A bifunctional kinase-phosphatase in bacterial chemotaxis.

addresses: Oxford Centre for Integrative Systems Biology and Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom.notes: PMCID: PMC2587623types: Journal Article; Research Support, Non-U.S. Gov'tCopyright © 2008, The National Academy of SciencesPhosphorylation-based signaling pathways employ dephosphorylation mechanisms for signal termination. Histidine to aspartate phosphosignaling in the two-component system that controls bacterial chemotaxis has been studied extensively. Rhodobacter sphaeroides has a complex chemosensory pathway with multiple homologues of the Escherichia coli chemosensory proteins, although it lacks homologues of known signal-terminating CheY-P phosphatases, such as CheZ, CheC, FliY or CheX. Here, we demonstrate that an unusual CheA homologue, CheA(3), is not only a phosphodonor for the principal CheY protein, CheY(6), but is also is a specific phosphatase for CheY(6)-P. This phosphatase activity accelerates CheY(6)-P dephosphorylation to a rate that is comparable with the measured stimulus response time of approximately 1 s. CheA(3) possesses only two of the five domains found in classical CheAs, the Hpt (P1) and regulatory (P5) domains, which are joined by a 794-amino acid sequence that is required for phosphatase activity. The P1 domain of CheA(3) is phosphorylated by CheA(4), and it subsequently acts as a phosphodonor for the response regulators. A CheA(3) mutant protein without the 794-amino acid region lacked phosphatase activity, retained phosphotransfer function, but did not support chemotaxis, suggesting that the phosphatase activity may be required for chemotaxis. Using a nested deletion approach, we showed that a 200-amino acid segment of CheA(3) is required for phosphatase activity. The phosphatase activity of previously identified nonhybrid histidine protein kinases depends on the dimerization and histidine phosphorylation (DHp) domains. However, CheA(3) lacks a DHp domain, suggesting that its phosphatase mechanism is different from that of other histidine protein kinases

Body composition in Nepalese children using isotope dilution: the production of ethnic-specific calibration equations and an exploration of methodological issues.

Background. Body composition is important as a marker of both current and future health. Bioelectrical impedance (BIA) is a simple and accurate method for estimating body composition, but requires population-specific calibration equations. Objectives. (1) To generate population specific calibration equations to predict lean mass (LM) from BIA in Nepalese children aged 7-9 years. (2) To explore methodological changes that may extend the range and improve accuracy. Methods. BIA measurements were obtained from 102 Nepalese children (52 girls) using the Tanita BC-418. Isotope dilution with deuterium oxide was used to measure total body water and to estimate LM. Prediction equations for estimating LM from BIA data were developed using linear regression, and estimates were compared with those obtained from the Tanita system. We assessed the effects of flexing the arms of children to extend the range of coverage towards lower weights. We also estimated potential error if the number of children included in the study was reduced. Findings. Prediction equations were generated, incorporating height, impedance index, weight and sex as predictors (R (2) 93%). The Tanita system tended to under-estimate LM, with a mean error of 2.2%, but extending up to 25.8%. Flexing the arms to 90° increased the lower weight range, but produced a small error that was not significant when applied to children <16 kg (p 0.42). Reducing the number of children increased the error at the tails of the weight distribution. Conclusions. Population-specific isotope calibration of BIA for Nepalese children has high accuracy. Arm position is important and can be used to extend the range of low weight covered. Smaller samples reduce resource requirements, but leads to large errors at the tails of the weight distribution

Two- and three-point functions in two-dimensional Landau-gauge Yang-Mills theory: Continuum results

We investigate the Dyson-Schwinger equations for the gluon and ghost propagators and the ghost-gluon vertex of Landau-gauge gluodynamics in two dimensions. While this simplifies some aspects of the calculations as compared to three and four dimensions, new complications arise due to a mixing of different momentum regimes. As a result, the solutions for the propagators are more sensitive to changes in the three-point functions and the ansaetze used for them at the leading order in a vertex a expansion. Here, we therefore go beyond this common truncation by including the ghost-gluon vertex self-consistently for the first time, while using a model for the three-gluon vertex which reproduces the known infrared asymptotics and the zeros at intermediate momenta as observed on the lattice. A separate computation of the three-gluon vertex from the results is used to confirm the stability of this behavior a posteriori. We also present further arguments for the absence of the decoupling solution in two dimensions. Finally, we show how in general the infrared exponent kappa of the scaling solutions in two, three and four dimensions can be changed by allowing an angle dependence and thus an essential singularity of the ghost-gluon vertex in the infrared.Comment: 24 pages; added references, improved choices of parameters for vertex models; identical to version published in JHE

Two-week dietary soy supplementation has an estrogenic effect on normal premenopausal breast.

