explain a common element of male sexual displays.     

Characterization of the minimum domain required for targeting budding yeast myosin II to the site of cell division

Background  

All eukaryotes with the exception of plants use an actomyosin ring to generate a constriction force at the site of cell division (cleavage furrow) during mitosis and meiosis. The structure and filament forming abilities located in the C-terminal or tail region of one of the main components, myosin II, are important for localising the molecule to the contractile ring (CR) during cytokinesis. However, it remains poorly understood how myosin II is recruited to the site of cell division and how this recruitment relates to myosin filament assembly. Significant conservation between species of the components involved in cytokinesis, including those of the CR, allows the use of easily genetically manipulated organisms, such as budding yeast (Saccharomyces cerevisiae), in the study of cytokinesis. Budding yeast has a single myosin II protein, named Myo1. Unlike most other class II myosins, the tail of Myo1 has an irregular coiled coil. In this report we use molecular genetics, biochemistry and live cell imaging to characterize the minimum localisation domain (MLD) of budding yeast Myo1.