An association has been reported between consumption of a high soy diet and a low incidence of breast cancer within populations of Southeast Asia. Phytoestrogens present in soy act as partial estrogen agonists or antagonists and can inhibit breast cancer cell proliferation in vitro. The effect of 14-day dietary soy supplementation with 60 g (45 mg isoflavones) on the normal breast of 84 premenopausal patients was determined. Serum concentrations of the isoflavanoids, genistein, daidzein, and equol, were raised in patients after soy supplementation (P < or = 0.025). Nipple aspirate (NA) levels of genistein and daidzein were higher than paired serum levels, both before (P < 0.001 and P = 0.001, respectively) and after soy supplementation (P < 0.001 and P = 0.049, respectively); however, there was no significant increase in NA isoflavone levels in response to soy. NA levels of apolipoprotein D were significantly lowered and pS2 levels raised in response to soy supplementation (P < or = 0.002), indicative of an estrogenic stimulus. No effect of soy supplementation on breast epithelial cell proliferation, estrogen and progesterone receptor status, apoptosis, mitosis, or Bcl-2 expression was detected. In conclusion, short term dietary soy has a weak estrogenic response on the breast, as measured by nipple aspirate apolipoprotein D and pS2 expression. No antiestrogenic effect of soy on the breast was detected

Tailored biocompatible polyurethane-poly(ethylene glycol) hydrogels as a versatile nonfouling biomaterial

Polyurethane-based hydrogels are relatively inexpensive and mechanically robust biomaterials with ideal properties for various applications, including drug delivery, prosthetics, implant coatings, soft robotics, and tissue engineering. In this report, a simple method is presented for synthesizing and casting biocompatible polyurethane-poly(ethylene glycol) (PU-PEG) hydrogels with tunable mechanical properties, nonfouling characteristics, and sustained tolerability as an implantable material or coating. The hydrogels are synthesized via a simple one-pot method using commercially available precursors and low toxicity solvents and reagents, yielding a consistent and biocompatible gel platform primed for long-term biomaterial applications. The mechanical and physical properties of the gels are easily controlled by varying the curing concentration, producing networks with complex shear moduli of 0.82–190 kPa, similar to a range of human soft tissues. When evaluated against a mechanically matched poly(dimethylsiloxane) (PDMS) formulation, the PU-PEG hydrogels demonstrated favorable nonfouling characteristics, including comparable adsorption of plasma proteins (albumin and fibrinogen) and significantly reduced cellular adhesion. Moreover, preliminary murine implant studies reveal a mild foreign body response after 41 days. Due to the tunable mechanical properties, excellent biocompatibility, and sustained in vivo tolerability of these hydrogels, it is proposed that this method offers a simplified platform for fabricating soft PU-based biomaterials for a variety of applications

A comparison of transgenic rodent mutation and in vivo comet assay responses for 91 chemicals.

A database of 91 chemicals with published data from both transgenic rodent mutation (TGR) and rodent comet assays has been compiled. The objective was to compare the sensitivity of the two assays for detecting genotoxicity. Critical aspects of study design and results were tabulated for each dataset. There were fewer datasets from rats than mice, particularly for the TGR assay, and therefore, results from both species were combined for further analysis. TGR and comet responses were compared in liver and bone marrow (the most commonly studied tissues), and in stomach and colon evaluated either separately or in combination with other GI tract segments. Overall positive, negative, or equivocal test results were assessed for each chemical across the tissues examined in the TGR and comet assays using two approaches: 1) overall calls based on weight of evidence (WoE) and expert judgement, and 2) curation of the data based on a priori acceptability criteria prior to deriving final tissue specific calls. Since the database contains a high prevalence of positive results, overall agreement between the assays was determined using statistics adjusted for prevalence (using AC1 and PABAK). These coefficients showed fair or moderate to good agreement for liver and the GI tract (predominantly stomach and colon data) using WoE, reduced agreement for stomach and colon evaluated separately using data curation, and poor or no agreement for bone marrow using both the WoE and data curation approaches. Confidence in these results is higher for liver than for the other tissues, for which there were less data. Our analysis finds that comet and TGR generally identify the same compounds (mainly potent mutagens) as genotoxic in liver, stomach and colon, but not in bone marrow. However, the current database content precluded drawing assay concordance conclusions for weak mutagens and non-DNA reactive chemicals

Biological and technical variables affecting immunoassay recovery of cytokines from human serum and simulated vaginal fluid: A multicenter study

The increase of proinflammatory cytokines in vaginal secretions may serve as a surrogate marker of unwanted inflammatory reaction to microbicide products topically applied for the prevention of sexually transmitted diseases, including HIV-1. Interleukin (IL)-1β and IL-6 have been proposed as indicators of inflammation and increased risk of HIV-1 transmission; however, the lack of information regarding detection platforms optimal for vaginal fluids and interlaboratory variation limit their use for microbicide evaluation and other clinical applications. This study examines fluid matrix variants relevant to vaginal sampling techniques and proposes a model for interlaboratory comparisons across current cytokine detection technologies. IL-1β and IL-6 standards were measured by 12 laboratories in four countries, using 14 immunoassays and four detection platforms based on absorbance, chemiluminescence, electrochemiluminescence, and fluorescence. International reference preparations of cytokines with defined biological activity were spiked into (1) a defined medium simulating the composition of human vaginal fluid at pH 4.5 and 7.2, (2) physiologic salt solutions (phosphate-buffered saline and saline) commonly used for vaginal lavage sampling in clinical studies of cytokines, and (3) human blood serum. Assays were assessed for reproducibility, linearity, accuracy, and significantly detectable fold difference in cytokine level. Factors with significant impact on cytokine recovery were determined by Kruskal−Wallis analysis of variance with Dunn’s multiple comparison test and multiple regression models. All assays showed acceptable intra-assay reproducibility; however, most were associated with significant interlaboratory variation. The smallest reliably detectable cytokine differences (P < 0.05) derived from pooled interlaboratory data varied from 1.5- to 26-fold depending on assay, cytokine, and matrix type. IL-6 but not IL-1β determinations were lower in both saline and phosphate-buffered saline as compared to vaginal fluid matrix, with no significant effect of pH. The (electro)chemiluminescence-based assays were most discriminative and consistently detected <2-fold differences within each matrix type. The Luminex-based assays were less discriminative with lower reproducibility between laboratories. These results suggest the need for uniform vaginal sampling techniques and a better understanding of immunoassay platform differences and cross-validation before the biological significance of cytokine variations can be validated in clinical trials. This investigation provides the first standardized analytic approach for assessing differences in mucosal cytokine levels and may improve strategies for monitoring immune responses at the vaginal mucosal interface

High-throughput amplicon sequencing reveals distinct communities within a corroding concrete sewer system

This study investigated the variation in microbially induced concrete corrosion communities at different circumferential locations of a real sewer pipe and the effects of a wastewater flooding event on the community. Three distinct microbial community groups were found in different corrosion samples. The physico-chemical properties of the corrosion layers and the microbial communities were distinct for the cross-sectional positions within the pipe, ie ceiling, wall and tidal zones. The microbial communities detected from the same positions in the pipe were consistent over the length of the pipe, as well as being consistent between the replicate pipes. The dominating ceiling communities were members of the bacterial orders Rhodospirillales, Acidithiobacillales, Actinomycetales, Xanthomonadales and Acidobacteriales. The wall communities were composed of members of the Xanthomonadales, Hydrogenophilales, Chromatiales and Sphingobacteriales. The tidal zones were dominated by eight bacterial and one archaeal order, with the common physiological trait of anaerobic metabolism. Sewage flooding within the sewer system did not change the tidal and wall communities, although the corrosion communities in ceiling samples were notably different, becoming more similar to the wall and tidal samples. This suggests that sewage flooding has a significant impact on the corrosion community in sewers